Tagungsband zur VAAM-Jahrestagung 2009 ... - bei der VAAM
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<strong>2009</strong><br />

Son<strong>der</strong>ausgabe<br />

BIO<br />

D13808F · ISSN 0947-0867<br />

www.biospektrum.de<br />

spektrum<br />

Das Magazin für Biowissenschaften<br />

<strong>Tagungsband</strong> <strong>zur</strong><br />

<strong>VAAM</strong>-<strong>Jahrestagung</strong> <strong>2009</strong><br />

Bochum, 8.–11. März <strong>2009</strong>

07.012 sign-berlin.de<br />

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Der LightMix ® Kit Norovirus erlaubt einen schnellen<br />

Nachweis <strong>der</strong> viralen RNA und eine Differenzierung<br />

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Vereinigung für Allgemeine und Angewandte Mikrobiologie (<strong>VAAM</strong>)<br />

<strong>Tagungsband</strong><br />

<strong>zur</strong> <strong>Jahrestagung</strong> <strong>2009</strong><br />

8. bis 11. März in Bochum<br />

Wissenschaftliche Leitung und Organisationskomitee<br />

Ruhr Universität Bochum:<br />

Ulrich Kück, Ralf Erdmann, Nicole Frankenberg-Dinkel, Sören Gatermann, Thomas Happe, Wolf-Hubert Kunau,<br />

Franz Narberhaus, Minou Nowrousian, Matthias Rögner<br />

3 Inhalt<br />

4 Grußwort des Rektors <strong>der</strong> Ruhr-Universität Bochum<br />

Elmar W. Weiler<br />

4 Grußwort des <strong>VAAM</strong>-Präsidenten<br />

Bernhard Hauer<br />

6 Grußwort des Organisationskomitees<br />

6 Einladung <strong>zur</strong> <strong>Jahrestagung</strong> <strong>der</strong> <strong>VAAM</strong> 2010<br />

8 Aus den Fachgruppen <strong>der</strong> <strong>VAAM</strong><br />

13 Instituts-Portrait: Mikrobiologie an <strong>der</strong> Ruhr-Universität in<br />

Bochum<br />

18 Einladung <strong>zur</strong> Mitglie<strong>der</strong>versammlung <strong>der</strong> <strong>VAAM</strong><br />

18 Mitteilungen des <strong>VAAM</strong>-Sekretariats<br />

19 General Information<br />

21 Unternehmen auf <strong>der</strong> Fachausstellung und Sponsoren<br />

22 Symposium by GATC Biotech AG<br />

22 Symposium by Eurofins MWG Operon<br />

23 Conference Program Annual Meeting <strong>2009</strong> of the <strong>VAAM</strong><br />

26 Mini-Symposia of the Special Groups<br />

31 Short Lectures<br />

39 Poster Overview<br />

40 How dead is dead? Survival and final inactivation of<br />

microorganisms<br />

42 Overview: Plenary Lectures, Special Groups, Mini Symposia, Short<br />

Lectures and Posters<br />

Plenary Lectures<br />

43 H Plenary Lectures<br />

Special Group Mini-Symposia and WorkshopPage<br />

46 FGA Biologie bakterieller Naturstoffproduzenten<br />

47 FGB Biotransformation<br />

47 FGC Functional Genomics<br />

47 FGD Fungal Biology and Biotechnology<br />

49 FGE Identification and Systematics<br />

50 FGF Microbial Pathogenicity<br />

51 FGG Regulation and Signalstransduction in Prokaryotes<br />

52 FGH Yeast<br />

Short Lectures<br />

53 KA Anaerobic Metabolism<br />

54 KB Archaea<br />

55 KC Cyanobacteria and Algae<br />

57 KD Developmental Microbiology<br />

58 KE Fungal Biology<br />

60 KF Green Biotechnology<br />

61 KG Host-Microbe Interactions<br />

62 KH Imaging Techniques in Microbiology<br />

63 KI Microbial Cell Biology<br />

65 KJ Microbial Communities<br />

66 KK Microbial Diversity<br />

68 KL Microbial Pathogens and Pathogenicity<br />

69 KM Photosynthesis and Bioenergetics<br />

71 KN Physiology<br />

72 KO Regulation<br />

73 KP Secondary Metabolism<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

74 KQ Sensory and Regulatory RNA<br />

75 KR Systems Biology<br />

77 KS Transport<br />

78 KT White Biotechnology<br />

80 KU Open Topics<br />

Posters<br />

81 PA Anaerobic Metabolism<br />

90 PB Archaea<br />

96 PC Special Group Biologie bakterieller Naturstoffproduzenten<br />

98 PD Special Group Biotransformation<br />

99 PE Cyanobacteria and Algae<br />

100 PF Developmental Microbiology<br />

101 PG Special Group Functional Genomics<br />

103 PH Fungal Biology<br />

110 PI Green Biotechnology<br />

111 PJ Host-Microbe Interactions<br />

116 PK Special Group Identification and Systematics<br />

117 PL Imaging Techniques in Microbiology<br />

117 PM Microbial Cell Biology<br />

122 PN Microbial Communities<br />

141 PO Microbial Diversity<br />

152 PP Microbial Pathogens and Pathogenicity<br />

161 PQ Photosynthesis and Bioenergetics<br />

164 PR Physiology<br />

170 PS Regulation<br />

183 PT Secondary Metabolism<br />

185 PU Sensory and Regulatory RNA<br />

188 PV Systems Biology<br />

190 PW Transport<br />

195 PX White Biotechnology<br />

205 PY Special Group Yeast<br />

206 PZ Open Topics<br />

How dead is dead<br />

218 HDID How dead is dead Lectures<br />

220 HDID P How dead is dead Posters<br />

224 Autorenverzeichnis (Author index)<br />

236 Personalia 2008<br />

238 Promotionen 2008 (PhD theses 2008)<br />

242 Impressum<br />

Zum Titelbild:<br />

From left to right:<br />

– Photobioreactor for the mass cultivation of cyanobacteria<br />

– Scanning electron micrograph of a conidiophor with conidiospores<br />

from Aspergillus niger<br />

– Structure of a bacterial RNA-thermometer<br />

– Purified phycoerythrin from Prochlorococcus marinus SS120,<br />

a highly fluorescent biliprotein<br />

3

4 GRUSSWORTE<br />

Grußwort des Rektors <strong>der</strong> Ruhr-Universität Bochum<br />

ó Herzlich Willkommen an <strong>der</strong> Ruhr-Universität!<br />

Ich freue mich, dass die Vereinigung<br />

für Allgemeine und Angewandte Mikrobiologie<br />

(<strong>VAAM</strong>) sich entschieden hat, ihre <strong>Jahrestagung</strong><br />

<strong>2009</strong> in Bochum stattfinden zu lassen.<br />

Sie sind zu Gast an einer <strong>der</strong> größten<br />

deutschen Universitäten – mit einem Fächerspektrum,<br />

das es in dieser Breite an kaum<br />

einer an<strong>der</strong>en deutschen Universität gibt. Der<br />

Campuscharakter <strong>der</strong> Ruhr-Universität macht<br />

es möglich, innerhalb weniger Minuten alle<br />

hier vertretenen Fachdisziplinen zu erreichen<br />

– beste Voraussetzungen für ein hohes Maß<br />

an Interdisziplinarität und fächerübergreifende<br />

Kooperationen.<br />

Im Jahre 2007 hat die Ruhr-Universität im<br />

Exzellenzwettbewerb des Bundes und <strong>der</strong><br />

Län<strong>der</strong> die Auszeichnung als „Exzellenzhochschule“<br />

nur äußerst knapp verfehlt. Dessen<br />

ungeachtet werden alle Anstrengungen<br />

unternommen, das Zukunftskonzept, mit dem<br />

sich die Universität in den Wettbewerb<br />

gestellt hat, möglichst ungeschmälert umzusetzen.<br />

Ziel ist es, die Ruhr-Universität in den<br />

nächsten Jahren dauerhaft und international<br />

sichtbar unter den großen forschungsstarken<br />

Universitäten zu etablieren.<br />

Gleichzeitig vollzieht sich am Ort <strong>der</strong> in den<br />

später 60-er und frühen 70-er Jahren größten<br />

Baustelle Europas ein erneut groß angelegtes<br />

Bauprojekt, die Campussanierung, die mit <strong>der</strong><br />

Errichtung eines neuen Gebäudes für die Ingenieurwissenschaften<br />

soeben ihren Anfang<br />

genommen hat. Am Ende des für einen Zeitraum<br />

von mehr als 10 Jahren geplanten Erneuerungsprozesses<br />

soll ein Campus stehen,<br />

<strong>der</strong> in seiner Funktionalität und Ausstrahlung<br />

unseren ehrgeizigen Vorstellungen <strong>der</strong> Universität<br />

als Ort von Forschung und Lehre in<br />

je<strong>der</strong> Hinsicht gerecht wird.<br />

Die Ruhr-Universität Bochum hat im Jahre<br />

2007 gemeinsam mit ihren Nachbarhochschulen,<br />

<strong>der</strong> Technischen Universität Dortmund<br />

und <strong>der</strong> Universität Duisburg-Essen,<br />

die „Universitätsallianz Metropole Ruhr“ ins<br />

Leben gerufen – <strong>der</strong> Verbund dieser (weiterhin<br />

selbstständigen) Universitäten liegt in<br />

seiner Größenordnung mit mehr als 90.000<br />

Studierenden und einer in ihrer Bündelung<br />

herausragenden Forschungsstärke auf dem<br />

Niveau <strong>der</strong> größten deutschen und europäischen<br />

Hochschullandschaften.<br />

Die Fakultät für Biologie und Biotechnologie<br />

gehört zu den Leistungsträgern dieser<br />

Universität; dies gilt in beson<strong>der</strong>em Maße für<br />

den Bereich <strong>der</strong> Mikrobiologie mit ihrer Beteiligung<br />

an mehreren Son<strong>der</strong>forschungsbereichen,<br />

einem DFG-Schwerpunktprogramm,<br />

sowie weiteren BMBF- und EU-Projekten. Beispielhaft<br />

ist hier, wie in <strong>der</strong> gesamten Fakultät,<br />

das hohe Maß an Vernetzung mit angrenzenden<br />

Disziplinen; beson<strong>der</strong>s hervorzuheben<br />

ist schließlich die exzellente Nachwuchsför<strong>der</strong>ung<br />

<strong>der</strong> Fakultät. Die Graduiertenschule<br />

<strong>der</strong> Fakultät für Biologie und Biotechnologie<br />

hat Vorbildcharakter für die im<br />

Grußwort des <strong>VAAM</strong>-Präsidenten<br />

ó Zur unserer <strong>Jahrestagung</strong> in Bochum <strong>2009</strong><br />

begrüße ich Sie alle recht herzlich! Ein interessantes<br />

Programm, das ein breites Spektrum<br />

<strong>der</strong> mo<strong>der</strong>nen Mikrobiologie abdeckt, hat uns<br />

nach Bochum geführt. Viele Doktoranden und<br />

junge Wissenschaftler erhalten hier einen Einblick<br />

in die vielfältigen Fragen und Themen,<br />

die unter dem Dach <strong>der</strong> <strong>VAAM</strong> zusammenkommen.<br />

Vielleicht finden sie sogar ihr zukünftiges<br />

Ar<strong>bei</strong>tsfeld o<strong>der</strong> einen neuen Ar<strong>bei</strong>tsplatz.<br />

Beteiligen Sie sich vor allem an den Fachgruppen,<br />

denn unsere Gesellschaft lebt von Ihrem<br />

Engagement und Ihren Impulsen.<br />

Neben <strong>der</strong> Begeisterung für die Mikroorganismen<br />

und ihre Erforschung freuen wir<br />

uns natürlich immer, auf <strong>der</strong> <strong>Jahrestagung</strong><br />

alte Freunde und Kollegen wie<strong>der</strong>zusehen.<br />

Dieses kommunikative Element einer <strong>Jahrestagung</strong><br />

ist nicht zu unterschätzen. Es wäre<br />

nicht das erste Mal, dass aus diesen persönlichen<br />

Begegnungen neue Projektideen und<br />

Konzepte entstehen.<br />

Dem Organisationskomitee möchte ich<br />

bereits an dieser Stelle für die Organisation<br />

<strong>der</strong> Tagung herzlich danken. Dazu zählen<br />

neben Ulrich Kück und <strong>der</strong> Firma Conventus<br />

vor allem das wissenschaftliche Komitee und<br />

die unzähligen freiwilligen Helfer, die erstmals<br />

ein Poster präsentierenden Diplomanden<br />

bis hin zu weit angereisten hochgeschätzten<br />

Kollegen, die Fachgruppen, die<br />

Geschäftsstelle und viele mehr. Wie im letzten<br />

Jahr bewährt, wird auch <strong>2009</strong> die Tagung<br />

von einem öffentlichen Vortrag eröffnet. Es<br />

wird eine unserer Aufgaben für die Zukunft<br />

sein, die Öffentlichkeit vermehrt an unserer<br />

Forschung und ihren Ergebnissen teilhaben<br />

zu lassen. Zum zweiten Mal wird <strong>der</strong> <strong>VAAM</strong>-<br />

Jahre 2006 in <strong>der</strong> ersten För<strong>der</strong>linie <strong>der</strong> Exzellenzinitiative<br />

erfolgreiche „Research School“<br />

gehabt, die bundesweit einzige Graduiertenschule,<br />

die das gesamte Fächerspektrum <strong>der</strong><br />

Universität umfasst.<br />

Ein beson<strong>der</strong>es Merkmal des diesjährigen<br />

Tagungsprogramms, das sich auch in <strong>der</strong><br />

Zusammensetzung des Organisationskomitees<br />

nie<strong>der</strong>schlägt, ist die Beteiligung <strong>der</strong> Medizinischen<br />

Fakultät <strong>der</strong> Ruhr-Universität mit ihrer<br />

hervorragend aufgestellten mikrobiologischen<br />

Abteilung – die Kooperation mit <strong>der</strong> Fakultät<br />

für Biologie und Biotechnologie (und darüber<br />

hinaus auch mit vielen an<strong>der</strong>en Fakultäten)<br />

ist für mich ein lebendiges Beispiel für die eingangs<br />

angesprochene interdisziplinäre Orientierung<br />

auf unserem Campus.<br />

Dem gesamten Organisationskomitee,<br />

namentlich dem Tagungspräsidenten, Herrn<br />

Prof. Dr. Kück, möchte ich für die Vorbereitung<br />

dieses „Großereignisses“ und allen denjenigen,<br />

die für einen reibungslosen Ablauf<br />

<strong>der</strong> Veranstaltung sorgen, sehr herzlich danken.<br />

Den Teilnehmerinnen und Teilnehmern<br />

<strong>der</strong> <strong>Jahrestagung</strong> <strong>der</strong> <strong>VAAM</strong> wünsche ich<br />

einen guten Aufenthalt in Bochum, eine<br />

erfolgreiche Tagung mit regem Austausch und<br />

anregenden Diskussionen. Ich freue mich,<br />

Sie am 8. März <strong>2009</strong> im Audimax <strong>der</strong> Ruhr-<br />

Universität begrüßen zu dürfen. ó<br />

Prof. Dr. Elmar W. Weiler<br />

Rektor <strong>der</strong> Ruhr-Universität Bochum<br />

Forschungspreis verliehen, <strong>der</strong> nicht zuletzt<br />

auch einem breiten Publikum die Bedeutung<br />

<strong>der</strong> mo<strong>der</strong>nen Mikrobiologie verdeutlicht.<br />

Dieses Jahr erwartet uns zudem ein einschneidendes<br />

Ereignis: Jan und Marion<br />

Andreesen, Gründungsmitglie<strong>der</strong> <strong>der</strong> ersten<br />

Stunde, scheiden nach über 30 Jahren aus<br />

dem aktiven Management <strong>der</strong> <strong>VAAM</strong> aus. Wie<br />

kein an<strong>der</strong>er haben sie unserer Gesellschaft<br />

ein Gesicht gegeben, sie geprägt, geleitet und<br />

zu dem gemacht, was die <strong>VAAM</strong> heute ist. Sie<br />

kennen alles und jeden, sind in <strong>der</strong> Mikrobiologie<br />

verwurzelt und ihr von ihrer Historie<br />

bis heute eng verbunden. Im Namen <strong>der</strong> ganzen<br />

<strong>VAAM</strong> vielen Dank für den unermüdlichen<br />

Einsatz und alles Gute für die Zukunft!<br />

Uns allen möge ihr Engagement und ihr<br />

soziales Miteinan<strong>der</strong> ein Vorbild sein. ó<br />

Prof. Dr. Bernhard Hauer<br />

<strong>VAAM</strong>-Präsident<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

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6 GRUSSWORTE<br />

Grußwort des Organisationskomitees<br />

ó Wir begrüßen Sie ganz herzlich als Teilnehmer<br />

<strong>der</strong> diesjährigen <strong>Jahrestagung</strong> <strong>der</strong><br />

Vereinigung für Allgemeine und Angewandte<br />

Mikrobiologie (<strong>VAAM</strong>) in Bochum. Die<br />

Ruhr-Universität Bochum, die größte universitäre<br />

Neugründung nach 1945, stellt eine<br />

klassische Campusuniversität dar, auf <strong>der</strong>en<br />

Gelände <strong>der</strong> Kongress unter Einbeziehung<br />

des Auditorium Maximum stattfindet. Die<br />

sehr große Zahl <strong>der</strong> angemeldeten Beiträge<br />

verspricht eine Rekordteilnehmerzahl, und<br />

es ist <strong>der</strong> Wunsch <strong>der</strong> Organisatoren, dass die<br />

Plenarvorträge <strong>der</strong> international renommierten<br />

Sprecherinnen und Sprecher sowie<br />

die zahlreichen Kurzvorträge und umfangreichen<br />

Posterpräsentationen vor allem auch<br />

junge Teilnehmer von <strong>der</strong> Faszination <strong>der</strong><br />

mikrobiologischen Forschung überzeugen.<br />

Die diesjährigen Tagungsthemen reflektieren<br />

weitgehend die mikrobiologischen<br />

Schwerpunkte, welche an <strong>der</strong> Ruhr-Universität<br />

vertreten sind. Die Grundlagenforschung<br />

wird exemplarisch durch die Themen „Sensory<br />

and Regulatory RNA“, „Cyanobacteria<br />

and Algae“, „Microbial Cell Biology“, „Fun-<br />

gal Biology“ und „Photosynthesis and Bioenergetics“<br />

repräsentiert, während sich die<br />

angewandte Mikrobiologie in den Themen<br />

„Green Biotechnology“, „White Biotechnology“<br />

sowie „Host-Microbe Interactions“ und<br />

„Microbial Pathogens and Pathogenicity“<br />

wi<strong>der</strong>spiegelt.<br />

Die Bochumer Mikrobiologie ist <strong>der</strong> Grundlagenforschung<br />

und <strong>der</strong> Biotechnologie unter<br />

Verwendung einer breiten Palette von Organismen<br />

gleichermaßen zugewandt. Neben<br />

Prokaryoten – dies schließt die photosynthetischen<br />

Cyanobakterien ein – werden in vielen<br />

Ar<strong>bei</strong>tsgruppen Hefen, Hyphenpilze und<br />

Algen als Versuchsorganismen eingesetzt.<br />

Diese Breite findet sich in zahlreichen Forschungsschwerpunkten<br />

an <strong>der</strong> Ruhr-Universität<br />

wie<strong>der</strong>, wie zum Beispiel in den Son<strong>der</strong>forschungsbereichen<br />

SFB 480 („Molekulare<br />

Biologie komplexer Leistungen von botanischen<br />

Systemen“, Sprecher U. Kück) und SFB<br />

642 („GTP- und ATP-abhängige Membranprozesse“,<br />

Sprecher K. Gerwert), sowie durch<br />

die Beteiligung an einem DFG-Schwerpunktprogramm<br />

„Sensorische und regulatorische<br />

RNAs in Prokaryoten“ (Koordinator F. Narberhaus).<br />

Die biotechnologisch orientierte<br />

Mikrobiologie wird unter an<strong>der</strong>em durch das<br />

BMBF-Verbundprojekt BioH2 (Projektleiter<br />

M. Rögner) und das EU-Projekt SOLAR-H2,<br />

sowie durch das Christian-Doppler Labor „Biotechnologie<br />

<strong>der</strong> Pilze“ (Projektleiter U. Kück)<br />

repräsentiert.<br />

Wir wünschen Ihnen allen eine interessante<br />

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Besuche des Botanischen Gartens, <strong>der</strong> umfangreichen<br />

universitären Kunstsammlung,<br />

des Bergbaumuseums o<strong>der</strong> des Bermuda-<br />

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8 AUS DEN FACHGRUPPEN DER <strong>VAAM</strong><br />

Fachgruppe: Archaea<br />

ó Die Fachgruppe traf sich im letzten Herbst<br />

in Schmitten <strong>bei</strong> Frankfurt zu einer Tagung<br />

zum Thema „Genome function and Gene regulation<br />

in Archaea“. Die Tagung wurde von Jörg<br />

Soppa organisiert (s. Bericht in BIOspektrum<br />

07/2008, S. 760 ).<br />

Mitglie<strong>der</strong> <strong>der</strong> Fachgruppe Archaea treffen<br />

sich auf <strong>der</strong> <strong>VAAM</strong>-Tagung in Bochum<br />

am Montag, den 09.03.<strong>2009</strong>; 17:30 Uhr,<br />

Raum HZO 80.<br />

Tagesordnungspunkte:<br />

– Diskussion gemeinsamer Forschungsaktivitäten<br />

– Wahl eines Sprechers o<strong>der</strong> einer Sprecherin<br />

ó<br />

Fachgruppe: Biotransformationen<br />

ó Die 1996 gegründete Fachgruppe „Biotransformationen“<br />

umfasst <strong>zur</strong> Zeit ca. 140<br />

Mitglie<strong>der</strong> aus Universitäten, Forschungseinrichtungen<br />

und <strong>der</strong> chemischen Industrie.<br />

Primäres Ziel <strong>der</strong> Fachgruppe ist es, einen<br />

regelmäßigen Kontakt und Austausch zwischen<br />

deutschen und europäischen Ar<strong>bei</strong>tsgruppen,<br />

die auf dem Gebiet <strong>der</strong> Biokatalyse<br />

tätig sind, zu gewährleisten und gemeinsame<br />

Fortbildungsveranstaltungen durchzuführen.<br />

Diese sind ebenso wie die Fachgruppe,<br />

an <strong>der</strong> sich neben Mikrobiolog/inn/en<br />

auch Chemiker/innen und Ingenieur/e/innen<br />

aktiv beteiligen, i.d.R. interdisziplinär ausgerichtet.<br />

Die wachsende Nachfrage nach ökonomischen,<br />

ökoeffizienten und ressourcenschonenden<br />

Prozessen in <strong>der</strong> Chemie-, Pharma-,<br />

Energie- und Lebensmittelindustrie erfor<strong>der</strong>t<br />

verstärkte Anstrengungen, Forschungsergebnisse<br />

in Produkte umzusetzen. An<br />

biokatalytischen Verfahren führt da<strong>bei</strong><br />

kein Weg vor<strong>bei</strong>, sie bilden die Grundlage<br />

<strong>der</strong> Industriellen Biotechnologie. Das Forschungsgebiet<br />

erlebt <strong>zur</strong> Zeit ein rasantes<br />

Wachstum, das <strong>der</strong> neu eingerichtete<br />

Gemeinschaftsausschuss „Biotransformation“,<br />

getragen von <strong>der</strong> <strong>VAAM</strong> und <strong>der</strong><br />

DECHEMA, durch die Adressierung folgen<strong>der</strong><br />

<strong>bei</strong>den Oberthemen weiter beschleunigen<br />

möchte: zum einen das Auffinden neuer<br />

Biokatalysatoren (Mikroorganismen und<br />

Enzyme) mittels Metagenomanalysen, High-<br />

Throughput-Systeme, Assayentwicklung,<br />

Expressionssysteme und zum an<strong>der</strong>en die<br />

Optimierung von Biokatalysatoren mittels<br />

Stammoptimierung und Protein-Design. Den<br />

Vorsitz des Gemeinschaftsausschusses führen<br />

Andreas Liese (TU Hamburg-Harburg)<br />

und Christoph Syldatk (Univ. Karlsruhe).<br />

Neben gemeinsamen Fachtagungen sollen<br />

beson<strong>der</strong>s junge Forscher durch Sommer-<br />

Fachgruppe: Funktionelle Genomanalyse<br />

ó Der 1998 gegründeten Fachgruppe Funktionelle<br />

Genomanalyse gehören über 200<br />

<strong>VAAM</strong>-Mitglie<strong>der</strong> an. Sie dient als Forum für<br />

den Austausch von Informationen über<br />

Methoden, Techniken, Verfahren und Ergebnissen<br />

mikrobieller Genomforschung. Das<br />

inhaltliche Themenspektrum <strong>der</strong> Fachgruppe<br />

umfasst da<strong>bei</strong> alle wesentlichen Technologien<br />

und Verfahren <strong>zur</strong> Entschlüsselung <strong>der</strong> Genfunktion<br />

aus DNA-Sequenzinformation, z. B.<br />

Bioinformatik, High-throughput Screening,<br />

Molecular Arrays, Protein Expression Profiling,<br />

Proteomics und Pathway Analysis. Der<br />

wissenschaftlicher Austausch in <strong>der</strong> Fachgruppe<br />

findet vorwiegend auf den Mini-Symposien<br />

<strong>bei</strong> den <strong>VAAM</strong>-<strong>Jahrestagung</strong>en statt. In<br />

diesem Jahr ist die Fachgruppe bereits zum<br />

10. Mal mit einem Mini-Symposium <strong>bei</strong> <strong>der</strong><br />

<strong>Jahrestagung</strong> vertreten. Nach dem sehr gut<br />

besuchen Symposium zum Thema ‚Microbial<br />

Pathway Genomics and Systems Biology’ <strong>bei</strong><br />

<strong>der</strong> vorigen <strong>Jahrestagung</strong> in Frankfurt, hat<br />

die Fachgruppe in diesem Jahr drei international<br />

bekannte Sprecher zum Thema ‚Evolution<br />

of the information processing machinery’<br />

nach Bochum eingeladen, die in ihren<br />

komplementären Vorträgen die Entwicklung<br />

<strong>der</strong> Informationsprozessierungssysteme in<br />

Archaea, Bakterien und Eukaryonten darstellen<br />

werden.<br />

Die Mitglie<strong>der</strong> <strong>der</strong> Fachgruppe sowie Interessenten<br />

an <strong>der</strong> künftigen Gestaltung <strong>der</strong><br />

Sprecherin:<br />

Felicitas Pfeifer,<br />

Universität Darmstadt<br />

Email: pfeifer@bio.tu-darmstadt.de<br />

schulen und spezielle Symposien für Doktoranden,<br />

Habilitanden und Juniorprofessoren<br />

geför<strong>der</strong>t werden. Ein Positionspapier,<br />

das gemeinsam mit dem DECHEMA/GVC-<br />

Gemeinschaftsausschuss Bioprozesstechnik<br />

erar<strong>bei</strong>tet werden soll, wird die Perspektiven<br />

<strong>der</strong> Biokatalyse darstellen und Handlungsoptionen<br />

für Forschungs- und Wirtschaftspolitik<br />

identifizieren. ó<br />

Andreas Schmid,<br />

Universität Dortmund für<br />

alle Sprecher <strong>der</strong> FG Biotransformationen<br />

(Andreas Schmid,<br />

Christoph Syldatk,<br />

Jürgen Eck,<br />

Uwe Bornscheuer,<br />

Bernhard Hauer)<br />

Email: Andreas.Schmid@bci.tu-dortmund.de<br />

Fachgruppenveranstaltungen werden <strong>zur</strong><br />

unmittelbar nach dem Symposium stattfindenden<br />

Mitglie<strong>der</strong>versammlung eingeladen<br />

(Raum HZO 70), <strong>bei</strong> <strong>der</strong> turnusgemäß die<br />

Wahl <strong>der</strong> Fachgruppenvertreter für die nächsten<br />

zwei Jahre stattfinden wird. ó<br />

Sprecher:<br />

Hans-Peter Klenk, DSMZ,<br />

Braunschweig<br />

Email: hpk@dsmz.de<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Fachgruppe: Hefen<br />

ó Die Fachgruppe Hefen veranstaltete<br />

im Mai 2008 zusammen<br />

mit dem französischen Genolevure-Konsortium<br />

eine Tagung mit<br />

dem Titel „Yeast and Filamentous<br />

Fungi“ in Straßburg. Insgesamt<br />

100 Teilnehmer nahmen an Plenarvorträgen,<br />

Workshops und<br />

Poster-Ausstellung teil, darunter<br />

jeweils etwa 30 Mitglie<strong>der</strong> des<br />

Genolevure-Konsortiums und<br />

Teilnehmer aus dem deutschsprachigen<br />

Raum sowie rund 40<br />

Kollegen aus europäischen Län<strong>der</strong>n<br />

(s. BIOspektrum 05/2008,<br />

Seite 533).<br />

Ziele <strong>der</strong> Tagung waren,<br />

1. Genomforschung, physiologischer<br />

und zellbiologischer Forschung<br />

stärker zu vernetzen,<br />

2. die wichtige Stellung von<br />

Hefen und filamentösen Pilzen<br />

als eukaryotische Modellorganismen<br />

aufzuzeigen, und<br />

3. Nachwuchswissenschaftlern<br />

ein internationales Podium für<br />

die Darstellung ihrer Ergebnisse<br />

zu geben.<br />

Die Plenarvorträge und die Poster-Ausstellung<br />

wurden durch<br />

zwei Workshops ergänzt: „Cell<br />

biology of yeasts and filamentous<br />

fungi: Growth control and nuclear<br />

dynamics“, das Peter Philippsen,<br />

Basel, unterstützt von Gerhard<br />

Braus, Göttingen, organisierte<br />

und „The future of genome<br />

analysis“, das Bernard Dujon,<br />

Paris, und Claude Gaillardin, Triveral-Grignon,<br />

gestalteten. Beide<br />

Workshops wurden von den Teilnehmern<br />

gerne angenommen<br />

und in <strong>der</strong> Abschlussdiskussion<br />

als sehr erfolgreich bewertet.<br />

Die Tagung wurde gemeinsam<br />

von Karl-Dieter Entian, Frankfurt<br />

und Jean-Luc Suociet gestaltet<br />

und das Straßburger Team sorgte<br />

für eine hervorragende Bewirtung<br />

mit einem stimmungsvollen<br />

Abendessen am Freitag. Durch<br />

die finanzielle Unterstützung des<br />

Genolevure-Konsortiums, <strong>der</strong><br />

<strong>VAAM</strong> sowie Karl Singer Instruments,<br />

UK, und <strong>der</strong> SRD GmbH,<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Oberursel, ist es gelungen, die<br />

Teilnahmegebühren von 60 N für<br />

Studenten und 100 N für Wissenschaftler<br />

sehr niedrig zu halten.<br />

Die wissenschaftlich sehr interessante<br />

gemeinsame Tagung fand<br />

ein positive Resonanz und wird<br />

vom 28.–30. Mai <strong>2009</strong> erneut in<br />

Straßburg stattfinden.<br />

AUS DEN FACHGRUPPEN DER <strong>VAAM</strong><br />

Bei <strong>der</strong> <strong>VAAM</strong>-Tagung <strong>2009</strong> in<br />

Bochum wird die Fachgruppe ein<br />

Fachgruppensymposium durchführen<br />

zu dem als Gastredner<br />

Prof. Roland Lill, Universität Marburg,<br />

mit dem Thema: Biogenesis<br />

of iron-sulfur proteins in eukaryotes<br />

and its impact on iron<br />

homeostasis, eingeladen ist.<br />

Im Anschluss an das Fachgruppensymposium<br />

findet eine<br />

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Neuwahl <strong>der</strong> Fachgruppensprecher<br />

statt (Raum HZO 10). ó<br />

Sprecher:<br />

Karl-Dieter Entian,<br />

Universität<br />

Frankfurt<br />

Email:<br />

entian@bio.unifrankfurt.de<br />

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10 AUS DEN FACHGRUPPEN DER <strong>VAAM</strong><br />

Fachgruppe: Lebensmittelmikrobiologie<br />

ó Die Fachgruppe ar<strong>bei</strong>tet eng mit <strong>der</strong> Fachgruppe<br />

„Lebensmittelmikrobiologie und<br />

Lebensmittelhygiene“ <strong>der</strong> DGHM zusammen.<br />

Die Mitglie<strong>der</strong> kommen aus Universitäten,<br />

Fachhochschulen, Forschungseinrichtungen<br />

des Bundes und <strong>der</strong> Län<strong>der</strong>, Industrie, Untersuchungsämtern<br />

und privaten Beratungsfirmen.<br />

Die Hauptaktivität <strong>der</strong> Fachgruppe<br />

besteht in <strong>der</strong> Organisation eines zweitägigen<br />

jährlich stattfindenden Fachsymposiums,<br />

welches abwechselnd von <strong>der</strong> Fachgruppe <strong>der</strong><br />

<strong>VAAM</strong> und <strong>der</strong> DGHM ausgerichtet wird. Das<br />

10. Fachsymposium Lebensmittelmikrobiologie<br />

richtete die DGHM-Fachgruppe vom 9.–<br />

11. April 2008 in <strong>der</strong> GENO-Akademie Stuttgart<br />

aus (s. BIOspektrum 4/2008, S. 413). An<br />

<strong>der</strong> Tagung nahmen 142 Lebensmittelmikrobiologen<br />

aus dem gesamten deutschsprachigen<br />

Raum teil. Im Rahmen des Symposiums<br />

wurde <strong>der</strong> Preis <strong>der</strong> Seliger-Stiftung 2008 an<br />

Herbert Schmidt, Universität Hohenheim, für<br />

seine herausragenden wissenschaftlichen<br />

Tätigkeiten, insbeson<strong>der</strong>e auf dem Gebiet <strong>der</strong><br />

Pathogenität von Escherichia coli, verliehen.<br />

Die Bandbreite <strong>der</strong> 40 Vorträge reichte von<br />

<strong>der</strong> Grundlagenforschung über angewandte<br />

Routineanalytik bis zu Präsentationen aus<br />

<strong>der</strong> diagnostischen Industrie.<br />

Für das Jahr <strong>2009</strong> ist vom 22.–24. Juni das<br />

11. Fachsymposium in Wildbad Kreuth<br />

geplant, welches dann wie<strong>der</strong> von <strong>der</strong> <strong>VAAM</strong>-<br />

Fachgruppe ausgerichtet wird. ó<br />

Sprecher:<br />

Siegfried Scherer,<br />

Zentralinstitut für<br />

Ernährungs- und Lebensmittelforschung<br />

(ZIEL) <strong>der</strong><br />

TU München<br />

Email: Siegfried.Scherer@wzw.tum.de<br />

Fachgruppe: Regulation und Signaltransduktion in Prokaryoten<br />

ó Die Fachgruppe „Regulation und Signaltransduktion<br />

in Prokaryoten“ organisiert während<br />

<strong>der</strong> <strong>VAAM</strong>-Tagung <strong>2009</strong> in Bochum ein<br />

Symposium zum Thema „trigger enzymes“.<br />

Trigger-Enzyme sind bifunktionelle Proteine,<br />

die sowohl enzymatische Prozesse im Metabolismus<br />

katalysieren als auch die Genregulation<br />

auf unterschiedlichen Ebenen kontrollieren.<br />

So beeinflussen Trigger-Enzyme als<br />

DNA-bindende Transkriptionsfaktoren direkt<br />

die Genexpression. An<strong>der</strong>e Trigger-Enzyme<br />

wirken als posttranskriptionale Regulatoren<br />

über Protein-RNA-Interaktionen o<strong>der</strong> beeinflussen<br />

die Phosphorylierung von Transkriptionsregulatoren.<br />

Schließlich gibt es Trigger-<br />

Enzyme, die über Protein-Protein-Wechsel-<br />

wirkungen die Aktivität von Transkriptionsfaktoren<br />

regulieren. Eine Spezialgruppe <strong>der</strong><br />

Trigger-Enzyme bilden die Trigger-Transportproteine,<br />

die im Zusammenspiel mit sensorischen<br />

Proteinen eine wesentliche Rolle <strong>bei</strong><br />

<strong>der</strong> Übertragung von Informationen über die<br />

Membran haben. Zur Thematik <strong>der</strong> Trigger-<br />

Enzyme ist es uns gelungen, zwei internationale<br />

Sprecher einzuladen. Linc Sonenshein,<br />

Tufts University Boston, USA, wird über die<br />

Aconitase als Trigger-Enzym in Bacillus sprechen.<br />

Donald F. Becker, University of Nebraska,<br />

Lincoln, USA, wird über die direkte Beziehung<br />

von Metabolismus und Genexpression<br />

am Beispiel <strong>der</strong> multifunktionellen Prolin-<br />

Dehydrogenase PutA in Escherichia coli berich-<br />

Fachgruppe: Wasser/Abwasser<br />

ó Im Anschluss an die <strong>Jahrestagung</strong> <strong>der</strong><br />

<strong>VAAM</strong> in Bochum findet am 12. März eine<br />

Konferenz statt: „How dead is dead? Survival<br />

and final inactivation of microorganisms“.<br />

Die Frage, wann Mikroorganismen wirklich<br />

tot sind, ist von großer ökologischer und<br />

gesundheitlicher Bedeutung. Die Antwort<br />

hängt ausschließlich von <strong>der</strong> Methode ab,<br />

mit <strong>der</strong> die Vitalität nachgewiesen wird. In<br />

dieser Konferenz geht es darum, die Grauzone<br />

unterhalb <strong>der</strong> klassischen Methode –<br />

Wachstum und Koloniebildung – zu beleuchten.<br />

Wenn keine Kolonien mehr gebildet werden,<br />

heißt das noch lange nicht, dass die<br />

Organismen auch tot sind. Vielmehr gibt es<br />

eine Reihe von verschiedenen Ebenen <strong>der</strong><br />

Vitalität, die sich mit an<strong>der</strong>en Methoden noch<br />

nachweisen lässt. Daraus ergibt sich auch<br />

<strong>der</strong> provokative Titel <strong>der</strong> Veranstaltung.<br />

Idealerweise wird die Konferenz mit einer<br />

mo<strong>der</strong>nen Definition des Todes von Mikroorganismen<br />

gekrönt.<br />

Organisiert wird die Veranstaltung, wie<br />

auch die <strong>Jahrestagung</strong> <strong>der</strong> <strong>VAAM</strong>, von Conventus.<br />

Informationen liefern die Website<br />

(www.conventus.de/hdid) und ein Flyer.<br />

Rita Colwell und Staffan Kjelleberg wurden<br />

für Plenarvorträge und die Mitar<strong>bei</strong>t im<br />

Programmkomitee gewonnen. Ursula Obst<br />

(TU Karlsruhe) und Martin Exner (Universität<br />

Bonn) werden ebenfalls Plenarvorträge<br />

halten. Die Ankündigung dieser Veranstaltung<br />

ist bereits auf großes Interesse gestoßen.<br />

Es wird mit einer Teilnehmerzahl von<br />

mindestens 200 gerechnet.<br />

ten. Das Programm des Symposiums wird mit<br />

Kurzvorträgen komplettiert, die aus den eingereichten<br />

Kurzvortrags- und Poster<strong>bei</strong>trägen<br />

ausgewählt werden.<br />

Im Anschluss an dieses Fachgruppensymposium<br />

findet die diesjährige Mitglie<strong>der</strong>versammlung<br />

unserer Fachgruppe statt (Audimax),<br />

zu <strong>der</strong> ich Sie herzlich einlade. ó<br />

Sprecherin:<br />

Kirsten Jung,<br />

Universität München,<br />

Email: jung@lmu.de<br />

Die Fachgruppe Wasser/Abwasser wird<br />

während <strong>der</strong> <strong>Jahrestagung</strong> <strong>der</strong> <strong>VAAM</strong> eine<br />

Mitglie<strong>der</strong>versammlung (Montag, 9.3.<strong>2009</strong>,<br />

17.30 Uhr, Raum HZO 90) abhalten, <strong>bei</strong> <strong>der</strong><br />

SprecherIn und StellvertreterIn neu gewählt<br />

werden. Die bisherigen Amtsinhaber (Hans-<br />

Curt Flemming und Ulrich Szewzyk) stehen<br />

für eine Wie<strong>der</strong>wahl <strong>zur</strong> Verfügung.<br />

Sprecher:<br />

Hans-Curt Flemming,<br />

Universität Duisburg-Essen<br />

Email: hanscurtflemming@compuserve.com<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

AUS DEN FACHGRUPPEN DER <strong>VAAM</strong><br />

Fachgruppe: Fungal Biology and Biotechnology/Experimentelle<br />

Mykologie<br />

ó Unsere Fachgruppe dient vor allem dem<br />

Ziel, jungen Nachwuchswissenschaftlern<br />

zweimal im Jahr ein Forum <strong>zur</strong> Diskussion<br />

eigener Ergebnisse zu bieten.<br />

Daher findet auch dieses Jahr im Rahmen<br />

<strong>der</strong> <strong>VAAM</strong>-Frühjahrstagung in Bochum ein<br />

Fachgruppensymposium statt. Das Symposium<br />

„Macromolecules secreted by fungi“<br />

wird unter Leitung von Jan Schirawski am<br />

Montag, den 9. März <strong>2009</strong> von 17:30 Uhr<br />

bis 19:30 stattfinden. Aus den zum Thema<br />

eingereichten Abstracts hat er sechs Kurzvorträge<br />

von Doktoranden ausgesucht. Zu<br />

Gast ist Franz Klis von <strong>der</strong> Universität<br />

Amsterdam mit einem Vortrag über „An in<br />

vitro model for mucosal infections reveals<br />

the dynamics of the cell wall proteome of<br />

the clinical fungus Candida albicans“.<br />

Im Anschluss an das Fachgruppensymposium<br />

wird die Fachgruppensitzung stattfinden<br />

(Raum HZO 20), in <strong>der</strong> Ideen für<br />

künftige Veranstaltungen diskutiert werden<br />

sollen, insbeson<strong>der</strong>e Themenvorschläge<br />

für ein Mini-Symposium <strong>zur</strong><br />

<strong>VAAM</strong>-Frühjahrstagung 2010 in Hannover.<br />

Für den Herbst 2010 steht bereits nach<br />

ersten Gesprächen mit Bernhard Hauer<br />

und Christoph Syldatk eine gemeinsame<br />

Tagung mit <strong>der</strong> Fachgruppe „Biotransformationen“<br />

<strong>zur</strong> Diskussion. Auch über das<br />

für den vergangenen Herbst von Eckhard<br />

Thines an <strong>der</strong> Technischen Universität Kaiserslautern<br />

in <strong>der</strong> Zeit 24.–26. September<br />

2008 geplante Symposium <strong>zur</strong> „Physiologie,<br />

Rolle und Funktion des Sekundärmetabolismus<br />

von Pilzen“, das wegen zahlreicher<br />

zeitgleicher Veranstaltungen und<br />

<strong>der</strong> daraus resultierenden zu geringen<br />

Nachfrage lei<strong>der</strong> abgesagt werden musste,<br />

soll diskutiert werden. Wer <strong>zur</strong> Sitzung<br />

nicht kommen kann, wird um Interessensbekundung<br />

unter stahmann@fh-lausitz.de<br />

gebeten.<br />

Der zweite Fachgruppenhöhepunkt dieses<br />

Jahres wird die Traditionstagung „Molekularbiologie<br />

<strong>der</strong> Pilze“ sein, die vom 27.–<br />

30. September <strong>2009</strong> an <strong>der</strong> Universität Münster<br />

stattfindet und von Paul und Bettina<br />

Tudzynski organisiert wird. ó<br />

Sprecher:<br />

Klaus-Peter Stahmann,<br />

Fachhochschule Lausitz<br />

Email: stahmann@fh-lausitz.de<br />

Ursula Kües,<br />

Universität Göttingen<br />

Email: ukuees@gwdg.de<br />

Fachgruppe: Umweltmikrobiologie<br />

ó Einladung <strong>zur</strong> Mitglie<strong>der</strong>versammlung:<br />

Am 09.03.<strong>2009</strong> findet um 19.30 Uhr in<br />

Raum HZO 80 die Jahresversammlung <strong>der</strong><br />

Fachgruppe Umweltmikrobiologie statt.<br />

Folgende Punkte sind vorgesehen:<br />

– Wahl des Sprechers<br />

– Findung von Themen für zukünftige<br />

Workshops<br />

– Allgemeines ó<br />

Sprecher:<br />

Karl-Heinz Engesser,<br />

Universität Stuttgart<br />

Email: karl-h.engesser@iswa.uni-stuttgart.de<br />

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12 AUS DEN FACHGRUPPEN DER <strong>VAAM</strong><br />

Fachgruppe: Mikrobielle Pathogenität<br />

ó Das Jahr 2008 war für die Fachgruppe<br />

Mikrobielle Pathogenität durch eine Reihe<br />

von Tagungen, die Initiierung eines neuen<br />

DFG-Schwerpunktes und die Wahl <strong>der</strong> Fachgruppensprecher<br />

zum Jahresende geprägt.<br />

Der im zweijährigen Rhythmus gemeinsam<br />

mit <strong>der</strong> gleichnamigen Fachgruppe <strong>der</strong> DGHM<br />

stattfindende Workshop für Nachwuchswissenschaftler<br />

fand mit lebhafter Beteiligung<br />

im Frühjahr in Bad Urach statt. Über die Yersinien-Tagung<br />

in Braunschweig wurde bereits<br />

ausführlich in Biospektrum (07/2008, Seite<br />

759) berichtet.<br />

2008 wurde <strong>der</strong> neue DFG-Schwerpunkt<br />

SPP1316 (Koordinator Prof. Michael Hensel,<br />

Erlangen) ‘Wirtsadaptierter Metabolismus<br />

von bakteriellen Infektionserregern’ initiiert,<br />

an dem auch verschiedene Vertreter <strong>der</strong> Fachgruppe<br />

beteiligt sind. Der Schwerpunkt<br />

umfasst insgesamt 22 geför<strong>der</strong>te Projekte und<br />

hat sich die Aufgabe gestellt, Stoffwechselleistungen<br />

von pathogenen Bakterien zu identifizieren<br />

und zu charakterisieren, die dem<br />

Krankheitserreger während <strong>der</strong> Infektion<br />

einen Vorteil bieten und für die Etablierung<br />

des Bakteriums in Wirt von essentieller<br />

Bedeutung sind. Eine bioanalytische und bioinformatische<br />

Plattform, die von den <strong>der</strong> TU<br />

München, <strong>der</strong> TU Braunschweig und <strong>der</strong> Universität<br />

Würzburg betrieben wird, unterstützt<br />

da<strong>bei</strong> den Schwerpunkt <strong>bei</strong> <strong>der</strong> Identifizierung,<br />

Auswertung und Modellierung <strong>der</strong> wirtsadaptierten<br />

Stoffwechselwege. Das gleiche<br />

Thema wird dieses Jahr auch durch das internationale<br />

Symposium ‘Metabolism meets<br />

virulence’ aufgenommen, das vom 4.–7.4.09<br />

in <strong>der</strong> Akademie Schloss Hohenkammer <strong>bei</strong><br />

München stattfinden wird (www.metabolismmeets-virulence.org).<br />

Kurz vor Jahresende<br />

wurde schließlich die Leitung <strong>der</strong> Fachgruppe<br />

per Email neu gewählt. Da<strong>bei</strong> wurden<br />

Andreas Peschel (Tübingen), <strong>der</strong> bisherige<br />

Stellvertreter, als Sprecher und Petra Dersch<br />

(Braunschweig) als neue stellvertretende<br />

Sprecherin gewählt. Beim anstehenden Fachgruppentreffen<br />

am Rande <strong>der</strong> <strong>VAAM</strong>-Jahres-<br />

Aufruf <strong>zur</strong> Neugründung <strong>der</strong> Fachgruppe<br />

„Symbiotische Interaktionen“<br />

ó Die komplexen Interaktionen zwischen<br />

symbiontischer Mikrobiota und ihren Wirten<br />

haben in den letzten Jahren an Bedeutung<br />

gewonnen und bilden eine neue Forschungsrichtung<br />

mit innovativen Fragestellungen und<br />

Methoden.<br />

Die Zunahme entsprechen<strong>der</strong> Publikationen<br />

dieser Fachrichtung in Journals wie<br />

Science und Nature belegt, dass das wissenschaftliche<br />

Interesse in angewandten und<br />

grundlegenden mikrobiologischen Disziplinen<br />

diverser Fachrichtung zunimmt. Dies<br />

zeigt sich auch in <strong>der</strong> Neugründung <strong>der</strong><br />

DGHM-Fachgruppe „Mikrobiota, Probiotika<br />

und Wirt“. Diese Fachgruppe richtet sich in<br />

ihrem Schwerpunkt humanmedizinisch aus.<br />

Wir als Mitglie<strong>der</strong> <strong>der</strong> <strong>VAAM</strong> halten es<br />

jedoch ergänzend für wichtig, diese Interaktionen<br />

grundlegend aufzuklären und zu<br />

verstehen. Dazu gehören z. B. die Aufklärung<br />

<strong>der</strong> Stoffwechselinteraktionen zwischen<br />

Mikrobiota und Wirt sowie <strong>der</strong> Mikrobiota<br />

untereinan<strong>der</strong>, die Bedeutung des<br />

„Quorum Sensing“ und die Aufklärung <strong>der</strong><br />

Interaktionen im bakteriellen Biofilm des<br />

gesunden Wirtes. Da<strong>bei</strong> ist auch die Erar<strong>bei</strong>tung<br />

erfolgreicher Methoden und Modelle<br />

ein relevanter Aspekt.<br />

Die neue Fachgruppe soll ein interdisziplinäres<br />

Gremium darstellen, dass sich mit<br />

diesen Fragestellungen in grundlegen<strong>der</strong> und<br />

angewandter Ausrichtung beschäftigt. Sie<br />

tagung in Bochum werden die Mitglie<strong>der</strong> und<br />

<strong>der</strong> neu gewählte Vorstand künftige Aktivitäten<br />

<strong>der</strong> deutschen Infektionsforschung<br />

besprechen (Raum HZO 30). Von beson<strong>der</strong>er<br />

Bedeutung dürften <strong>2009</strong> Konzepte für neue<br />

DFG-Verbünde wie dem passend zum Darwin-Jahr<br />

beantragte Schwerpunkt ‘Mikroevolution<br />

bakterieller Krankheitserreger’<br />

(Koordination: Regine Hakenbeck, Kaiserslautern)<br />

sein. ó<br />

Sprecher:<br />

Andreas Peschel, Universität Tübingen<br />

Email: andreas.peschel@uni-tuebingen.de<br />

Petra Dersch, Universität Braunschweig<br />

Email: p.<strong>der</strong>sch@tu-bs.de<br />

soll alle relevanten symbiotischen Interaktionen<br />

in <strong>der</strong> Biologie umfassen. Hiermit soll<br />

ein produktiver Austausch zwischen den<br />

erfahrenen und jüngeren Mitglie<strong>der</strong>n <strong>der</strong><br />

<strong>VAAM</strong> ermöglicht werden. Die damit verbundene<br />

Netzwerkbildung trägt dazu da<strong>bei</strong>,<br />

dieses wichtige Thema sowohl in wissenschaftlicher<br />

als auch in industriell angewandter<br />

Richtung zu etablieren und zu vertreten.<br />

Ansprechpartner: Dr. Andreas Schwiertz,<br />

Email: andreas.schwiertz@mikrooek.de<br />

Die Fachgruppe soll auf <strong>der</strong> <strong>VAAM</strong>-<strong>Jahrestagung</strong><br />

in Bochum neu gegründet werden.<br />

Ein erstes Treffen findet am Montag, den<br />

9.3. um 19.30 Uhr in Raum HZO 90 statt. ó<br />

Bring your copy of the BIOspektrum Son<strong>der</strong>ausgabe <strong>2009</strong>;<br />

at the meeting it will cost 10 Euro<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

INSTITUTSPORTRAIT<br />

Mikrobiologie an <strong>der</strong> Ruhr-Universität<br />

in Bochum<br />

ó Die Ruhr-Universität Bochum wurde<br />

1965 gegründet. Allerdings fand eine Aufnahme<br />

von Forschung und Lehre in den<br />

naturwissenschaftlichen Fächern erst im<br />

Jahr 1969 statt. Seitdem hat sich die mikrobiologische<br />

Forschung in vielfältiger Weise<br />

entwickelt; Schwerpunktforschung findet<br />

in diesem Bereich vor allen Dingen in den<br />

Fakultäten für Biologie und Biotechnologie<br />

sowie Medizin statt. Bemerkenswert<br />

ist da<strong>bei</strong> die Breite <strong>der</strong> verwendeten Mikroorganismen;<br />

dies schließt neben den Bakterien<br />

vor allen Dingen auch pilzliche<br />

Organismen sowie Cyanobakterien und<br />

Algen ein. Mikrobiologische Forschung,<br />

die durch ein breites Methodenspektrum<br />

gekennzeichnet ist, spiegelt sich in verschiedensten<br />

Zentren und Schwerpunktbildungen<br />

wi<strong>der</strong>, wie zum Beispiel im SFB<br />

480 „Molekulare Biologie komplexer Leistungen<br />

von botanischen Systemen“ sowie<br />

dem SFB 642 „GTP- und ATP-abhängige<br />

Membranprozesse“, dem Protein- und dem<br />

Medizinischen Proteom-Center sowie dem<br />

Christian-Doppler-Labor für „Biotechnologie<br />

<strong>der</strong> Pilze“. Auch in dem im Jahr 2008<br />

gegründeten Protein Research Center wird<br />

die mikrobiologische Forschung einen<br />

wesentlichen Teil <strong>der</strong> Aktivitäten einnehmen.<br />

Die Bochumer Mikrobiologinnen und<br />

Mikrobiologen haben gerne die Aufgabe<br />

übernommen, die <strong>VAAM</strong>-<strong>Jahrestagung</strong><br />

<strong>2009</strong> aus<strong>zur</strong>ichten.<br />

Prof. Dr. Ulrich Kück, Lehrstuhl für<br />

Allgemeine und Molekulare Botanik,<br />

Fakultät für Biologie und Biotechnologie<br />

Die Forschungsschwerpunkte am Lehrstuhl<br />

sind dem Bereich <strong>der</strong> molekularen<br />

Genetik zu<strong>zur</strong>echnen. Da<strong>bei</strong> werden Fragen<br />

<strong>zur</strong> Organellen-Biogenese und <strong>zur</strong><br />

organismischen und zellulären Differenzierung<br />

mit ein- und mehrzelligen eukaryotischen<br />

Mikroorganismen durchgeführt.<br />

Grundsätzlich können drei große<br />

Forschungsbereiche unterschieden werden:<br />

1. Die Chloroplasten-Biogenese am<br />

Beispiel <strong>der</strong> plastidären RNA-Prozessierung<br />

in <strong>der</strong> Grünalge Chlamydomonas reinhardtii.<br />

Bei diesem Einzeller wird die<br />

Abhängigkeit <strong>der</strong> plastidären Genexpression<br />

von kernkodierten Faktoren am Beispiel<br />

<strong>der</strong> psaA-Genexpression in den Chlo-<br />

roplasten mit genetischen und biochemischen<br />

Methoden untersucht. Die reife<br />

psaA-mRNA wird durch das Trans-Spleißen<br />

verschiedener Primär-Transkripte<br />

unter Beteiligung von mindestens 15<br />

nukleären und mehreren plastidärkodierten<br />

Faktoren gebildet. Unter Nutzung von<br />

vorwärts und rückwärts gerichteten nukleären<br />

Ansätzen werden Komponenten identifiziert,<br />

die am Trans-Spleißvorgang beteiligt<br />

sind. Diese Proteinkomponenten sind<br />

Teil eines hochmolekularen Protein-RNA-<br />

Komplexes, <strong>der</strong> möglicherweise einem<br />

plastidären Spleißosom entspricht. 2. Zelldifferenzierung<br />

<strong>bei</strong> Pilzen, genetische<br />

Grundlage <strong>der</strong> multizellulären Entwicklung<br />

<strong>bei</strong> Eukaryoten. Bei diesem Projekt<br />

wird die Organdifferenzierung <strong>bei</strong><br />

Hyphenpilzen analysiert. Die Analyse von<br />

Entwicklungsmutanten identifiziert Faktoren,<br />

die an <strong>der</strong> Zelldifferenzierung beteiligt<br />

sind. Interessanterweise konnten<br />

sowohl Komponenten von konservierten<br />

Signaltransduktionswegen (Pheromon-<br />

GPCR-vermittelte Signaltransduktion) als<br />

auch neue, bisher uncharakterisierte Proteine<br />

identifiziert werden, die möglicherweise<br />

Teil eines makromolekularen Proteinkomplexes<br />

sind, <strong>der</strong> Signale aus verschiedenen<br />

Transduktionswegen miteinan<strong>der</strong><br />

verknüpft. Aufbauend auf diesen<br />

Ergebnissen können allgemeine Schlüsse<br />

auf Signaltransduktionswege gezogen werden,<br />

die auch <strong>bei</strong> an<strong>der</strong>en eukaryotischen<br />

Organismen relevant für die Zelldifferenzierung<br />

sind. 3. Molekulargenetik von biotechnologisch<br />

relevanten Pilzen. In diesem<br />

Projekt werden industriell genutzte<br />

Hyphenpilze mit molekulargenetischen<br />

Methoden gezielt verän<strong>der</strong>t. Da<strong>bei</strong> geht es<br />

zum einen um die Entwicklung von Verfahren<br />

<strong>zur</strong> optimalen Konstruktion rekombinanter<br />

Stämme, zum an<strong>der</strong>en um die<br />

Analyse <strong>der</strong> Genexpression, um den pilzlichen<br />

Sekundärmetabolismus gezielt qualitativ<br />

o<strong>der</strong> quantitativ zu verän<strong>der</strong>n.<br />

Prof. Dr. Matthias Rögner, Lehrstuhl<br />

für Biochemie <strong>der</strong> Pflanzen, Fakultät<br />

für Biologie und Biotechnologie<br />

Schwerpunkt des Lehrstuhls für Biochemie<br />

<strong>der</strong> Pflanzen ist die Untersuchung<br />

zeit- und ortsabhängiger Prozesse des<br />

Membranproteinnetzwerks von Cyano-<br />

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14 INSTITUTSPORTRAIT<br />

bakterien, ausgehend von <strong>der</strong> Geninduktion<br />

bis <strong>zur</strong> Proteindegradation. Im Mittelpunkt<br />

steht das Verständnis <strong>der</strong> Effizienz und <strong>der</strong><br />

Anpassung lichtgesteuerter Prozesse <strong>der</strong><br />

Photosynthese sowie <strong>der</strong>en Anwendung <strong>zur</strong><br />

Biowasserstoffproduktion aus Wasser. Die<br />

Realisierung dieses Ziels erfolgt sowohl in<br />

natürlichen (Konstruktion einer „Design-Zelle“<br />

<strong>zur</strong> photosynthetischen H 2 -Produktion)<br />

als auch in semiartifiziellen Systemen (Konstruktion<br />

einer „Biobatterie“ mit immobilisierten<br />

Komponenten). Voraussetzung hierfür<br />

ist ein grundlegendes Verständnis <strong>der</strong> zellulären<br />

Elektronentransportprozesse, des<br />

molekularen Mechanismus <strong>der</strong> lichtgesteuerten<br />

Wasserspaltung durch Photosystem 2<br />

sowie <strong>der</strong>en Optimierung durch die Entwicklung<br />

neuer Photobioreaktormodule am<br />

Lehrstuhl.<br />

Prof. Dr. Franz Narberhaus, Lehrstuhl für<br />

Biologie <strong>der</strong> Mikroorganismen, Fakultät<br />

für Biologie und Biotechnologie<br />

Am Lehrstuhl werden vier verschiedene Themen<br />

bear<strong>bei</strong>tet. In einem Projekt werden<br />

RNA-abhängige Regulationsmechanismen,<br />

insbeson<strong>der</strong>e die Kontrolle <strong>der</strong> Translation<br />

durch RNA-Thermometer im 5’-untranslatierten<br />

Bereich bakterieller Hitzeschock- o<strong>der</strong><br />

Virulenzgenen, untersucht. Darüber hinaus<br />

werden kleine regulatorische RNAs in Agrobacterium<br />

tumefaciens identifiziert und charakterisiert.<br />

In einem an<strong>der</strong>en Projekt geht<br />

es um die Bedeutung des Membranlipids<br />

Phosphatidylcholin für die symbiontische und<br />

pathogene Interaktion von Bakterien mit<br />

Pflanzen. Der Abbau zellulärer Substrate<br />

durch die essentielle, membranständige FtsH-<br />

Protease stellt einen weiteren Schwerpunkt<br />

dar. Eine beson<strong>der</strong>e Rolle spielt da<strong>bei</strong> die Kontrolle<br />

<strong>der</strong> Lipopolysaccharid-Biosynthese in<br />

Escherichia coli. Die Ar<strong>bei</strong>tsgruppe um Dr.<br />

Bernd Masepohl untersucht die Regulation<br />

<strong>der</strong> Stickstoff-Fixierung und den Molybdänund<br />

Kupfer-Stoffwechsel in dem phototrophen<br />

Bakterium Rhodobacter capsulatus.<br />

Prof. Dr. Sören Gatermann, Abteilung für<br />

Medizinische Mikrobiologie, Medizinische<br />

Fakultät<br />

Die Abteilung für Medizinische Mikrobiologie<br />

beschäftigt sich mit <strong>der</strong> Erforschung bakterieller<br />

Virulenz, <strong>der</strong> Entwicklung neuer<br />

diagnostischer Techniken in <strong>der</strong> Medizinischen<br />

Mikrobiologie sowie <strong>der</strong> Entdeckung<br />

und Erkennung von Antibiotika-Resistenzmechanismen<br />

<strong>bei</strong> medizinisch relevanten<br />

Bakterien. Die Pathogenitätsforschung kon-<br />

zentriert sich auf Virulenzfaktoren und -mechanismen<br />

<strong>bei</strong> Staphylokokken, wo<strong>bei</strong> Staphylococcus<br />

saprophyticus <strong>der</strong> Modellorganismus<br />

ist. Bei diesem Erreger wurden verschiedene<br />

Enzyme (z.B. Urease, Lipase, Serin-<br />

Desaminase) und Oberflächenfaktoren (z.B.<br />

SdrI, Aas) als Virulenzfaktoren entdeckt und<br />

werden charakterisiert. Für die medizinische<br />

Diagnostik wurden Verfahren <strong>der</strong> Nukleinsäuredetektion<br />

von Erregern von Atemwegsund<br />

Darminfektionen entwickelt und es<br />

konnte in <strong>der</strong> Region erstmalig <strong>der</strong> hochvirulente<br />

Ribotyp 027 von Clostridium difficile<br />

nachgewiesen werden. Bei den Resistenzmechanismen<br />

werden Verfahren <strong>zur</strong> Detektion<br />

von extended-spectrum Beta-Laktamasen<br />

weiterentwickelt und die Prävalenz verschiedener<br />

ESBL-Typen bestimmt sowie das<br />

Vorkommen von Chinolon-modifizierenden<br />

Enzymen untersucht.<br />

Prof. Dr. Ralf Erdmann, Institut für<br />

Physiologische Chemie, Abt. Systembiochemie,<br />

Medizinische Fakultät<br />

Im Fokus <strong>der</strong> Forschung <strong>der</strong> Abteilung steht<br />

die Untersuchung <strong>der</strong> Biogenese von Peroxisomen,<br />

ubiquitären Organellen eukaryotischer<br />

Zellen, <strong>der</strong>en enzymatische Ausstattung<br />

in Abhängigkeit von Zell- o<strong>der</strong> Gewebetyp,<br />

Entwicklungszustand o<strong>der</strong> äußeren<br />

Bedingungen variiert. Angeborene Störungen<br />

<strong>der</strong> Biogenese von Peroxisomen sind meist<br />

in den ersten Lebensjahren tödlich. Die Biogenese<br />

<strong>der</strong> Peroxisomen umfasst die Synthese<br />

<strong>der</strong> peroxisomalen Membran, die Proliferation<br />

und Vererbung <strong>der</strong> Organellen, <strong>der</strong>en<br />

regulierten Abbau und den Proteinimport in<br />

die peroxisomale Matrix. Im Unterschied zu<br />

an<strong>der</strong>en Organellen importieren Peroxisomen<br />

Proteine im gefalteten o<strong>der</strong> oligomerisierten<br />

Zustand auf bislang völlig unbekannte Weise.<br />

Die Aufklärung <strong>der</strong> molekularen Mechanismen<br />

des peroxisomalen Proteinimports ist<br />

ein wesentliches Ziel aktueller Ar<strong>bei</strong>ten.<br />

Prof. Dr. Nicole Frankenberg-Dinkel,<br />

Physiologie <strong>der</strong> Mikroorganismen,<br />

Fakultät für Biologie und Biotechnologie<br />

Zentrales Forschungsthema unserer Ar<strong>bei</strong>tsgruppe<br />

sind offenkettige Tetrapyrrole (Biline).<br />

Da<strong>bei</strong> interessieren wir uns nicht nur für<br />

<strong>der</strong>en Biosynthese, son<strong>der</strong>n auch für ihre<br />

Funktion in Bakterien. Während man in<br />

Cyanobakterien große Mengen an offenkettigen<br />

Tetrapyrrolen in den Lichtsammelkomplexen,<br />

den Phycobilisomen findet, gibt es<br />

nur einige heterotrophe Bakterien, die offenkettige<br />

Tetrapyrrole als Chromophore in Rot-<br />

lichtrezeptoren, den Phytochromen besitzen.<br />

Am Modellsystem Pseudomonas aeruginosa<br />

untersuchen wir die Funktion des bakteriellen<br />

Phytochroms BphP. Neben proteinbiochemischen<br />

Ar<strong>bei</strong>ten nutzen wir hier Transkriptom-<br />

und Proteomanalysen, um diese Fragestellung<br />

zu beantworten.<br />

Die Biosynthese <strong>der</strong> offenkettigen Tetrapyrrole<br />

untersuchen wir an den <strong>bei</strong>den beteiligten<br />

Enzymklassen, den Hämoxygenasen<br />

und den Ferredoxin-abhängigen Bilinreduktasen.<br />

Hier<strong>bei</strong> untersuchen wir nicht nur<br />

rekombinante Enzyme aus Bakterien, son<strong>der</strong>n<br />

auch Vertreter aus höheren Pflanzen<br />

und marinen Viren (Cyanophagen). Beson<strong>der</strong>s<br />

auf dem Gebiet <strong>der</strong> Bilinreduktasen<br />

haben wir im vergangenen Jahr durch die Entdeckung<br />

eines neuen Mitglieds dieser Enzymfamilie<br />

große Fortschritte erzielt. Das neu entdeckte<br />

Enzym aus einem Cyanophagen zeigt<br />

unerwartete Katalyseeigenschaften, da es die<br />

Aktivität von zwei Wirtsenzymen vereint. In<br />

Zusammenar<strong>bei</strong>t mit Prof. Dr. Eckhard Hofmann<br />

(AG Proteinkristallographie, Ruhr-Universität<br />

Bochum) ist es uns gelungen, die Kristallstruktur<br />

dieses Enzyms aufzuklären, und<br />

somit erste Einblicke in die Katalyse zu gewinnen.<br />

Im Vor<strong>der</strong>grund <strong>der</strong> momentanen Ar<strong>bei</strong>ten<br />

stehen die Aufklärung des Reaktionsmechanismus<br />

sowie die Evolution des Phagenenzyms.<br />

In einem dritten Schwerpunkt beschäftigen<br />

wir uns seit kurzem mit <strong>der</strong> Reifung von<br />

Phycobiliproteinen. Im Interesse steht da<strong>bei</strong><br />

die Anknüpfung offenkettiger Tetrapyrrole<br />

an die Phycobiliproteine <strong>der</strong> cyanobakteriellen<br />

Photosynthese. Die Struktur und Funktion<br />

<strong>der</strong> daran beteiligten Phycobilin-Lyasen,<br />

eine bislang sehr schlecht untersuchte<br />

Enzymklasse, steht hier im Fokus unserer<br />

Ar<strong>bei</strong>ten.<br />

Prof. Dr. Thomas Happe, Photobiotechnologie,<br />

Fakultät für Biologie und<br />

Biotechnologie<br />

Die Ar<strong>bei</strong>tsgruppe befasst sich mit grundlegenden<br />

Fragestellungen <strong>zur</strong> Physiologie, Biochemie<br />

und Biotechnologie photosynthetischer<br />

Mikroorganismen unter anaeroben<br />

Bedingungen. Als Modellorganismus dient<br />

da<strong>bei</strong> die einzellige Grünalge Chlamydomonas<br />

reinhardtii. Diese Alge produziert unter<br />

anaeroben Bedingungen lichtabhängig molekularen<br />

Wasserstoff und bildet gleichzeitig<br />

einen komplexen fermentativen Stoffwechsel<br />

mit Gärungsprodukten wie D-Laktat, Ethanol<br />

und Formiat aus. Dieser von uns als Photofermentation<br />

bezeichnete Metabolismus wur-<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

4t Matthes + Traut · Darmstadt<br />

de in den letzten Jahren eingehend charakterisiert.<br />

Eine Reihe von O 2 -sensiblen Enzymen<br />

wie die Pyruvat-Formiat-Lyase (PFL) o<strong>der</strong><br />

die Hydrogenase werden nur in Abwesenheit<br />

von Sauerstoff gebildet. Eine neue Klasse von<br />

[FeFe]-Hydrogenasen (HydA) katalysiert die<br />

H 2 -Produktion in den Algen und ist über den<br />

Elektronendonor Ferredoxin (PetF) an die<br />

photosynthetische Elektronentransportkette<br />

gekoppelt. In <strong>der</strong> Vergangenheit ist es uns<br />

gelungen, ein sehr effektives Expressionsund<br />

Aufreinigungssystem für Metalloproteine<br />

zu etablieren. Dieses System wird dazu<br />

benutzt, die Struktur, die Funktion und die<br />

Wechselwirkung von photosynthetischen<br />

Hydrogenasen, <strong>der</strong>en Maturationsfaktoren<br />

und eine neue Klasse von anaeroben Ferredoxinen<br />

aufzuklären. Neben <strong>der</strong> Grundlagenforschung<br />

beschäftigen wir uns auch mit<br />

biotechnologischen Konzepten für eine photobiologische<br />

Wasserstoffproduktion und Laktatgewinnung<br />

aus Grünalgen.<br />

Probleme mit<br />

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unspezifischen Bindungen,<br />

Kreuzreaktivitäten und<br />

Matrixeffekten <strong>bei</strong><br />

Immunoassays. Seine<br />

Einsatzgebiete sind neben<br />

dem ELISA, EIA o<strong>der</strong><br />

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Jun.-Prof. Dr. Julia Bandow, Mikrobielle<br />

Antibiotikaforschung, Fakultät für<br />

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Die Ar<strong>bei</strong>tsgruppe konzentriert ihre Forschungsaktivitäten<br />

im Wesentlichen auf zwei<br />

Antibiotika-relevante Fragestellungen, zum<br />

einen die Aufklärung von Wirkmechanismen<br />

neuer antibiotisch aktiver Substanzen, zum<br />

an<strong>der</strong>en <strong>der</strong> Suche nach neuen Antibiotika-<br />

Targets. Am Beispiel <strong>der</strong> Gram-positiven und<br />

Gram-negativen Modellorganismen B. subtilis<br />

und E. coli wird die bakterielle Antwort auf die<br />

Hemmung essentieller Zellfunktionen durch<br />

Antibiotika untersucht. Da<strong>bei</strong> erlaubt die zelluläre<br />

Antwort Rückschlüsse darauf, welche<br />

Mechanismen <strong>der</strong> Antibiotikawirkung<br />

zugrunde liegen. Durch Konditionalmutanten<br />

wird weiterhin die zelluläre Reaktion auf<br />

die Hemmung essentieller Zellfunktionen<br />

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Die Ar<strong>bei</strong>tsgruppe beschäftigt sich mit <strong>der</strong><br />

zellulären Thiol-Disulfid Homeostase und<br />

den Auswirkungen des oxidativen Stresses<br />

auf Escherichia coli. Der Fokus <strong>der</strong> Gruppe<br />

richtet sich auf die Untersuchung oxidativer<br />

Thiol-Modifikationen <strong>der</strong> Aminosäure<br />

Cystein mit verschiedenen globalen Methoden.<br />

Diese Methoden können genutzt werden<br />

um Substrat-Proteine von Oxido-Reduktasen<br />

wie z.B. Thioredoxin o<strong>der</strong> DsbA zu definieren.<br />

Des weiteren wird <strong>der</strong> Einfluss verschiedener<br />

oxidativer Stressoren auf den<br />

Thiol-Disulfid-Status zellulärer Proteine<br />

untersucht. Die da<strong>bei</strong> gefundenen redox-sensitiven<br />

Proteine werden biochemisch weiter<br />

charakterisiert und erweisen sich zum Teil<br />

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16 INSTITUTSPORTRAIT<br />

Jun.-Prof. Dr. Bettina Warscheid, Cellular<br />

Proteomics, Medizinische Fakultät<br />

Der Forschungsschwerpunkt <strong>der</strong> Gruppe liegt<br />

in <strong>der</strong> Entwicklung und Etablierung quantitativer<br />

Methoden für die funktionelle Proteomanalyse.<br />

Als Schlüsselmethode wird die<br />

hochauflösende Massenspektrometrie in<br />

Kombination mit Techniken des stabilen Isotopenlabelling<br />

o<strong>der</strong> Label-freier Ansätze eingesetzt.<br />

Ein wesentlicher thematischer Fokus<br />

ist die proteomische Charakterisierung von<br />

Membranproteinkomplexen in Saccharomyces<br />

cerevisiae. So erfolgen in Kooperation mit<br />

Prof. Dr. Ralf Erdmann (Abteilung für Systembiochemie)<br />

Studien <strong>zur</strong> Biogenese und<br />

Proliferation von Peroxisomen. Die umfassende<br />

Aufdeckung subzellulärer Proteinnetzwerke<br />

sowie Einblicke in ihre Dynamik<br />

und Regulation soll zu einem tieferen Verständnis<br />

<strong>der</strong> da<strong>bei</strong> zugrunde liegenden molekularen<br />

Prozesse führen.<br />

PD Dr. Minou Nowrousian, Entwicklungsbiologie<br />

<strong>der</strong> Pilze, Fakultät für Biologie<br />

und Biotechnologie<br />

Die Gruppe untersucht Morphogeneseprozesse<br />

<strong>bei</strong> Pilzen mit Hilfe von Functional<br />

Genomics-Analysen. Pilze sind eukaryontische<br />

Mikroorganismen, die während ihrer<br />

sexuellen Entwicklung komplexe, vielzellige<br />

Strukturen, die sogenannten Fruchtkörper,<br />

bilden. Die Gruppe untersucht die genetische<br />

Basis dieser Differenzierungsprozesse. Zu<br />

diesem Zweck analysieren wir Mutanten des<br />

Hyphenpilzes Sordaria macrospora, die in frühen<br />

Stadien <strong>der</strong> Fruchtkörperentwicklung<br />

gehemmt sind, und vergleichen die Genexpression<br />

in den Mutanten mit <strong>der</strong> des Wildtyps<br />

mittels Mikroarray-Analysen und quantitativer<br />

Real-Time-PCR. Weiterhin vergleichen<br />

wir die Genexpression in verschiedenen<br />

Pilzen (S. macrospora, Neurospora crassa,<br />

Pyronema confluens) untereinan<strong>der</strong>, um evolutionär<br />

konservierte Expressionsmuster zu<br />

identifizieren. Gene mit gleicher Regulation<br />

in verschiedenen Spezies können dazu<br />

genutzt werden, eine zentrale Gruppe von<br />

Genen zu definieren, die an <strong>der</strong> pilzlichen<br />

Morphogenese beteiligt sind.<br />

PD Dr. Ansgar Poetsch, Proteomforschung,<br />

Fakultät für Biologie und<br />

Biotechnologie<br />

Die Gruppe setzt Proteomik ein, um biotechnologisch<br />

wichtige Mikroorganismen, insbeson<strong>der</strong>e<br />

Corynebacterium glutamicum zu<br />

untersuchen. Da<strong>bei</strong> liegt <strong>der</strong> Schwerpunkt<br />

auf Methodenentwicklung und Analysen <strong>zur</strong><br />

physiologischen Adaptation des bakteriellen<br />

Membranproteoms, da im Gegensatz zu cytosolischen<br />

Proteinen für die überwiegende<br />

Zahl an Membranproteinen die Funktion und<br />

die Regulation nicht ausreichend bekannt<br />

sind. In bisherigen Ar<strong>bei</strong>ten konnte die<br />

Zusammensetzung des Membranproteoms<br />

von C. glutamicum Produktionsstämmen und<br />

Verän<strong>der</strong>ungen im Vergleich zum Wildtyp,<br />

sowie <strong>der</strong> Einfluss von verschiedenen Kohlenstoffquellen<br />

auf Kompomenten <strong>der</strong> Membran<br />

(Transportproteine, Energiestoffwechsel)<br />

definiert werden.<br />

SFB 480: Molekulare Biologie komplexer<br />

Leistungen von botanischen Systemen<br />

Sprecher: Prof. Dr. Ulrich Kück<br />

(www.ruhr-uni-bochum.de/sfb480/)<br />

Die Forschungsschwerpunkte im SFB 480<br />

haben das Ziel, das Wechselspiel zwischen<br />

Nukleinsäuren, Proteinen und nie<strong>der</strong>molekularen<br />

Signalmolekülen <strong>bei</strong> <strong>der</strong> Umsetzung<br />

<strong>der</strong> genetischen Information im Verlaufe von<br />

Entwicklungsprozessen zu verstehen. Durchweg<br />

werden Experimentalsysteme <strong>der</strong> Pflanzenwissenschaften<br />

genutzt, um mit unterschiedlichsten<br />

experimentellen Ansätzen Fragestellungen<br />

<strong>der</strong> Molekularen Botanik und<br />

Mikrobiologie zu beantworten. Die Vielfalt<br />

reicht hier<strong>bei</strong> von biochemischen und biophysikalischen<br />

über molekulargenetische bis<br />

zu cytologischen, phytochemischen und<br />

immunologischen Methoden. An<strong>der</strong>s als Tiere<br />

besitzen pflanzliche und mikrobielle Organismen<br />

Zellwände, die <strong>bei</strong> <strong>der</strong> Zelldifferenzierung<br />

und Organogenese geson<strong>der</strong>te<br />

Mechanismen und regulatorische Prozesse<br />

notwendig machen. Photoautotrophe Pflanzen<br />

und Algen sind auf zellulärer Ebene durch<br />

den Besitz von Plastiden ausgezeichnet, <strong>der</strong>en<br />

Biogenese und Wechselwirkung mit an<strong>der</strong>en<br />

Zellkomponenten nur unvollständig molekular<br />

verstanden ist. Insbeson<strong>der</strong>e <strong>der</strong> Aufbau<br />

hochmolekularer Proteinkomplexe <strong>der</strong> Thylakoidmembran<br />

ist abhängig von <strong>der</strong> koordinierten<br />

Interaktion verschiedener molekularer<br />

Komponenten. Ähnlich den Plastiden<br />

besitzen auch Peroxisomen eine für die Zelldifferenzierung<br />

determinierende Rolle. Ihre<br />

Differenzierung zu Glyoxysomen o<strong>der</strong> „Woronin<br />

Bodies“ trägt zu <strong>der</strong> physiologischen<br />

Funktion pflanzlicher und mikrobieller Zellen<br />

<strong>bei</strong>. Schließlich besitzen Höhere Pflanzen<br />

typische kleine GTP-Bindeproteine (ROPs),<br />

welche für die Kontrolle <strong>der</strong> pflanzlichen Entwicklung<br />

entscheidend sind. Ähnlich findet<br />

man <strong>bei</strong> Pilzen Komponenten konservierter<br />

Siganalkaskaden als Teil zellulärer Netzwer-<br />

ke, die mit neuen, bisher nicht charakterisierten<br />

Proteinen interagieren und so Zelldifferenzierungsprozesse<br />

steuern. Die folgenden<br />

Fragestellungen fassen die Aktivitäten<br />

des SFB480 zusammen: 1. Welche Regelmechanismen<br />

bestimmen <strong>bei</strong> multizellulären<br />

Lebewesen die organismische Differenzierung?<br />

2. Welche intrazellulären Prozesse sind<br />

an <strong>der</strong> Biogenese und Wechselwirkung von<br />

Organellen beteiligt? 3. Wie sehen molekulare<br />

Reaktionsmechanismen aus, die in Molekülverbänden<br />

o<strong>der</strong> in Proteinkomplexen und<br />

Einzelmolekülen zu beobachten sind?<br />

Bei <strong>der</strong> Beantwortung dieser komplexen<br />

Fragestellungen wird im Rahmen des SFB 480<br />

die Bandbreite <strong>der</strong> verfügbaren Experimentalsysteme<br />

von Einzellern bis zu vielzelligen<br />

Organismen mit photoautotropher o<strong>der</strong><br />

heterotropher Lebensweise genutzt.<br />

SFB 642: GTP- und ATP-abhängige<br />

Membranprozesse<br />

Sprecher: Prof. Dr. Klaus Gerwert<br />

(www.sfb642.rub.de/de/index.htm)<br />

Die im SFB 642 untersuchten GTP- und ATPabhängigen<br />

Membranprozesse bieten eine<br />

ausgezeichnete Gelegenheit, die Lücke zwischen<br />

molekularer und systemischer Biologie<br />

zu schließen. Um die gemeinsamen molekularen<br />

Reaktionsmechanismen von GTPund<br />

ATP-abhängigen Membranprozessen herauszuar<strong>bei</strong>ten,<br />

sollen die Raumstrukturen<br />

<strong>der</strong> beteiligten Proteine, die Ligandenbindung,<br />

die Reaktionskinetiken und die Protein-Protein-Interaktionen<br />

untersucht werden. Weiterhin<br />

soll geklärt werden, wo und wann Proteine<br />

direkt in die Membran eingelagert o<strong>der</strong><br />

aber über Lipidanker an die Membran gebunden<br />

werden. Schließlich soll die Rolle <strong>der</strong> Proteine<br />

und ihrer Modifikationen im biologischen<br />

System, sowohl in Zellkulturen als auch<br />

im Tiermodell, untersucht werden. Dazu hat<br />

sich eine Gruppe von ausgewiesenen Experten<br />

unterschiedlicher Fachrichtungen<br />

zusammengefunden, die im SFB 642 Stateof-the-Art-Methoden<br />

<strong>der</strong> Strukturbiologie, Biophysik,<br />

chemischen Biologie, Systembiologie<br />

und Zellbiologie etabliert haben.<br />

Wichtige Fragen gilt es im Kontext des<br />

SFB 642 zu beantworten: Welche Strukturelemente<br />

und dynamischen Verän<strong>der</strong>ungen<br />

eines Proteins sind verantwortlich für die<br />

Aktivierung unterschiedlicher Signalwege?<br />

Welche Multienzymkomplexe werden gebildet?<br />

Welche Rolle spielt die Einbettung eines<br />

Proteins im biologischen System und wie ist<br />

es reguliert? Wie kann die Funktion beeinflusst<br />

werden? Wie reagiert das Netzwerk<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

auf Intervention mit kleinen Molekülen? Die<br />

Beantwortung dieser Fragen wird zu einem<br />

besseren Verständnis <strong>der</strong> molekularen<br />

Grundlagen GTP- und ATP-abhängiger Signalwege<br />

und Transportprozesse führen. Da<br />

Mutationen von hieran beteiligten Proteinen<br />

Krankheiten auslösen können, haben viele<br />

<strong>der</strong> im SFB 642 untersuchten Fragen auch<br />

eine hohe medizinische Relevanz. Ein wichtiges<br />

Ziel vieler Teilprojekte in <strong>der</strong> nächsten<br />

För<strong>der</strong>periode wird sein, von den bereits<br />

sehr detaillierten In-vitro-Untersuchungen<br />

in die nächst höhere Hierarchiestufe zu den<br />

Prozessen an <strong>der</strong> Membran zu gehen und<br />

die Ergebnisse mit den zell-/systembiologischen<br />

Analysen zu verknüpfen, sodass ein<br />

detailliertes Bild <strong>der</strong> Transduktionsnetzwerke<br />

entsteht. Da<strong>bei</strong> sollen zum einen die<br />

Netzwerke verschiedener Guaninnukleotidbinden<strong>der</strong><br />

Proteine an <strong>der</strong> Membran rekonstituiert<br />

werden, zum an<strong>der</strong>en sind aber<br />

auch Experimente im Kontext <strong>der</strong> lebenden<br />

Zelle geplant.<br />

Schwerpunktprogramm SPP 1258:<br />

Sensorische und regulatorische RNAs<br />

in Prokaryoten<br />

Koordinator: Prof. Dr. Franz Narberhaus,<br />

Lehrstuhl für Biologie <strong>der</strong> Mikroorganismen<br />

Erst im letzten Jahrzehnt wurde die große<br />

Bedeutung regulatorischer RNAs in Pro- und<br />

Eukaryoten erkannt. Ziel des Schwerpunktprogramms<br />

ist die Untersuchung <strong>der</strong> Struktur<br />

und Funktion regulatorischer RNA-Moleküle,<br />

die an <strong>der</strong> Kontrolle wichtiger zellulärer<br />

Prozesse in Bakterien und Archaea beteiligt<br />

sind. Folgende Mechanismen stehen im Zentrum<br />

des Interesses: (A) Kleine nichtkodierende<br />

RNAs, die spezifisch an mRNAs o<strong>der</strong><br />

Proteine binden und <strong>der</strong>en biologische Aktivität<br />

modulieren; (B) Riboschalter (Riboswit-<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

ches), die durch hochaffine Bindung eines<br />

zellulären Metaboliten die Konformation einer<br />

mRNA verän<strong>der</strong>n und dadurch die Expression<br />

regulieren; (C) RNA-Thermometer, die<br />

durch temperaturinduzierte Konformationsän<strong>der</strong>ungen<br />

die Zugänglichkeit <strong>der</strong> Ribosomenbindestelle<br />

von Hitzeschock- und Virulenzgenen<br />

kontrollieren.<br />

An dem im Jahr 2007 etablierten Schwerpunktprogramm<br />

sind deutschlandweit 25<br />

Ar<strong>bei</strong>tsgruppen beteiligt. Das interdisziplinäre<br />

Team umfasst Wissenschaftler und Wissenschaftlerinnen<br />

aus <strong>der</strong> Molekularen<br />

Mikrobiologie, Genetik, Bioinformatik, Strukturbiologie<br />

und organischen Chemie.<br />

Christian Doppler Labor „Biotechnologie<br />

<strong>der</strong> Pilze“<br />

Leiter: Prof. Dr. Ulrich Kück<br />

(www.ruhr-uni-bochum.de/cd-labor)<br />

Das Forschungsinteresse des Christian<br />

Doppler Labors für „Biotechnologie <strong>der</strong> Pilze“<br />

ist es, Hyphenpilze durch genetische<br />

Rekombination für die pharmazeutische Produktion<br />

zu optimieren. Hierdurch wird ein<br />

Brückenschlag zwischen Grundlagenforschung<br />

und industrieller Anwendung geschaffen.<br />

Geforscht wird in drei Kernbereichen,<br />

dazu gehört die Entwicklung molekularer<br />

Tools für die gezielte Manipulation, die Identifizierung<br />

neuer regulatorischer Faktoren<br />

des Sekundärmetabolismus und die funktionelle<br />

Analyse des Gesamt-Genoms. Zu den<br />

Versuchsorganismen gehören die Antibiotika-Produzenten<br />

Penicillium chrysogenum<br />

(Penicillin) und Acremonium chrysogenum<br />

(Cephalosporin C), <strong>der</strong> Produzent des Immunsupressivums<br />

Cyclosporin A Tolypocladium<br />

inflatum, sowie die Produzenten von Statinen<br />

(Cholesterinsenker) Aspergillus terreus und<br />

Penicillium citrinum.<br />

INSTITUTSPORTRAIT<br />

17<br />

BMBF-Verbundprojekt „Bio-H 2 “ – Grundlagen<br />

für einen biotechnologischen und<br />

biomimetischen Ansatz <strong>der</strong> Wasserstoffproduktion<br />

Sprecher: Prof.Dr. Matthias Rögner,<br />

Lehrstuhl für Biochemie <strong>der</strong> Pflanzen<br />

(www.ruhr-uni-bochum.de/bioh2/)<br />

Das BMBF-Verbundprojekt „BioH 2 “ verfolgt<br />

die praktische Umsetzung eines Konzeptes,<br />

nach welchem in einem zyklischen Prozess<br />

Wasserstoff durch natürliche, selbstreplizierende<br />

o<strong>der</strong> biomimetische Systeme aus Wasser<br />

erzeugt und <strong>bei</strong> Verbrennung (z.B. unter<br />

Energieverwertung in einer Brennstoffzelle)<br />

wie<strong>der</strong> in Wasser überführt wird. Die <strong>zur</strong> H 2 -<br />

Erzeugung benötigte Energie wird hier<strong>bei</strong> von<br />

<strong>der</strong> Sonne geliefert und die benötigten<br />

Elektronen und Protonen werden aus <strong>der</strong><br />

photobiologischen Spaltung von H 2 O gewonnen.<br />

Durch gezielte Modifizierung des Energiemetabolismus’<br />

einer Cyanobakterienzelle<br />

wird hierfür ein biologisches Modellsystem<br />

entwickelt, welches geeignete genetisch modifizierte<br />

Komponenten des photosynthetischen<br />

Elektronentransportes mit Komponenten <strong>der</strong><br />

Wasserstoffproduktion – insbeson<strong>der</strong>e einer<br />

auf Sauerstofftoleranz optimierten hochaktiven<br />

Hydrogenase – koppelt. Den „proof of<br />

principle“ liefert ein biomimetisches Modellsystem,<br />

in welchem die biologischen Komponenten<br />

auf Goldelektroden immobilisiert und<br />

in einer Art „Biobatterie“ gekoppelt und getestet<br />

werden. Parallel hierzu werden in Zus.ar<strong>bei</strong>t<br />

mit <strong>der</strong> Industrie kostengünstige Photobioreaktoren<br />

entwickelt, die sich für eine spätere<br />

Massenanzucht H 2 -produzieren<strong>der</strong> Cyanobakterien<br />

im technischen Maßstab eignen.<br />

Das Netzwerk ist interdisziplinär zusammengesetzt<br />

und umfasst 7 universitäre Ar<strong>bei</strong>tsgruppen<br />

(3 <strong>der</strong> Universität Bochum sowie je<br />

eine von HU und FU Berlin, Univ. Bielefeld<br />

und Univ. Köln) sowie 2 MPI-Gruppen (MPI<br />

Mülheim und MPI Marburg). ó<br />

Bring your copy of the BIOspektrum<br />

Son<strong>der</strong>ausgabe <strong>2009</strong>;<br />

at the meeting it will cost 10 Euro

18 ALLGEMEINES<br />

Einladung <strong>zur</strong> Mitglie<strong>der</strong>versammlung <strong>der</strong> <strong>VAAM</strong><br />

ó Hiermit lade ich alle Mitglie<strong>der</strong> <strong>der</strong> <strong>VAAM</strong><br />

<strong>zur</strong> Mitglie<strong>der</strong>versammlung ein. Sie wird am<br />

Dienstag, den 10. März <strong>2009</strong>, um 17.30 Uhr<br />

im Audimax <strong>der</strong> Universität Bochum stattfinden.<br />

Vorläufige Tagesordnung:<br />

1. Festlegung <strong>der</strong> Tagesordnung und Genehmigung<br />

<strong>der</strong> Nie<strong>der</strong>schrift <strong>der</strong> Mitglie<strong>der</strong>versammlung<br />

vom 10. März 2008 in<br />

Frankfurt (siehe BIOspektrum 3/08, Seiten<br />

300 und 301)<br />

2. Bericht aus dem Vorstand, u.a. Haushalt<br />

2008 und Haushaltsplan <strong>2009</strong>, Umstrukturierung<br />

Geschäftsstelle/Sekretariat, Mitglie<strong>der</strong>werbung,<br />

Ort und Zeit <strong>der</strong> nächsten<br />

<strong>Jahrestagung</strong>, Aktivitäten <strong>der</strong> Fachgruppen,<br />

VBIO<br />

3. Bericht <strong>der</strong> Kassenprüfer<br />

4. Entlastung des Vorstandes<br />

5. Wahl des Präsidiums (Präsident, 1. Vizepräsident,<br />

Schatzmeister, Schriftführer)<br />

und drei <strong>der</strong> sechs Mitglie<strong>der</strong> des Beirates<br />

(geheime Wahl während <strong>der</strong> Mitglie<strong>der</strong>versammlung)<br />

6. Ehrenmitglie<strong>der</strong>wahl<br />

7. Verschiedenes<br />

Verleihung <strong>der</strong> <strong>VAAM</strong>-Promotionspreise<br />

<strong>2009</strong><br />

Hiermit bitte ich alle Mitglie<strong>der</strong>, Vorschläge<br />

<strong>zur</strong> Wahl des Präsidiums und des Beirates<br />

<strong>bei</strong>m Präsidenten ein<strong>zur</strong>eichen (bis 14 Tage<br />

vor <strong>der</strong> Mitglie<strong>der</strong>versammlung), wo<strong>bei</strong> Vorschläge<br />

für das Präsidium von zehn <strong>VAAM</strong>-<br />

Mitglie<strong>der</strong>n und für den Beirat von drei Mit-<br />

Mitteilungen des <strong>VAAM</strong>-Sekretariats<br />

Ende April <strong>2009</strong> wird das <strong>VAAM</strong>-Sekretariat<br />

in Bovenden altersbedingt nach 24 Jahren aufgelöst.<br />

Die dortigen Aufgaben werden nach<br />

Frankfurt in die gemeinsame Geschäftsstelle<br />

mit <strong>der</strong> GBM verlagert und auf mehrere Personen<br />

verteilt: <strong>VAAM</strong>-Geschäftsstelle, Mörfel<strong>der</strong><br />

Landstrasse 125, 60598 Frankfurt/M.<br />

Mitglie<strong>der</strong>verwaltung:<br />

Bitte teilen Sie alle Än<strong>der</strong>ungen (Adresse,<br />

Bankverbindung, Studentenstatus) schriftlich<br />

dem Sekretariat mit (Brief, Fax o<strong>der</strong><br />

eMail).<br />

Falsche Bankverbindungen (auch <strong>bei</strong><br />

Namensän<strong>der</strong>ungen <strong>der</strong> Bank) führen zu kostenpflichtigen<br />

Retouren, die auf das jeweilige<br />

Mitglied (10 N) umgelegt werden müssen. Das<br />

BIOspektrum wird <strong>bei</strong> getätigtem Umzug<br />

(trotz bezahltem Nachsendeservice !) wegen<br />

des sehr verbilligten Postzeitungsdienstes<br />

nicht nachgesandt. Daher erhält <strong>der</strong> Spektrum-Verlag<br />

vor jedem Versand eines Heftes<br />

eine aktualisierte Adressdatei vom <strong>VAAM</strong>-<br />

Sekretariat. Bitte achten Sie darauf, dass Ihr<br />

Name auf Ihrem Briefkasten steht.<br />

Mitglie<strong>der</strong>verzeichnis:<br />

Die <strong>VAAM</strong> bietet ihren Mitglie<strong>der</strong>n ein<br />

elektronisches Mitglie<strong>der</strong>verzeichnis an, das<br />

über die Homepage passwortgeschützt zu<br />

erreichen ist. Än<strong>der</strong>ungen können von den<br />

Mitglie<strong>der</strong>n nicht selbst vorgenommen werden.<br />

In dem Verzeichnis sind nur die Mitglie<strong>der</strong><br />

aufgeführt, die schriftlich ihre Einwilligung<br />

gegeben haben. Da<strong>bei</strong> wird <strong>der</strong> Dienstadresse<br />

höchste Priorität zuerkannt, auch<br />

wenn <strong>der</strong> Versand des „BIOspektrum“ an eine<br />

an<strong>der</strong>e Adresse erfolgt. Ein Ausdruck <strong>der</strong><br />

Daten ist nicht möglich, um einen Missbrauch<br />

zu verhin<strong>der</strong>n.<br />

Mitgliedsbescheinigung:<br />

Die nicht spezifizierte und nicht unterschriebene<br />

Mitgliedsbescheinigung 2008 können<br />

Sie wie in den vergangenen Jahren von<br />

<strong>der</strong> Homepage herunterladen. Diese gilt dann<br />

zusammen mit dem Abbuchungsbeleg als<br />

Nachweis für das Finanzamt.<br />

Einzug des Mitglieds<strong>bei</strong>trags per Kreditkarte/Lastschrift:<br />

Die <strong>VAAM</strong> kann weiterhin ihren im Ausland<br />

lebenden Mitglie<strong>der</strong>n den Einzug des <strong>2009</strong><br />

erhöhten Jahres<strong>bei</strong>trags per Kreditkarte<br />

(VISA, Mastercard) anbieten. Dafür benötigen<br />

wir neben <strong>der</strong> aktuellen Laufzeit und <strong>der</strong><br />

eingestanzten Kreditkartennummer die auf<br />

glie<strong>der</strong>n unterschrieben sein müssen. Ich<br />

möchte auch darauf hinweisen, dass <strong>der</strong> Vorstand<br />

<strong>der</strong> <strong>VAAM</strong> den jetzigen 1. Vizepräsidenten<br />

entsprechend <strong>der</strong> Geschäftsordnung<br />

(siehe Homepage <strong>der</strong> <strong>VAAM</strong>) <strong>zur</strong> Wahl zum<br />

Präsidenten vorschlagen wird. Ordentliche<br />

und studentische Mitglie<strong>der</strong> haben auf <strong>der</strong><br />

Mitglie<strong>der</strong>versammlung gleiches Stimmrecht.<br />

Reisekostenzuschüsse für studentische Mitglie<strong>der</strong><br />

können <strong>bei</strong> fristgerecht eingegangenen<br />

Anträgen und <strong>bei</strong> Vorliegen <strong>der</strong> sonstigen<br />

Voraussetzungen nur persönlich ab<br />

Dienstag, den 10. März <strong>2009</strong>, 14.00 Uhr bis<br />

Mittwoch, den 11. März <strong>2009</strong>, 13.00 Uhr im<br />

Tagungsbüro abgeholt werden.<br />

Hubert Bahl<br />

Schriftführer<br />

<strong>der</strong> Rückseite ausgedruckte zusätzliche<br />

Sicherheitszahl (drei Zahlen). Die kostengünstigste<br />

Lösung bleibt jedoch <strong>der</strong> Einzug<br />

per Lastschrift über ein Girokonto in Deutschland,<br />

das nicht auf den gleichen Namen<br />

lauten, aber uns namentlich benannt werden<br />

muss. Eine Überweisung aus dem Euro-<br />

Währungsgebiet ist ebenfalls möglich und<br />

erfor<strong>der</strong>t nur die Nennung unserer BIC-<br />

(GENODEF1GOE) und IBAN-Nummer<br />

(DE6926090050 3900150400). Eine „normale“<br />

Auslandsüberweisung von außerhalb <strong>der</strong><br />

EU ist nach wie vor mit sehr hohen Gebühren<br />

belastet und von daher nicht akzeptabel.<br />

Reisekostenzuschüsse für studentische<br />

Mitglie<strong>der</strong> können <strong>bei</strong> fristgerecht eingegangenen<br />

Anträgen und <strong>bei</strong> Vorliegen <strong>der</strong><br />

sonstigen Voraussetzungen nur persönlich<br />

am Dienstag, den 10. März <strong>2009</strong> von 14.00<br />

bis 17.00 Uhr und am Mittwochvormittag im<br />

Tagungsbüro in Bochum abgeholt werden.<br />

Marion Andreesen, <strong>VAAM</strong>-Sekretariat<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

General Information<br />

Annual Conference <strong>2009</strong> of the <strong>VAAM</strong><br />

Location of the meeting<br />

Ruhr-Universität Bochum<br />

Audimax/Zentrales Hörsaalgebäude<br />

Universitätsstraße 150<br />

44801 Bochum<br />

Germany<br />

Address for correspondence<br />

Conventus Congressmanagement &<br />

Marketing GmbH<br />

Jutta Vach<br />

Markt 8<br />

07743 Jena<br />

Germany<br />

Phone +49 (0)3641 35 33 15<br />

Fax +49 (0)3641 35 33 21<br />

E-Mail: vaam<strong>2009</strong>@conventus.de<br />

Internet: www.vaam<strong>2009</strong>.de<br />

Opening times<br />

Sunday 08.03.<strong>2009</strong> 13:00 – 19:30<br />

Monday 09.03.<strong>2009</strong> 07:30 – 18:00<br />

Tuesday 10.03.<strong>2009</strong> 07:30 – 19:00<br />

Wednesday 11.03.<strong>2009</strong> 08:00 – 13:30<br />

Travelling to Bochum<br />

By plane<br />

Bochum offers air passengers different possibilities<br />

of international and national air<br />

transport. Düsseldorf airport can be reached<br />

in about 45 minutes by car or in 33 minutes<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

by train (ICE). It only takes 60 minutes to go<br />

to the airports of Münster/Osnabrück and<br />

Cologne/Bonn. Closer is the airport in Dortmund<br />

(about 30 minutes).<br />

By car<br />

You can reach Bochum by motorway A 43,<br />

interchange „Bochum-Witten“, take the exit<br />

at „Bochum-Querenburg/Universität“ (exit<br />

no. 19). You will reach the Universitätsstraße<br />

by taking the direction to Universität/Zentrum,<br />

exit at „Uni-Mitte“ (about 2 km). P3 and<br />

P4 are reserved for the conference participants.<br />

Free parking is also sign posted. The<br />

postal address is Universitätsstraße 150,<br />

44801 Bochum.<br />

By train<br />

Bochum is also well-connected with<br />

the intercity network of the Deutsche Bahn<br />

AG. Bochum main station is served by 60<br />

EuroCity (EC) and InterCity (IC) trains every<br />

day.<br />

Public transport<br />

From Bochum main station take the U35<br />

towards Bochum Querenburg (Hustadt) and<br />

get out at stop „Ruhr-Universität“. (Ticket<br />

needed: Preisstufe A).<br />

On weekdays the subway U35 leaves<br />

every 5 minutes and reaches the university<br />

Bochum City: Die Ruhr-Universität Bochum im Großraum Rhein-Ruhr. Mit freundlicher Genehmigung<br />

<strong>der</strong> AG Geomatik im Geographischen Institut <strong>der</strong> Ruhr-Universität-Bochum, <strong>2009</strong><br />

ALLGEMEINES<br />

19<br />

within 9 minutes. Other transport connections<br />

reach and leave the university directly:<br />

– Subway U35 towards Schloß Strünkede via<br />

Herne<br />

– Schnellbus SB67 towards Wuppertal Central<br />

Station via Sprockhövel (every hour)<br />

– Bus 320 towards Witten (every 20 min)<br />

– Bus 339 towards Witten-Center (every hour)<br />

– Bus 346 towards Bochum-Oberdahlhausen<br />

(every 20 min)<br />

– Bus 370 towards S-Bahnhof Dortmund Lütgendortmund<br />

via BO-Langendreer (every<br />

hour)<br />

– Bus 372 towards BO-Langendreer Nord<br />

(every hour)<br />

– Bus 377 towards BO-Langendreer Nord<br />

(every 20 min)<br />

Timetables are available on the website of the<br />

local transportation company „Verkehrsverbund<br />

Rhein-Ruhr (VRR)“, www.vrr.de<br />

Hotel reservation<br />

We have reserved a contingent of rooms at<br />

special rates in Bochum. Please find the reservation<br />

fax on our conference homepage<br />

www.vaam<strong>2009</strong>.de.<br />

Hotel rooms may also be looked through:<br />

www.bochum.de<br />

Registration<br />

Online registration is possible till March<br />

5 through the conference homepage<br />

www.vaam<strong>2009</strong>.de. Registrations after this<br />

date are possible only on site. Beside cash<br />

payments we also accept credit cards at the<br />

conference reception desk (Master/Euro,<br />

VISA, American Express and JBC) as well as<br />

EC-Cards.<br />

Coat check<br />

You will find the wardrobe at the Audimax.<br />

Mixer<br />

The Mixer will take place on Tuesday<br />

10.03.<strong>2009</strong> at 20:00 at the Mensa, Ruhr University<br />

Bochum. Accompanying persons may<br />

purchase a ticket for the mixer at the conference<br />

reception desk.<br />

Annual Meeting<br />

The Annual General Meeting of the <strong>VAAM</strong><br />

will take place on Tuesday 10.03.<strong>2009</strong>, 17:30

20 ALLGEMEINES<br />

Plan of site (Audimax and HZO)<br />

www.ruhr-uni-bochum.de<br />

in the Audimax of the Ruhr University<br />

Bochum.<br />

Presentation of the Honory<br />

Award, PhD Awards, and poster<br />

prizes<br />

The presentation of the Honory Award will<br />

take place on 09.03.<strong>2009</strong> at 09:50, Audimax,<br />

Ruhr University Bochum.<br />

The presentation of the PhD Awards will<br />

take place on 10.03.<strong>2009</strong> at 17:30, Audimax,<br />

Ruhr University Bochum.<br />

The presentation of the poster prizes will<br />

take place on 11.03.<strong>2009</strong> at 12:10, Audimax,<br />

Ruhr University Bochum.<br />

Trade Exhibition<br />

The trade exhibition will be located at the<br />

Audimax, Ruhr University Bochum. The exhibitors<br />

are looking forward to welcoming you<br />

at their booth!<br />

Short lectures<br />

Short lectures are to be held in English. Data<br />

projectors are available in each of the lecture<br />

halls. In each lecture hall there will be an<br />

assistant for technical support. We ask all lecturers<br />

to make use of the computer facilities<br />

located at HZO 90 to check their presentations<br />

in advance.<br />

Primary support will be given to<br />

the following formats:<br />

– Windows based PowerPoint<br />

– PowerPoint presentations saved on CD or<br />

USB memory stick<br />

Please submit your presentation in room HZO<br />

90 at least 120 minutes before your lecture<br />

will start. You are asked to clearly label your<br />

CD/memory stick and the file with your short<br />

lecture code number and the name of the person<br />

giving the talk. All presentations will be<br />

loaded onto our computers and will be deleted<br />

after the talks.<br />

Posters<br />

Posters are to be presented in English and in<br />

the format DIN A0 (84,1 cm x 118,9 cm).<br />

Authors are asked to attach to the posters the<br />

time when they will be available for discussion.<br />

Mounting materials will be provided.<br />

The posters may be mounted from 14:00<br />

on Sunday, 08.03.<strong>2009</strong> and should not be<br />

removed before 11:00 on Wednesday,<br />

12.03.<strong>2009</strong>.<br />

The poster sessions will be held on:<br />

Monday 09.03.<strong>2009</strong> 13:45 – 15:15<br />

Tuesday 10.03.<strong>2009</strong> 15:30 – 17:30<br />

Conference fees<br />

Member N 140,–<br />

Day ticket<br />

N 90,–<br />

Non member<br />

Student, technical assistant,<br />

N 220,– N 120,–<br />

unemployed (member)<br />

Student, technical assistant,<br />

N 90,– N 40,–<br />

unemployed (non member) N 120,– N 70,–<br />

Mixer – Participants included N 30,–<br />

Mixer – Accompanying Person N 30,– N 30,–<br />

www.<strong>VAAM</strong>.de<br />

Aktuelles über<br />

• Wissenschaft im Allgemeinen und Mikrobiologie im Beson<strong>der</strong>en<br />

• Tagungen und Workshops<br />

• Institute und Fachgruppen<br />

und vieles mehr finden Sie auf <strong>der</strong> <strong>VAAM</strong>-Homepage.<br />

Schauen Sie doch mal rein!<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

ALLGEMEINES<br />

Fachausstellung Mikrobiologie<br />

Unternehmen auf <strong>der</strong> Fachausstellung und Sponsoren<br />

(Stand: 19.01.<strong>2009</strong>)<br />

Die Vereinigung für Allgemeine und Angewandte Mikrobiologie bedankt sich <strong>bei</strong> den Unternehmen <strong>der</strong> Fachausstellung<br />

und den Sponsoren für die Unterstützung <strong>der</strong> <strong>VAAM</strong> <strong>Jahrestagung</strong> <strong>2009</strong><br />

˘ AdvoGen ConsulT – Ben<strong>der</strong> & Kauch<br />

GbR (Bochum)<br />

www.advogenconsult.de<br />

˘ AGOWA GmbH (Berlin)<br />

www.agowa.de<br />

˘ Analytik Jena AG/bio solutions (Jena)<br />

www.analytik-jena.de<br />

˘ AppliChem GmbH (Darmstadt)<br />

www.applichem.de<br />

˘ Axel Semrau GmbH & Co. KG<br />

(Spröckhövel)<br />

www.axelsemrau.de<br />

˘ Bio-Budget Technologies GmbH<br />

(Krefeld)<br />

www.bio-budget.com<br />

˘ Bioline GmbH (Luckenwalde)<br />

www.bioline.com<br />

˘ bioMérieux Deutschland GmbH<br />

(Nürtingen)<br />

www.biomerieux.de<br />

˘ Biometra GmbH (Göttingen)<br />

www.biometra.de<br />

˘ BIO-RAD Laboratories GmbH (München)<br />

www.bio-rad.com<br />

˘ Biozym Scientific GmbH (Hessisch<br />

Oldendorf)<br />

www.biozym.com<br />

˘ Bruker Daltonik GmbH (Bremen)<br />

www.bruker-daltonik.de<br />

˘ Carl Roth GmbH + Co. KG (Karlsruhe)<br />

www.carlroth.de<br />

˘ Cfm Oskar Tropitzsch – Iris Biotech<br />

GmbH (Marktredwitz)<br />

www.iris-biotech.de<br />

˘ Coring System Diagnostix GmbH<br />

(Gernsheim)<br />

www.coring.de<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

˘ DFG – Deutsche Forschungsgemeinschaft<br />

(Bonn)<br />

www.dfg.de<br />

˘ DSMZ – Deutsche Sammlung von<br />

Mikroorganismen und Zellkulturen<br />

GmbH (Braunschweig)<br />

www.dsmz.de<br />

˘ Eurofins MWG GmbH (Ebersberg)<br />

www.the-mwg.com<br />

˘ G. Kisker GbR – Produkte für die<br />

Biotechnologie (Steinfurt)<br />

www.kisker-biotech.com<br />

˘ GATC Biotech AG (Konstanz)<br />

www.gatc-biotech.com<br />

˘ IBA GmbH (Göttingen)<br />

www.iba-go.com<br />

˘ Implen GmbH (München)<br />

www.implen.de<br />

˘ Infors GmbH (Stuttgart)<br />

www.infors-ht.com<br />

˘ Intas Science Imaging Instruments<br />

GmbH (Göttingen)<br />

www.intas.de<br />

˘ IUL Instruments GmbH (Königswinter)<br />

www.iul-instruments.de<br />

˘ LGC Standards GmbH (Wesel)<br />

www.lgcstandards.com<br />

˘ Macherey Nagel GmbH & Co.KG (Düren)<br />

www.mn-net.com<br />

˘ Merck Biosciences GmbH<br />

(Schwalbach/Ts.)<br />

www.merckbiosciences.de<br />

˘ MoBiTec GmbH (Göttingen)<br />

www.mobitec.de<br />

˘ MP Biomedicals GmbH (Eschwege)<br />

www.mpbio.com<br />

21<br />

˘ New England BioLabs (Frankfurt/Main)<br />

www.neb-online.de<br />

˘ Partec GmbH (Münster)<br />

www.partec.de<br />

˘ Pearson Education Deutschland GmbH<br />

(München)<br />

www.pearson-education.de<br />

˘ QIAGEN GmbH (Hilden)<br />

www.qiagen.com<br />

˘ SEQLAB Sequence Laboratories GmbH<br />

(Göttingen)<br />

www.seqlab.de<br />

˘ Serva Electrophoresis GmbH<br />

(Heidelberg)<br />

www.serva.de<br />

˘ Spektrum Akademischer Verlag GmbH<br />

BIOspektrum<br />

(Heidelberg)<br />

www.spektrum-verlag.de<br />

www.biospektrum.de<br />

˘ SÜD-LABORBEDARF GmbH (Gauting)<br />

www.suedlabor.de<br />

˘ Thermo Scientific (Erembodegem, BE)<br />

www.perbio.com<br />

˘ Toepffer Laborsysteme GmbH<br />

(Göppingen)<br />

˘ Transgenomic Ltd. (Berlin)<br />

www.transgenomic.com<br />

˘ USB Europe GmbH (Staufen)<br />

www.usbweb.com

22 ALLGEMEINES<br />

Symposium by GATC Biotech AG (Konstanz)<br />

Monday, March 9, 12:45 – 13:45, HZO 40<br />

Case studies on Next Gen sequencing<br />

technologies and bioinformatic analysis<br />

tools<br />

Dr. Andrea Bolte<br />

Performance of Next Generation sequencing<br />

systems in daily routine<br />

Dr. Kerstin A. Stangier<br />

Case studies using different bioinformatic<br />

tools in Next Gen sequencing projects<br />

N.N.<br />

Sequencing and Resequencing of bacterial<br />

genomes – case studies<br />

ó Projects performed by GATC show that the<br />

use of one technology alone does not deliver<br />

the best results for all projects. Rather a combination<br />

of two or three technologies provides<br />

a more complete, cost-effective analysis. In<br />

addition to sequencing, bioinformatic analysis<br />

is critically important for gaining an in-depth<br />

un<strong>der</strong>standing of the biological significance<br />

of the sequence data. The combination, analysis<br />

and visualisation of these data are key challenges<br />

to the successful application of the Next<br />

Generation sequencing technologies.<br />

Symposium by Eurofins MWG Operon (Ebersberg)<br />

Tuesday, March 10, 11:40 – 13:00, HZO 40<br />

Genome Sequencing and Transcriptom<br />

Analysis using Next Generation Sequencing.<br />

Presentation of selected results<br />

from prokaryotic, fungal and eukaryotic<br />

genomes.<br />

Dr. Georg Gradl, Dr. Axel Strittmatter, Eurofins<br />

MWG Operon, Ebersberg, Germany<br />

ó We have successfully introduced the<br />

Roche Genome Sequencer GS 20/454 technology<br />

in December 2006 and meanwhile<br />

upgraded to the GS FLX system with Titanium<br />

series chemistry. Build around this platform<br />

we offer a broad portfolio for de novo<br />

and re-sequencing of bacterial and fungal<br />

genomes and bioinformatics analysis like<br />

strain comparison and annotation. Sequencing<br />

with barcodes allows us multiplexing of<br />

samples like e.g. BAC clones or phages. Transcriptom<br />

analysis by Next Generation<br />

sequencing becomes more and more important.<br />

We offer the construction and sequencing<br />

of expression profile, normalised cDNA<br />

and small RNA libraries that are designed<br />

for the GS FLX technology.<br />

Qualitative and quantitative expression profiling<br />

is offered with specially designed 3’-fragment<br />

libraries. Thanks to the long reads of up<br />

to 450 bp coding and non coding information<br />

can be used for annotation and mapping to the<br />

chromosomal locus. Non coding RNA libraries<br />

with longer than the standard 29 bp allow the<br />

analysis of non coding RNAs that are not seen<br />

with alternative sequencing technologies.<br />

A selected number of examples will be presented.<br />

ó<br />

To ensure a successful sequencing project<br />

and to maximise the information obtained, it<br />

is necessary to choose the best Next Generation<br />

technology or combination of technologies<br />

followed by bioinformatic analysis using<br />

a pipeline consisting of state-of-the-art analysis<br />

tools. ó<br />

Karrieresymposium<br />

Dienstag, 10. 3. <strong>2009</strong><br />

15.30 – 17.00 Uhr<br />

Raum HZO 40<br />

Marvin Karos, Ludwigshafen<br />

BASF-SE, Rekrutierung Naturwissenschaftler und Ingenieure<br />

Biologie – Bewerbung – Beruf<br />

„Einstieg und Entwicklung in <strong>der</strong> Industrie“<br />

Carmen Zirngibl, Düsseldorf<br />

Geschäftsführerin und Grün<strong>der</strong>in <strong>der</strong><br />

DCZ Life Science Personalmanagement GmbH<br />

Chancen – Herausfor<strong>der</strong>ungen – Perspektiven<br />

„ Quest for Talents in den Life Sciences“<br />

Nicole Nolting, Hamburg<br />

Vorstandsreferentin und Wissenschaftskoordinatorin<br />

am Heinrich-Pette-Institut<br />

Karrieremöglichkeiten über die Grenzen des Labors hinweg<br />

„Die drei ??? und die verlassene Bench“<br />

Im Anschluss: Diskussionsrunde<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

CONFERENCE PROGRAMME<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

CONFERENCE PROGRAMME<br />

<strong>VAAM</strong> <strong>2009</strong> <strong>Jahrestagung</strong> Bochum (08.03.–11.03.<strong>2009</strong>)<br />

˘Sunday, 08.03.<strong>2009</strong><br />

14:00 – 19:30 Registration and mounting of the posters Audimax, HZO<br />

PUBLIC LECTURE Audimax<br />

16:00 – 16:30 Welcome Address<br />

Bürgermeisterin <strong>der</strong> Stadt Bochum Frau Gabriela Schäfer<br />

Rektor <strong>der</strong> Ruhr-Universität Bochum Prof. Dr. Elmar W. Weiler<br />

Dekan <strong>der</strong> Fakultät für Biologie und Biotechnologie Prof. Dr. Franz Narberhaus<br />

Prodekan <strong>der</strong> Medizinischen Fakultät Prof. Dr. Klaus Überla<br />

16:30 – 17:15 H 01: W. Goebel<br />

Biocenter (Microbiology), University of Würzburg, Würzburg, Germany<br />

„Wie sich bakterielle Krankheitserreger in Animalzellen vermehren“<br />

Chairperson: S. Gatermann<br />

17:15 – 18:00 Coffee break<br />

KEYNOTE LECTURES Audimax<br />

18:00 – 18:40 H 02: A. Goldberg<br />

Department of Cell Biology, Harvard Medical School, Cambridge, Boston, USA<br />

“Functions of the Proteasome: From Protein Degradation and Immune Surveillance<br />

to Cancer Therapy”<br />

Chairperson: R. Erdmann<br />

18:40 – 19:20 H 03: D. Haas<br />

Department of Fundamental Microbiology, University of Lausanne,<br />

Lausanne, Switzerland<br />

“Small regulatory RNAs make a great difference to Pseudomonas”<br />

Chairperson: F. Narberhaus<br />

19:30 Welcome Reception Audimax<br />

˘Monday, 09.03.<strong>2009</strong><br />

KEYNOTE LECTURES Audimax<br />

08:30 – 09:10 H 04: H. Hennecke<br />

Institute of Microbiology, ETH Zürich, Zürich, Switzerland<br />

“What it takes to be a nitrogen-fixing root-nodule bacterium”<br />

Chairperson: F. Narberhaus<br />

09:10 – 09:50 H 05: S. Hultgren<br />

Department of Molecular Microbiology, Washington University School of Medicine,<br />

St. Louis, USA<br />

“E. coli Biofilms, Bottlenecks, and Host Responses in Urinary Tract Infections”<br />

Chairperson: S. Gatermann<br />

09:50 – 10:30 <strong>VAAM</strong> Honory Award to: Dr. Marcel Kuypers<br />

Max-Planck-Institute for Marine Microbiology, Bremen, Germany<br />

“Sizing Up the Uncultivated Majority”<br />

Audimax<br />

10:30 – 11:00 Coffee break<br />

11:00 – 12:45 Short lectures I Audimax, HZO<br />

12:45 – 13:45 Lunch<br />

12:45 – 13:45 Symposium by GATC Biotech AG (Konstanz)<br />

Dr. Andrea Bolte, Dr. Kerstin A. Stangier<br />

“Case studies on Next Gen sequencing technologies and bioinformatic analysis tools”<br />

HZO 40<br />

13:45 – 15:15 Poster Session I HZO<br />

15:15 – 17:00 Short lectures II Audimax, HZO<br />

17:00 – 17:30 Coffee break<br />

17:30 – 19:30 Special Groups Mini Symposia Audimax, HZO<br />

17:30 – 19:30 Annual Meetings of the Special Groups Audimax, HZO<br />

08:30 – 18:00 Trade Exhibition Audimax<br />

23

24 CONFERENCE PROGRAMME<br />

CONFERENCE PROGRAMME<br />

<strong>VAAM</strong> <strong>2009</strong> <strong>Jahrestagung</strong> Bochum (08.03.–11.03.<strong>2009</strong>)<br />

˘Tuesday, 10.03.<strong>2009</strong><br />

KEYNOTE LECTURES Audimax<br />

08:30 – 09:10 H 06: L. Eberl<br />

Institute of Plant Biology, Department of Microbiology, University of Zurich,<br />

Zürich, Switzerland<br />

“The formation of surface-associated communities by Burkhol<strong>der</strong>ia sp. is dependent<br />

on cell-to-cell communication”<br />

Chairperson: F. Narberhaus<br />

09:10 – 09:50 H 07: J. C. Dunlap<br />

Department of Genetics, Dartmouth Medical School, Hanover, USA<br />

“Proteomics and Epigenetics: Dissection of the Eukaryotic Circadian Clock in Neurospora”<br />

Chairperson: M. Nowrousian<br />

09:50 – 10:20 Coffee break<br />

10:20 – 11:00 H 08: S. Merchant<br />

Department of Chemistry and Biochemistry, The Molecular Biology Institute at UCLA,<br />

Los Angeles, USA<br />

“Functional analysis of trace nutrient homeostasis in Chlamydomonas using next<br />

generation sequencers”<br />

Chairperson: N. Frankenberg-Dinkel<br />

11:00 – 11:40 H 09: E. Flores<br />

Instituto de Bioquímica Vegetal y Fotosíntesis, Sevilla, Spain<br />

“Cell differentiation and multicellularity in heterocyst-forming cyanobacteria”<br />

Chairperson: T. Happe<br />

11:40 – 13:00 Lunch<br />

11:40 – 13:00 Symposium by Eurofins MWG Operon (Ebersberg)<br />

Dr. Georg Gradl, Dr. Axel Strittmatter, Eurofins MWG Operon, Ebersberg, Germany<br />

“Genome Sequencing and Transcriptom Analysis using Next Generation Sequencing.<br />

Presentation of selected results from prokaryotic, fungal and eukaryotic genomes.”<br />

HZO 40<br />

13:00 – 15:00 Short lectures III Audimax, HZO<br />

15:00 – 15:30 Coffee break<br />

15:30 – 17:30 Poster Session II HZO<br />

15:30 – 17:00 Karrieresymposium HZO 40<br />

17:30 <strong>VAAM</strong> Annual General Meeting<br />

Presentation of the PhD Awards<br />

Sponsored by: BASF AG, Bayer Schering Pharma AG, Evonik Degussa GmbH,<br />

Lonza Group Ltd, New England Biolabs GmbH,<br />

Sanofi Aventis Pharma Deutschland GmbH.<br />

Audimax<br />

08:30 – 18:00 Trade Exhibition Audimax<br />

20:00 Mixer Mensa<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

CONFERENCE PROGRAMME<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

CONFERENCE PROGRAMME<br />

<strong>VAAM</strong> <strong>2009</strong> <strong>Jahrestagung</strong> Bochum (08.03.–11.03.<strong>2009</strong>)<br />

˘Wednesday, 11.03.<strong>2009</strong><br />

KEYNOTE LECTURES Audimax<br />

09:00 – 09:40 H 10: N. Pfanner<br />

Institute for Biochemistry and Molecular Biology, University of Freiburg,<br />

Freiburg, Germany<br />

“The mitochondrial machinery for import and assembly of proteins”<br />

Chairperson: R. Erdmann<br />

09:40 – 10:20 H 11: Z. Reich<br />

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel<br />

“Cyanobacterial thylakoid membrane networks: Connectedness, permeability,<br />

and (dis-) similarity to higher-plant networks”<br />

Chairperson: M. Rögner<br />

10:20 – 10:50 Coffee break<br />

10:50 – 11:30 H 12: B. Friedrich<br />

Institut für Mikrobiologie, Humboldt-Universität zu Berlin, Berlin, Germany<br />

“The challenge of biological hydrogen conversion in the presence of air”<br />

Chairperson: M. Rögner<br />

11:30 – 12:10 H 13: D. Archer<br />

The School of Biology, The University of Nottingham, Nottingham, UK<br />

“Molecular basis of resistance to weak acids in fungi”<br />

Chairperson: U. Kück<br />

12:10 – 12:30 Poster Awards Audimax<br />

Sponsored by: Ruhr-Universität Bochum<br />

12:30 – 12:45 Closing Remarks Audimax<br />

08:30 – 13:00 Trade Exhibition Audimax<br />

25

26 SPECIAL GROUPS<br />

ACTIVITIES OF THE SPECIAL GROUPS<br />

Mini-Symposia of the Special Groups: Monday, March 9, 17:30–19:30<br />

˘Special Group Biologie bakterieller Naturstoffproduzenten<br />

Organisation: W. Wohlleben, University of Tübingen, Germany<br />

HZO 60<br />

FGA 1 17:30 T. Schnei<strong>der</strong>*, G. Sahl<br />

Institute for Medical Microbiology, Immunology and Parasitology – Pharmaceutical Microbiology Section,<br />

University of Bonn, Bonn, Germany<br />

“Cell wall biosynthesis as a target of new antibiotic compounds”<br />

FGA 2 17:55 H. Brötz-Oesterhelt<br />

Antibacterial Research, AiCuris GmbH & Co. KG, Wuppertal, Germany<br />

“A novel antibacterial class acting by reprogramming Clp protease”<br />

FGA 3 18:20 C. Hertweck<br />

Leibniz Institute for Natural Product Research and Infection Biology (HKI), Jena, Germany<br />

“Mycotoxin Biosynthesis by Endofungal Bacteria – Insights into an overlooked system”<br />

FGA 4 18:45 J. Vogelmann, J. Guezguez, T. Roth, G. Muth*<br />

Mikrobiologie/Biotechnologie, Universität Tübingen, Tübingen, Germany<br />

“Molecular Characterization of the DNA- translocation systems mediating conjugal plasmid transfer in<br />

Streptomyces”<br />

FGA 5 19:10 D. Claessen<br />

University of Groningen, The Netherlands<br />

“Formation of fimbriae during attachment of Streptomyces coelicolor: roles of cellulose and amyloids”<br />

˘Special Group Biotransformation<br />

Topic: „Challenges and New Trends in Biotransformation“<br />

Organisation: U. Bornscheuer, Greifswald University, Germany; J. Eck, B.R.A.I.N. AG, Zwingenberg, Germany<br />

HZO 50<br />

FGB 1 17:30 D. Janssen<br />

University of Groningen, The Netherlands<br />

„Evolving dehalogenases: from environmental cleanup to applied biocatalysis“<br />

FGB 2 18:00 W. Kroutil<br />

Karl-Franzens University Graz, Austria<br />

„Deracemisation via Enzyme Cascades“<br />

FGB 3 18:30 M. Höhne<br />

Greifswald University, Germany<br />

„Synthesis of optically active 3-aminopyrrolidines and -piperidines with omega-transaminase“<br />

FGB 4 19:00 P. Neubauer<br />

Technische Universtät Berlin/Berlin Institute of Technology, Germany<br />

„EnBase – Microplate based high-cell-density fermentation for high-throughput and high-content screening<br />

of biocatalysts“<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

ACTIVITIES OF THE SPECIAL GROUPS<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

SPECIAL GROUPS<br />

Mini-Symposia of the Special Groups: Monday, March 9, 17:30–19:30<br />

˘Special Group Functional Genomics<br />

Topic: “Evolution of Information Processing Systems”<br />

Organisation: H.-P. Klenk, DSMZ, Braunschweig, Germany<br />

HZO 70<br />

FGC 1 17:30 C. Brochier-Armanet<br />

Institut de Biologie Structurale et de Microbiologie, Marseille, France<br />

“LUCA and the universal tree of life”<br />

FGC 2 18:10 S. Gribaldo<br />

Unite de Biologie Moleculaire chez les Extremophiles (BMGE), Département de Microbiologie – Institut Pasteur,<br />

Paris, France<br />

“The Thaumarchaeota: a key to archaeal history?”<br />

FGC 3 18:50 J. McInerney<br />

Departement of Biology, The National University of Ireland Maynooth, County Kildare, Ireland<br />

“The transition from prokaryote to eukaryote”<br />

19:30 Annual Meeting of the Special Group Functional Genomics<br />

Discussion of Work program <strong>2009</strong>/2010<br />

Election of speaker and co-speaker of the special interest group<br />

˘Special Group Fungal Biology and Biotechnology<br />

Topic: „Macromolecules secreted by fungi“<br />

Organisation: J. Schirawski, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany<br />

HZO 20<br />

FGD 1 17:30 T. Brefort*, B. Franzki, J. Schirawski, H. Ghareeb, C. Mengel, E. Meyer, V. Vincon, R. Kahmann<br />

Department of Organismic Interactions, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany<br />

“A highly diverse pathogenicity island for secreted proteins modulates biotrophy of Ustilago maydis and<br />

Sporisorium reilianum”<br />

FGD 2 17:45 P. Wiemann 1 *, W. Brown 2 , U. Humpf 3 , B. Tudzynski 1<br />

1 Institut für Botanik, Westfälische Wilhelms-Universität Münster, Münster, Germany<br />

2 Mycotoxin Research Unit, U.S. Department of Agriculture-ARS, Peoria, United States<br />

3 Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster, Münster, Germany<br />

“How does VeA effect secondary metabolism in Fusarium fujikuroi?”<br />

FGD 3 18:00 L.M. Blank 1 *, M. Fraatz 2 , A. Schmid 1 , H. Zorn 2<br />

1 Biochemical and Chemical Engineering, TU Dortmund, Dortmund, Germany<br />

2 Institute of Food Chemistry and Food Biotechnology, Justus Liebig University Giessen, Giessen, Germany<br />

“Quantitative physiology of the basidiomycete Pleurotus sapidus”<br />

FGD 4 18:15 R. Wu*, H. Luo, L.L.P. Vrijmoed<br />

Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, Hong Kong<br />

“Studies on the biodegradation of anthracene and benz[a]anthracene by two Fusarium sp. stains isolated<br />

from mangrove sediment”<br />

FGD 5 18:30 B. Cherdchim*, M. Navarro-González, A. Majcherczyk, U. Kües<br />

Molecular Wood Biotechnology and Technical Mycology, Georg-August-University Göttingen, Göttingen,<br />

Germany<br />

“Wood extractives as a whole protect grand fir (Abies grandis) wood against enzymatic degradation by<br />

white-rot fungi but specific compounds induce laccases for attack on lignin”<br />

FGD 6 18:45 S. Barig*, R. Alisch, S. Nieland, K.-P. Stahmann<br />

Technische Mikrobiologie, Fachbereich Bio-, Chemie- und Verfahrenstechnik, Fachhochschule Lausitz,<br />

Senftenberg, Germany<br />

“Secreted but cell wall associated lipases by Phialemonium spec. AW02 allow easy down-stream processing”<br />

27

28 SPECIAL GROUPS<br />

ACTIVITIES OF THE SPECIAL GROUPS<br />

Mini-Symposia of the Special Groups: Monday, March 9, 17:30–19:30<br />

FGD 7 19:00 Invited Lecture<br />

F. Klis*, G. Sosinska, A. Sorgo, P. De Groot, E. Man<strong>der</strong>s, H. Dekker, L. de Koning, C. De Koster, S. Brul<br />

Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands<br />

“An in vitro model for mucosal infections reveals the dynamics of the cell wall proteome of the clinical fungus<br />

Candida albicans”<br />

19:30 Annual Meeting of the Special Group Fungal Biology and Biotechnology/Experimentelle Mykologie<br />

˘Special Group Identification and Systematics<br />

Organisation: B. J. Tindall, DSMZ, Braunschweig, Germany<br />

HZO 40<br />

FGE 1 17:30 F. O. Glöckner*, H. Teeling, M. Weber, J. Waldmann<br />

Max Planck Institute for Marine Microbiology, Microbial Genomics and Bioinformatics<br />

Group, Bremen, Germany<br />

“Needles in the haystack: Binning and phylogenetic classification in microbial metagenomic communities”<br />

FGE 2 18:00 D. W. Ussery 1 *, T. M. Wassenaar 2<br />

1 Center for Biological Sequences, Danish Technical University, Lyngby, Denmark,<br />

2 Molecular Microbiology and Genomics Consultants, Zotzenheim, Germany<br />

“The genus Burkhol<strong>der</strong>ia: analysis of 54 genomic sequences”<br />

FGE 3 18:30 E.R.B. Moore 1 *, L. Svensson 1 , C. Unosson 1 , N. Karami 2<br />

1 Culture Collection University of Göteborg (CCUG), University of Göteborg, Göteborg, Sweden, 2 Department<br />

of Clinical Bacteriology, University of Göteborg, Göteborg, Sweden<br />

“Nucleic acid sequencing for evaluating bacterial species and systematics: applications for identification”<br />

FGE 4 19:00 P. Yarza 1 *, M. Richter 1 , J. Peplies 2 , J. Euzeby 3 , R. Amann 4 , H. Schleifer 5 , W. Ludwig 5 , F. O. Glöckner 6 ,<br />

R. Rosselló-Móra 1<br />

1 Marine Microbiology Group, Instituto Mediterraneo de Estudios Avanzados, Esporles (Mallorca), Spain,<br />

2 Ribocon GmbH, Ribocon GmbH, Bremen, Germany, 3 Société de Bacteriologie Systématique et Vétérinaire<br />

SBSV, École Nationale Vétérinaire de Touluse (ENVT), Toulouse, France, 4 Department of Molecular Ecology,<br />

Max Planck Institute for Marine Microbiology, Bremen, Germany, 5 Lehrstuhl für Mikrobiologie, Technische<br />

Universität München, Freising, Germany, 6 Microbial Genomics Group, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

“The All-Species Living Tree project”<br />

19:30 Annual Meeting of the Special Group Identification and Systematics<br />

˘Special Group Pathogenicity<br />

Topic: “Microbial pathogenicity meets physiology”<br />

Organisation: A. Peschel, University of Tübingen, Germany; P. Dersch, University of Braunschweig, Germany<br />

HZO 30<br />

FGF 1 17:30 P. Nitsche-Schmitz 1 *, K. Dinkla 1 , V. Barroso 1 , S. Reißmann 1 , H. Linge 2 , J.M. Frick 2 , M. Rohde 1 ,<br />

G.S. Chhatwal 1<br />

1 Mikrobielle Pathogenität, Helmholtz-Zentrum für Infektionsforschung, Braunschweig, Germany,<br />

2 Dept. of Clinical Sciences, BMC, Lund University, Lund, Sweden<br />

“Identification of a streptococcal octa-peptide motif involved in acute rheumatic fever”<br />

FGF 2 17:50 J. Schilling, B. Shutinoski, K. Wagner, G. Heusipp*<br />

ZMBE, Institut für Infektiologie, Westf. Wilhelms-Universität Münster, Münster, Germany<br />

“The Pyp regulatory network of Yersinia enterocolitica controls expression of pili and type-II secretion<br />

systems”<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

ACTIVITIES OF THE SPECIAL GROUPS<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

SPECIAL GROUPS<br />

Mini-Symposia of the Special Groups: Monday, March 9, 17:30–19:30<br />

FGF 3 18:10 B. Waidner1 *, M. Specht2 , F. Dempwolff2 , K. Häberer1 , V. Speth3 , M. Kist1 , P.L. Graumann2 1Institut für med. Mikrobiologie und Hygiene, Uniklinik Freiburg, Freiburg, Germany<br />

2Institut für Mikrobiologie, Universität Freiburg, Freiburg, Germany, 3Institut für Zellbiologie, Universität Freiburg,<br />

Freiburg, Germany<br />

“Helicobacter pylori contains a novel system of cytoskeletal elements that is essential for the maintenance<br />

of cell shape and for the development of motility”<br />

FGF 4 18:30 C. Haagsma 1 , K. Andries 2 , A. Koul 2 , H. Lill 1 , D. Bald 1 *<br />

1 Structural Biology, Department of Molecular Cell Biology, VU University Amsterdam, Amsterdam, Netherlands,<br />

2 Pharmaceutical Research and Development, Johnson & Johnson, Beerse, Belgium<br />

“Respiratory ATP synthesis: an Achilles’ heel in mycobacteria?”<br />

FGF 5 18:50 C. Otzen*, M. Brock<br />

Microbial Biochemistry and Physiology, Hans-Knöll-Institute, Jena, Germany<br />

“Propionyl-CoA metabolism in Candida albicans”<br />

FGF 6 19:10 B. Winterberg 1 *, U. Linne 2 , R. Kahmann 1 , J. Schirawski 1<br />

1 Max-Planck-Institut für Terrestrische Mikrobiologie, Abteilung Organismische Interaktionen, Marburg, Germany,<br />

2 Fachbereich Biochemie, Philipps-Universität Marburg, Marburg, Germany<br />

“Regulation of si<strong>der</strong>ophore biosynthesis in Ustilago maydis during the infection of maize”<br />

19:30 Annual Meeting of the Special Group Pathogenicity<br />

Future activities<br />

˘Special Group Regulation and Signalstransduction in Prokaryotes<br />

Topic: „Trigger Enzymes“<br />

Organisation: K. Jung, Ludwig-Maximilians-Universität, Martinsried, Germany<br />

Audimax<br />

FGG 1 17:30 D. Becker<br />

Biochemistry Department, University of Nebraska-Lincoln, Lincoln, United States<br />

“Redox control of proline metabolism”<br />

FGG 2 18:00 L. Sonenshein*, W. Serio, K. Pechter<br />

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, United States<br />

“The RNA-binding regulatory activity of Bacillus subtilis aconitase”<br />

FGG 3 18:30 F.M. Rothe, M. Lehnik-Habrink, J. Stülke, F.M. Commichau*<br />

Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

“Functional analysis of Rny: A novel player involved in RNA metabolism of Bacillus subtilis”<br />

FGG 4 18:45 L. Tetsch*, A. Dönhöfer, T. Friedrich, K. Jung<br />

Biozentrum <strong>der</strong> LMU München, Mikrobiologie, Ludwig-Maximilians-Universität, Martinsried, Germany<br />

“Activation of the membrane-integrated transcriptional activator CadC of Escherichia coli involves the<br />

opening of a periplasmic disulfide bridge”<br />

FGG 5 19:00 J. Bauer 1 *, W. Erker 2 , F. Liao 2 , T. Basché 2 , G. Unden 1<br />

1 Institut für Mikrobiologie und Weinforschung, AG Unden, Johannes-Gutenberg Universität Mainz, Mainz,<br />

Germany, 2 Institut für Physikalische Chemie, Johannes-Gutenberg Universität Mainz, Mainz, Germany<br />

“DctA of Escherichia coli interacts with the DcuSR two component system”<br />

19:30 Annual Meeting of the Special Group Regulation<br />

29

30 SPECIAL GROUPS<br />

ACTIVITIES OF THE SPECIAL GROUPS<br />

Mini-Symposia of the Special Groups: Monday, March 9, 17:30–19:30<br />

˘Special Group Yeast<br />

Topic: „Yeast as a model in mo<strong>der</strong>n life science“<br />

Organisation: B. Schäfer, Department of Biology IV (Microbiology & Genetics), RWTH Aachen University, Aachen,<br />

Germany; K.-D. Entian, Institute for Molecular Biosciences, University of Frankfurt, Frankfurt, Germany<br />

HZO 10<br />

FGH 1 17:30 R. Lill<br />

Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg, Germany<br />

“Biogenesis of cellular iron-sulfur proteins: The essential and minimal function of mitochondria”<br />

FGH 2 18:00 K.-D. Entian*, B. Meyer, J. Wöhnert<br />

Institute for Molecular Biosciences, University of Frankfurt, Frankfurt, Germany<br />

„Untersuchung <strong>der</strong> Funktion <strong>der</strong> nucleolären essentiellen Methyltransferase Nep1 in <strong>der</strong> eukaryotischen<br />

Ribosomenbiogenese“<br />

FGH 3 18:25 B. Schäfer<br />

Department of Biology IV (Microbiology & Genetics), RWTH Aachen University, Aachen, Germany<br />

“Fission yeast as a model to investigate mitochondrial RNA turn over”<br />

FGH 4 18:50 R. Klassen*, S. Wemhoff, J. Krause, F. Meinhardt<br />

Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische Wilhelms-Universität, Münster, Germany<br />

“DNA apurinic site repair confers resistance to yeast anticodon-nuclease killer toxins”<br />

FGH 5 19:05 D. Gerhards 1 *, S. Schnell 2 , M. Grossmann 1 , C. von Wallbrunn 1<br />

1 Section of Microbiology and Biochemistry, Geisenheim Research Center, Geisenheim, Germany, 2 Institute of<br />

Applied Microbiology, Justus-Liebig-University Giessen, Gießen, Germany<br />

“Investigations of the metacaspase YCA1 for better detection of apoptosis in Saccharomyces cerevisiae<br />

during alcoholic fermentation of grape must.”<br />

19:20 Annual Meeting of the Special Group Yeast<br />

˘Special Group Archea<br />

HZO 80<br />

17:30 Annual Meeting<br />

˘Special Group Environmental Microbiology<br />

HZO 80<br />

19:30 Annual Meeting<br />

˘Special Group Water/Waste Water<br />

HZO 90<br />

17:30 Annual Meeting<br />

˘New Special Group Symbiotic Interactions<br />

HZO 90<br />

19:30 Formation of the new Special Group<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Monday, 09.03.<strong>2009</strong> 11:00–12:45<br />

HZO 60<br />

HZO 50<br />

HZO 40<br />

HZO 30<br />

HZO 20<br />

HZO 10<br />

Audimax<br />

Systems Biology<br />

Photosynthesis<br />

and<br />

Bioenergetics<br />

Transport<br />

Archaea<br />

Microbial<br />

Pathogens and<br />

Pathogenicity<br />

Anaerobic<br />

Metabolism<br />

Regulation<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KR 01 – KR 06<br />

KM 01 – KM 06<br />

KS 01 – KS 06<br />

KB 01 - KB 06<br />

KL 01 – KL 06<br />

KA 01 – KA 06<br />

KO 01 – KO 06<br />

Monday, 09.03.<strong>2009</strong> 15:15–17:00<br />

HZO 60<br />

HZO 50<br />

HZO 40<br />

HZO 30<br />

HZO 20<br />

HZO 10<br />

Audimax<br />

Developmental<br />

Microbiology<br />

Green<br />

Biotechnology<br />

Imaging<br />

Techniques in<br />

Microbiology<br />

Sensory and<br />

Regulatory RNA<br />

Secondary<br />

Metabolism<br />

Host-Microbe<br />

Interactions<br />

Physiology<br />

OVERVIEW · SHORT LECTURES 31<br />

KD 01 – KD 06<br />

KF 01 – KF 06<br />

KH 01 – KH 06<br />

KQ 01 – KQ 06<br />

KP 01 – KP 06<br />

KG 01 – KG 06<br />

KN 01 – KN 06<br />

Tuesday, 10.03.<strong>2009</strong> 13:00–15:00<br />

HZO 60<br />

HZO 50<br />

HZO 40<br />

HZO 30<br />

HZO 20<br />

HZO 10<br />

Audimax<br />

Cyanobacteria<br />

and Algae<br />

Microbial Cell<br />

Biology<br />

Fungal Biology<br />

White<br />

Biotechnology<br />

Microbial<br />

Diversity<br />

Open Topics<br />

(Microbial<br />

Molecular Tools)<br />

Microbial<br />

Communities<br />

KC 01 – KC 07<br />

KI 01 – KI 07<br />

KE 01 – KE 07<br />

KT 01 – KT 07<br />

KK 01 – KK 07<br />

KU 01 – KU 07<br />

KJ 01 – KJ 07

32 SHORT LECTURES<br />

Regulation<br />

Session I Monday, 09.03.<strong>2009</strong>, 11:00 – 12:45<br />

Lecture Hall Audimax<br />

Chair: M. Schobert<br />

Co-Chair: B. Masepohl<br />

KO01<br />

11:00<br />

N. BOES, A. STEEN, K. SCHREIBER,<br />

M. SCHEER, M. SCHOBERT*<br />

Impact of stringent response on survival<br />

and usp gene expression in<br />

Pseudomonas aeruginosa<br />

KO02<br />

11:25<br />

I. HITKOVA, S. LINNERBAUER, K. JUNG,<br />

R. HEERMANN*<br />

Acyl-homoserine lactone mediated regulation<br />

and LuxR receptors in the insect<br />

pathogen Photorhabdus luminescens<br />

KO03<br />

11:40<br />

X. ZHOU, R. FLEISCHER, N. KRAUß,<br />

P. SCHEERER, S. HUNKE*<br />

Structural basis for the regulatory function<br />

of the CpxP adaptor protein of<br />

Escherichia coli<br />

KO04<br />

11:55<br />

J. HEINRICH, K. SCHÄFER, K. HEIN,<br />

T. WIEGERT*<br />

Analysis of ECF sigma factor regulation<br />

through regulated intramembrane proteolysis<br />

in Bacillus subtilis<br />

KO05<br />

12:10<br />

S. METZ*, G. KLUG<br />

Light-dependent gene regulation in<br />

Rhodobacter sphaeroides – Investigating<br />

the interplay between the AppA/<br />

PpsR- and PrrB/PrrA-system<br />

KO06<br />

12:25<br />

N. REHM*, E. HIERY, T. GEORGI, M. BOTT,<br />

A. BURKOVSKI<br />

Nitrogen control in Corynebacterium<br />

glutamicum: impact on glutamine metabolism<br />

Anaerobic Metabolism<br />

Lecture Hall HZO 10<br />

Chair: R. G. Sawers<br />

Co-Chair: A. Hemschemeier<br />

KA 01<br />

11:00<br />

G. SAWERS*, B. SOBOH<br />

Biosynthesis of the [NiFe]-Hydrogenases<br />

of Escherichia coli<br />

KA02<br />

11:25<br />

S. MANN*, O. RIEBE, F. HILLMANN,<br />

H. BAHL<br />

Microoxic growth and metabolism of<br />

Clostridium acetobutylicum<br />

KA03<br />

11:40<br />

M. KERN*, J. SIMON<br />

Nitrate respiration in Wolinella succinogenes:<br />

Role of the multi-functional<br />

NapGHF menaquinol dehydrogenase<br />

complex<br />

KA04<br />

11:55<br />

C. BÜCKING*, F. POPP, S. KERZENMACHER,<br />

J. GESCHER<br />

Involvement and specificity of outer<br />

membrane cytochromes in extracellular<br />

electron transfer reactions<br />

KA05<br />

12:10<br />

S. STRIPP*, M. HAUMANN, F. ARMSTRONG,<br />

T. HAPPE<br />

Oxygen deactivation in [FeFe] Hydrogenases-<br />

two hydrogen producing enzymes<br />

studied by protein film voltammetry and<br />

X-ray absorption spectroscopy<br />

KA06<br />

12:25<br />

F. TEN BRINK*, O. EINSLE, B. SCHINK,<br />

P.M. KRONECK<br />

Exploring the active site of the W, FeS<br />

enzyme acetylene hydratase<br />

Microbial Pathogens and<br />

Pathogenicity<br />

Lecture Hall HZO 20<br />

Chair: A. Peschel<br />

Co-Chair: S. Gatermann<br />

KL01<br />

11:00<br />

A. PESCHEL<br />

The Gram-positive sugar toppings –<br />

teichoic acids and related cell wall glycopolymers<br />

in staphylococcal physiology<br />

and host interaction<br />

KL02<br />

11:25<br />

T. KOHLER*, C. WEIDENMAIER, A. PESCHEL<br />

The role of wall teichoic acid of S. aureus<br />

in resistance against antimicrobial<br />

fatty acids from the human skin<br />

KL03<br />

11:40<br />

F. SZABADOS*, S. RODEPETER, B. HECK,<br />

A. ALBRECHT, B. KLEINE, M. KAASE,<br />

S. GATERMANN<br />

In contrast to human isolates, animal<br />

isolates of S. saprophyticus subsp.<br />

saprophyticus are not internalized into<br />

human urinary blad<strong>der</strong> carcinoma cell<br />

line 5637<br />

KL04<br />

11:55<br />

E. PACHULEC, S. JAIN, W. SALGADO-<br />

PABÓN, J. DILLARD, M. VALEVICH,<br />

M. HELLER, C. VAN DER DOES*<br />

The type IV DNA secretion system of<br />

Neisseria gonorrhoeae<br />

KL05<br />

12:10<br />

C. EWERS*, T. HOMEIER, L.H. WIELER<br />

The genomic mutS-rpoS region of extraintestinal<br />

pathogenic E. coli (ExPEC):<br />

a minimal predictor of phylogenetic<br />

background and in vivo pathogenicity<br />

KL06<br />

12:25<br />

B. WÄCHTLER, F. DALLE, B. HUBE*<br />

Dissection of the invasion process of<br />

Candida albicans<br />

Archaea<br />

Lecture Hall HZO 30<br />

Chair: R. Hensel<br />

Co-Chair: N. Frankenberg-Dinkel<br />

KB01<br />

11:00<br />

B. HUBER, F. THOMAS, R. DIAZ-BONE,<br />

R. HENSEL*<br />

Unexpected properties of a<br />

methanogenic enzyme: Methylcobalamin-CoM-Methyltransferase<br />

(MtbA) of<br />

Methanosarcina mazei transforms bismuth<br />

into volatile trimethylbismuth<br />

KB02<br />

11:25<br />

A. BELLACK*, R. RACHEL, R. WIRTH<br />

Flagella of Pyrococcus furiosus: a structural<br />

and functional characterization<br />

KB03<br />

11:40<br />

E. ZELLER*, J. ARMACHE, P. CRAMER,<br />

M. THOMM<br />

Detailed analysis of structural elements<br />

in TFB and RNA polymerase during transcription<br />

initiation<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KB04<br />

11:55<br />

V. ALBERS<br />

Assembly and function of archaeal cell<br />

surface structures<br />

KB05<br />

12:10<br />

S. STANDFEST*, M. HÜGLER, D. WISCHER,<br />

H. CYPIONKA, M. KÖNNEKE<br />

Inside in the ecophysiology of marine<br />

ammonia-oxidizing Archaea<br />

KB06<br />

12:25<br />

K. BEBLO*, H. HUBER, R. RACHEL, G. REITZ,<br />

P. RETTBERG<br />

Radiation resistance of hyper/thermophilic<br />

Archaea and thermophilic<br />

deep-branching Bacteria<br />

Transport<br />

Session I Monday, 09.03.<strong>2009</strong>, 11:00 – 12:45<br />

Lecture Hall HZO 40<br />

Chair: P. Rehling<br />

Co-Chair: R. Erdmann<br />

KS01<br />

11:00<br />

P. REHLING<br />

Mitochondrial protein biogenesis<br />

KS02<br />

11:25<br />

M. BIEN, S. LONGEN, N. MESECKE,<br />

K. BIHLMAIER, J.M. HERRMANN, J. RIEMER*<br />

Oxidative Protein Folding in the Intermembrane<br />

Space of Mitochondria<br />

KS03<br />

11:40<br />

C. CIZMOWSKI*, E. HAMBRUCH,<br />

W. STANLEY, M. WILLMANNS, W. SCHLIEBS,<br />

H.W. PLATTA, W. GIRZALSKY, R. ERDMANN<br />

Peroxisomal matrix protein import: Critical<br />

steps of the receptor cycle<br />

KS04<br />

11:55<br />

K. BOERNGEN*, N. MOEKER, S. MORBACH,<br />

R. KRAEMER<br />

YggB (MscS) of Corynebacterium gluatmicum:<br />

Mechanosensitive channel<br />

and/or glutamate exporter<br />

KS05<br />

12:10<br />

C. VÖLLMECKE, G. SCHRÖTER, S. DREES,<br />

M. ZOLTNER, S. ALBERS, A. DRIESSEN,<br />

M. LÜBBEN*<br />

Two copper translocating ATPases of the<br />

thermoacidophilic archaeon Sulfolubus<br />

solftataricus<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KS06<br />

12:25<br />

U. LINDENSTRAUSS, T. BRÜSER*<br />

Tat-dependent transport of protein substrates<br />

with long unstructured linker<br />

peptides between the signal peptide and<br />

a folded domain<br />

Photosynthesis and<br />

Bioenergetics<br />

Lecture Hall HZO 50<br />

Chair: A. Wilde<br />

Co-Chair: M. Nowaczyk<br />

KM01<br />

11:00<br />

D. DIENST, I. AXMANN, S. LEGEWIE,<br />

J. GEORG, W.R. HESS, A. WILDE*<br />

The regulatory potential of non-coding<br />

RNAs in photosynthetic processes<br />

KM02<br />

11:25<br />

M. NOWACZYK*, N. GRASSE, J. SANDER,<br />

P. STRIEBECK, K. LEISCHNER, M. RÖGNER<br />

Transient PS2 subcomplexes in Thermosynechococcus<br />

elongatus<br />

KM03<br />

11:40<br />

S. REXROTH, C. MULLINEAUX, M. RÖGNER,<br />

F. KOENIG*<br />

Segregation of membrane domains in<br />

Gloeobacter violaceus<br />

KM04<br />

11:55<br />

B. CARIUS*, M. SAEGER, P. BOLLIN,<br />

H. GRAMMEL<br />

Intracellular redox potential and photosynthetic<br />

membrane production in Rhodospirillum<br />

rubrum: Correlation with<br />

culture – redox, substrate concentrations<br />

and oxygen supply<br />

KM05<br />

12:10<br />

K. KASTER*, K. PAREY, R.K. THAUER<br />

Electron bifurcation catalyzed by the<br />

MvhADG-HdrABC complex from Methanothermobacter<br />

marburgensis<br />

KM06<br />

12:25<br />

T. VORBURGER, A. STEIN, G. KAIM,<br />

J. STEUBER*<br />

Functional role of a conserved aspartic<br />

acid residue in the motor of the Na + -<br />

driven flagellum from Vibrio cholerae<br />

SHORT LECTURES 33<br />

Systems Biology<br />

Lecture Hall HZO 60<br />

Chair: J. Kämper<br />

Co-Chair: M. Nowrousian<br />

KR01<br />

11:00<br />

M. VRANES, K. HEIMEL, R. WAHL,<br />

A. ZAHIRI, G. DÖHLEMANN, R. KAHMANN,<br />

J. KÄMPER*<br />

Transcriptomics in the Ustilago-maize<br />

pathosystem<br />

KR02<br />

11:25<br />

I. BISCHOFS*, J. HUG, A. LIU, D. WOLF, A.<br />

ARKIN<br />

Quorum signal integration and subpopulation<br />

signalling in sporulating B. subtilis<br />

communities<br />

KR03<br />

11:40<br />

M. ZAPARTY*, B. SIEBERS, AND THE<br />

SULFOSYS CONSORTIUM<br />

SulfoSYS – Sulfolobus Systems Biology:<br />

towards a Silicon Cell Model for the central<br />

carbohydrate metabolism of the Archaeon<br />

Sulfolobus solfataricus un<strong>der</strong><br />

temperature variation<br />

KR04<br />

11:55<br />

B.A. HENSE*, J. MÜLLER, C. KUTTLER<br />

Origin and primary function of the luciferase<br />

reaction in bacteria<br />

KR05<br />

12:10<br />

O. KNIEMEYER*, M. VÖDISCH,<br />

K. SCHERLACH, R. WINKLER,<br />

C. HERTWECK, U. HORN, A.A. BRAKHAGE<br />

Proteomic analysis of the response of<br />

the human-pathogenic fungus Aspergillus<br />

fumigatus to hypoxia<br />

KR06<br />

12:25<br />

A. POETSCH*, D. SCHLUESENER,<br />

F. FISCHER, U. HAUSSMANN,<br />

C. TROETSCHEL, D. WOLTERS, J. LIU<br />

Comparison of composition and adaptation<br />

of the Corynebacterium glutamicum<br />

proteome un<strong>der</strong> different physiological<br />

conditions

34 SHORT LECTURES<br />

Physiology<br />

Session II Monday, 09.03.<strong>2009</strong>, 15:15 – 17:00<br />

Lecture Hall Audimax<br />

Chair: J. Steuber<br />

Co-Chair: L. Leichert<br />

KN01<br />

15:15<br />

M.S. CASUTT, T. HUBER, J. STEUBER*<br />

Localization and quantification of the<br />

flavin cofactors of the Na+-translocating<br />

NADH:quinone oxidoreductase from<br />

Vibrio cholerae<br />

KN02<br />

15:40<br />

C. FLECK*, M. BROCK<br />

Ach1p from Saccharomyces cerevisiae<br />

and the connection to acetate metabolism<br />

KN03<br />

15:55<br />

T.J. ERB*, B.E. ALBER, G. FUCHS<br />

The Ethylmalonyl-CoA Pathway – a Story<br />

of New Reactions and Substrates<br />

KN04<br />

16:10<br />

M. BAUMGART*, M. BOTT<br />

Studies of aconitase in Corynebacterium<br />

glutamicum<br />

KN05<br />

16:25<br />

Y. KOHLMANN*, A. POHLMANN, A. OTTO,<br />

D. BECHER, B. FRIEDRICH, M. HECKER<br />

Physiological insights into hydrogenbased<br />

lifestyle<br />

KN06<br />

16:40<br />

L. LEICHERT*, F. GEHRKE, V. GUDISEVA,<br />

M. ILBERT, A.K. WALKER, R. STRAHLER,<br />

U. JAKOB<br />

Quantifying the Thiol Redox Proteome<br />

Host-Microbe Interactions<br />

Lecture Hall HZO 10<br />

Chair: M. Hensel<br />

Co-Chair: S. Gatermann<br />

KG01<br />

15:15<br />

M. HENSEL<br />

New approaches to the intracellular<br />

lifestyle of Salmonella enterica in eukaryotic<br />

cells<br />

KG02<br />

15:40<br />

M. BURIAN*, C. WOLZ, C. GOERKE<br />

Staphylococcus aureus gene expression<br />

and -regulation during persistent colonization<br />

of the human nares<br />

KG03<br />

15:55<br />

D. OPITZ*, M. CLAUSEN, B. MAIER<br />

Dynamics of type IV pili of Neisseria<br />

gonorrhoeae during infection<br />

KG04<br />

16:10<br />

S. ZEHNER*, M. WENZEL, L. FRIEDRICH,<br />

M. GÖTTFERT<br />

Transport of a Bradyrhizobium japonicum<br />

secreted protein into nodule cells<br />

and its impact on symbiosis<br />

KG05<br />

16:25<br />

P.K. WÜST*, M.A. HORN, H.L. DRAKE<br />

Concomitant Fermentation and Denitrification<br />

along the Earthworm Alimentary<br />

Canal<br />

KG06<br />

16:40<br />

J. PREISING*, H. NIESS, C.U. RIEDEL<br />

Anti-inflammatory activity of a B. bifidum<br />

strain in Rag1 –/– colitic mice<br />

Secondary Metabolism<br />

Lecture Hall HZO 20<br />

Chair: P. Tudzynski<br />

Co-Chair: B. Hoff<br />

KP01<br />

15:15<br />

P. TUDZYNSKI<br />

Molecular aspects of ergot alkaloid<br />

biosynthesis<br />

KP02<br />

15:40<br />

B. HOFF*, I. ZADRA, H. KÜRNSTEINER,<br />

U. KÜCK<br />

Molecular analysis of the velvet complex<br />

in Penicillium chrysogenum and its role<br />

in regulating secondary metabolism and<br />

morphogenesis<br />

KP03<br />

15:55<br />

M. KAI*, B. PIECHULLA<br />

Volatiles of Serratia odorifera: Emission<br />

profile analysis and growth<br />

promotion/inhibition of Arabidipsis<br />

thaliana<br />

KP04<br />

16:10<br />

S. NIELAND*, P. STAHMANN<br />

Overproduction of riboflavin is not needed<br />

for mycelial growth of Ashbya<br />

gossypii but leads to protection of its<br />

hyaline spores against UV-light<br />

KP05<br />

16:25<br />

J. SELVIN<br />

Antagonistic potential of Streptomyces<br />

dendra sp. nov associated with marine<br />

sponge Dendrilla nigra<br />

KP06<br />

16:40<br />

K. SCHERLACH*, A. FUNK, S. BERGMANN,<br />

J. SCHÜMANN, V. SCHROECKH,<br />

A. A. BRAKHAGE, C. HERTWECK<br />

Triggering Cryptic Natural Product<br />

Biosynthesis in Microorganisms<br />

Sensory and Regulatory RNA<br />

Lecture Hall HZO 30<br />

Chair: J. Vogel<br />

Co-Chair: B. Klinkert<br />

KQ01<br />

15:15<br />

J. VOGEL<br />

How small RNAs target multiple mRNAs<br />

to rewire gene expression post transcription<br />

KQ02<br />

15:40<br />

B. BERGHOFF*, J. GLAESER, C. SHARMA,<br />

J. VOGEL, G. KLUG<br />

Small non-coding RNAs in Rhodobacter<br />

sphaeroides and the singlet oxygen<br />

stress response<br />

KQ03<br />

15:55<br />

K. BÖHME*, K. HEROVEN, P. DERSCH<br />

YmoA activates expression of the virulence<br />

regulator gene rovA in Yersinia<br />

pseudotuberculosis through the Csr<br />

regulatory system<br />

KQ04<br />

16:10<br />

N. HEIDRICH*, A. RAINE, G.H. WAGNER<br />

CRISPR – a novel prokaryotic immune<br />

system that provides acquired resistance<br />

against phages<br />

KQ05<br />

16:25<br />

L. GAUBIG*, F. NARBERHAUS<br />

The ibpAB operon of Escherichia coliregulation<br />

after heat shock<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Session II Monday, 09.03.<strong>2009</strong>, 15:15 – 17:00<br />

KQ06<br />

16:40<br />

E. EVGUENIEVA-HACKENBERG<br />

The influence of RNase E and RNase J on<br />

maturation and stability of non-coding<br />

RNAs in S. meliloti<br />

Imaging techniques in<br />

microbiology<br />

Lecture Hall HZO 40<br />

Chair: R. Fischer<br />

Co-Chair: I. Engh<br />

KH01<br />

15:15<br />

R. FISCHER*, N. ZEKERT<br />

Analysis of the microtubule cytoskeleton<br />

in Aspergillus nidulans using fluorescent<br />

proteins<br />

KH02<br />

15:40<br />

I. ENGH*, M. NOWROUSIAN, U. KÜCK<br />

Live cell imaging in filamentous fungi<br />

KH03<br />

15:55<br />

A. MÜLLER*, A. MCDOWALL, G.J. JENSEN,<br />

W.M. CLEMONS, JR.<br />

Electron cryotomography of Campylobacter<br />

jejuni<br />

KH04<br />

16:10<br />

A. DESCH*, B. AVERHOFF<br />

In vivo localisation of the DNA translocator<br />

in Acinetobacter baylyi ADP1<br />

KH05<br />

16:25<br />

M. PULS*, J. POTZKEI, F. CIRCOLONE,<br />

T. EGGERT, T. DREPPER<br />

Fluoresent Proteins for Anaerobic Applications<br />

KH06<br />

16:40<br />

K. SCHIWON*, K. ARENDS, E. GROHMANN<br />

GFP based monitoring tools for isolation<br />

of new mobile genetic elements and assessment<br />

of conjugative plasmid transfer<br />

in biofilms<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Green Biotechnology<br />

Lecture Hall HZO 50<br />

Chair: T. Börner<br />

Co-Chair: M. Rögner<br />

KF01<br />

15:15<br />

T. BÖRNER<br />

Blue-green biotechnology<br />

KF02<br />

15:40<br />

E. DITTMANN*, Y. ZILLIGES, C. KEHR,<br />

S. MIKKAT, M. HAGEMANN, N. TANDEAU<br />

DE MARSAC<br />

Posttranslational modification by the<br />

nonribosomal peptide microcystin affects<br />

protein stability in the toxic<br />

cyanobacterium Microcystis<br />

KF03<br />

15:55<br />

M. WINKLER*, P. KNÖRZER, T. HAPPE<br />

Structure function relationship in<br />

Chlorophyta type [FeFe]-hydrogenase<br />

KF04<br />

16:10<br />

H. KWON, N. WASCHEWSKI, G. BERNÁT,<br />

M. BROEKMANS, S. REXROTH*,<br />

M. RÖGNER<br />

Construction of a continuous photobiological<br />

reactor system for cyanobacterial<br />

H2-production KF05<br />

16:25<br />

I. HIRSCHMANN*, M. TERASHIMA,<br />

D. PETROUTSOS, D. KRAWIETZ, T. HAPPE,<br />

M. HIPPLER<br />

Analysis of the anaerobic response in<br />

Chlamydomonas reinhardtii regarding<br />

the impact of putative regulatory proteins<br />

KF06<br />

16:40<br />

A. IDOINE*, R. BOCK, J. RUPPRECHT<br />

Organelle transcriptomics and translatomics<br />

of Chlamydomonas reinhardtii<br />

SHORT LECTURES 35<br />

Developmental Microbiology<br />

Lecture Hall HZO 60<br />

Chair: F. Kempken<br />

Co-Chair: W. Schliebs<br />

KD01<br />

15:15<br />

M. MERCKER, K. KOLLATH-LEIß,<br />

S. ALLGAIER, N. WEILAND, F. KEMPKEN*<br />

The BEM46-like protein appears to be<br />

essential for hyphal development upon<br />

ascospore germination in Neurospora<br />

crassa and is targeted to the Endoplasmic<br />

Reticulum<br />

KD02<br />

15:40<br />

D. BRUST*, A. HAMANN, H.D. OSIEWACZ<br />

Cyclophilin D: a link between apoptosis<br />

and lifespan control in the ascomycete<br />

Podospora anserina<br />

KD03<br />

15:55<br />

J. HELLER*, N. SEGMÜLLER, P. TUDZYNSKI<br />

Comparison of genes controlled by the<br />

MAP kinases BcSak1 and Bmp3 of Botrytis<br />

cinerea during oxidative stress<br />

KD04<br />

16:10<br />

E. VOLLMEISTER*, C. HAAG, S. BAUMANN,<br />

J. KÖNIG, M. FELDBRÜGGE<br />

The RNA-binding protein Khd4 is important<br />

for pathogenicity in Ustilago maydis<br />

KD05<br />

16:25<br />

D. SCHACHTSCHABEL*, M. SCHLICHT,<br />

D. MENZEL, F. BALUSKA, W. BOLAND<br />

Apocarotenoids-Signaling Compounds of<br />

Zygomycetes and Plants?<br />

KD06<br />

16:40<br />

G. HENSEL<br />

Development of experimental tools for<br />

the investigation of the barley–pow<strong>der</strong>y<br />

mildew pathosystem

36 SHORT LECTURES<br />

Session III Tuesday, 10.03.<strong>2009</strong>, 13:00 – 15:00<br />

Microbial Communities<br />

Lecture Hall Audimax<br />

Chair: K. Thormann<br />

Co-Chair: J. Bandow<br />

KJ01<br />

13:00<br />

K. THORMANN<br />

Communities of Shewanella oneidensis<br />

MR-1: what can we learn?<br />

KJ02<br />

13:25<br />

N. GRANTCHAROVA, U. RÖMLING*<br />

Biofilm formation in Salmonella<br />

typhimurium: expression analysis of<br />

CsgD at the single cell level<br />

KJ03<br />

13:40<br />

C. HUNG*, J. PINKNER, S. HULTGREN<br />

Life Above Water for Uropathogenic<br />

Escherichia coli<br />

KJ04<br />

13:55<br />

T. HEIMERL, N. WASSERBURGER,<br />

C. MEYER, T. BURGHARDT, B. JUNGLAS,<br />

H. HUBER, R. WIRTH, R. RACHEL*<br />

3D Structure of the Archaea Ignicoccus<br />

hospitalis and Nanoarchaeum equitans,<br />

as determined by serial section electron<br />

microscopy<br />

KJ05<br />

14:10<br />

M. TAUBERT*, N. JEHMLICH, F. SCHMIDT,<br />

M. VON BERGEN, H. RICHNOW, C. VOGT<br />

Comparison of methods for simultaneous<br />

identification of bacterial species<br />

and determination of metabolic activity<br />

by protein-based stable isotope probing<br />

(Protein-SIP) experiments<br />

KJ06<br />

14:25<br />

L. SCHREIBER*, A. MEYERDIERKS,<br />

K. KNITTEL, B. FUCHS, R. AMANN<br />

Sulfate reducing bacteria associated<br />

with ANME2-aggregates<br />

KJ07<br />

14:40<br />

A. HÜTZ*, M. MAYER, K. SCHUBERT,<br />

J. OVERMANN<br />

Chemotaxis toward inorganic phosphate<br />

– an adaptive strategy of marine bacteria<br />

with implications for nutrient cycling<br />

in the ocean<br />

Open Topics (Microbial<br />

Molecular Tools)<br />

Microbial Diversity<br />

Lecture Hall HZO 10<br />

Lecture Hall HZO 20<br />

Chair: F.O. Glöckner<br />

Chair: S. Pöggeler<br />

Co-Chair: D. Begerow<br />

Co-Chair: D. Janus<br />

KK01<br />

KU01<br />

13:00<br />

13:00<br />

E. PRÜßE*, C. QUAST, K. DIETRICH,<br />

S. PÖGGELER*, S. ELLEUCHE<br />

Protein splicing of fungal inteins<br />

KU02<br />

K. KNITTEL, B. FUCHS, W. LUDWIG,<br />

J. PEPLIES, F.O. GLÖCKNER<br />

The SILVA project for comprehensive,<br />

quality checked and aligned ribosomal<br />

13:25<br />

RNA databases<br />

B. ZORIN*, Y. LU, I. SIZOVA, P. HEGEMANN<br />

Nuclear gene targeting in Chlamydomonas<br />

as exemplified by disruption of<br />

the PHOT gene<br />

KU03<br />

KK02<br />

13:25<br />

K. KOCH*, D. WAGNER<br />

Methanogenic community in Terrestrial<br />

and Submarine Permafrost of the<br />

13:40<br />

Siberian Laptev Sea Region<br />

H.-P. KLENK*, G. HASZPRUNAR, W.<br />

WÄGELE, B. GEMEINHOLZER<br />

Progress in the formation of the DNA<br />

Bank Network<br />

KU04<br />

KK03<br />

13:40<br />

F. BEHNAM*, M. HORN, M. TAYLOR,<br />

S. LUECKER, S. WHALAN, T. RATTEI,<br />

N. WEBSTER, M. WAGNER<br />

13:55<br />

Deep sequencing reveals exceptional di-<br />

M. VERA*, T. ROHWERDER, V. BONNEFOY, versity and alternative lifestyles of ma-<br />

W. SAND<br />

rine bacterial sponge symbionts<br />

Initial steps for the characterization of<br />

biofilm formation by the bioleaching<br />

acidophilic bacterium Acidithiobacillus<br />

ferrooxidans following a microarray<br />

transcriptome analysis<br />

KU05<br />

KK04<br />

13:55<br />

S. LIEBNER*, T. STÜHRMANN, J. HARDER,<br />

J. ZEYER, D. WAGNER<br />

Microbial diversity in Arctic polygonal<br />

tundra soils<br />

14:10<br />

S. THOLEN*, K. GUNKA, J. STÜLKE<br />

Darwin or Lamarck: Genetic analysis of<br />

a suppressor mutation in Bacillus subtilis<br />

KU06<br />

KK05<br />

14:10<br />

M. STIEGLMEIER, C. MOISSL-EICHINGER*<br />

New insights into the microbial diversity<br />

in spacecraft assembly clean rooms and<br />

the impact on planetary protection<br />

14:25<br />

P. BIJTENHOORN*, C. SCHIPPER,<br />

C. HORNUNG, M. QUITSCHAU, S. GROND,<br />

W. STREIT<br />

Metagenomic-<strong>der</strong>ived quorum-quenching<br />

clones interfering with P. aeruginosa<br />

biofilm formation<br />

KU07<br />

KK06<br />

14:25<br />

R. WENTER*, G. WANNER, D. SCHÜLER,<br />

J. OVERMANN<br />

Ultrastructure, tactic behaviour and potential<br />

for sulfate reduction of a novel<br />

multicellular magnetotactic prokaryote<br />

from North Sea sediments<br />

14:40<br />

B. REINARTZ, S. OELJEKLAUS, I. MICHELS,<br />

C. STEPHAN, M. EISENACHER, W.<br />

SCHLIEBS, R. ERDMANN, H.E. MEYER, B.<br />

WARSCHEID*<br />

Quantitative proteomics approach to the<br />

establishment of interaction networks<br />

of peroxisomal membrane proteins in<br />

Saccharomyces cerevisiae<br />

KK07<br />

14:40<br />

A. YURKOV*, M. KEMLER, D. BEGEROW<br />

Assessment of the yeast diversity in<br />

soils un<strong>der</strong> different land use management<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Session III Tuesday, 10.03.<strong>2009</strong>, 13:00 – 15:00<br />

White Biotechnology<br />

Lecture Hall HZO 30<br />

Chair: V. F. Wendisch<br />

Co-Chair: K.-P. Stahmann<br />

KT01<br />

13:00<br />

V.F. WENDISCH<br />

Corynebacterium glutamicum as platform<br />

for production of fine chemicals:<br />

carbon control and access to new carbon<br />

substrates<br />

KT02<br />

13:25<br />

L.M. BLANK*, J. RUEHL, B. EBERT,<br />

B. BÜHLER, A. SCHMID<br />

Solvent-tolerant Pseudomonas: platform<br />

organisms for whole-cell biocatalysis?<br />

KT03<br />

13:40<br />

M. FAIRHEAD, K. GRIEDER, L. THÖNY-<br />

MEYER*<br />

Expression of a novel bacterial tyrosinase<br />

in Escherichia coli<br />

KT04<br />

13:55<br />

E. ARANDA*, M. KLUGE, R. ULLRICH,<br />

M. HOFRICHTER, G. KAYSER<br />

Conversion of polycyclic aromatic and<br />

heterocyclic hydrocarbons by extracellular<br />

fungal peroxygenases<br />

KT05<br />

14:10<br />

C. ELEND*, A. BASNER, W.R. STREIT,<br />

G. ANTRANIKIAN<br />

Improving thermal stability of a cold-active<br />

lipase by directed evolution<br />

KT06<br />

14:25<br />

S. GHANEGAONKAR*, G. SPRENGER,<br />

C. ALBERMANN<br />

Metabolic Engineering of Escherichia<br />

coli towards the Production of Tocotrienol<br />

KT07<br />

14:40<br />

K.-P. STAHMANN*, K. SCHNITZLEIN,<br />

M. HUDDAR, S. BARIG<br />

Monoseptic cultivation un<strong>der</strong> non-sterile<br />

conditions in 350 litre scale<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Fungal Biology<br />

Lecture Hall HZO 40<br />

Chair: H.D. Osiewacz<br />

Co-Chair: J. Kamerewerd<br />

KE01<br />

13:00<br />

H.D. OSIEWACZ<br />

Mitochondrial quality control systems:<br />

role in fungal development and lifespan<br />

control<br />

KE02<br />

13:25<br />

S. ELLEUCHE*, S. PÖGGELER<br />

A mitochondrial β-class carbonic anhydrase<br />

is involved in sexual reproduction<br />

of the filamentous fungus Sordaria<br />

macrospora<br />

KE03<br />

13:40<br />

S. BLOEMENDAL*, I. ENGH, S. SEILER,<br />

U. KÜCK<br />

Cross species yeast two-hybrid analyses<br />

unravel cellular networks of fungal development<br />

KE04<br />

13:55<br />

J. WETZEL*, O. SCHEIBNER,<br />

A. BURMESTER, C. SCHIMEK,<br />

J. WOESTEMEYER<br />

4-Dihydrotrisporin dehydrogenase from<br />

Mucor mucedo, an enzyme of the sex<br />

hormone pathway<br />

KE05<br />

14:10<br />

D. WAGNER*, A. SCHMEINCK,<br />

B. TUDZYNSKI<br />

The role of the bZIP transcription factor<br />

MeaB in the nitrogen metabolite repression<br />

of Fusarium fujikuroi<br />

KE06<br />

14:25<br />

M. PANAKOVA*, N. MAASSEN,<br />

M. ZIMMERMANN, M. BÖLKER, U. KLINNER<br />

Formation of itaconic acid by the fungus<br />

Ustilago maydis MB215<br />

KE07<br />

14:40<br />

C. LIERS*, C. BOBETH, R. ULLRICH,<br />

M. HOFRICHTER<br />

Novel DyP-type peroxidases from the jelly<br />

fungus Auricularia auricula-judae<br />

SHORT LECTURES 37<br />

Microbial Cell Biology<br />

Lecture Hall HZO 50<br />

Chair: J. Herrmann<br />

Co-Chair: W.-H. Kunau<br />

KI01<br />

13:00<br />

J. HERRMANN*, M. PRESTELE, S. FUNES,<br />

H. BAUERSCHMITT, M. OTT<br />

The mitochondrial translation machinery:<br />

From protein synthesis to membrane<br />

integration<br />

KI02<br />

13:25<br />

J. DEFEU SOUFO, C. REIMOLD, U. LINNE,<br />

J. GESCHER, P. GRAUMANN*<br />

Translation elongation factor ER-Tu is<br />

part of the bacterial cytoskeleton<br />

KI03<br />

13:40<br />

V.J. SCHUENEMANN*, S.M. KRALIK,<br />

R. ALBRECHT, S.K. SPALL, K.N. TRUSCOTT,<br />

D.A. DOUGAN, K. ZETH<br />

Structural basis of N-end rule substrate<br />

recognition in Escherichia coli by the<br />

ClpAP adaptor protein ClpS<br />

KI04<br />

13:55<br />

S. VAN BAARLE*, M. BRAMKAMP<br />

Spatial control of division site selection<br />

in Bacillus subtilis<br />

KI05<br />

14:10<br />

T. WALDMINGHAUS*, K. SKARSTAD<br />

Binding of SeqA to the Escherichia coli<br />

chromosome<br />

KI06<br />

14:25<br />

T. BURGHARDT, F. SIEDLER, R. WIRTH,<br />

H. HUBER, R. RACHEL*<br />

Proteins in the contact site of the two<br />

hyperthermophilic archaea, I. hospitalis<br />

and N. equitans<br />

KI07<br />

14:40<br />

S. HALBEDEL*, R. BREITLING,<br />

L.W. HAMOEN<br />

Mechanisms of subcellular DivIVA targeting<br />

in Bacillus subtilis

�<br />

38 SHORT LECTURES<br />

Session III Tuesday, 10.03.<strong>2009</strong>, 13:00 – 15:00<br />

Cyanobacteria and Algae<br />

Lecture Hall HZO 60<br />

Chair: K. Forchhammer<br />

Co-Chair: T. Happe<br />

KC01<br />

13:00<br />

G. RASCH, M. DRATH, J. ESPINOSA,<br />

P. MICHEL, K. FORCHHAMMER*<br />

Towards a global un<strong>der</strong>standing of nitrogen<br />

starvation acclimation in unicellular<br />

cyanobacteria<br />

KC02<br />

13:25<br />

S. KLÄHN, A. DIEDERICH, E. SIMON,<br />

S. ANACKER, M. HAGEMANN*<br />

The protein Ssl3076 represses the saltregulated<br />

ggpS gene involved in synthesis<br />

of the compatible solute glucosylglycerol<br />

in Synechocystis sp. strain PCC<br />

6803.<br />

Bioanalytik für Einsteiger<br />

NEU!!!<br />

Reinhard Renneberg / Darja Süßbier<br />

Bioanalytik für<br />

Einsteiger<br />

1. Aufl. 2008, 284 S.,<br />

600 Abb., geb.<br />

� (D) 34,95 / � (A) 35,93 / CHF 54,50<br />

ISBN 978-3-8274-1831-9<br />

KC03<br />

13:40<br />

M. SCHOTTKOWSKI*, L. SHAO,<br />

B. RENGSTL, E. ANKELE, J. NICKELSEN<br />

Cyanobacterial TPR proteins and their<br />

role in photosynthetic functions<br />

KC04<br />

13:55<br />

A. HEMSCHEMEIER*, J. JACOBS,<br />

D. KRAVIETZ, G. PHILIPPS, T. HAPPE<br />

Chlamydomonas and Escherichia – fraternal<br />

twins in fermentation?<br />

KC05<br />

14:10<br />

S. GLANZ*, U. KÜCK<br />

Towards un<strong>der</strong>standing the spliceosome-mediated<br />

RNA trans-splicing in<br />

the chloroplast of Chlamydomonas reinhardtii<br />

„Das Buch ist unglaublich… keine langweilige Sammlung von<br />

Kochrezepten wie Analytikbücher, son<strong>der</strong>n ein Abenteuerroman…<br />

Eine Reise durch die Bioanalytik-Welt. An je<strong>der</strong> Ecke<br />

eine Überraschung!“<br />

Prof. i.R. Georges M. Halpern, University of California at Davis<br />

Sie denken <strong>bei</strong> dem Fachbegriff „Bioanalytik“ an mo<strong>der</strong>ne<br />

und aufwendige biochemische o<strong>der</strong> molekularbiologische<br />

Labormethoden, um Molekülstrukturen aufzuklären und <strong>der</strong>en<br />

Funktionen zu ergründen? Durchaus korrekt, doch geht<br />

Reinhard Rennebergs Buch weit darüber hinaus: Der Autor<br />

vermittelt mit einer Vielzahl an vierfarbigen Grafiken und Fotos<br />

sowie abwechslungsreichen Texten eine hochaktuelle, aber<br />

auch alltagstaugliche Gesamtschau <strong>der</strong> Bioanalytik, die neben<br />

Wissenschaftlern/-innen auch Laien und Studierenden einen<br />

gut verständlichen Einstieg bietet. Drogen- und HIV-Tests, die<br />

Blutzucker-Bestimmung <strong>bei</strong> Diabetes, die Messung <strong>der</strong> körperlichen<br />

Fitness und <strong>der</strong> Check des Auftretens eines Herzinfarkts<br />

sind einige <strong>der</strong> beschriebenen spannenden und lebensnahen<br />

Aspekte. Wie das erfolgreiche Werk des Autors „Biotechnologie<br />

für Einsteiger“ soll auch dieses Buch zeigen „Wissenschaft<br />

kann Spaß machen!“ und die Neugier auf mehr wecken – und<br />

das schon alleine <strong>bei</strong>m Durchblättern.<br />

Bequem bestellen: direkt <strong>bei</strong> www.spektrum-verlag.de<br />

per E-Mail: SDC-bookor<strong>der</strong>@springer.com<br />

�<br />

�<br />

�<br />

�<br />

KC06<br />

14:25<br />

O. VOYTSEKH*, S. SEITZ, D. ILIEV,<br />

M. MITTAG<br />

The circadian RNA-binding protein<br />

CHLAMY1 can integrate temperature information<br />

KC07<br />

14:40<br />

J. NEUPERT*, D. KARCHER, R. BOCK<br />

Generation of Chlamydomonas strains<br />

that efficiently express nuclear transgenes<br />

Dazu:<br />

Bild-DVD, Renneberg, Bioanalytik für<br />

Einsteiger mit allen Grafiken des Buches<br />

1.Aufl. 2008,<br />

� (D) 25,– / � (A) 25,21 / CHF 37,–<br />

ISBN 978-3-8274-2087-9<br />

telefonisch: + 49 6221 345-0<br />

per Fax: + 49 6221 345-4229<br />

NEU!!!

Poster Overview<br />

Arranged by topics<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

POSTER OVERVIEW 39<br />

ó The poster session will be held in the “Hörsaalzentrum Ost”. The posters may be mounted from 14:00 on Sunday 08.03.<strong>2009</strong> and should<br />

not be removed before 11:00 on Wednesday 11.03.<strong>2009</strong>.<br />

The Poster sessions will be held on:<br />

Monday, 09.03.<strong>2009</strong> 13:45 – 15:15<br />

Tuesday, 10.03.<strong>2009</strong> 15:30 – 17:30<br />

Authors are asked to attach to the posters the time when they will be available for discussion. Fixing material will be provided – please use<br />

ONLY the materials indicated. ó<br />

Abbrev. Topic Poster No.<br />

PA Anaerobic Metabolism PA01 – PA44<br />

PB Archaea PB01 – PB26<br />

PC Special Group Biologie bakterieller Naturstoffproduzenten PC01 – PC07<br />

PD Special Group Biotransformation PD01 – PD06<br />

PE Cyanobacteria and Algae PE01 – PE08<br />

PF Developmental Microbiology PF01 – PF04<br />

PG Special Group Functional Genomics PG01 – PG07<br />

PH Fungal Biology PH01 – PH34<br />

PI Green Biotechnology PI01 – PI05<br />

PJ Host-Microbe Interactions PJ01 – PJ25<br />

PK Special Group Identification and Systematics PK01 – PK03<br />

PL Imaging Techniques in Microbiology PL01 – PL04<br />

PM Microbial Cell Biology PM01– PM26<br />

PN Microbial Communities PN01 – PN88<br />

PO Microbial Diversity PO01 – PO50<br />

PP Microbial Pathogens and Pathogenicity PP01 – PP45<br />

PQ Photosynthesis and Bioenergetics PQ01 – PQ11<br />

PR Physiology PR01 – PR30<br />

PS Regulation PS01 – PS61<br />

PT Secondary Metabolism PT01 – PT11<br />

PU Sensory and Regulatory RNA PU01 – PU14<br />

PV Systems Biology PV01 – PV08<br />

PW Transport PW01– PW23<br />

PX White Biotechnology PX01 – PX46<br />

PY Special Group Yeast PY01 – PY05<br />

PZ Open Topics PZ01 – PZ58

40 MEETING<br />

Thursday, 12.03.<strong>2009</strong><br />

University of Bochum, Hörsaalzentrum Ost, HZO 40<br />

How dead is dead? Survival and final inactivation of<br />

microorganisms<br />

ó Viability of microorganisms is traditionally<br />

determined by cultivation techniques<br />

based on the microbial ability to multiply as<br />

colonies on solid agar media or to grow in<br />

liquid culture. These methods are widely used<br />

for the quantitative analysis and identification<br />

of microorganisms in clinical and natural<br />

(water, soil) environments as well as in<br />

water, food and beverages. Particularly after<br />

disinfection and other processing measures,<br />

it is important to verify that the target organisms<br />

are dead. However, culture-based<br />

methods can grossly un<strong>der</strong>estimate the<br />

08:30 – 09:00 Registration<br />

amount of viable cells actually present – they<br />

may be “not so dead” after all. The viability<br />

can be demonstrated by a range of alternative<br />

culture-independent methods which can<br />

reveal remaining cell integrity and physiological<br />

activity, resulting in the recovery of cells<br />

which no longer grow on media designed for<br />

their detection. Inactivation of microorganisms<br />

can lead to various levels of stress response,<br />

induce repair mechanisms and eventually<br />

lead to survival from which cells may<br />

recover until final “death” can be confirmed<br />

– which depends genuinely on the method<br />

applied. The state of microorganisms after<br />

transition from cuturable to nonculturable is<br />

characterized by various descriptions such<br />

as injured, dormant or viable-but-nonculturable<br />

(VBNC). In this conference, methodologies<br />

for the investigation of the relationship<br />

between culturability and viability as well as<br />

explanations for the un<strong>der</strong>lying mechanisms<br />

of these phenomena are discussed and it is<br />

attempted to give a proper definition of death<br />

of microorganisms. ó<br />

09:00 – 09:15 Introduction (Hans-Curt Flemming)<br />

Chair: Hans-Curt Flemming<br />

09:15 – 10:00 HDID 01:<br />

The Viable but not Culturable Paradigm<br />

Rita Colwell<br />

University of Maryland and Johns Hopkins University Bloomberg School of Public Health, Collage Park and Baltimore,<br />

United States<br />

10:00 – 10:45 HDID 02<br />

Physiological aspects of the viable but non-culturable response<br />

Diane McDougald 1 *, Zoe Moore 2 , Staffan Kjelleberg 1<br />

1 Centre for Marine Bio-Innovation, University of New South Wales, Sydney, Australia<br />

2 Ecowise Environmental, University of New South Wales, Sydney, Australia<br />

10:45 – 11:15 Coffee break, posters<br />

Chair: Rita Colwell<br />

11:15 – 12:00 HDID 03<br />

Indicators to pursuit living deads<br />

Ursula Obst*<br />

Institute for Functional Interfaces (JFG), Forschungszentrum Karlsruhe, Karlsruhe, Germany<br />

12:00 – 12:45 HDID 04<br />

Non-growing cells and maintenance metabolism in dense cultures of E. coli in membrane bioreactors<br />

Ulrich Szewzyk*, Claudia Keil, Susann Müller<br />

Institute for Environmental Microbiology, Technische Universität Berlin, Berlin, Germany<br />

12:45 – 14:00 Lunch break, posters<br />

Chair: Ursula Obst<br />

14:00 – 14:20 HDID 05<br />

A physiological approach to determine the survival of sub-seafloor prokaryotes un<strong>der</strong> energy deprivation.<br />

Falko Mathes*, Henrik Sass, R. John Parkes<br />

School of Earth and Ocean Sciences, Cardiff University, Cardiff, United Kingdom<br />

14:20 – 14:40 HDID 06<br />

The case for safe disinfection of drinking water with chlorine: breaking the nucleic acids and repair systems<br />

Erell Le Guen 1 *, Meng-Huot Phe 1 , Mohamad Hajj Chehade 1 , Christophe Merlin 2 , Manuel Dossot 1 , Jean-Claude Block 1<br />

1 LCPME, UMR 7564 CNRS – Nancy-University, Villers-lès-Nancy, France<br />

2 LCPME, UMR 7564 CNRS – Nancy University, Vandoeuvre-lès-Nancy, France<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

�<br />

Thursday, 12.03.<strong>2009</strong><br />

MEETING 41<br />

14:40 – 15:00 HDID 07<br />

Community analysis and taxonomic identification of drinking water bacteria with respect to live/dead status<br />

Leila Kahlisch*, Karsten Henne, Josefin Draheim, Lothar Groebe, Ingrid Brettar, Manfred Höfle<br />

Dept. Vaccinology & Applied Microbiology, HZI-Helmholtz Center for Infection Research, Braunschweig, Germany<br />

15:00 – 15:40 Coffee break, posters<br />

Chair: Ulrich Szewzyk<br />

15:40 – 16:00 HDID 08<br />

Use of propidium monoazide for live-dead distinction<br />

Andreas Nocker<br />

Quality of Life, Netherlands Organisation for Applied Scientific Research (TNO), Zeist, Netherlands<br />

16:00 – 16:20 HDID 09<br />

Transcriptional activity around bacterial cell death reveals molecular biomoarkers for cell viability<br />

Remco Kort*, Bart J. Keijser, Martien P.M. Caspers, Frank H. Schuren, Roy Montijn<br />

Netherlands Organisation for Applied Scientific Research (TNO), Zeist, The Netherlands<br />

16:20 – 17:00 HDID 10<br />

Definition of death – relevance for public health and risk regulation<br />

Martin Exner*<br />

Institue for Hygiene and Public Health, University of Bonn, Bonn, Germany<br />

17:00 – 17:15 What do we know now? (Hans-Curt Flemming)<br />

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42 ABSTRACTS<br />

Abstracts: Overview Plenary Lectures, Special Group<br />

Mini-Symposia, Short Lectures and Posters<br />

Plenary Lectures Page<br />

H Plenary Lectures 43<br />

Special Group Mini-Symposia and Workshop Page<br />

FGA Biologie bakterieller Naturstoffproduzenten 46<br />

FGB Biotransformation 47<br />

FGC Functional Genomics 47<br />

FGD Fungal Biology and Biotechnology 47<br />

FGE Identification and Systematics 49<br />

FGF Microbial Pathogenicity 50<br />

FGG Regulation and Signalstransduction in Prokaryotes 51<br />

FGH Yeast 52<br />

Short Lectures Page<br />

KA Anaerobic Metabolism 53<br />

KB Archaea 54<br />

KC Cyanobacteria and Algae 55<br />

KD Developmental Microbiology 57<br />

KE Fungal Biology 58<br />

KF Green Biotechnology 60<br />

KG Host-Microbe Interactions 61<br />

KH Imaging Techniques in Microbiology 62<br />

KI Microbial Cell Biology 63<br />

KJ Microbial Communities 65<br />

KK Microbial Diversity 66<br />

KL Microbial Pathogens and Pathogenicity 68<br />

KM Photosynthesis and Bioenergetics 69<br />

KN Physiology 71<br />

KO Regulation 72<br />

KP Secondary Metabolism 73<br />

KQ Sensory and Regulatory RNA 74<br />

KR Systems Biology 75<br />

KS Transport 77<br />

KT White Biotechnology 78<br />

KU Open Topics (Microbial Molecular Tools) 80<br />

Posters Page<br />

PA Anaerobic Metabolism 81<br />

PB Archaea 90<br />

PC Special Group Biologie bakterieller Naturstoffproduzenten 96<br />

PD Special Group Biotransformation 98<br />

PE Cyanobacteria and Algae 99<br />

PF Developmental Microbiology 100<br />

PG Special Group Functional Genomics 101<br />

PH Fungal Biology 103<br />

PI Green Biotechnology 110<br />

PJ Host-Microbe Interactions 111<br />

PK Special Group Identification and Systematics 116<br />

PL Imaging Techniques in Microbiology 117<br />

PM Microbial Cell Biology 117<br />

PN Microbial Communities 122<br />

PO Microbial Diversity 141<br />

PP Microbial Pathogens and Pathogenicity 152<br />

PQ Photosynthesis and Bioenergetics 161<br />

PR Physiology 164<br />

PS Regulation 170<br />

PT Secondary Metabolism 183<br />

PU Sensory and Regulatory RNA 185<br />

PV Systems Biology 188<br />

PW Transport 190<br />

PX White Biotechnology 195<br />

PY Special Group Yeast 205<br />

PZ Open Topics 206<br />

How dead is dead Page<br />

HDID How dead is dead Lectures 218<br />

HDID P How dead is dead Posters 220<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

H 01<br />

Live of intracellular bacterial pathogens within mammalian<br />

cells<br />

E. Eylert 1 , R. Stoll 2 , S. Mertins 2 , B. Joseph 2 , J. Schär 2 , W. Eisenreich 1 , W.<br />

Goebel *2<br />

1 Institute of Biochemistry, TU München, München, Germany<br />

2 Biocenter (Microbiology), University of Würzburg, Würzburg, Germany<br />

Intracellular bacterial pathogens are able to efficiently replicate either in<br />

specialized phagosomal compartments or in the cytosol of the infected host<br />

cells. Well-studied members of the first group include the human pathogens<br />

Salmonella enterica, Legionella pneumophila and Mycobacterium tuberculosis,<br />

whereas Shigella spp, the closely related enteroinvasive E. coli (EIEC) and<br />

Listeria monocytogenes are the best characterized human pathogens of the<br />

second group. An impressive knowledge exists by now concerning the genetics<br />

and biochemistry of the specific virulence factors enabling these pathogens to<br />

reach their specific cellular compartments and to survive intracellularly. We<br />

know also much about the host cells responses induced by these pathogens and<br />

their ability to overcome adverse host reactions. Our knowledge on the<br />

metabolic adaptation processes between the pathogen and the host cell which<br />

are apparently decisive for the efficient intracellular replication of these<br />

bacterial pathogens is, however still rather limited. There is also little known on<br />

how the intracellular bacterial metabolism affects the expression of the<br />

virulence genes that are decisive for the intracellular life cycle of these bacteria.<br />

We have started to study this important problem using the cytosolically<br />

replicating Listeria monocytogenes and enteroinvasive E. coli (EIEC) as model<br />

systems. For this goal we constructed suitable mutants and analysed their<br />

metabolism in comparison to the wild-type strain within different mammalian<br />

host cells by genome-based high-throughput techniques and NMR- or MSbased<br />

13 C-isotopologue analysis. Although these two intracellular pathogens<br />

use the same host cell compartment for growth, the results obtained so far<br />

indicate that their metabolic adaptation strategies are quite different and clearly<br />

aim to optimise the expression of the virulence genes which are crucial for the<br />

intracellular life style.<br />

These mteabolic studies have also identified metabolic enzymes and<br />

transporters which are indispensable for intracellular bacterial growth and<br />

hence may represent useful targets for the screening of antibacterial drugs<br />

directed specifically against these bacterial pathogens.<br />

H 02<br />

Functions of the Proteasome: From Protein Degradation<br />

and Immune Surveillance to Cancer Therapy<br />

A. Goldberg *1<br />

1 Department of Cell Biology, Harvard Medical School, Boston, United States<br />

Most proteins in mammalian cells are degraded by the ubiquitin-proteasome<br />

pathway in which linkage of substrates to a chain of ubiquitin molecules marks<br />

the protein for rapid degradation by the 26S proteasome. This large 2.4kDa<br />

complex contains 6 ATPase subunits in a ring and uses ATP to unfold the<br />

proteins and to translocate them into the 20S core proteasome, where they are<br />

digested to small peptides. To clarify the functions of the proteasomeassociated<br />

ATPases, we studied the archaeal 20S proteasome and the PAN<br />

ATPase, the archaeal homolog of the 26S ATPases. Substrates enter the 20S<br />

proteasome through a narrow gated channel in its outer α-ring. The N-termini<br />

of the a-subunits function as an ATPase-regulated gate that in its closed form<br />

prevents nonspecific entry of protein substrates. ATP binding allows gate<br />

opening, but ATP hydrolysis to ADP restores the closed form. Thus, ATP<br />

binding alone can activate multiple key steps in proteasome function (complex<br />

formation, gate opening, and translocation of unfolded proteins), but<br />

degradation of globular proteins requires energy-dependent unfolding. The<br />

association of the ATPase with the 20S particle and activation of gate-opening<br />

requires a small conserved C-terminal tripeptide (HbYX) motif. When the<br />

ATPases bind ATP, their C-termini bind to intersubunit pockets in the<br />

proteasome’s a-ring and act like „keys-in-a-lock“ to trigger gate-opening and<br />

substrate entry. Using single-particle cryo-EM, we have been able to visualize<br />

the binding of the C-termini and this gate-opening mechanism.<br />

Much has been learned in recent years about the functions of the proteasome<br />

using inhibitors of the 20S’s peptidase sites.Blocking proteasome function<br />

eventually induces apoptosis, especially in cancer cells, and one such inhibitor,<br />

Bortezomib (Velcade, PS341), has been approved by the FDA and is widely<br />

used for treatment of multiple myeloma. For several reasons, this cancer is<br />

particularly sensitive to proteasome inhibition. Bortezomib acts synergistically<br />

with many other chemotherapeutic agents and is now in trials against diverse<br />

cancers. The proteasome’s six active sites function synergistically in degrading<br />

proteins. At therapeutic levels, Velcade inhibits only the 20S’s chymotrypsinlike<br />

sites and reduce protein degradation only partially, which explains why<br />

proteasome inhibitors are not generally toxic.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Peptides released by proteasomes range from 2-24 residues in length. Although<br />

most are rapidly digested by cytosolic peptidases to amino acids, some are<br />

transported into the ER delivered to the cell surface and presented to the<br />

immune system on MHC Class I molecules.This process enables cytotoxic T<br />

cells to screen for virally infected cells and cancers. In immune tissues and<br />

disease states, g-interferon enhances the efficiency of antigen presentation by<br />

inducing alternative forms of proteasomes and the unusual ER peptidase,<br />

ERAP1, which trims proteasome products to the final 8-9 residue antigenic<br />

peptides.<br />

H 03<br />

Small regulatory RNAs make a great difference to<br />

Pseudomonas<br />

D. Haas *1<br />

1 Department of Fundamental Microbiology, University of Lausanne, Lausanne,<br />

Switzerland<br />

Small RNAs (sRNAs) of prokaryotes are usually 40 - 600 nucleotides in length<br />

and most have regulatory functions. As a rule, sRNAs do not encode<br />

polypeptides, but important exceptions exist. To date, close to 100 sRNAs have<br />

been reported in Escherichia coli and about 40 in Pseudomonas spp. Among<br />

the latter, four sRNAs are homologues of well-known E. coli sRNAs: tmRNA<br />

(transfer-messenger RNA for tagging truncated polypeptides), 6S RNA<br />

(regulator of RNA polymerase), 4.5S RNA (RNA component of signal<br />

recognition particle), and 10Sb RNA (RNA component of RNase P). Whereas<br />

annotation of these sRNA genes in Pseudomonas spp. can be accomplished by<br />

homology searches, most other Pseudomonas sRNAs cannot be identified by<br />

this approach, because (i) their sequences have too strongly diverged or (ii)<br />

they fulfil functions that enteric bacteria do not have. In group (i), the sRNAs<br />

RsmY, RsmZ, PrrF1,2 of P. aeruginosa are functional homologues of the E.<br />

coli sRNAs CsrB, CsrC and RyhB, respectively. RsmY and RsmZ (as well as<br />

RsmX of P. fluorescens) are antagonists of the RNA-binding protein RsmA, a<br />

translational repressor of secondary metabolism. The expression of rsmXYZ<br />

depends on the GacS/GacA two-component system, the ambient temperature<br />

and the function of the tricarboxylic acid cycle. PrrF1 and PrrF2 are repressed<br />

by the Fur protein with iron and are important Hfq-dependent, translational<br />

regulators of iron homeostasis, as shown by Vasil and coworkers. In group (ii),<br />

RgsA and PhrS are translational regulators of oxidative stress response and<br />

quorum sensing, repectively.<br />

H 04<br />

What it takes to be a nitrogen-fixing root-nodule bacterium<br />

H. Hennecke *1<br />

1 Institute of Microbiology, ETH Zürich, Zürich, Switzerland<br />

Life of a rhizobial endosymbiont is the result of massive physiologic<br />

adaptations to the host root-nodule environment. This is evidenced by the<br />

profound gene expression changes that occur in symbiotic bacteroids as<br />

compared with cells grown in culture. One of the important signals sensed by<br />

rhizobia in nodules is the extremely low oxygen partial pressure. This cue leads<br />

in Bradyrhizobium japonicum to the induction of hundreds of genes regulated<br />

primarily by two low-oxygen responsive signal transduction systems, the<br />

FixLJ-FixK2 and the RegSR-NifA cascades. Genes for microoxic respiration<br />

and genes for nitrogen fixation are amongst the respective regulated targets.<br />

Other genes are induced in response to host-specific nutrient supplies, e.g.<br />

genes for carbon source and trace element utilization. Many of the induced<br />

genes are essential for symbiosis. Yet, mutation of a substantial number of<br />

genes, often highly induced in symbiosis, does not result in noticeable<br />

phenotypes. This can be explained in some cases by gene redundancy, but<br />

remains a mystery in other cases. Also poorly un<strong>der</strong>stood is why certain stress<br />

signals (e.g., reactive oxygen species) are integrated in the aforementioned<br />

regulatory circuits.<br />

H 05<br />

E. coli Biofilms, Bottlenecks, and Host Re-sponses in<br />

Urinary Tract Infections<br />

S. Hultgren *1<br />

1 Department of Molecular Microbiology, Washington University School of<br />

Medicine, St. Louis, United States<br />

The rise in antibiotic resistant pathogens, emergence of new diseases, and<br />

involvement of<br />

bacterial pathogens in diseases formerly thought to be due to non-infectious<br />

agents have rekindled the need to un<strong>der</strong>stand the „molecular logic“ of virulent<br />

bacteria. We have uncovered the fine details of a molecular machine, called the<br />

chaperone/usher pathway, used by diverse pathogenic bacteria to assemble<br />

43

44<br />

adhesive fibers called pili on their surfaces. Pili initiate hostpathogen<br />

interactions critical in the pathogenic processes of a wide range of bacteria. We<br />

discovered that periplasmic chaperones serve as folding templates for pilus<br />

subunits, actively shaping the final structure of pilus subunits in a mechanism<br />

that represents a surprising twist of the classic Anfinsen postulate. We have<br />

used multidisciplinary approaches including functional genomics, molecular<br />

biology, genetics, immunology, cell biology, biochemistry, X-ray<br />

crystallography, and multiple imaging technologies in or<strong>der</strong> to advance our<br />

un<strong>der</strong>standing of E. coli pathogenesis. Using uropathogenic Escherichia coli<br />

(UPEC) as a model system, we discovered that UPEC form biofilms in the<br />

blad<strong>der</strong> to subvert innate host defenses. UPEC entry into superficial umbrella<br />

cells lining the blad<strong>der</strong> lumen is a critical event in disease. We found that<br />

bacterial entry into umbrella cells activates a complex genetic cascade leading<br />

to the formation of intracellular bacterial communities (IBCs) that un<strong>der</strong>go a<br />

defined maturation and differentiation program involving the expression of type<br />

1 pili. We found that the host response has implications for normal epithelial<br />

renewal and blad<strong>der</strong> cancer. In addition, we discovered that curli fibers<br />

produced by E. coli are amyloid fibers important in biofilm formation. Using E.<br />

coli curli biogenesis as a model, we developed small molecular weight<br />

compounds that block curli formation in vivo, and in vitro. Thus, these same<br />

compounds blocked Alzheimer’sassociated β-amyloid formation and bacterial<br />

biofilms. Our work is spawning new insights into basic principles of molecular<br />

biology related to protein folding and macromolecular assembly and is<br />

providing a paradigm to un<strong>der</strong>stand infectious diseases, their relationship to<br />

cancer and better strategies for treatment and prevention.<br />

H 06<br />

The formation of surface-associated communities by<br />

Burkhol<strong>der</strong>ia sp. is dependent on cell-to-cell communication<br />

L. Eberl *1<br />

1 Department of Microbiology, Institute of Plant Biology, University of Zürich,<br />

Zürich, Switzerland<br />

Taxonomic studies of the past few years have shown that Burkhol<strong>der</strong>ia<br />

cepacia-like organisms comprise a very heterogenous group of strains,<br />

collectively referred to as the B. cepacia complex (Bcc). Strains of the Bcc are<br />

ubiquitously distributed in nature and have been frequently isolated from water,<br />

the plant rhizosphere, the hospital environment, and industrial settings. B.<br />

cepacia complex strains have an enormous biotechnological potential and have<br />

been used for bioremediation of recalcitrant xenobiotics and biocontrol<br />

purposes. At the same time, however, Bcc strains have emerged as<br />

opportunistic pathogens of humans, particularly those with cystic fibrosis.<br />

An important factor that contributes to the great versatility of these bacteria is<br />

their ability to form surface associated consortia, so-called biofilms, on various<br />

surfaces. To investigate the factors required for surface colonization random<br />

mini-Tn5 insertion mutants were generated and analyzed both on the genetic<br />

and phenotypic level. The identified genes fell into several classes: (i) genes<br />

encoding for surface proteins, (ii) genes involved in the biogenesis and<br />

maintenance of an integral outer membrane, (iii) genes encoding regulatory<br />

factors, and (iv) genes involved in cell-to-cell communication (quorum sensing,<br />

QS). To investigate the role of QS in biofilm formation we mapped the QS<br />

regulon employing both transcriptomics and proteomics. QS-regulated genes<br />

that may link cell signaling with the formation of surface-associated consortia<br />

are currently analyzed in better detail.<br />

H 07<br />

Proteomics and Epigenetics: Dissection of the Eukaryotic<br />

Circadian Clock in Neurospora<br />

C.L. Baker 1 , W.J. Belden 1 , A.N. Kettenbach 1 , S.A. Gerber 1 , J.J. Loros 1 , J.C.<br />

Dunlap *1<br />

1 Department of Genetics, Dartmouth Medical School, Hanover, United States<br />

Transcription/translation feedback loops are central to circadian rhythms in<br />

eukaryotes. In fungi and animals the feedback loop comprises a heterodimer of<br />

PAS-domain proteins that activates transcription of genes encoding negative<br />

regulator(s) that, in turn, feed back to close the loop. Slow and progressive<br />

phosphorylation of the negative elements over the next 14 hours leads to their<br />

destabilization and precipitous turnover. In Neurospora, the PAS domain<br />

heterodimer is WC-1/WC-2 and the negative element protein is FRQ. We<br />

won<strong>der</strong>ed, „What are the events at the frq promoter whereby WC-1/WC-2 lead<br />

to it’s rhythmic expression, and what is the spatial and temporal profile of FRQ<br />

phosphorylation?“<br />

FRQ is phosphorylated at nearly 95 sites making it one of the most heavily<br />

modified proteins known. We used SILAC, a method of stable isotope labeling,<br />

and tandem MSMS to follow the course of FRQ phosphorylation, as well as to<br />

follow the assembly and disassembly of the core circadian interactome over a<br />

day. Phosphorylation is not uniform but is clustered with clusters appearing in a<br />

temporal pattern. Expression of frq is complex, as it encodes alternatively<br />

spliced sense transcripts as well as a long (> 4knt) antisense transcript, qrf. The<br />

frq and qrf promoters show chromatin rearrangement in response to light as<br />

well as time-of-day. Deletion of all 19 genes encoding ATP-dependent<br />

chromatin-remodeling enzymes revealed only 2 genes, clockswitch (csw-1, a<br />

homolog of yeast Fun30, mouse Etl1 and human SMARCAD) and chd2 (a<br />

homolog of mammalian mi-2, chd2 and yeast Chd1), required for remodeling<br />

and for normal circadian clock function. ChIP localizes them to frq, along with<br />

WC-1/WC-2. frq is methylated and is hypermethylated in Δchd2, and<br />

methylation requires both a functional circadian clock as well as the frq<br />

antisense transcript, qrf.<br />

H 08<br />

Functional analysis of trace nutrient homeostasis in<br />

Chlamydomonas using next generation sequencers<br />

S. Merchant *1,3,4 , J. Kropat 1 , M. Castruita 1 , D. Casero 2 , S. Karpowicz 1 , S.<br />

Cokus 2 , M. Pellegrini 2,3,4<br />

1 Department of Chemistry and Biochemistry, Molecular, Cell and<br />

Developmental Biology, Molecular Biology Institute and the Institute for<br />

Genomics and Proteomics, UCLA, Los Angeles, United States<br />

The first row transition metals like iron, copper and manganese are nutritionally<br />

essential because they are used in bioenergetic systems to catalyze redox<br />

reactions, which are the basis for life. The bioavailability of these elements has<br />

changed over the few billion years since the origin of life so that the growth<br />

potential of many organisms can be limited by mineral nutrition. Accordingly,<br />

organisms have devised mechanisms to survive transient and even sustained<br />

deficiency. The mechanisms involved in maintaining copper and iron<br />

homeostasis in Chlamydomonas will be presented. Use of state of the art RNA-<br />

Seq methodology to characterize the Chlamydomonas transcriptome will also<br />

be presented. The analyses indicate previously unknown modifications of the<br />

photosynthetic apparatus and the potential for modification of bioenergetic<br />

pathways.<br />

H 09<br />

Cell differentiation and multicellularity in heterocystforming<br />

cyanobacteria<br />

E. Flores *1<br />

1 Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC and Universidad de<br />

Sevilla, Sevilla, Spain<br />

The bacterial world displays an outstanding biochemical and morphological<br />

diversity. The cyanobacteria perform oxygenic photosynthesis, and many<br />

filamentous cyanobacteria, such as those of the genera Anabaena and Nostoc,<br />

are true multicellular organisms in which growth requires the activity of two<br />

interdependent cell types, photosynthetic vegetative cells and nitrogen-fixing<br />

heterocysts. Un<strong>der</strong> nitrogen deprivation, heterocysts differentiate from some<br />

vegetative cells in the filament. This process requires the N-control<br />

transcription factor NtcA, which activates the expression of many genes during<br />

the differentiation process and in the mature heterocyst. Transcription from the<br />

promoters of some of these genes can be effected in vitro by the Anabaena<br />

RNA polymerase supported by NtcA and 2-oxoglutarate, a signal of the C to N<br />

balance in cyanobacteria. The mature heterocyst exchanges metabolites and<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

egulatory compounds with nearby vegetative cells. Because the cell-envelope<br />

outer membrane runs along the filament without entering the intercellular septa,<br />

the cyanobacterial filament consists of a string of cells that share the periplasm.<br />

Intercellular transfer of some compounds could take place through the<br />

continuous periplasm, but cell-to-cell joining structures that could mediate<br />

intercellular molecular exchange are also present. The SepJ (FraG) protein,<br />

which consists of a large N-terminal periplasmic domain and a C-terminal<br />

integral membrane domain homologous to some bacterial permeases, is<br />

conspicuously located at the cell poles in the intercellular septa. Additionally,<br />

two Fra proteins needed for proper localization of SepJ at the cell poles have<br />

been identified. The SepJ and Fra proteins are required for filament integrity<br />

and could constitute a cell-to-cell joining complex essential for multicellularity.<br />

H 10<br />

The mitochondrial machinery for import and assembly of<br />

proteins<br />

N. Pfanner *1 , N. Wiedemann 1 , A. Chacinska 1 , M. van <strong>der</strong> Laan 1 , C. Meisinger 1<br />

1<br />

Institute for Biochemistry and Molecular Biology, University of Freiburg,<br />

Freiburg, Germany<br />

About 1,000 different proteins are transported from the cytosol into<br />

mitochondria via the main entry gate, the translocase of the outer membrane<br />

(TOM complex). The precursor proteins are then distributed into the<br />

mitochondrial subcompartments by different pathways. (i) The presequence<br />

pathway transfers preproteins into the matrix, inner membrane and<br />

intermembrane space of mitochondria. The presequence translocase of the inner<br />

membrane (TIM23 complex) is composed of functional modules to switch<br />

between matrix translocation and membrane insertion of preproteins. (ii) The<br />

carrier pathway directs multispanning hydrophobic proteins into the inner<br />

membrane, using the Tim9-Tim10 chaperone complex of the intermembrane<br />

space and the carrier translocase of the inner membrane (TIM22 complex). (iii)<br />

Many intermembrane space proteins are imported by a redox-regulated<br />

machinery (MIA), involving disulfide-linked intermediates between the<br />

intermembrane space receptor Mia40 and precursor proteins. (iv) Protein<br />

insertion into the mitochondrial outer membrane involves different pathways<br />

for beta-barrel proteins (SAM complex) and alpha-helical proteins.<br />

H 11<br />

Cyanobacterial thylakoid membrane networks:<br />

Connectedness, permeability, and (dis-) similarity to<br />

higher-plant networks<br />

R. Nevo 1 , D. Charuvi 1,2 , E. Shimoni 3 , S.G. Chuartzman 1 , O. Rav-Hon 1 , R.<br />

Schwarz 4 , A. Kaplan 5 , I. Ohad 5 , V. Brumfeld 6 , Z. Reich *1<br />

1<br />

Department of Biological Chemistry, Weizmann Institute of Science, Rehovot,<br />

Israel<br />

2<br />

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture,<br />

The Hebrew University of Jerusalem, Rehovot, Israel<br />

3<br />

Electron Microscopy Unit, Weizmann Institute of Science, Rehovot, Israel<br />

4<br />

Faculty of Life Science, Bar-Ilan University, Ramat-Gan, Israel<br />

5<br />

The Institute of Life Science and Avron-Even-Ari Minerva Center for<br />

Photosynthesis Research, The Hebrew University of Jerusalem, Jerusalem,<br />

Israel<br />

6<br />

Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel<br />

Cyanobacterial thylakoid membrane networks are the most ancient oxygenic<br />

photosynthetic membranes known. As such, they provide a valuable window<br />

into the fundamental designs employed by evolution in the construction of<br />

these lamellar systems, which enabled and still sustain aerobic life on Earth. I<br />

will begin my talk by addressing the basic requisites and constraints imposed<br />

on thylakoid membranes by their function as well as by the general homeostasis<br />

of their hosting cells or organelles. I will then describe how these requisites and<br />

constraints are met in cyanobacteria, as revealed by electron microscope<br />

tomography studies. This will be followed by a comparison with the most<br />

evolved thylakoid networks existing, those of higher plants, and by a short<br />

commentary on what it takes to form and remodel thylakoid membrane<br />

networks.<br />

[1] Nevo, R., Charuvi, D., Chuartzman, S. G., Shimoni, E., Tsabari, O. and<br />

Reich, Z. Architecture and plasticity of thylakoid membrane networks. In<br />

Lipids in photosynthesis (Wada, H. and Murata, N., Eds.), Springer-Verlag,<br />

<strong>2009</strong>.<br />

[2] Brumfeld, V., Charuvi, D., Nevo, R., Chuartzman, S. G., Tsabari, O., Ohad,<br />

I., Shimoni, E. and Reich, Z. A note on 3D models of higher-plant thylakoid<br />

networks. Plant Cell. In press (available as Plant Cell Advance Online<br />

Publication).<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

[3] Chuartzman, S. G., Nevo, R., Shimoni, E., Charuvi, D., Kiss, V., Ohad, I.,<br />

Brumfeld, V. and Reich Z. Thylakoid membrane remodelling during state<br />

transitions in Arabidopsis. Plant Cell. 20, 1029-1039, 2008.<br />

[4] Nevo, R., Charuvi, D., Shimoni, E., Schwarz, R., Kaplan, A., Ohad, I. and<br />

Reich, Z. Thylakoid membrane perforations and connectivity enable<br />

intracellular trafficking in cyanobacteria. EMBO J. 26, 1467-1473, 2007.<br />

[5] Ohad, I., Nevo, R., Brumfeld, V., Reich, Z., Tsur, T., Yair, M. and Kaplan,<br />

Inactivation of photosynthetic electron flow during desiccation of desert<br />

biological sand crusts and Microcoleus sp.-enriched isolates. Photochem.<br />

Photobiol. Sci. 4, 977-982, 2005.<br />

[6] Shimoni, E., Rav-Hon, O., Ohad, I., Brumfeld, V. and Reich, Z. Threedimensional<br />

organization of chloroplast thylakoid membranes revealed by<br />

electron tomography. Plant Cell 17, 2580-2586, 2005.<br />

H 12<br />

Challenge of biological hydrogen conversion in the presence<br />

of air<br />

B. Friedrich *1<br />

1<br />

Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Berlin,<br />

Germany<br />

Hydrogen, an attractive prokaryotic energy source, is released into the anoxic<br />

environment by fermentation where it gets immediately consumed by anaerobic<br />

microbes. Thus, only traces of hydrogen escape to oxic habitats where aerobic<br />

hydrogen-oxidizers, the "Knallgas" bacteria thrive. These bacteria host [NiFe]<br />

hydrogenases that tolerate ambient oxygen during catalysis. The well studied<br />

Ralstonia eutropha H16 (Re) contains three physiologically distinct [NiFe]<br />

hydrogenases: (i) a membrane-bound enzyme which is exposed to the<br />

periplasm; (ii) a hexameric soluble enzyme which reduces NAD + at the expense<br />

of hydrogen and (iii) a hydrogen-sensing regulatory protein which signals the<br />

presence of hydrogen to the cell. Oxygen may affect hydrogenases on various<br />

molecular levels. In this presentation it is demonstrated that several oxygenprotecting<br />

mechanisms exist in Re. The hydrogen sensor acquires oxygen<br />

resistance by preventing access of oxygen to the NiFe active site. The NADreducing<br />

hydrogenase shields its NiFe cofactor against oxidation by<br />

modification of the ligands bound to the Ni ion. The membrane-bound<br />

hydrogenase has an extremely high affinity for hydrogen which confers<br />

tolerance to oxygen. Oxygen tolerance of this hydrogenase relies also on an<br />

intricate protein-assisted maturation process which guarantees a precise<br />

assembly of the redox cofactors in the presence of air. The ability of aerobic<br />

hydrogenases to be catalytically active in air is an essential prerequisite for the<br />

industrial application of hydrogenases, e.g. in biological fuel cells or for<br />

biosolar production of hydrogen.<br />

H 13<br />

Molecular basis of resistance to weak acids in fungi<br />

D. Archer *1<br />

1 School of Biology, University of Nottingham, Nottingham, United Kingdom<br />

Sorbic acid (2,4-hexadienoic acid) and other weak acids may be added to some<br />

foods and beverages as preservatives, <strong>bei</strong>ng most effective at acidic pH. While<br />

sorbic acid may combat spoilage by yeasts such as Saccharomyces cerevisiae<br />

and moulds such as Aspergillus niger, both of these fungal species are able to<br />

decarboxylate sorbic acid to the volatile product 1,3-pentadiene. The capacity<br />

for decarboxylation is limited in S. cerevisiae and does not constitute a<br />

resistance mechanism but the decarboxylation of sorbic acid provides resistance<br />

in filamentous fungi and, especially so, during the germination of conidia.<br />

Decarboxylation requires the activities of both a phenylacrylic acid<br />

decarboxylase, encoded by padA, and a putative 4-hydroxybenzoic acid (3octaprenyl-4-hydroxybenzoic<br />

acid) decarboxylase encoded by ohbA1(ubiD1).<br />

Other homologues of padA1 (padA2 and padA3) were identified in the genome<br />

of A. niger but these were not essential for decarboxylation. The padA1 and<br />

ohbA1 genes were found to be in close proximity to each other on chromosome<br />

6 in the A. niger genome, separated by only one gene. Further bioinformatic<br />

analysis revealed conserved synteny at this locus in several Aspergillus species<br />

and other ascomycete fungi, although the intervening gene was sometimes<br />

absent and the relative orientations of the genes differed. The intervening gene<br />

between padA1 and ohbA1 in A. niger appears to encode a fungal Zn(2)-Cys(6)<br />

binuclear cluster protein (PF00172). Both padA1 and ohbA1 are absent from the<br />

genomes of A. fumigatus and A. clavatus and, as a consequence, neither species<br />

is capable of decarboxylating sorbic acid. Knowledge of the mechanisms of<br />

both toxicity and resistance will help in the design of more effective anti-fungal<br />

strategies in foods.<br />

Acknowledgements: I thank my colleagues Andrew Plumridge, Malcolm<br />

Stratford and Petter Melin, collaborators from DSM Food Specialities (Hein<br />

Stam, Jacques Stark, Hans Roubos and Hildegard Menke) and Defra/BBSRC<br />

for funding.<br />

45

46<br />

FGA 01<br />

Cell wall biosynthesis as a target of new antibiotic<br />

compounds<br />

T. Schnei<strong>der</strong> *1 , H.G. Sahl 1<br />

1 Institute for Medical Microbiology, Immunology and Parasitology –<br />

Pharmaceutical Microbiology Section, University of Bonn, Bonn, Germany<br />

Bacterial cell wall biosynthesis is economically the most important target<br />

pathway for antibiotic intervention. On the molecular level, penicillin-binding<br />

proteins and Lipid II, the cell wall building unit attached to a membrane carrier,<br />

are the most important targets; however, many antibiotics other than β-lactams<br />

and glycopeptides have been described to act on further molecular target sites<br />

within this pathway. Most lantibiotics target the final bactoprenol-bound cell<br />

wall precursor Lipid II. Binding of the precursor prevents incorporation into the<br />

growing peptidoglycan polymer, however, some lantibiotics use Lipid II<br />

additionally as an anchor molecule for pore formation. Such combined<br />

activities result in most potent antibiotic action.<br />

We found the lipopeptide antibiotic friulimicin to form a stoichiometric<br />

complex with the membrane carrier bactoprenolphosphate itself, which is not<br />

targeted by other antibiotic in use. Since bactoprenolphosphate also serves as a<br />

carrier of teichoic acid and carbohydrate capsule building blocks, friulimicin<br />

appears to cause an extensive cell surface stress.<br />

Yet another mode of cell wall biosynthesis inhibition seems to be applied by<br />

host defense peptides which, unlike conventional antibiotics that act via defined<br />

target molecules are assumed to act unspecifically by permeabilising the cell<br />

membrane.<br />

In contrast to this widely held view, we report for the first time the bacterial cell<br />

wall precursor Lipid II as the molecular target of the fungal defensin plectasin.<br />

Also, the human β-defensin 3 (hBD3), appears to interfere with Lipid IIdependent<br />

synthetic reactions and possibly disturbes the coordinated<br />

interactions within the highly dynamic, multi-enzyme cell wall biosynthesis<br />

machinery.<br />

FGA 02<br />

A novel antibacterial class acting by reprogramming Clp<br />

protease<br />

H. Brötz-Oesterhelt *1<br />

1 Antibacterial Research, AiCuris GmbH & Co. KG, Wuppertal, Germany<br />

Bacterial resistance against antibiotics increases worldwide highlighting the<br />

need for novel antibiotics devoid of cross-resistance to drugs in therapeutic<br />

application. New natural product <strong>der</strong>ived acyldepsipetides designated ADEPs<br />

are highly active against most nosocomial Gram-positive problem pathogens<br />

(e.g. staphylococci, enterococci, and streptococci) with antibacterial in vitro<br />

activities surpassing that of many marketed antibiotics and impressive efficacy<br />

in rodent models of bacterial infection. The ADEPs act on an unprecedented<br />

target, the bacterial caseinolytic protease ClpP, and thoroughly reprogram its<br />

activity. Here, we demonstrate that binding of the ADEPs to ClpP abrogates its<br />

interaction with cooperating Hsp100 ATPases, thereby preventing the protease<br />

from performing its physiological tasks in protein quality control and regulatory<br />

proteolysis. Instead, ClpP is redirected to the ribosome, where it degrades<br />

nascent polypeptide chain in an uncontrolled manner, leading to inhibition of<br />

bacterial cell division and death. Due to this novel mechanism of action the<br />

ADEPs show undiminished activity against multi-resistant bacterial isolates,<br />

demonstrating their potential for the future treatment of Gram-positive<br />

infections. As ClpP is essential for virulence factor expression in several Grampositive<br />

species but not essential for bacterial growth per se, potential<br />

application of the ADEP should be consi<strong>der</strong>ed in combination therapy to<br />

prevent rapid resistance development. As combination partner ADEPs may be<br />

able to exert a double role in controlling the infection process by inhibiting<br />

bacterial virulence as well a bacterial growth.<br />

FGA 03<br />

Mycotoxin Biosynthesis by Endofungal Bacteria - Insights<br />

into an Overlooked Symbiosis<br />

C. Hertweck *1<br />

1<br />

Dept. Biomolecular Chemistry, Leibniz Institute for Natural Product Research<br />

and Infection Biology (HKI), Jena, Germany<br />

Pathogenic fungi generally exert their destructive effects through virulence<br />

factors. An important example is the macrocyclic polyketide rhizoxin, the<br />

causative agent of rice seedling blight, from the fungus Rhizopus microsporus.<br />

The phytotoxin efficiently binds to rice β-tubulin, which results in inhibition of<br />

mitosis and cell cycle arrest.<br />

By a series of experiments we could unequivocally demonstrate that rhizoxin is<br />

not biosynthesized by the fungus itself, but by endosymbiotic bacteria of the<br />

genus Burkhol<strong>der</strong>ia. Our unexpected findings unveil a remarkably complex<br />

symbiotic-pathogenic alliance that extends the fungus–plant interaction to a<br />

third, bacterial key player. In addition, we were able to culture the symbionts to<br />

produce antitumoral rhizoxin <strong>der</strong>ivatives, and to elucidate the molecular basis<br />

for the biosynthesis of the toxin. A second example for the formation of a<br />

„mycotoxin“ by endofungal bacteria has been discovered: the cyclopeptide<br />

rhizonin is not a fungal metabolite.<br />

The importance of this rare symbiotic interaction is highlighted by the<br />

unexpected observation that in the absence of bacterial endosymbionts the<br />

fungal host is not capable of vegetative reproduction. Formation of sporangia<br />

and spores is only restored upon re-introduction of endobacteria. The fungus<br />

has become totally dependent on endofungal bacteria, which in return provide a<br />

highly potent toxin for defending the habitat and accessing nutrients from<br />

decaying plants.<br />

Recent progress in studying the toxin-producing fungal-bacterial alliance will<br />

be reported.<br />

FGA 04<br />

Molecular Characterization of the DNA- translocation<br />

systems mediating conjugal plasmid transfer in<br />

Streptomyces<br />

J. Vogelmann 1 , J. Guezguez 1 , T. Roth 1 , G. Muth *1<br />

1 Mikrobiologie/Biotechnologie, Universität Tübingen, Tübingen, Germany<br />

A single plasmid encoded protein (TraB) is sufficient to promote conjugal<br />

plasmid transfer in the gram positive soil bacterium Streptomyces. TraB is a<br />

multimeric protein that belongs to the FtsK/SpoIIIE family of septal DNA<br />

translocator proteins involved in segregation of double stranded chromosomal<br />

DNA during cell division (FtsK) and sporulation (SpoIIIE).<br />

TraB proteins encoded by different Streptomyces plasmids are membrane<br />

associated ATPases that have a highly specific DNA binding activity and<br />

interact via a C-terminal wHTH motif with a specific plasmid sequence clt,<br />

containing characteristic 8 bp direct repeats. Binding of TraB-pSVH1 to clt<br />

does not involve processing of the plasmid DNA, indicating translocation of a<br />

double stranded DNA molecule during Streptomyces conjugation.<br />

Conjugation in mycelial streptomycetes also involves subsequent spreading of<br />

the transferred plasmid in the recipient mycelium via septal crosswalls. Plasmid<br />

spreading requires five to six plasmid encoded proteins (Spd) in addition to<br />

TraB. SpdB2 is an oligomeric integral membrane protein that has DNA binding<br />

activity. SpdB2 was shown to assemble to a pore structure in artificial<br />

membranes. When voltage was applied, plasmid DNA was translocated through<br />

the SpdB2 pores, demonstrating that the SpdB2 pores were large enough to<br />

translocate double stranded ccc- plasmid DNA.<br />

Bacterial two hybrid analysis, in vivo crosslinking and pulldown assays<br />

revealed interactions of TraB and many Spd proteins. This suggests a large<br />

DNA translocation complex at the septal crosswalls with TraB acting as the<br />

motor protein that pumps ds-DNA through the SpdB2 channel.<br />

FGA 05<br />

Formation of fimbriae during attachment of Streptomyces<br />

coelicolor: roles for cellulose and amyloids!<br />

D. Claessen *1<br />

1 Department of Microbial Physiology, University of Groningen, Groningen,<br />

Netherlands<br />

Streptomyces coelicolor differentiates on solid media by forming aerial hyphae<br />

that septate into chains of spores. We here show a specialized form of<br />

differentiation that only occurs when hyphae attach to surfaces. Attachment<br />

coincides with the formation of a network of fimbriae that is tightly associated<br />

with the adhering hyphae. These fimbriae have a diameter of about 30-100 nm,<br />

and are virtually absent in a mutant strain lacking the chaplin proteins, which<br />

were previously shown to assemble into amyloid-like fibrils on the cell surface<br />

of aerial structures. In addition, the amyloid-binding dye Congo red abolished<br />

attachment and prevented formation of the fimbrial network, possibly due to<br />

interference with chaplin assembly. Interestingly, firm attachment mediated by<br />

these fimbriae not only requires chaplins, but possibly also cellulose. Deletion<br />

of a cellulose synthase-like protein strongly reduced attachment although the<br />

formation of fimbriae appeared unaffected. Similarly, growth of the wild-type<br />

strain in the presence of cellulase prevented attachment without affecting<br />

formation of fimbriae. Staining of adhering wild-type hyphae with calcofluor<br />

white, which binds to polysaccharides such as cellulose, revealed its<br />

accumulation at sites where the fimbriae adhere to the cell surface. We<br />

therefore propose a model in which fimbriae are mainly composed of<br />

assembled chaplins, which are tightly anchored to the cell walls of adhering<br />

hyphae by cellulose.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

FGB 01<br />

Evolving dehalogenases: from environmental cleanup to<br />

applied biocatalysis<br />

D. Janssen *1<br />

1 University of Groningen, Groningen, Netherlands<br />

No abstract submitted<br />

FGB 02<br />

Deracemisation via Enzyme Cascades<br />

W. Kroutil *1<br />

1 Karl-Franzens University Graz, Graz, Austria<br />

No abstract submitted<br />

FGB 03<br />

Synthesis of optically active 3-aminopyrrolidines and -<br />

piperidines with omega-transaminase<br />

M. Höhne *1<br />

1 Greifswald University, Greifswald, Germany<br />

No abstract submitted<br />

FGB 04<br />

EnBase - Microplate based high-cell-density fermentation<br />

for high-throughput and high-content screening of<br />

biocatalysts<br />

P. Neubauer *1<br />

1 Technische Universtät Berlin/Berlin Institute of Technology, Berlin, Germany<br />

No abstract submitted<br />

FGC 01<br />

LUCA and the universal tree of life<br />

C. Brochier-Armanet *1<br />

1 Laboratoire de Chimie Bacterienne, Institut de Biologie Structurale et de<br />

Microbiologie, Marseille, France<br />

150 years ago, Charles Darwin hypothesized that all the present day organisms<br />

share a common evolutionary origin. Over the XXth century, research in<br />

molecular biology has confirmed Darwin’s prediction. The common origin of<br />

all present-day organisms was also confirmed by early molecular phylogenetic<br />

analyses. Among these, the pioneer works of George Fox and Carl Woese in<br />

the late 70´s have opened, with the discovery of Archaea, a new research field<br />

dedicated to the Last Universal Common Ancestor (LUCA). The ever-growing<br />

availability of genomic sequence data, together with a more extensive<br />

exploration of biological diversity through metagenomics and microbial<br />

ecology, has radically changed our vision of life diversity and evolution.<br />

Through a number of selected examples, I will illustrate how our conception of<br />

the universal tree of life has changed over the last decade.<br />

FGC 02<br />

The Thaumarchaeota: a key to archaeal history?<br />

S. Gribaldo *1<br />

1 Unite de Biologie Moleculaire chez les Extremophiles, Département de<br />

Microbiologie - Institut Pasteur, Paris, Germany<br />

Cultivated archaea are classified into two major phyla, the Euryarchaeota and<br />

the Crenarchaeota. However, a highly diversified group of uncultivated<br />

mesophilic archaea have been discovered and classified as ‘mesophilic<br />

crenarchaeota’ since they appear related to hyperthermophilic crenarchaeota in<br />

SSU rRNA trees. It is now growingly recognized that „mesophilic<br />

crenarchaeota“ are a major component of deep oceans and soil biotopes and<br />

play an important role in global nitrogen and carbon cycles. The recent<br />

availability of complete genomic sequences from two representatives of<br />

mesophilic crenarchaeota now allows more detailed analyses. These indicate<br />

that mesophilic crenarchaeota are not closely related to hyperthermophilic<br />

crenarchaeota and may represent a deep branch in the archaeal phylogeny.<br />

Moreover, they harbour important differences with hyperthermophilic<br />

crenarchaeota in terms of genomic content (including genes involved in main<br />

cellular processes). We suggest that ‘mesophilic crenarchaeota’ should be<br />

consi<strong>der</strong>ed as a new and ancient archaeal phylum, which we have named the<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Thaumarchaeota. We anticipate that future studies on Thaumarchaeota will<br />

provide crucial information on the diversity and evolution of the third domain<br />

of life.<br />

FGC 03<br />

The transition from prokaryote to eukaryote<br />

J. McInerney *1<br />

1<br />

Departement of Biology, The National University of Ireland, Maynooth,<br />

Ireland<br />

There are more than 70 published hypotheses concerning the origin of the<br />

eukaryotic cell. Many of these hypotheses cite certain lines of evidence, but<br />

some are simply models and are based on plausible assumptions about how this<br />

transition might have happened. With such a large number of hypotheses, there<br />

is clearly some conflict and indeed, for more than 100 years, eukaryote origins<br />

have been the subject of contentious debate. In this talk, I will review our<br />

progress in this field. I shall outline how we have used a phylogenetic supertree<br />

approach in or<strong>der</strong> to untangle the evolutionary signals in eukaryotic genes. I<br />

shall also talk about our analysis of mitochondrial origins and the prokaryotic<br />

sister group of the mo<strong>der</strong>n mitochondrion. I will then go further to outline how<br />

the events of Eukaryogenesis are still seen today in the expression levels of<br />

genes in yeast, the likelihood that they have a lethal phenotype upon knockout<br />

and their centrality in metabolic networks.<br />

FGD 01<br />

A highly diverse pathogenicity island for secreted proteins<br />

modulates biotrophy of Ustilago maydis and Sporisorium<br />

reilianum<br />

T. Brefort *1 , B. Franzki 1 , J. Schirawski 1 , H. Ghareeb 1 , C. Mengel 1 , E. Meyer 1 ,<br />

V. Vincon 1 , R. Kahmann 1<br />

1 Department of Organismic Interactions, Max Planck Institute for terrestrial<br />

Microbiology, Marburg, Germany<br />

The biotrophic fungi Ustilago maydis and Sporisorium reilianum cause smut<br />

diseases in maize. During biotrophic growth both fungi penetrate the cuticle<br />

and establish a mycelial network inside the plant host. While U. maydis induces<br />

spore-filled tumors on all aerial parts of its host, spore formation of S.<br />

reilianum is restricted to the inflorescence and does not involve tumor<br />

induction. Effectors facilitating these biotrophic interactions have remained<br />

elusive in both organisms.<br />

We have identified a 43-kb genomic locus in U. maydis comprising 24 genes<br />

for novel secreted proteins that are highly induced during biotrophic<br />

development. Genetic analysis revealed that at least three effector genes play<br />

additive, crucial roles for maintenance of biotrophy and tumor formation.<br />

Yeast-two-hybrid screens suggest that these effectors interfere with plant<br />

defense responses and development. Comparison of this locus in U. maydis and<br />

S. reilianum revealed very low sequence identity between the encoded effectors<br />

shared by the two pathogens, while flanking genes are conserved and syntenic.<br />

In addition, we found species-specific expansions and contractions in some of<br />

the encoded gene families as well as genes unique to either U. maydis or S.<br />

reilianum. Deletion analyses of the S. reilianum locus revealed that it codes for<br />

at least two virulence effectors, one of them <strong>bei</strong>ng S. reilianum-specific.<br />

Currently, we pursue interspecies complementation experiments to elucidate<br />

functional conservation. Our data suggest that this locus represents a<br />

pathogenicity island harboring a suite of secreted effectors that cooperatively<br />

facilitate the biotrophic interaction of smut fungi with their host plant maize.<br />

FGD 02<br />

How does VeA effect secondary metabolism in Fusarium<br />

fujikuroi?<br />

P. Wiemann *1 , D.W. Brown 2 , H.U. Humpf 3 , B. Tudzynski 1<br />

1 Institut für Botanik, Westfälische Wilhelms-Universität Münster, Münster,<br />

Germany<br />

2<br />

Mycotoxin Research Unit, U.S. Department of Agriculture-ARS, Peoria,<br />

United States<br />

3<br />

Institut für Lebensmittelchemie, Westfälische Wilhelms-Universität Münster,<br />

Münster, Germany<br />

Fusarium fujikuroi is known as a rice pathogen causing hyperelongation of<br />

stalks and leaves due to production of gibberellic acids (GAs). Besides GAs, F.<br />

fujikuroi may also synthesize other toxins like fumonisins, fusarin C and<br />

bikaverin. Although the clustered genes responsible for synthesis of these<br />

secondary metabolites are well characterized, our un<strong>der</strong>standing of their<br />

regulatory mechanisms is incomplete.<br />

In this study, we identified and characterized the F. fujikuroi veA gene and<br />

examined its role as a global regulator of secondary metabolism. Comparison of<br />

47

48<br />

wild type and veA deletion strains in pathogenicity assays revealed no<br />

hyperelongation of rice when infected with the deletion mutant, suggesting a<br />

down-regulation of GA genes. To identify more genes influenced by VeA we<br />

examined changes in gene expression of wild type and veA knock-out strains by<br />

use of a F. verticillioides oligo microarray. We found that among the downregulated<br />

genes are those involved in sexual development as well as genes<br />

needed for GA and fumonisin synthesis. Up-regulated genes included those<br />

involved in bikaverin synthesis. No changes were found for genes involved in<br />

fusarin C production. Northern blot analysis confirmed the microarray data.<br />

Examination of veA-regulated genes led to the identification of a putative<br />

ortholog of laeA, a critical regulator of secondary metabolism in Aspergillus.<br />

The impact of this gene on Fusarium secondary metabolism is currently un<strong>der</strong><br />

investigation. Our results further prove the cross-species use of the F.<br />

verticillioides microarray to elucidate the diverse effects VeA has on secondary<br />

metabolism in F. fujikuroi.<br />

FGD 03<br />

Quantitative physiology of the basidiomycete Pleurotus<br />

sapidus<br />

L.M. Blank *1 , M. Fraatz 2 , A. Schmid 1 , H. Zorn 2<br />

1 Biochemical and Chemical Engineering, TU Dortmund, Dortmund, Germany<br />

2 Institute of Food Chemistry and Food Biotechnology, Justus Liebig University<br />

Giessen, Giessen, Germany<br />

Basidiomycetes have the potential to contribute significantly to overcome the<br />

arguably most prominent limitation of industrial biotechnology; the use of nonfood<br />

biopolymers (e.g. cellulose, hemi-cellulose, and lignin) as renewable<br />

sources for the production of fine chemicals. Since basidiomycetes fulfill this<br />

role in the natural carbon cycle, researchers are interested in the enzymes that<br />

allow for the extracellular degradation of lignocelluloses. However, the use of<br />

basidiomycetes as whole cell biocatalysts is rather complicated, and often<br />

hardly reproducible. To extend the experimental toolbox for basidiomycetes,<br />

we report the development of the first minimal medium that enables<br />

exponential growth of our model basidiomycete Pleurotus sapidus in<br />

submerged cultures. With this minimal medium in hand, we investigated the<br />

physiology of P. sapidus during its growth on glucose using 13C-tracer based<br />

metabolic flux analysis. The results indicate that the network structure of<br />

central carbon metabolism is similar to the network of the hemiascomycete<br />

Saccharomyces cerevisiae. However, the function of the metabolic network<br />

differed consi<strong>der</strong>ably between these two species. In contrast to S. cerevisiae,<br />

glucose was significantly catabolized after phosphorylation via the pentose<br />

phosphate pathway. The synthesized pyruvate fueled the TCA cycle, while no<br />

by-product formation was observed. The results are discussed in the context of<br />

ongoing developments for the highly interesting group of organisms: the fungi<br />

of the class of basidiomycetes.<br />

FGD 04<br />

Studies on the biodegradation of anthracene and<br />

benz[a]anthracene by two Fusarium sp. stains isolated from<br />

mangrove sediment<br />

Y.R. Wu *1 , Z.H. Luo 1 , L.L.P. Vrijmoed 1<br />

1 Department of Biology and Chemistry, City University of Hong Kong, Hong<br />

Kong, Hong Kong<br />

Our studies described two fungal isolates capable of degrading anthracene<br />

(ANT) and benz[a]anthracene (BAA) which are, respectively, the three-ring<br />

and four-ring polycyclic aromatic hydrocarbons (PAHs) listed by US<br />

Environmental Protection Agency as one of the priority pollutants. These two<br />

strains, isolated from the mangrove sediments in Ma Wan of Hong Kong SAR,<br />

were identified as Fusarium sp. according to their microscopic morphology and<br />

18S rRNA sequences, which could both grow in the culture medium using<br />

ANT and BAA as the sole carbon source and energy. Strain MAS2 was able to<br />

degrade 20 mg•l-1 ANT, while strain MBS1 could degrade over 12 mg•l-1<br />

BAA provided during the 40 days of incubation. Total six metabolic products<br />

were detected during the degradation process based on the solid-phase<br />

microextraction (SPME) technology, which suggested that the isolated fungi<br />

degraded both ANT and BAA via their respective quinone substances to<br />

generate phthalic acid. Moreover, the extracellular enzymes, manganesedependent<br />

peroxidase and laccase, were also found in these two fungi with a<br />

higher activity level, showing that the enzymes should play an important role<br />

for the substrate transformation. In conclusion, this work indicated that<br />

Fusarium sp., as a potential genus, could solely grow and utilize ANT and<br />

BAA with a relatively higher degradation ability, suggesting its environmental<br />

application in the PAHs bioremediation.<br />

FGD 05<br />

Wood extractives as a whole protect grand fir (Abies<br />

grandis) wood against enzymatic degradation by white-rot<br />

fungi but specific compounds induce laccases for attack on<br />

lignin<br />

B. Cherdchim *1 , M. Navarro-González 1 , A. Majcherczyk 1 , U. Kües 1<br />

1 Molecular Wood Biotechnology and Technical Mycology, Georg-August-<br />

University Göttingen, Göttingen, Germany<br />

Abies grandis is a fast growing coniferous tree with a high potential for<br />

sustainable wood production and applications in the wood products industry.<br />

For defining possible indoor and outdoor usages of the A. grandis wood,<br />

knowledge on its behaviour with fungal degra<strong>der</strong>s is required. In wood block<br />

decay tests, A. grandis wood was easily degraded by brown-rot fungi but<br />

showed a good resistance against white-rot and full resistance against soft-rot<br />

species. Wood extracted with water and acetone however lost resistance. Wood<br />

extractives were shown to inhibit growth of the white-rots Trametes versicolor<br />

and Pleurotus ostreatus. Moreover, wood extractives in concentrationdependent<br />

manner induced production of extracellular laccases but no other<br />

oxidative enzymes. We identified the extractable wood compounds using gas<br />

chromatography and mass spectrometry (GC-MS) and tested the importance of<br />

these compounds in wood degradation processes as potential degradation<br />

signals and as mediators in laccase oxidation of wood lignin. Some of the<br />

natural phenolic compounds have a potential as natural inducers in<br />

biotechnological laccase production. Laccase isoenzymes of the two fungi<br />

induced by extractives are analyzed by native gel electrophoresis and identified<br />

by MS-analysis of peptides from specific proteolytic digests. As the extractives,<br />

raw A. grandis wood particles also induce laccase production. Within the wood,<br />

the fungal laccases appear to modify lignin within the plant cell walls as<br />

deduced from a novel staining method.<br />

FGD 06<br />

Secreted but cell wall associated lipases by Phialemonium<br />

spec. AW02 allow easy down-stream processing<br />

S. Barig *1 , R. Alisch 1 , S. Nieland 1 , K.P. Stahmann 1<br />

1 Technische Mikrobiologie, Fachbereich Bio-, Chemie- und Verfahrenstechnik,<br />

Fachhochschule Lausitz, Senftenberg, Germany<br />

About one hundred lipase producing microorganisms are known today. For<br />

industrial production the recombinant production e.g. of a Thermomyces lipase<br />

in Aspergillus oryzae is state of the art. Such a system has two disadvantages.<br />

Firstly, the production needs sterile technique, secondly, the extracellular<br />

enzyme is freely diffusing and has to be concentrated by cost-intensive<br />

measures e.g. ultrafiltration. To establish a robust production system without<br />

sterile technique selective conditions were adjusted to screen for new lipase<br />

producers. With mineral salts medium, low pH, nitrate as nitrogen, soybean oil<br />

as sole source of carbon and energy, and a stepwise increase of the temperature<br />

from 20°C up to 34°C the filamentous fungus AW02 was isolated from<br />

compost. CBS in Utrecht, The Netherlands, classified the strain by morphology<br />

and sequencing of ITS as Phialemonium spec., closely related with the known<br />

genus Acremonium.<br />

In shake flasks AW02 produced up to 10 9 mitotic spores per millilitre. Since<br />

they have a hydrophilic surface they can be easily used as inoculum.<br />

Interestingly, more than 90% of the activity cleaving para-nitrophenyl<br />

palmitate, triolein or triacetin was found to be cell wall associated. Only by<br />

strong shear stress 50% of the activity was liberated. Solubililty allowed<br />

analytical isoelectric focussing revealing two bands at pH 4 and 6 visualized as<br />

lipases by activity staining. The strong cell wall adherance in combination with<br />

the tendency of AW02 to biofilm formation allowed a comfortable<br />

donwstream–processing by simply harvesting plastic cubes which were used as<br />

growth support.<br />

FGD 07<br />

An in vitro model for mucosal infections reveals the<br />

dynamics of the cell wall proteome of the clinical fungus<br />

Candida albicans<br />

F. Klis *1 , G. Sosinska 1 , A. Sorgo 1 , P. De Groot 1 , E. Man<strong>der</strong>s 1 , H. Dekker 1 , L. de<br />

Koning 1 , C. De Koster 1 , S. Brul 1<br />

1 Swammerdam Institute for Life Sciences, University of Amsterdam,<br />

Amsterdam, Netherlands<br />

The cell wall of Candida albicans contains at any time more than twenty<br />

different covalently linked mannoproteins varying widely in function and<br />

probably also their precise location in the wall. To mimic mucosal infections,<br />

we developed an in vitro system based on the use of low-agarose plates<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

containing mucin as the sole nitrogen source. Un<strong>der</strong> these conditions, biomats<br />

were formed that extended with a constant radial growth rate of about 25-30<br />

µm/h. At pH 4, which is representative for the vaginal pH, the cells largely<br />

grew as yeast and pseudohyphal cells, and invasive growth was very limited,<br />

whereas at pH 7, which is representative for oral infections, invasive growth<br />

into the agarose layer predominated.<br />

Quantification of the cell wall proteomes of pH 4- and pH 7-grown biomats<br />

was realized by mixing the cell cultures grown un<strong>der</strong> the two conditions with a<br />

15 N metabolically labeled reference cell culture, followed by LC-FTMS mass<br />

spectrometric 14 N/ 15 N peptide ratio measurements in the tryptic lysate. The<br />

identification and quantification of 24 cell wall proteins showed that the cell<br />

wall proteome of C. albicans is highly dynamic. This was reflected in the<br />

strong up-regulation at pH 7 of three adhesion proteins (Als1, Als3, and Hwp1),<br />

an iron-acquisition-protein (Rbt5), a defense protein (the superoxide dismutase<br />

Sod5), two proteins involved in cell wall formation (Phr1 and Sim1) and two<br />

cell wall proteins with unknown function (Hyr1 and Ihd1/Pga36). The<br />

proteome quantification results were consistent with immunological analysis<br />

and with transcript profiling data. Our results show that the switch from yeastto-hyphal<br />

growth and from noninvasive to invasive growth is accompanied by<br />

the incorporation of a different set of cell wall proteins. We propose that these<br />

proteins prepare the cells for the new environmental stress conditions related to<br />

invasive growth.<br />

FGE 01<br />

Needles in the haystack: Binning and phylogenetic<br />

classification in microbial metagenomic communities<br />

F.O. Glöckner *1 , H. Teeling 1 , M. Weber 1 , J. Waldmann 1<br />

1 Microbial Genomics and Bioinformatics Group, Max Planck Institute for<br />

Marine Microbiology, Bremen, Germany<br />

Metagenomics, which is defined as the “functional and sequenced-based<br />

analysis of the collective microbial genomes contained in an environmental<br />

sample” has become the tool of choice to address the functional microbial<br />

diversity in the environment. To organize the metagenomic fragments into<br />

organism „bins“ and provide phylogenetic assignments for them is still an<br />

active area of research. Several approaches have been proposed, while the most<br />

common ones map phylogenetic marker genes, or all genes, to taxonomic<br />

groups based on best BLAST-hits. Gene independent approaches use intrinsic<br />

genomic signatures, based on oligonucleotide frequencies, for fragment<br />

correlation (binning) or phylogenetic classification. The currently available<br />

techniques can be classified into supervised (classification) and unsupervised<br />

(clustering, binning) methods. Supervised means that sequence fragments are<br />

classified based on their intrinsic DNA signatures to one or many classes that<br />

have been modelled based on prior knowledge (e.g. all bacterial genomes). A<br />

recent example for this approach is Phylophytia. Unsupervised methods like<br />

TETRA or Self Organizing Maps (SOM) do not need training sets and can<br />

produce sequence clusters independent of prior phylogenetic assignments. In a<br />

subsequent mapping step phylogenetic marker genes, often present at least on<br />

one fragment in the clusters, are used to assign the sequence clusters back to an<br />

organism. The talk will give an overview about the different methods, as well<br />

as their advantages and disadvantages when applied to metagenomic samples.<br />

FGE 02<br />

The genus Burkhol<strong>der</strong>ia: analysis of 54 genomic sequences<br />

D.W. Ussery *1 , T.M. Wassenaar 2<br />

1 Center for Biological Sequences, Danish Technical University, Lyngby,<br />

Denmark<br />

2 Molecular Microbiology and Genomics Consultants, Zotzenheim, Germany<br />

The genus Burkhol<strong>der</strong>ia consists of a number of very diverse species, both in<br />

terms of lifestyle (which varies from category B pathogens to apathogenic soil<br />

bacteria and plant colonisers) and their genetic contents. We have used 54<br />

publicly available Burkhol<strong>der</strong>ia genome sequences to explore the true diversity<br />

within this genus. A BLAST matrix visualizes the fraction of conserved genes<br />

in pairwise comparisons. In contrast, a BLAST atlas shows which genes are<br />

actually conserved in a number of genomes, located and visualized with<br />

reference to a chosen genome. The pan- and core genomes of (groups of)<br />

species result in insights in the conserved and variable fraction of genomes, and<br />

can enforce (or question) historic, taxonomic groupings. There are more gene<br />

families in the Burkhol<strong>der</strong>ia pan-genome than genes in the human genome. Our<br />

analysis indicates that B. mallei is closely related to the Pseudomallei group,<br />

and could, based on genome content, be included in that group. Approximately<br />

80% of genes in a B. pseudomallei genome and 60% of a B. mallei genome are<br />

conserved. The remaining genes comprise the variable fraction, where variation<br />

in virulence potential is most likely coded.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

FGE 03<br />

Nucleic acid sequencing for evaluating bacterial species and<br />

systematics: applications for identification<br />

E.R.B. Moore *1 , L. Svensson 1 , C. Unosson 1 , N. Karami 2<br />

1<br />

Culture Collection University of Göteborg (CCUG), University of Göteborg,<br />

Göteborg, Sweden<br />

2<br />

Department of Clinical Bacteriology, University of Göteborg, Göteborg,<br />

Sweden<br />

Identification of prokaryotes in the complexity of microbial diversity is<br />

increasingly problematic for clinical diagnoses and environmental<br />

microbiology. DNA sequence-based analyses of bacteria have enabled the<br />

identification of microorganisms in the environment and are <strong>bei</strong>ng adopted as<br />

routine in clinical analyses. DNA-based methods are suited to analyses of<br />

fastidious organisms, as well as those presenting health risks during cultivation.<br />

Comparative 16S rRNA gene sequence analyses are able to estimate<br />

phylogenetic relationships, although it is recognised that such analyses are not<br />

able to provide definitive species identifications. Among the most difficult<br />

problems for clinical diagnostics is the identification of organisms within<br />

"complexes" of closely related species, comprising pathogenic and nonpathogenic<br />

species with limited differentiating characteristics, e.g., 16S rRNA<br />

gene sequence dissimilarities among such organisms are often less than 1.0%.<br />

However, these species complexes are comprised of organisms with different<br />

pathogenic and virulence potential and it is essential to be able to obtain<br />

reliable identifications.<br />

A "polyphasic" multi-locus sequence analysis (MLSA) strategy can be<br />

established for the identification of bacteria, including "first-phase"<br />

comparisons of partial 16S rRNA gene sequences, for identification to the subgenus<br />

level, and subsequent, "second-phase" analyses of one or more conserved<br />

house-keeping genes, for identification to the species level. However, it is<br />

recognised that house-keeping genes are not equally useful for all taxa. An<br />

average nucleotide index (ANI), based upon sequence similarities among<br />

house-keeping genes, may be applied for establishing expected gene sequence<br />

"cut-offs" that differentiate the species of a given taxon. Thus, a potential key to<br />

effective bacterial identification depends upon the selection of conserved genes<br />

with levels of resolution high enough to differentiate the most closely related<br />

species.<br />

FGE 04<br />

The All-Species Living Tree project<br />

P. Yarza *1 , M. Richter 1 , J. Peplies 2 , J. Euzeby 3 , R. Amann 4 , K.H. Schleifer 5 , W.<br />

Ludwig 5 , F.O. Glöckner 6 , R. Rosselló-Móra 1<br />

1 Marine Microbiology Group, Instituto Mediterraneo de Estudios Avanzados,<br />

Esporles (Mallorca), Spain<br />

2 Ribocon GmbH, Ribocon GmbH, Bremen, Germany<br />

3 Société de Bacteriologie Systématique et Vétérinaire SBSV, École Nationale<br />

Vétérinaire de Touluse (ENVT), Toulouse, France<br />

4 Department of Molecular Ecology, Max Planck Institute for Marine<br />

Microbiology, Bremen, Germany<br />

5 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising,<br />

Germany<br />

6 Microbial Genomics Group, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

One of the most important premises for circumscribing a prokaryotic species is<br />

the inference of monophyly of the taxon. In this regard, ribosomal SSU gene<br />

sequence analyses have become the gold standard for genealogical inference.<br />

Since this gene sequence has also become the tool for cultivation-independent<br />

analysis of the diversity of microbial communities, we are attending to an<br />

exponential growth in the 16S rRNA databases. Actually, the number of SSU<br />

entries is about two or<strong>der</strong>s of magnitude higher than that of the validly<br />

published species.<br />

Within this framework, the Systematic and Applied Microbiology journal<br />

together with the ARB, SILVA and LPSN projects have started the All-Species<br />

Living Tree project (P. Yarza, et al. 2008) to provide a useful tool especially<br />

designed for the microbial taxonomist community.<br />

Our main goal is to produce: (I) an updated and curated SSU database of all<br />

type strains for which sequences are available; (II) an optimized and universally<br />

usable alignment; and (III) reconstruct a tree harboring species genealogies as a<br />

single set and exempt of non-type material. The tree provided in the first release<br />

was a result of the calculation of a single dataset containing 9,975 sequences.<br />

Of them, 6,728 represented a single type strain, and 3,247 additional sequences<br />

were added to give robustness to the reconstruction. A complete set of<br />

supplementary tables, figures, alignments and ARB databases are online<br />

available at http://www.arb-silva.de/living-tree/.<br />

49

50<br />

The All-Species Living Tree project is <strong>bei</strong>ng upgraded twice a year by adding<br />

the new validly published species that appear in Validation and Notification<br />

lists of the IJSEM. In or<strong>der</strong> to provide an entry point for the scientific<br />

community, a contact email address has been created for requests and<br />

recommendations (living-tree@arb-silva.de).<br />

FGF 01<br />

Identification of a streptococcal octa-peptide motif involved<br />

in acute rheumatic fever<br />

D.P. Nitsche-Schmitz *1 , K. Dinkla 1 , V. Barroso 1 , S. Reißmann 1 , H. Linge 2 , I.M.<br />

Frick 2 , M. Rohde 1 , G.S. Chhatwal 1<br />

1<br />

Mikrobielle Pathogenität, Helmholtz-Zentrum für Infektionsforschung,<br />

Braunschweig, Germany<br />

2<br />

Dept. of Clinical Sciences, BMC, Lund University, Lund, Sweden<br />

Acute rheumatic fever is known as a serious autoimmune sequela of pharyngitis<br />

caused by certain group A streptococci. One mechanism of ARF pathogenesis<br />

is formation of an autoantigenic complex with human collagen IV. In some<br />

geographic regions with high incidence of ARF pharyngeal carriage of group C<br />

and group G streptococci prevails. Examination of such strains revealed the<br />

presence of M-like surface proteins that bind human collagen. Using a peptide<br />

array and recombinant proteins with targeted amino acid substitutions, we<br />

could demonstrate that formation of collagen complexes during streptococcal<br />

infections depends on an octa-peptide consensus motif, which is present in all<br />

collagen binding M- and M-like proteins of different beta-hemolytic<br />

streptococcal species. Mice immunized with streptococcal proteins that contain<br />

the collagen binding octa-peptide motif, developed high serum titers of anticollagen<br />

antibodies. In sera of rheumatic fever patients such a collagen<br />

autoimmunity was accompanied by specific reactivity against the collagenbinding<br />

proteins, linking the observed effect to clinical cases. Taken together,<br />

the data demonstrate that the identified octa-peptide motif, through its action on<br />

collagen plays a crucial role in the pathogenesis of rheumatic fever. Eradication<br />

of streptococci expressing proteins with the collagen binding motif appears<br />

advisable for controlling rheumatic fever.<br />

FGF 02<br />

The Pyp regulatory network of Yersinia enterocolitica<br />

controls expression of pili and type-II secretion systems<br />

J. Schilling 1 , B. Shutinoski 1 , K. Wagner 1 , G. Heusipp *1<br />

1 ZMBE, Institut für Infektiologie, Westf. Wilhelms-Universität Münster,<br />

Münster, Germany<br />

The coordinated expression of virulence genes in pathogenic bacteria is tightly<br />

regulated. In a previous genetic screen, we identified three genes (pypA, pypB<br />

and pypC; protein involved in the regulation of Yersinia hreP expression A, B,<br />

C), whose products control the expression of the virulence-associated HreP<br />

protease in the human pathogen Yersinia enterocolitica. Further analysis shows<br />

that the pypB gene that encodes the ToxR-like transcriptional regulator PypB is<br />

part of the Tad locus of Yersiniae. Tad loci are widespread among prokaryotes<br />

and encode proteins for the biogenesis of Flp pili, a subclass of type-IVb pili.<br />

We could show that PypB induces transcription of the tad locus, resulting in the<br />

expression of pili on the bacterial surface. These pili mediate microcolony<br />

formation and are involved in the initial adhesion to eukaryotic cells. Similarly,<br />

the pypC gene is associated with an operon encoding a type-II secretion system.<br />

Interestingly, Y. enterocolitica possesses a second type-II secretion system that<br />

is also associated with a PypC-like regulator that we termed PypC2. PypC2<br />

activates expression of chiY, encoding a putative secreted chitin-binding protein<br />

that is a substrate for a type-II secretion system of Vibrio cholerae, where it<br />

mediates adhesion to zooplankton as well as eukaryotic cells. Furthermore, we<br />

could show that H-NS and the Yersinia virulence regulator RovA influence the<br />

Pyp regulatory network. Our data provide an insight into a complex regulatory<br />

network controlling various virulence-associated phenotypes in Y.<br />

enterocolitica.<br />

FGF 03<br />

Helicobacter pylori contains a novel system of cytoskeletal<br />

elements that is essential for the maintenance of cell shape<br />

and for the development of motility<br />

B. Waidner *1 , M. Specht 2 , F. Dempwolff 2 , K. Häberer 1 , V. Speth 3 , M. Kist 1 ,<br />

P.L. Graumann 2<br />

1 Institut für med. Mikrobiologie und Hygiene, Uniklinik Freiburg, Freiburg,<br />

Germany<br />

2 Institut für Mikrobiologie, Universität Freiburg, Freiburg, Germany<br />

3 Institut für Zellbiologie, Universität Freiburg, Freiburg, Germany<br />

MreB-like proteins have been shown to be essential for the maintenance of rod<br />

shape in several bacteria, while intermediate filament (IF)-like protein<br />

crescentin affects cell curvature in the vibrio Caulobacter crescentus. It has<br />

been unclear how spiral-shaped bacteria obtain the shape. We have found that<br />

the human pathogen Helicobacter pylori has a novel type of machinery for the<br />

maintenance of cell morphology. Actin-like MreB protein is dispensable for<br />

viability in H. pylori, and does not affect not cell shape, but chromosome<br />

segregation. Helical cell shape depends on coiled coil-rich proteins (Cscc1-4),<br />

which form extended filamentous structures in vitro and in vivo, and are<br />

differentially required for maintenance of cell morphology, and for the<br />

assembly of polar flagella. Consistent with a cytoskeleton-like structure, Cscc<br />

proteins localized in a distinct helical pattern within H. pylori cells. Thus, IFlike<br />

Cscc proteins, but not MreB, affect cell morphology, while both<br />

cytoskeletal components affect the development of pathogenicity factors or cell<br />

cycle progression. The high variability of Cscc encoding genes are the likely<br />

reason for the high diversity between different H. pylori strains.<br />

FGF 04<br />

Respiratory ATP synthesis: an Achilles´ heel in<br />

mycobacteria?<br />

A.C. Haagsma 1 , K. Andries 2 , A. Koul 2 , H. Lill 1 , D. Bald *1<br />

1<br />

Structural Biology, Department of Molecular Cell Biology, VU University<br />

Amsterdam, Amsterdam, Netherlands<br />

2<br />

Pharmaceutical Research and Development, Johnson & Johnson, Beerse,<br />

Belgium<br />

Infections with Mycobacterium tuberculosis lead to nearly 2 million deaths per<br />

year with about 2 billion people latently infected. Multi-drug resistant strains as<br />

well as mycobacteria staying dormant within the host strongly demand<br />

development of drugs with new targets.<br />

Previously, we validated ATP synthase as the target of Diarylquinolines<br />

(DARQs) [1], a novel class of antibiotics highly active against Mycobacterium<br />

tuberculosis. In a biochemical assay, DARQs inhibited ATP synthase at<br />

nanomolar concentrations. BIAcore studies with DARQ-coated chips revealed<br />

strong binding for purified ATP synthase. Point mutations in hydrophobic<br />

subunit-c lead to decreased binding affinity.<br />

ATP synthase in mycobacteria is not only essential for growth, but also<br />

required in the physiologically dormant state associated with latent<br />

tuberculosis. Dormant mycobacteria are active in ATP synthesis and their ATP<br />

production is potently blocked by DARQs, leading to bacterial killing [2].<br />

DARQ lead compound TMC207 displays >20,000-fold higher affinity for<br />

mycobacterial ATP synthase (IC50 = 10 nM) compared human mitochondrial<br />

ATP synthase (IC50 >200 microM). These results suggest that TMC207 is a<br />

very specific inhibitor of the mycobacterial ATP synthase and may not elicit<br />

ATP synthesis-related toxicity in mammalian cells [3].<br />

These results indicate that respiratory ATP synthesis may be a critical weakness<br />

of (dormant) mycobacteria and suggest that inhibition of energy production,<br />

although the enzymes involved are strongly conserved between prokaryotes and<br />

eukaryotes, is a promising approach for antibacterial drug discovery.<br />

Recent progress on molecular mechanism of DARQ action, on possible<br />

adaptation mechanisms of mycobacterial ATP synthase to dormant conditions,<br />

as well as on applicability of this approach to combat other bacterial infections<br />

will be discussed.<br />

[1] Koul et al. (2007) Nat. Chem. Biol. 3, 323-4<br />

[2] Koul et al. (2008) J. Biol. Chem. 283, 25273-80<br />

[3] Haagsma et al. submitted<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

FGF 05<br />

Propionyl-CoA metabolism in Candida albicans<br />

C. Otzen *1 , M. Brock 1<br />

1 Microbial Biochemistry and Physiology, Hans-Knöll-Institute, Jena, Germany<br />

Candida albicans secretes various aspartic proteases, so-called SAPs, during<br />

host infection, which may be used for nutrient acquisition by protein<br />

degradation from host tissues. Although the degradation of several amino acids<br />

leads to intermediates of central metabolic pathways, other metabolites are<br />

formed, which are toxic to the cell. One of the toxic metabolites is propionyl-<br />

CoA, which is formed during the degradation of valine, isoleucine and<br />

methionine but also from the degradation of odd-chain fatty acids. Propionyl-<br />

CoA disturbes several central metabolic functions and, thereby, reduces the<br />

growth rate of the cells. Therefore, accumulation of propionyl-CoA has to be<br />

avoided. Several bacteria as well as filamentous fungi and the yeast<br />

Saccharomyces cerevisiae use the methylcitrate cycle for the efficient removal<br />

of propionyl-CoA. However, the genome of C. albicans does not contain the<br />

genes needed for a functional methylcitrate cycle. To investigate the enzymes<br />

of C. albicans involved in propionyl-CoA degradation, we are currently<br />

performing 2-D-gel experiments and RNA microarrys by using samples<br />

<strong>der</strong>iving from various growth conditions. Comparisons of the protein patterns<br />

and gene expression analyses are performed to identify the pathway responsible<br />

for propionyl-CoA degradation. The deletion of the selected genes will reveal<br />

whether the identified enzymes are required for propionyl-CoA degradation and<br />

additionally will help to study their impact on virulence.<br />

FGF 06<br />

Regulation of si<strong>der</strong>ophore biosynthesis in Ustilago maydis<br />

during the infection of maize<br />

B. Winterberg *1 , U. Linne 2 , R. Kahmann 1 , J. Schirawski 1<br />

1 Max-Planck-Institut für Terrestrische Mikrobiologie, Abteilung Organismische<br />

Interaktionen, MArburg, Germany<br />

2 Fachbereich Biochemie, Philipps-Universität Marburg, Marburg, Germany<br />

The phytopathogenic fungus Ustilago maydis induces tumors on its host plant<br />

maize. To acquire the essential element iron, U. maydis has two high affinity<br />

iron uptake systems that are repressed by the transcription factor Urbs1 un<strong>der</strong><br />

high iron conditions. The permease based system is necessary for<br />

pathogenicity. The other system based on the synthesis of the two si<strong>der</strong>ophores,<br />

ferrichrome and ferrichrome A, is dispensable for virulence.<br />

Here we show that spores generated by si<strong>der</strong>ophore biosynthesis mutants<br />

display a meiosis defect during germination indicating that si<strong>der</strong>ophores are<br />

needed for iron storage in spores.<br />

To investigate the expression of si<strong>der</strong>ophore biosynthetic genes of U. maydis<br />

during plant infection, we used quantitative real time PCR. This analysis<br />

revealed that si<strong>der</strong>ophore biosynthesis is repressed in U. maydis until the<br />

developmental stage of spore formation. We postulated that the transcription<br />

factor Yap1 that is involved in H2O2-stress response might repress si<strong>der</strong>ophore<br />

biosynthesis. Using Northern blot analysis we were able to show that<br />

expression of si<strong>der</strong>ophore biosynthetic genes is reduced in the presence of<br />

H2O2. We propose the following model of transcriptional regulation of<br />

si<strong>der</strong>ophore biosynthesis during biotrophic development. U maydis infection<br />

leads to the activation of Yap1 by plant-<strong>der</strong>ived H2O2, which represses<br />

si<strong>der</strong>ophore biosynthesis during early stages of plant invasion. Due to the<br />

downregulation of Yap1 activity si<strong>der</strong>ophore biosynthesis occurs during later<br />

stages of fungal development in planta. Our results provide new insight into the<br />

intimate communication between U. maydis and its host plant that is<br />

characterized by mutual perception and response<br />

FGG 01<br />

Redox control of proline metabolism<br />

D. Becker *1<br />

1<br />

Biochemistry Department, University of Nebraska-Lincoln, Lincoln, United<br />

States<br />

PutA (proline utilization A) from Escherichia coli is a remarkable trifunctional<br />

flavoprotein that switches between two mutually exclusive functions as a<br />

transcriptional repressor and membrane-bound proline catabolic enzyme. As a<br />

membrane-associated enzyme, PutA catalyzes the two-step conversion of<br />

proline to glutamate by coordinating the activities of separate flavin-dependent<br />

proline dehydrogenase (PRODH) and NAD-dependent pyrroline-5-carboxylate<br />

dehydrogenase domains. In certain prokaryotes such as E. coli, PutA also<br />

contains a ribbon-helix-helix (RHH) DNA binding domain at the N-terminus<br />

which enables PutA to function as an autogenous transcriptional repressor of<br />

the proline utilization (put) genes putA and putP (encodes a high affinity<br />

proline transporter). A 419-bp regulatory region separates the putA and putP<br />

genes and contains five GTTGCA PutA binding motifs or operators. The roles<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

of these operators in repressing the expression of putA and putP have been<br />

elucidated by cell-based expression assays and X-ray crystallography studies.<br />

Evaluation of put control DNA sequences from other bacteria suggests that the<br />

GTTGCA motif is the fundamental transcriptional control element of the PutA<br />

autogenous repression system. The mechanism by which PutA switches<br />

between DNA-binding and membrane-bound enzymatic activity is redox<br />

dependent. Important molecular interactions in the PRODH active site that<br />

un<strong>der</strong>lie redox-dependent functional switching of PutA involve the FAD 2’-OH<br />

ribityl group and the FAD N(5)-Arg431 hydrogen bond pair. New insights into<br />

how redox signals are transmitted out of the PRODH active site to the<br />

regulatory and membrane binding domains of PutA will also be discussed.<br />

FGG 02<br />

The RNA-binding regulatory activity of Bacillus subtilis<br />

aconitase<br />

A.L. Sonenshein *1 , A.W. Serio 1 , K. Pechter 1<br />

1 Department of Molecular Biology and Microbiology, Tufts University School<br />

of Medicine, Boston, United States<br />

Some bacterial aconitases are very similar to a mammalian aconitase, IRP-1.<br />

IRP-1 is not involved in Krebs cycle function, but has a non-enzymatic activity<br />

as an iron regulatory protein. IRP-1 binds to the 5’ or 3’ UTRs of mRNAs<br />

whose products are involved in iron uptake and storage, affecting either the<br />

translatability or stability of the mRNAs. To bind to RNA, IRP-1 must lose its<br />

4Fe-4S cluster, as occurs during iron limitation or oxidative stress, allowing the<br />

entry of an RNA stem-loop structure into a cleft in the protein. In B. subtilis, a<br />

single aconitase protein acts both as a Krebs cycle enzyme and as an RNA<br />

binding protein. Both activities are important for spore formation. A mutant<br />

lacking enzymatic activity was blocked at a very early stage in sporulation<br />

because of the accumulation of citrate and inhibition of activation of Spo0A,<br />

the master regulator of sporulation. By contrast, the lack of RNA binding<br />

activity caused a block late in sporulation, corresponding to the stage of<br />

synthesis and assembly of coat proteins around the developing spore. The<br />

mRNA for GerE, a regulatory protein that controls the synthesis of coat<br />

proteins, was shown to be a direct target for aconitase. gerE mRNA and GerE<br />

protein were much less abundant in late stage cells of the RNA-bindingdefective<br />

mutant than in the wild-type. Moreover, purified aconitase bound<br />

tightly to the 3’ region of the gerE mRNA in vitro; deletion of a specific stemloop<br />

structure in the 3’-UTR reduced accumulation of GerE protein in<br />

sporulating cells. Other sporulation-related targets of aconitase are <strong>bei</strong>ng sought<br />

by immunoprecipitation and affinity purification strategies. In addition,<br />

aconitase plays a regulatory role in the iron limitation response. More than a<br />

dozen mRNAs induced by iron limitation were altered in abundance in an RNA<br />

binding-defective mutant. For at least one of these mRNAs, tight binding of<br />

aconitase was seen in vitro.<br />

FGG 03<br />

Functional analysis of Rny: A novel player involved in RNA<br />

metabolism of Bacillus subtilis<br />

F.M. Rothe 1 , M. Lehnik-Habrink 1 , J. Stülke 1 , F.M. Commichau *1<br />

1 Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

Glycolysis is one of the key metabolic pathways. Interestingly, many glycolytic<br />

enzymes of Bacillus subtilis are encoded by essential genes [1] suggesting that<br />

glycolytic enzymes are doing more than catalyzing their reactions. Indeed, we<br />

found that glycolytic enzymes interact with essential proteins explaining their<br />

indispensability [2]. Among the interaction partners of glycolytic enzymes is<br />

the essential protein Rny that is involved in the maturation of the gapA operon<br />

mRNA (2). Bacterial-two hybrid (B2H) analyses revealed that Rny interacts<br />

with the phosphofructokinase, the enolase, the polynucleotide phosphorylase<br />

and the essential ribonuclease J1 suggesting the presence of a degradosome-like<br />

complex in B. subtilis [2]. Rny is a membrane protein that possesses a putative<br />

coiled-coil region as well as KH and HD domains that are involved in binding<br />

of nucleic acids and cofactors, respectively [3]. These domains are located in<br />

the C-terminal region of Rny (3). Since Rny interacts with essential enzymes<br />

we wanted to analyze the impact of the different domains on these interactions.<br />

Using the B2H system we found that the membrane domain and the HD<br />

domain of Rny are important for all essential interactions. Moreover, the<br />

membrane domain of Rny is important to complement a B. subtilis strain<br />

depleted of Rny. Taken together, our results unequivocally show that<br />

membrane localization and the HD domain of Rny are essential for in vivo<br />

function and interactions with the other degradosome components.<br />

[1] Kobayashi et al. (2003) PNAS, 100: 4678-4683<br />

[2] Commichau et al., submitted<br />

[3] Hunt et al. (2006) Microbiology, 152: 2895-2907<br />

51

52<br />

FGG 04<br />

Activation of the membrane-integrated transcriptional<br />

activator CadC of Escherichia coli involves the opening of a<br />

periplasmic disulfide bridge<br />

L. Tetsch *1 , A. Dönhöfer 1 , T. Friedrich 1 , K. Jung 1<br />

1 Biozentrum <strong>der</strong> LMU München, Mikrobiologie, Ludwig-Maximilians-<br />

Universität, 82152 Martinsried, Germany<br />

The Cad system, part of the acid stress response of E. coli, is composed of the<br />

lysine decarboxylase CadA, the lysine/cadaverine antiporter CadB and the<br />

membrane-integrated transcriptional activator CadC. The decarboxylation of<br />

lysine to cadaverine results in the consumption of a cytoplasmic proton and<br />

thereby in an increase of the internal pH. CadC regulates expression of the<br />

cadBA operon and induces transcription when cells are exposed to an acidic<br />

(pH 5.8) and lysine-rich environment. Whereas CadC senses lysine indirectly<br />

via an interaction with the lysine permease LysP, the pH is sensed by the<br />

periplasmic domain of CadC. Our studies revealed that the periplasmic sensory<br />

domain of CadC contains a disulfide bridge which has to be opened as a<br />

prerequisite for CadC activity. Moreover, a hydrophobic stretch of amino acids<br />

(Tyr219 to Val239) located between these two cysteines was shown to be<br />

essential for CadC activity. CadC-dependent cadBA expression was monitored<br />

in various dsb mutants. Deletion of the disulfide reductase DsbC reduced<br />

cadBA expression, whereas deletion of the disulfide oxidase DsbA caused a<br />

significant increase in cadBA expression. These results are consistent with the<br />

idea that DsbA and DsbC affect CadC activity by closing and opening of the<br />

disulfide bridge. In our model a pH shift sensed by the periplasmic domain<br />

triggers an opening of the disulfide bridge mediated by the disulfide reductase<br />

DsbC. Subsequently, the hydrophobic stretch of amino acids might immerse<br />

into the cytoplasmic membrane forcing binding of the cytoplasmic domain to<br />

the DNA.<br />

FGG 05<br />

DctA of Escherichia coli interacts with the DcuSR two<br />

component system<br />

J. Bauer *1 , W. Erker 2 , Y.F. Liao 2 , T. Basché 2 , G. Unden 1<br />

1 Institut für Mikrobiologie und Weinforschung, AG Unden, Johannes-<br />

Gutenberg Universität Mainz, Mainz, Germany<br />

2 Institut für Physikalische Chemie, Johannes-Gutenberg Universität Mainz,<br />

Mainz, Germany<br />

The facultative anaerobic bacterium Escherichia coli can use C4-dicarboxylates<br />

as carbon and energy source un<strong>der</strong> aerobic and anaerobic conditions. In the<br />

absence of oxygen fumarate is used as electron acceptor in fumarate respiration.<br />

The anaerobic uptake, exchange or efflux of C4-dicarboxylates is catalyzed by<br />

three independent anaerobic transport systems: DcuA, DcuB and DcuC [1].<br />

Expression of genes for the anaerobic fumarate respiration like frdABCD<br />

(fumarate reductase), fumB (fumarase) and dcuB (fumarate:succinate antiporter<br />

DcuB) is regulated by the DcuSR two component system.<br />

During aerobic growth of E. coli the dicarboxylate transport carrier DctA<br />

catalyses the uptake of succinate and other C4-dicarboxylates in symport with<br />

protons. The C4-dicarboxylates are oxidized in the citric acid cycle to CO2 and<br />

the reducing equivalents are reoxidized in aerobic respiration. The expression<br />

of dctA is activated by the DcuSR two component system [2] in the presence of<br />

C4-dicarboxylates. Deletion of dctA has an effect on dctA expression [3].<br />

Interaction of DctA with the sensor histidine kinase DcuS was tested by<br />

Fluorescence resonance energy transfer (FRET) [5]. Fusions of the membrane<br />

proteins DctA and DcuS with variants of GFP (Green Fluorescent Protein) were<br />

coexpressed in E. coli and the FRET efficiency was determined in vivo. The<br />

DcuS-CFP and DctA-YFP fusion proteins showed high FRET efficiency,<br />

indicating direct interaction of DctA with DcuS.<br />

[1] Six et. al. (1994) J. Bacteriol. 176:6470-6478<br />

[2] Davies et. al. (1999) J. Bacteriol. 181:5624-5635<br />

[3] Golby et. al. (1999) J. Bacteriol. 181:1238-1248<br />

[4] Kleefeld et.al. (<strong>2009</strong>) J. Biol. Chem. 284:265-275<br />

[5] Truong and Ikura (2001) Struc. Biol. 11:573-578<br />

FGH 01<br />

Biogenesis of cellular iron-sulfur proteins: The essential and<br />

minimal function of mitochondria<br />

R. Lill *1<br />

1<br />

Institut für Zytobiologie und Zytopathologie, Philipps-Universität Marburg,<br />

Marburg, Germany<br />

No abstract submitted<br />

FGH 02<br />

Untersuchung <strong>der</strong> Funktion <strong>der</strong> nucleolären essentiellen<br />

Methyltransferase Nep1 in <strong>der</strong> eukaryotischen<br />

Ribosomenbiogenese<br />

K.D. Entian *1 , B. Meyer 1 , J. Wöhnert 1<br />

1<br />

University of Frankfurt, Frankfurt, Germany<br />

No abstract submitted<br />

FGH 03<br />

Fission yeast as a model to investigate mitochondrial RNA<br />

turn over<br />

B. Schäfer *1<br />

1<br />

Department of Biology IV (Microbiology & Genetics), RWTH Aachen<br />

University, Aachen, Germany<br />

Controlled degradation of RNA is one key step in the regulation of gene<br />

expression. In Escherichia coli this process is promoted by a multiprotein<br />

complex called degradosome, composed of RNase E, a DEAD box RNA<br />

helicase and two other proteins. The main components of the eubacterial<br />

degradosome are also present in mitochondria as organelles of presumed alphaproteobacterial<br />

origin. In mitochondria of the budding yeast Saccharomyces<br />

cerevisiae, SUV3 (encoding the RNA helicase) and DSS1 (encoding the 3’ to<br />

5’ exoribonuclease) were found to be the homologs of the E. coli degradosome<br />

genes. The mitochondrial Exosome complex buit up by Suv3p and Dss1p is<br />

strictly RNA-specific and degrades single-stranded or partially double-stranded<br />

RNA with 3’ single-stranded tails. In human mitochondria only the hSuv3<br />

protein is present but is involved here in mt DNA replication and in apoptosis.<br />

An ortholog of the yeast DSS1 gene is lacking in the human genome.<br />

In a comprehensive functional genomics screen in S. pombe, we could identify<br />

an almost complete set of genes of the mitochondrial transcription machinery.<br />

The detailed characterization of these proteins uncovered new molecular<br />

mechanisms used to control steady state levels of transcripts in mitochondria.<br />

[1] Schäfer, B.; Hansen, M.; Lang, B.F. (2005). Transcription and RNA<br />

processing in fission yeast mitochondria. RNA 11: 785-795.<br />

[2] Schäfer, B. (2005). RNA maturation in mitochondria of S. cerevisiae and S.<br />

pombe. Gene 354: 80-85.<br />

[3] Wiesenberger, G.; Haller, G.; Speer F.; Schleiffer, A.; Bonnefoy, N. and<br />

Schäfer, B. (2007). RNA degradation in fission yeast mitochondria is<br />

stimulated by a member of a new family of proteins that are conserved in lower<br />

eucaryotes. J Mol Biol 367:681-691.<br />

FGH 04<br />

DNA apurinic site repair confers resistance to yeast<br />

anticodon-nuclease killer toxins<br />

R. Klassen *1 , S. Wemhoff 1 , J. Krause 1 , F. Meinhardt 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität, Münster, Germany<br />

Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT)<br />

cause target cell killing by anticodon-nuclease (ACNase) activity directed<br />

against tRNA Glu and tRNA Gln , respectively. Toxin mediated cell killing also<br />

involves induction of DNA damages, in particular replication <strong>der</strong>ived DNA<br />

double strand breaks (DSBs). Here we show, that in addition to homologous<br />

recombination, the base excision repair (BER) and the DNA damage avoidance<br />

mechanism postreplication repair (PRR) promote resistance to both of the<br />

toxins. The protective function of BER is restricted to endonucleases acting on<br />

apurinic (AP) sites as none of the known DNA glycosylases was involved.<br />

Since prevention of tRNA cleavage by removal of the wobble uridine<br />

modification 5-methoxy-carbonyl-methyl (mcm 5 ) rescues ACNase<br />

hypersensitivity associated with the absence of BER, such pathway is involved<br />

subsequent to tRNA cleavage. Genetic analyses of PaT hypersensitivity in<br />

various BER and PRR defective mutants suggest naturally arising AP sites to<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

accumulate in toxin treated cells, thereby inducing stalling of replication forks,<br />

the latter <strong>bei</strong>ng subsequently processed by either fork collapse and DSB repair<br />

or by lesion-bypass involving template switching. Alternatively, DNA<br />

polymerase ζ may facilitate AP site translesion synthesis at the expense of<br />

increased mutations.<br />

FGH 05<br />

Investigations of the metacaspase YCA1 for better detection<br />

of apoptosis in Saccharomyces cerevisiae during alcoholic<br />

fermentation of grape must.<br />

D. Gerhards *1 , S. Schnell 2 , M. Grossmann 1 , C. von Wallbrunn 1<br />

1 Section of Microbiology and Biochemistry, Geisenheim Research Center,<br />

Geisenheim, Germany<br />

2 Institute of Applied Microbiology, Justus-Liebig-University Giessen, Gießen,<br />

Germany<br />

The superfamily of caspases are known to be a main activator of apoptosis in<br />

metazoan organisms, which is crucial for homeostasis. In the yeast species<br />

Saccharomyces cerevisiae, a gene has been identified, which codes for<br />

metacaspase YCA1 and shows similarities to human caspases.<br />

Many investigations demonstrated the influence of apoptosis inducing factors<br />

un<strong>der</strong> respiratory conditions. Using commercial vine yeast (S. c. VIN13) and<br />

deviated mutants this work points out the differences in apoptosis of induced<br />

and non-induced programmed cell death (PCD) with 3 mM hydrogen peroxide<br />

un<strong>der</strong> aerobic and strictly anaerobic conditions. Also differences of cultivation<br />

and alcoholic fermentation in synthetic medium and grape must are shown.<br />

Comparisons of yeast cells lacking the metacaspase encoding gene (Δyca1),<br />

YCA1 overexpression mutants and the wild-type (WT) cells, demonstrate the<br />

influence of the metacaspase. The impact was monitored by measuring the<br />

apoptotic marker DNA-fragmentation, by Terminal Deoxynucleotidyl<br />

Transferase-mediated dUTP Nick End Labelling (TUNEL) staining.<br />

As expected, hydrogen peroxide induced PCD showed higher rates of apoptotic<br />

cells in the overexpressed mutant than the WT and particularly in the strain<br />

lacking YCA1. This confirms the proposition of the influence of this gene<br />

during apoptosis. The non-induced control showed in all cases little signals of<br />

apoptotic cells.<br />

Measuring PCD un<strong>der</strong> conditions of alcoholic fermentation, the results lead to<br />

the conclusion, that S. cerevisiae shows apoptosis in a caspase-independent<br />

manner.<br />

KA 01<br />

Biosynthesis of the [NiFe]-Hydrogenases of Escherichia coli<br />

G. Sawers *1 , B. Soboh 1<br />

1 Institute of Microbiology, Martin-Luther University, Halle (Saale), Germany<br />

Hydrogenases catalyse the reversible oxidation of molecular hydrogen and they<br />

are of importance to the metabolism of a variety of microbes. Moreover, as<br />

hydrogen represents a potential route to supporting an future alternative of<br />

energy economy, it is important that we un<strong>der</strong>stand the biology of these<br />

complex metalloproteins in its entirety. Although the E. coli genome has the<br />

coding capacity for the synthesis of four [NiFe]-hydrogenases, only three have<br />

ever been detected and biochemically characterised. All three are synthesised<br />

and are functional anaerobically: two are hydrogen-oxidising enzymes while<br />

the third forms part of the multisubunit hydrogen-evolving formate<br />

hydrogenlyase (FHL) complex. The active site iron atom of [NiFe]hydrogenases<br />

is characterised by having one CO and two CN- ligands. The<br />

metabolic precursor of the CN- ligands is carbamoyl phosphate while the nature<br />

of the CO ligand’s origin remains unclear. A series of accessory proteins,<br />

termed Hyp, is involved in active site biosynthesis and assembly of the<br />

complete NiFe-cofactor.<br />

The FHL complex is an excellent model system not only for the study of<br />

hydrogenase active site biosynthesis and assembly but also for the structural<br />

and functional characterisation of energy-converting hydrogenases (Ech), of<br />

which FHL is a prototype. Ech comprise a core of six subunits that share amino<br />

acid sequence similarity with the subunits that form the catalytic core of the<br />

NADH:quinone oxidoreductase. Our current un<strong>der</strong>standing of the biosynthesis<br />

and biochemistry of these enzymes will be presented.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KA 02<br />

Microoxic growth and metabolism of Clostridium<br />

acetobutylicum<br />

M.S. Mann *1 , O. Riebe 1 , F. Hillmann 1 , H. Bahl 1<br />

1 Institute of Biological Sciences/Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

Obligately anaerobic microorganisms by definition cannot use molecular<br />

oxygen as terminal electron acceptor for growth. The solvent producer<br />

Clostridium acetobutylicum is regarded as a classical example as its growth and<br />

fermentation metabolism is immediately halted when entering a fully aerobic<br />

environment. However, it is known that C. acetobutylicum counters oxygen or<br />

reactive <strong>der</strong>ivatives via NADH dependent reduction (1). This system<br />

contributes to survival un<strong>der</strong> oxidative stress and allows growth un<strong>der</strong><br />

microoxic conditions. Two highly expressed flavodiironproteins were<br />

characterized as rubredoxin dependent O2-reductases which are proposed to<br />

play key roles in the maintenance of an anaerobic environment, despite the<br />

continuous influx of O2(2). We are currently analyzing the physiological<br />

impacts of O2 as an alternative electron acceptor in C. acetobutylicum. First<br />

results indicated a divergent spectrum of fermentation products in response to<br />

microoxia, while un<strong>der</strong> the same conditions the growth rate and consumption of<br />

glucose were not affected. Thus, we propose that the O2 response of this<br />

obligate anaerobe is not limited to detoxification of O2 or its reactive<br />

<strong>der</strong>ivatives and integrates an adaption of central metabolic pathways.<br />

KA 03<br />

Nitrate respiration in Wolinella succinogenes: Role of the<br />

multifunctional NapGHF menaquinol dehydrogenase<br />

complex<br />

M. Kern *1 , J. Simon 1<br />

1 Department of Microbiology and Genetics, TU Darmstadt, Darmstadt,<br />

Germany<br />

Growth of the Epsilonproteobacterium Wolinella succinogenes by nitrate<br />

respiration depends on the periplasmic nitrate reductase system encoded by the<br />

napAGHBFLD locus [1]. The Nap system was shown to be functionally<br />

independent of a cytochrome c menaquinol dehydrogenase of the NapC/NrfH<br />

family. Instead, the predicted iron-sulfur cluster proteins NapG and NapH were<br />

identified to catalyse menaquinol oxidation [2-4]. Both proteins form a<br />

membrane-bound complex in which the polytopic integral membrane protein<br />

NapH anchors NapG on the periplasmic side of the membrane [3]. Here, the<br />

function of another proposed iron-sulfur cluster protein (NapF) was<br />

investigated which was assumed to play a role in maturation of nitrate-reducing<br />

NapA in other organisms.<br />

NapF from W. succinogenes was found to be a cytoplasmic protein that is<br />

attached to the membrane via NapH, indicating formation of a NapGHF<br />

complex. A NapF-deficient mutant was severely impaired in NapA maturation<br />

and exhibited decreased nitrate reductase activity. Site-directed modification of<br />

conserved poly-cysteine motifs suggests that only one of the four iron-sulfur<br />

centres of NapF is functionally essential. A model is presented in which NapF<br />

mediates electron transfer from membranous menaquinol via NapH to<br />

immature cytoplasmic NapA in or<strong>der</strong> to promote biogenesis of NapA and/or its<br />

Tat-dependent membrane translocation.<br />

[1] Simon et al. (2003) Mol Microbiol 49: 69-79<br />

[2] Kern et al. (2007) Microbiology 153: 3739-3747<br />

[3] Kern and Simon (2008) Mol Microbiol 69:1137-1152<br />

[4] Simon and Kern (2008) Biochem Soc Trans 36: 1011-1016<br />

KA 04<br />

Involvement and specificity of outer membrane<br />

cytochromes in extracellular electron transfer reactions<br />

C. Bücking *1 , F. Popp 1 , S. Kerzenmacher 2 , J. Gescher 1<br />

1<br />

Institut für Biologie II/ Mikrobiologie, Albert-Ludwigs-Universität, Freiburg,<br />

Germany<br />

2<br />

Institut für Mikrosystemstechnik, Albert-Ludwigs-Universität, Freiburg,<br />

Germany<br />

Dissimilatory metal reducing bacteria (DMRB) have established the formation<br />

of electron transport chains to metals as terminal electron acceptors. Due to the<br />

often low solubility of those metallic electron acceptors un<strong>der</strong> neutrophilic<br />

conditions DMRB have to perform electron transfer reactions onto the surface<br />

of the cell. The γ-proteobacterium Shewanella oneidensis has been established<br />

as a model organism to study this process.<br />

53

54<br />

It uses an extended respiratory pathway consisting of c-type cytochrome<br />

proteins to promote electron transfer onto insoluble electron acceptors. The<br />

final electron transfer reaction is believed to be catalyzed by outer membrane<br />

cytochromes (OMC). Shewanella oneidensis genome analysis revealed five<br />

putative genes for OMC, three of those have an unknown function.<br />

To assess function and specificity of individual OMC, we constructed a<br />

deletion mutant in all five outer membrane cytochromes. In this mutant we<br />

expressed single OMC and measured reduction rates as well as electrogenic<br />

activity in a microbial fuel cell (MFC) setup. Our data shows for the first time<br />

that MtrF, similar to the known key-player OmcB, is a terminal reductase <strong>bei</strong>ng<br />

capable of transferring electrons to AQDS (anthraquinone-2,6-disulfonate),<br />

ferric citrate and ferrihydrite with rates comparable to those of OmcB.<br />

Surprisingly, the OMC-mutant retained low-level reduction activity. Electronshuttling<br />

compounds that have recently been proposed to participate in electron<br />

transfer could possibly explain this behavior. These shuttles are believed to<br />

interact solely with OMC [1,2].<br />

However, MFC experiments point towards the involvement of membranepermeable<br />

substances in electron shuttling processes.<br />

[1] Marsili, E. et al. (2008) Proc. Natl. Acad. Sci. 105(10), 3968-73.<br />

[2] von Canstein, H. et al.. (2008) Appl. Environ. Microbiol. 74(3), 615-23.<br />

KA 05<br />

Oxygen Deactivation in [FeFe] Hydrogenases- two<br />

Hydrogen producing Enzymes studied by Protein Film<br />

Voltammetry and X-ray Absorption Spectroscopy<br />

S. Stripp *1 , G. Goldet 2 , O. Sanganas 3<br />

1<br />

Lehrstuhl Biochemie <strong>der</strong> Pflanzen, AG Photobiotechnology, Ruhr Universität<br />

Bochum, Bochum, Germany<br />

2<br />

Inorganic Chemistry Laboratory, University of Oxford, Oxford, United<br />

Kingdom<br />

3<br />

Institut für Experimentalphysik, Freie Universität Berlin, Berlin, Germany<br />

[FeFe] hydrogenases catalyse the production and consumption of molecular<br />

hydrogen with an overpotential close to zero. Therefore, hydrogenases are a<br />

possible alternative to platinum as a catalyst of H2 production. Oxygen<br />

sensitivity of [FeFe] hydrogenases is a big problem when it comes to<br />

biotechnological applications however. It is of great interest to un<strong>der</strong>stand the<br />

deactivation mechanisms of O2 to design novel enzyme variants or active site<br />

analogues less sensitive to aerobic degradation.<br />

The [FeFe] hydrogenases CrHydA1 from photosynthetic green algae<br />

Chlamydomonas reinhardtii and CaHydA from the anaerobic bacterium<br />

Clostridium acetobutylicum exhibit remarkably high H2 evolution activities. By<br />

protein film voltammetry (PFV), we found that CrHydA1 catalyses both uptake<br />

and evolution of H2 while the bacterial CaHydA shows only minor uptake<br />

activity. Despite their high turnover rates, notorious O2 sensitivity for these<br />

enzymes is far lower than expected. Now we are able to present a kinetic<br />

analysis that proves CrHydA1 deactivating ten times slower than well<br />

characterized [FeFe] hydrogenase DdH of Desulvovibrio desulfuricans.<br />

However, CaHydA is even less sensitive to oxygen by a factor of 100. We use<br />

X-ray absorption spectroscopy (XAS) to follow O2 deactivation at the algal<br />

active site "H-cluste" (a unique iron sulphur compound) with a resolution of<br />

about 0.02 Å and propose O2 degrading the accessory parts of the active site<br />

rather than the catalytic [2Fe2S] domain. Deactivation kinetics for both [FeFe]<br />

hydrogenases support this notion, and a comparison of H2-competing CO<br />

inhibition and O2 deactivation gains prove that there are indeed different<br />

binding sites for CO / H2 and O2.<br />

KA 06<br />

Exploring the active site aof the W,FeS enzyme acetylene<br />

hydratase<br />

F. ten Brink *1 , O. Einsle 2 , B. Schink 1 , P.M. Kroneck 1<br />

1<br />

FB Biologie, Universität Konstanz, Konstanz, Germany<br />

2<br />

Institut für Organische Chemie und Biochemie, Universität Freiburg,<br />

Freiburg, Germany<br />

Acetylene is only a minor component of our atmosphere (0.02 ppbv, mainly<br />

anthropogenic origin). In CH4/N2 rich atmospheres (e.g. Titan, early Earth), it<br />

might have been produced photochemically, and might have constituted a<br />

valuable carbon/energy source [1]. Hereby, acetylene is first converted to<br />

acetaldehyde catalyzed by acetylene hydratase (AH); AH belongs to the<br />

DMSOR family, it hosts a [4Fe-4S] cluster and a W(MGD)2 site [2]. At the<br />

active W site (1.2 Å resolution) there are several amino acids which might be<br />

important for catalysis, Asp13, Lys48. Cys141, Ile142: Asp13 for substrate<br />

positioning and activation, Lys48 for electron transfer between the W and the<br />

FeS center, Cys141 for W coordination, and Ile142, as part of a hydrophobic<br />

ring, for shielding the active site. Furthermore, Ile142 might also help to<br />

position the substrate. To test these assumptions, an expression system was<br />

established in E. coli. Expression un<strong>der</strong> anaerobic conditions helped to produce<br />

several soluble variants of AH. Their enzymatic activity was less compared to<br />

the wildtype enzyme isolated from P. acetylenicus but could be significantly<br />

enhanced by fusion of a chaperone binding sequence at the N-terminal end of<br />

the AH gene. In a second approach, crystals of the W-AH from P. acetylenicus<br />

and its Mo isoform were prepared un<strong>der</strong> elevated pressure of acetylene to study<br />

its binding to the W center.<br />

[1] Oremland R.S. and Voytek M.A., Astrobiology, 8, 45-58 (2008)<br />

[2] Seiffert G.B., Abt D., tenBrink F., Fischer D., Einsle O. and Kroneck<br />

P.M.H., Handbook of Metalloproteins, Vol. 4 (<strong>2009</strong>)<br />

KB 01<br />

Unexpected properties of a methanogenic enzyme:<br />

Methylcobalamin-CoM-Methyltransferase (MtbA) of<br />

Methanosarcina mazei transforms bismuth into volatile<br />

trimethylbismuth<br />

B. Huber *1 , F. Thomas 1 , R. Diaz-Bone 2 , R. Hensel 1<br />

1 Mikrobiologie I, Universität Duisburg-Essen, Essen, Germany<br />

2 Institut für Umweltanalytik, Universität Duisburg-Essen, Essen, Germany<br />

It is well known that a plenitude of biologically produced methylated metals<br />

and metalloids can be detected in the environment. These biogenic <strong>der</strong>ivatives<br />

are mostly volatile and – with only few exceptions – more toxic than their<br />

inorganic educts due to their greater hydrophobicity which results in higher<br />

solubility in and permeability through cell membranes. In recent studies we<br />

could prove that members of methanoarchaea play a predominant role in their<br />

synthesis. However, knowledge regarding the mechanism of organometal(loid)<br />

biosynthesis is still scarce.<br />

The methanogen Methanosarcina mazei, which uses various methyltransferases<br />

for methane formation depending on the carbon source (CO2, methylamines,<br />

methanol, and methylsulfide), is used as a model organism in this study. We<br />

could show that Methylcobalamin-CoM-Methyltransferase (MtbA), which<br />

catalyzes the methyltransfer from trimethylamine to Coenzyme M via a<br />

corrinoid cofactor, is also able to methylate bismuth to trimethylbismuth.<br />

Macromolecular and kinetic investigations with the recombinant enzyme were<br />

performed to define the molecular prerequisites for that unexpected activity and<br />

to get insight into the mechanism of methyltransfer to metal ions.<br />

KB 02<br />

Flagella of Pyrococcus furiosus: a structural and functional<br />

characterization<br />

A. Bellack *1 , R. Rachel 2 , R. Wirth 1<br />

1 Institute of Microbiology, University of Regensburg, Regensburg, Germany<br />

2 Institute of Microbiology and Electron microscopy, University of Regensburg,<br />

Regensburg, Germany<br />

Flagellation is a widespread feature among Prokaryotes. While the physical<br />

structure of both bacterial and archaeal flagella is highly similar, recent<br />

biochemical and genetic analyses indicate that these motility structures are<br />

unique for each prokaryotic domain. Nowadays, the ultrastructure and assembly<br />

of bacterial flagella are well un<strong>der</strong>stood, but data on the anchoring and motor<br />

proteins of archaeal flagella are scarce - not least due to an absent detectable<br />

homology to the bacterial counterparts.<br />

Our group has chosen Pyrococcus furiosus as a model organism to study<br />

function and structure of archaeal flagella. The flagellum consists of three<br />

flagellins with FlaB0 as the major flagellin and FlaB1/FlaB2 as minor proteins.<br />

Co-transcription experiments of the fla-operon revealed several proteins<br />

involved in the flagella assembly, but a functional characterization of those<br />

proteins was not successful yet.<br />

In this work we established a protocol for lysis of stationary Pyrococcus cells<br />

un<strong>der</strong> hypotonic conditions allowing the detection of flagella-associated<br />

proteins in membrane fractions. In further steps membrane proteins were<br />

successfully solubilized using various detergents and subjected to MALDI-TOF<br />

analyses or immunoprecipitation of flagella was done (together with chemically<br />

cross-linked proteins). Electron microscopy was a powerful tool to evaluate the<br />

effective purification of membranes and attached flagella without loss of<br />

ultrastructure.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KB 03<br />

Detailed analysis of structural elements in TFB and RNA<br />

polymerase during transcription initiation<br />

M.E. Zeller *1 , K.J. Armache 2 , P. Cramer 2 , M. Thomm 1<br />

1 Lehrstuhl für Mikrobiologie, Universität Regensburg, Regensburg, Germany<br />

2 Genzentrum München, Ludwig-Maximilians-Universität, München, Germany<br />

The minimal transcription initiation complex in archaea consists of RNA<br />

polymerase (RNAP) and 2 general transcription factors, TATA-binding protein<br />

(TBP) and transcription factor B (TFB). We analyzed a set of point and deletion<br />

mutants in TFB and RNAP of Pyrococcus furiosus, so as to learn more about<br />

the sophisticated interaction between RNAP and TFB - the prerequisite for<br />

open complex formation and initiation of RNA synthesis.<br />

Unexpectedly, we found that the linker region of TFB is essential for promoter<br />

DNA melting. Moreover, initial promoter opening is necessary for the rescue of<br />

TFB mutants by transcription factor E. In addition, we now can confine a<br />

stretch of amino acids in the B-finger region of TFB that is responsible for<br />

stabilization of the open complex in close proximity to the transcription start<br />

site. Furthermore, a complementary mutational analysis of reconstituted [1]<br />

RNAPs with single amino acid substitutions in the coiled coil structure of the<br />

clamp domain (CCC) [2] revealed that the residues in this region play a key<br />

role during promoter opening.<br />

Taken together, our and previous data suggest that initial promoter DNA<br />

melting requires both, the linker region of TFB and the CCC patch of RNAP.<br />

Subsequent stabilization of the open complex is mediated (i) by TFE, which<br />

binds to the non template DNA strand [3] and (ii) by a stretch of amino acids in<br />

the B-finger region of TFB that assures correct positioning of the transcribed<br />

DNA strand at the active site within the polymerase cleft.<br />

[1] Naji S, Gruenberg S & Thomm M, J Biol Chem 282,11047-57 (2007)<br />

[2] Cramer P, Bushnell DA, Kornberg RD, Science 292(5523):1863-76 (2001)<br />

[3] Gruenberg S, Bartlett MS, Naji S, Thomm M, J Biol Chem 282(49):35482-<br />

90 (2007)<br />

KB 04<br />

Assembly and function of archaeal cell surface structures<br />

S.V. Albers *1<br />

1 Molecular Biology of Archaea, Max-Planck-Institute for terrestrial<br />

Microbiology, Marburg, Germany<br />

Prokaryotes possess various kinds of cell surface organelles, serving versatile<br />

biological roles depending on the environmental niche of the organism. The<br />

formation of these structures involve fascinating machineries, as not only do the<br />

protein components need to travel across the cytoplasmic membrane like all<br />

secreted proteins, they also need to do so in a precisely coordinated manner for<br />

proper assembly. In archaea only the assembly of flagella has been studied in<br />

some detail, but reports about pili or other the assembly of other surface<br />

structures of archaea, such as Cannulae or hami, are missing.<br />

The thermoacidophilic archaeon Sulfolobus solfataricus contains a large<br />

number of sugar binding proteins and putative pilins that are synthesized as<br />

precursors with a class III signal peptide. Such signal peptides are commonly<br />

used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into<br />

extracellular macromolecular surface appendages. In S. solfataricus we have<br />

now identified three systems that assemble different kinds of cell surface<br />

structures: I.) the flagellum, II.) the bindosome, a binding protein containing<br />

appendage and III.) UV induced pili.<br />

All three systems contain at least one cytoplasmic ATPase and an integral<br />

membrane protein that possibly form the core of the assembly systems. In case<br />

of the flagellum only one flagellin protein is present, whereas in the UV pili<br />

system two pilins are expressed. In the bindosome a whole variety of sugarbinding<br />

proteins is assembled. Deletion mutant analysis of different genes<br />

present in these systems demonstrated that they are involved in the assembly of<br />

the specific surface structure. Using a virus-based vector system<br />

complementation of the phenotypes was observed and showed for two systems<br />

that the assembly of these structures is indeed dependent on ATP-hydrolysis.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KB 05<br />

Inside in the ecophysiology of marine ammonia-oxidizing<br />

Archaea<br />

S. Standfest *1 , M. Hügler 2 , D. Wischer 1 , H. Cypionka 1 , M. Könneke 1<br />

1<br />

ICBM, Universität Oldenburg, Oldenburg, Germany<br />

2<br />

Leibniz-Institut für Meereswissenschaften, IFM-GEOMAR, Kiel, Kiel,<br />

Germany<br />

The aerobic oxidation of ammonia to nitrite is the first and rate-limiting step<br />

during nitrification and represents a key process within the nitrogen cycle. The<br />

perception that ammonia-oxidizing bacteria (AOB) are the only catalysts of this<br />

reaction has been changed due to the isolation of an ammonia-oxidizing<br />

archaeon (AOA) from a marine tropical fish tank. Like AOB, Nitrosopumilus<br />

maritimus grows chemolithoautotrophically by oxidizing ammonia to nitrite<br />

and with CO2 as sole carbon source [1]. However, despite the akin catabolic<br />

metabolism physiological properties of AOA differ clearly from those of AOB.<br />

As essential process we focus our study on the carbon fixation pathway of N.<br />

maritimus by combining microbiological and biochemical methods. Using the<br />

same growth conditions, we found that the specific growth yield of N.<br />

maritimus was three times as high as that of the AOB Nitrosococcus oceanus<br />

indicating that AOA use other metabolic pathways than their bacterial<br />

counterparts. In fact, whole genome data and initial biochemical analysis<br />

revealed distinct evidences that N. maritimus uses a modified 3hydroxypropionate<br />

pathway while AOB use the Calvin-Benson-Cycle for CO2fixation.<br />

Further comparative studies are currently <strong>bei</strong>ng performed with the<br />

novel mesophilic AOA strain Jan1 that we have recently isolated from the<br />

German Wadden Sea, a natural marine environment. Strain Jan1 shares high<br />

phylogenetic relatedness with N. maritimus but exhibits other physiological<br />

characteristics that might reflect adaptation to varying conditions in its natural<br />

habitat.<br />

[1] Könneke et al., Nature 2005<br />

KB 06<br />

Radiation resistance of hyper/thermophilic Archaea and<br />

thermophilic deep-branching Bacteria<br />

K. Beblo *1 , H. Huber 2 , R. Rachel 3 , G. Reitz 1 , P. Rettberg 1<br />

1<br />

Institute of Aerospace Medicine, Radiation Biology Departement, German<br />

Aerospace Center (DLR), Cologne, Germany<br />

2<br />

Institute for Microbiology and Archaea Center, University of Regensburg,<br />

Regensburg, Germany<br />

3<br />

Center for Electronmicroscopy, University of Regensburg, Regensburg,<br />

Germany<br />

Due to the ability of thermophilic and hyperthermophilic Bacteria and Archaea<br />

to life in different extreme habitats on Earth (e.g. boiling acidic springs, black<br />

smoker chimneys, hyper-salinic brines), one could suggest that these organisms<br />

can also outlast other harsh conditions for example conditions prevailing in<br />

space. In space and on other planets without an atmosphere radiation doses are<br />

much higher than on Earth’s surface.<br />

In this study we investigated the ability of several non-spore forming<br />

hyperthermophilic Archaea as well as deep-branching Bacteria to survive high<br />

fluences of UV radiation (254 nm) and high doses of ionizing radiation up to 10<br />

kGy. UV-C radiation as well as ionizing radiation, both affecting directly the<br />

DNA are known to be very harmful to living organisms. Nevertheless results<br />

show that all of the tested vegetative cells do survive after UV-exposure in nonabsorbing<br />

liquid media. If the cells were dried in a monolayer before radiation<br />

treatment, we could demonstrate an interacting negative effect on the survival<br />

after desiccation and UV-radiation treatment. Furthermore we found in the<br />

group of hyper/thermophilic microorganisms some species which can survive<br />

exposures to very high doses of ionizing radiation. Thus making them<br />

interesting model organisms for the study of resistance mechanisms during real<br />

space exposure.<br />

KC 01<br />

Towards a global un<strong>der</strong>standing of nitrogen starvation<br />

acclimation in unicellular cyanobacteria<br />

G. Rasch 1 , M. Drath 1 , J. Espinosa 1 , K.P. Michel 2 , K. Forchhammer *1<br />

1<br />

Mikrobiologie, Universität Tübingen, Tübingen, Germany<br />

2<br />

Lehrstuhl für Molekulare Zellphysiologie, Universität Bielefeld, Bielefeld,<br />

Germany<br />

Acclimation of cyanobacteria to various nitrogen conditions is pivotal for their<br />

survival in natural habitats. Non-diazotrophic cyanobacteria acclimate to the<br />

absence of combined nitrogen by an or<strong>der</strong>ed degradation of photosynthetic<br />

pigments, termed chlorosis. They are able to survive in the chlorotic state for<br />

55

56<br />

extended periods of time. Recently we showed that Synechocystis PCC 6803<br />

employs an alternate methionine aminopeptidase (Map-2), which appears to be<br />

involved in the maturation of a more stress resistant PSII system, to survive<br />

chlorisis. On a global scale, a major regulator of the nitrogen response in<br />

cyanobacteria is transcription factor NtcA, which can act either as repressor or<br />

activator of nitrogen-regulated genes. Recently, the small protein PipX was<br />

discovered, which is required for high-level expression of several NtcA<br />

dependent genes un<strong>der</strong> conditions of nitrogen starvation. Interaction between<br />

PipX an NtcA is regulated by the PII signalling transduction protein, and the<br />

entire NtcA-PipX-PII network responds to the level of 2-oxoglutarate, the<br />

signal for the cellular N-supply. In addition to NtcA, another putative<br />

transcriptional regulator, NblR, is involved in regualtion of chlorosis in<br />

Synechococcus elongatus (strain PCC 7942). The presumable target of NblR,<br />

the nblA gene (encoding a factor promoting phycobiliprotein degradation), is<br />

also regulated by NtcA, pointing towards an overlap between NtcA- and NblR<br />

function. In or<strong>der</strong> to elucidate their roles in depth, microarray analysis was<br />

performed with a focus on the initial response to nitrogen starvation. The result<br />

shows an intriguing correlation between NtcA-and NblR-dependent activation<br />

of gene expression during nitrogen starvation.<br />

KC 02<br />

The protein Ssl3076 represses the salt-regulated ggpS gene<br />

involved in synthesis of the compatible solute<br />

glucosylglycerol in Synechocystis sp. strain PCC 6803.<br />

S. Klähn 1 , A. Die<strong>der</strong>ich 1 , E. Simon 1 , S. Anacker 1 , M. Hagemann *1<br />

1 Plant Physiology, University Rostock, Rostock, Germany<br />

Acclimation to high salt concentration involves concerted changes of gene<br />

expression. Transcriptomics revealed up-regulation of up to 300 genes [1] and<br />

proteomics showed accumulation of about 40 proteins [2] in Synechocystis. For<br />

the majority of the salt-regulated genes the mechanism un<strong>der</strong>lying the induction<br />

process is not known. The high-through put analyses did not identify a saltdependent<br />

transcriptional regulator. The ggpS-gene encoding glucosylglycerolphosphate<br />

synthase was found among the highest and stable up-regulated<br />

genes. Moreover, it is specifically induced by salt and not by other stresses such<br />

as heat or high light. The ggpS promoter was mapped about 400 bp upstream<br />

from the translational start point. The whole promoter and the 5’-upstream<br />

region were fused with promoter-less gfp. Defined deletions were introduced<br />

into the promoter and 5’-UTR. Stepwise deletion of promoter resulted in<br />

increased promoter activity un<strong>der</strong> low salt conditions; however, small saltinduction<br />

was still observed. Particularly, deletions in a small ORF ssl3076<br />

overlapping the ggpS promoter and the 5’-UTR abolished salt regulation. These<br />

results indicated that the Ssl3076 protein seems to be involved as a repressor in<br />

salt-sensing, which was supported by defined mutations and RT-PCR analyses.<br />

[1] Marin et al., PNAS 100:9061-9066, 2003<br />

[2] Fulda et al., Proteomics 6:2733-2745, 2006<br />

KC 03<br />

Cyanobacterial TPR proteins and their role in<br />

photosynthetic functions<br />

M. Schottkowski *1 , L. Shao 1 , B. Rengstl 1 , E. Ankele 2 , J. Nickelsen 1<br />

1<br />

Molekulare Pflanzenwissenschaften, LMU München, Planegg-Martinsried,<br />

Germany<br />

2<br />

Biochemie und Physiologie <strong>der</strong> Pflanzen, LMU München, Planegg-<br />

Martinsried, Germany<br />

The biogenesis of the thylakoid membrane is mediated by several trans-acting<br />

factors in chloroplasts and cyanobacteria. The family of the so-called<br />

tetratricopeptide repeat (TPR) proteins, which mediate protein-protein<br />

interactions, has been shown to be involved in various steps during the<br />

assembly of photosynthetic components. Here we introduce TPR proteins of<br />

Synechocystis sp. PCC 6803 which play a role for photosynthesis in<br />

cyanobacteria.<br />

The first is the periplasmic PratA (slr2048) factor which is involved in the early<br />

steps of photosystem II (PSII) assembly. Our data provide evidence for an<br />

interaction of PratA with the soluble C-terminus of the D1 protein of PSII.<br />

Interestingly, PratA is located to a special membrane subfraction which might<br />

represent a connecting region between plasma- and thylakoid membrane and,<br />

thus, the place of initiation of PSII assembly (Schottkowski et al. 2008 J Biol<br />

Chem, in press). Preliminary data suggest that the protein encoded by ORF<br />

slr0151 is also involved in this early step of thylakoid membrane biogenesis.<br />

A third TPR factor encoded by ORF slr1644 interacts in Yeast-Two-Hybrid<br />

analysis with the light-dependent protochloropyllide oxidoreductase (POR). In<br />

the respective deletion mutant, both POR content and chlorophyll synthesis are<br />

affected.<br />

KC 04<br />

Chlamydomonas and Escherichia – fraternal twins in<br />

fermentation?<br />

A. Hemschemeier *1 , J. Jacobs 1 , D. Kravietz 1 , G. Philipps 1 , T. Happe 1<br />

1 Biochemie <strong>der</strong> Pflanzen, Photobiotechnologie, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

The microalga Chlamydomonas reinhardtii has been used as a model to study<br />

oxygenic photosynthesis as well as structure and function of flagellae for a long<br />

time. Its "half plant – half beast" phenotype has recently been verified by the<br />

analysis of its genomic sequence. However, the alga shares many features with<br />

bacteria, too. Un<strong>der</strong> anaerobic conditions, it utilizes enzymes typically found in<br />

bacteria. C. reinhardtii has a [FeFe]-hydrogenase coupled to the photosynthetic<br />

electron transport chain and a formate and ethanol producing fermentative<br />

metabolism initiated by pyruvate formate lyase (Pfl), an enzyme typically<br />

found in prokaryotes like Escherichia coli. Both enzymes are active in the<br />

photofermentative metabolism of the alga, which is induced by sulphur<br />

depletion in illuminated cultures. We have examined the putative Pfl1 protein<br />

of C. reinhardtii and proved its formate producing activity by heterologous<br />

expression of the algal PFL1 cDNA in E. coli. Recently, we identified a C.<br />

reinhardtii Pfl1-mutant strain showing a remarkable flexibility of its<br />

fermentative strategies since it utilizes alternative pathways to maintain the<br />

cellular redox and energy balance. Moreover, Pfl1 deficient algae show a<br />

significant reduction of hydrogenase activity. Genetic complementation of the<br />

Pfl1-mutant as well as the external addition of formate restore its hydrogenase<br />

activity to the wild type level, indicating a direct and regulatory influence of the<br />

pyruvate fermentation pathway on the hydrogen metabolism of the alga.<br />

KC 05<br />

Towards un<strong>der</strong>standing the spliceosome-mediated RNA<br />

trans-splicing in the chloroplast of Chlamydomonas<br />

reinhardtii<br />

S. Glanz *1 , U. Kück 1<br />

1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

The unicellular green alga C. reinhardtii is widely used for analyzing nuclearencoded<br />

factors that are thought to promote the maturation of chloroplast<br />

precursor RNAs and are presumably part of a postulated chloroplast<br />

spliceosome. As an example, the expression of the psaA gene, in particular the<br />

trans-splicing process of its precursor RNAs, is studied. Here, we present<br />

molecular genetic and biochemical approaches that enable the isolation of novel<br />

nuclear-encoded factors.<br />

In forward genetic approaches, we used restriction enzyme-mediated<br />

integration to generate novel trans-splicing mutants. Genomic complementation<br />

of these mutants identified proteins with similarities to poly(ADP-ribose)<br />

polymerases (Rat1) and aminoacyl-tRNA synthetases (Raa4) that directly<br />

interact with psaA intron RNAs. The RNA-binding property was demonstrated<br />

by electrophoretic mobility shift assays using different organellar group II<br />

intron domains. In the second approach, UV-crosslinking and the yeast threehybrid<br />

system was used to isolate intron RNA-binding proteins that show<br />

homologies to nucleosome assembly proteins (cNAPL), 3-hydroxyisobutyratedehydrogenases<br />

(31 kDa protein), and the α-subunit of chaperonin Cpn60<br />

(Cpn60). The chloroplast localization of some of these splicing factors was<br />

determined by laser scanning confocal fluorescence microscopy.<br />

The availability of several trans-splicing mutants from C. reinhardtii and in<br />

vivo studies using the yeast two-hybrid system will further identify components<br />

that are most probably part of a chloroplast spliceosome.<br />

KC 06<br />

The circadian RNA-binding protein CHLAMY1 can<br />

integrate temperature information<br />

O. Voytsekh *1 , S. Seitz 1 , D. Iliev 2 , M. Mittag 1<br />

1 Institute of General Botany and Plant Physiology, Friedrich-Schiller-<br />

University Jena, Jena, Germany<br />

2 BMC B13, Stem Cell Center, Lund, Sweden<br />

Circadian rhythms are biological rhythms that persist with a period of about 24<br />

h un<strong>der</strong> constant conditions of light and temperature. Their physiological<br />

properties are well conserved in different organisms. In the green alga<br />

Chlamydomonas reinhardtii several circadian rhythms have been<br />

physiologically characterized such as phototaxis or chemotaxis (summarized in<br />

1). One component of the circadian system of C. reinhardtii represents the<br />

RNA-binding protein CHLAMY1 [2] that consists of the two subunits, C1 and<br />

C3. CHLAMY1 binds specifically to (UG)≥7-repeat sequences situated in the<br />

3´-UTRs of several mRNAs encoding, for example, proteins of nitrogen<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

metabolism. Introduction of such (UG)≥7-repeat sequences in the 3’-UTR of a<br />

luciferase reporter causes circadian expression of the reporter [3]. Up- and<br />

down-regulation of C1 or C3 showed that these two subunits are involved in the<br />

maintenance of acrophase and period of circadian rhythms [4]. Moreover, both<br />

subunits can integrate temperature information a prerequisite for entrainment<br />

by temperature cycles and/or temperature compensation [5]. While C1 gets<br />

hyper-phosphorylated at low temperature, c3 expression is up-regulated at this<br />

temperature. The temperature-dependent regulation of C1 and C3 is altered in<br />

the long period mutant per1. Moreover, temperature entrainment is disturbed in<br />

per1. Our data suggest that C1, C3 and PER1 are part of a functional network<br />

that is involved in temperature entrainment.<br />

[1] Mittag et al., 2005, Plant Physiol. 137: 399-409<br />

[2] Zhao et al., 2004, Euk. Cell 3: 815-825<br />

[3] Kiaulehn et al., 2007, J. Biol. Rhythms 22: 275-277<br />

[4] Iliev et al., 2006, Plant Physiol. 142: 797-806<br />

[5] Voytsekh et al., 2008, Plant Physiol. 147: 2179-2193.<br />

KC 07<br />

Generation of Chlamydomonas strains that efficiently<br />

express nuclear transgenes<br />

J. Neupert *1 , D. Karcher 1 , R. Bock 1<br />

1<br />

Organelle Biology and Biotechnology, Max-Planck-Institute of Molecular<br />

Plant Physiology, Potsdam-Golm, Germany<br />

The unicellular green alga Chlamydomonas reinhardtii is a well established<br />

model organism in basic research and has become an attractive production<br />

system in biotechnology. Despite the availability of efficient nuclear<br />

transformation technologies in Chlamydomonas , transgene expression levels<br />

are usually very poor. The low level of nuclear transgene expression poses a<br />

serious limitation to the usage of Chlamydomonas in basic research and<br />

biotechnology, including molecular farming. In or<strong>der</strong> to overcome this<br />

problem, we established a genetic screen that facilitates the isolation of algal<br />

strains that show high transgene expression levels. We were able to isolate two<br />

independent expression strains, UVM4 and UVM11. Foreign protein<br />

accumulation levels in our expression strains are nearly uniformly high in all<br />

transgenic clones suggesting the absence of position effects in these strains. The<br />

analysis of the transformants revealed a transcriptional nature of the transgene<br />

suppression mechanism present in wildtype Chlamydomonas and inactivated<br />

in UVM4 and UVM11. The possibility to express transgenes to high levels will<br />

greatly facilitate post-genomics research in Chlamydomonas and will also<br />

boost the exploitation of the alga as an inexpensive production host for<br />

biopharmaceuticals and other valuable compounds.<br />

KD 01<br />

The BEM46-like protein appears to be essential for hyphal<br />

development upon ascospore germination in Neurospora<br />

crassa and is targeted to the Endoplasmic Reticulum<br />

M. Mercker 1 , K. Kollath-Leiß 1 , S. Allgaier 2 , N. Weiland 3 , F. Kempken *4<br />

1 Institut für Angewandte Mathematik, Universität Heidelberg, Heidelberg,<br />

Germany<br />

2 Botanisches Institut, Abteilung für Botanik mit Schwerpunkt Genetik und<br />

Molekularbiologie, Christian-Albrechts-Universität zu Kiel, Kiel, Germany<br />

3 Neugenesis Corporation, Burlingame, United States<br />

4 Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel,<br />

Kiel, Germany<br />

The bud emergence (BEM)46 proteins are evolutionarily conserved members<br />

of the α/β-hydrolase super family, but their exact role remains unknown. To<br />

better un<strong>der</strong>stand the cellular role of BEM46 and its homologs, we used the<br />

model organism Neurospora crassa in conjunction with bem46 RNAi, overexpression<br />

vectors, and repeat induced point (RIP) mutation analyses. We<br />

clearly demonstrated that BEM46 is required for cell type-specific hyphal<br />

growth, which indicates a role for BEM46 in maintaining polarity. Vegetative<br />

hyphae, perithecia, and ascospores developed normally, but hyphae<br />

germinating from ascospores exhibited a loss-of-polarity phenotype. We also<br />

found that the BEM46 protein is targeted to the perinuclear endoplasmic<br />

reticulum (ER) and also localizes at or close to the plasma membrane. Our<br />

findings show that BEM46 can be used as a new ER marker for filamentous<br />

fungi, the first forN. crassa. Our data suggest that BEM46 plays a role in a<br />

signal transduction pathway involved in determining or maintaining cell typespecific<br />

polarity. This implies a higher degree of fungal hyphae differentiation<br />

than previously expected. This work also has implications for higher eukaryotic<br />

cells with polarized growth, such as pollen tubes or neuronal cells.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KD 02<br />

Cyclophilin D: a link between apoptosis and lifespan<br />

control in the ascomycete Podospora anserina<br />

D. Brust *1 , A. Hamann 1 , H.D. Osiewacz 1<br />

1 Institute of Molecular Biosciences, Department of Biosciences and Cluster of<br />

Excellence “Macromolecular Complexes”, J.W. Goethe University, Frankfurt,<br />

Germany<br />

Podospora anserina is a filamentous ascomycete displaying a limited lifespan.<br />

After a period of linear growth, the growth rate declines, ending up with the<br />

death of the peripheral hyphal tips. Recently, apoptotic processes were<br />

identified, which are involved in lifespan control of P. anserina.<br />

Proteome analyses demonstrated an increased level of the human cyclophilin D<br />

homolog in senescent P. anserina mitochondria, which is supposed to be part of<br />

the mitochondrial permeability transition pore (mPTP). This finding raises the<br />

question of whether the release of mitochondrial pro-apoptotic factors by mPTP<br />

formation triggers apoptosis in P. anserina. In or<strong>der</strong> to address this question<br />

PaCYPD levels were modulated and the impact of these genetic manipulations<br />

were analysed. The constitutive over-expression of PaCypD resulted in a<br />

decreased growth rate, decreased fertility and a dramatic reduction in lifespan.<br />

In contrast, deletion of PaCypD did not influence theses parameters.<br />

Furthermore, it was demonstrated that cyclosporin A (CsA), a specific inhibitor<br />

of human cyclophilin D, specifically binds to PaCYPD. Juvenile PaCypD overexpressors<br />

were found to contain mainly punctuate mitochondria as they are<br />

characteristic for senescent cultures of the wild-type. Moreover, mitochondria<br />

of PaCypD over-expressing strains were found to loose cytochrome c, a process<br />

that is a hallmark of apoptosis in mammalian cells. Based on our data we<br />

speculate about a crucial role of mPTP opening in the sequence of events that<br />

finally bring life of senescent P. anserina cultures to an end.<br />

KD 03<br />

Comparison of genes controlled by the MAP kinases<br />

BcSak1 and Bmp3 of Botrytis cinerea during oxidative<br />

stress<br />

J. Heller *1 , N. Segmüller 1 , P. Tudzynski 1<br />

1 Instiut für Botanik und Botanischer Garten, Westfälische-Wilhelms<br />

Universität, Münster, Germany<br />

Phytopathogenic fungi face consi<strong>der</strong>able oxidative stress by the so-called<br />

"oxidative burst", an early plant defense reaction, in which the plant produces<br />

ROS extensively. B. cinerea can produce ROS itself and thus contribute to the<br />

ROS status during infection. Furthermore ROS can serve as messenger<br />

molecules in signal transduction. We focus on signal chain components<br />

involved in oxidative stress signaling trying to determine their role in either<br />

ROS generation or ROS decomposition.<br />

The stress-activated mitogen-activated protein kinase (MAPK) BcSak1 of B.<br />

cinerea seems to be involved in both, the oxidative stress response and the<br />

sensing of intracellular ROS as messenger molecules. We could show that it is<br />

transcriptionally upregulated and activated during oxidative stress mediated by<br />

H2O2 and that it is necessary for essential differentiation processes like<br />

conidiation, sclerotia development and pathogenicity [1].<br />

In addition Bmp3, another MAP kinase of B. cinerea, which is a homologue of<br />

yeast Slt2, seems to play a role in maintaining the ROS equilibrium in the<br />

fungus. This MAP kinase controls the genes for the O2 - -producing enzymes<br />

BcNoxA and BcNoxB transcriptionally and the deletion of bmp3 leads to<br />

sensitivity against H2O2 [2].<br />

Macroarray hybridization was performed to identify target genes of BcSak1 and<br />

Bmp3 that are expressed differentially in the wild type strain and in both<br />

mutants during normal and oxidative stress conditions. While several ROS<br />

scavenging genes were found to be regulated by BcSak1 the role of Bmp3 as<br />

regulator of ROS scavenging genes seems to be of minor importance.<br />

[1] Segmüller et al. (2007) Euk. Cell. 6: 211–221<br />

[2] Segmüller et al. (2008) MPMI 21: 1443-1459<br />

57

58<br />

KD 04<br />

The RNA-binding protein Khd4 is important for<br />

pathogenicity in Ustilago maydis<br />

E. Vollmeister *1 , C. Haag 1 , S. Baumann 1 , J. König 1 , M. Feldbrügge 1<br />

1 Max-Planck-Institute for terrestrial Microbiology, Marburg, Germany<br />

Posttranscriptional regulation is an essential mechanism in organising cellular<br />

processes, e.g. in the plant pathogen Ustilago maydis. Loss of the RNA-binding<br />

protein Khd4 results in defects in cell morphology and pathogenicity suggesting<br />

an important role in regulating such crucial cellular processes. However,<br />

binding specificity, target mRNAs as well as a defined role of Khd4 within<br />

posttranscriptional regulation are presently unknown. Here, we demonstrate<br />

that Khd4 recognises the sequence AUACCC (KIS, Khd4 interacting sequence)<br />

via KH domain 3 and 4. To discover potential target mRNAs whose expression<br />

is dependent on Khd4, we compared khd4 deletion strains with wild type in<br />

microarray experiments. We identified a subset of 72 <strong>der</strong>egulated mRNAs. The<br />

vast majority of transcripts displayed an increased expression in khd4 deletion<br />

strains. Furthermore, AUACCC is enriched in the first 150 nucleotides of the 3’<br />

untranslated region of these transcripts. Biological function analysis revealed<br />

that Khd4 mutants failing to bind AUACCC display the khd4 deletion<br />

phenotype, e.g. disturbed cell morphology and reduced virulence. In essence,<br />

Khd4 is a sequence-specific RNA-binding protein that regulates a distinct<br />

subset of mRNAs most likely on the level of mRNA stability indicating an<br />

important role of posttranscriptional regulation in orchestrating cell<br />

morphological programmes in U. maydis.<br />

KD 05<br />

Apocarotenoids-Signaling Compounds of Zygomycetes and<br />

Plants?<br />

D. Schachtschabel *1 , M. Schlicht 2 , K.D. Menzel 3 , F. Baluska 2 , W. Boland 1<br />

1<br />

Bioorganische Chemie (Boland), Max-Planck-Institut für chemische Ökologie,<br />

Jena, Germany<br />

2<br />

IZMB, Universität Bonn, Bonn, Germany<br />

3<br />

Bio-Pilot-Plant, Hans Knöll Institut, Jena, Germany<br />

Fungi of the division Zygomycetes are common heterotrophic microorganisms<br />

which naturally occur on terrestrial habitats. For sexual reproduction, the<br />

zygomycete fungi interact via an elaborate series of carotene <strong>der</strong>ived<br />

compounds, namely trisporic acids and their biosynthetic precursors.[1] The<br />

compounds are used for mediating the recognition between zygomycetes and<br />

some of their mycoparasites.[2] However, details of their metabolism and the<br />

biological significance of the various intermediates remained unclear.<br />

Therefore we generated a trisporoid library including deuterium labeled<br />

intermediates by a combination of synthesis and biotransformation using liquid<br />

cultures of the species Blakeslea trispora [3]. These references enabled us to<br />

study the biosynthesis and the biological function of individual trisporoids in<br />

more detail [4]. The results prompted us to postulate a new sequence of<br />

molecular interaction between both mating partners, which includes two<br />

different metabolic pathways. One to initiate and stimulate the hormone<br />

production with a newly discovered trisporic acid and the second for the<br />

intrinsic production of the known trisporic acids.<br />

Since apocarotenoids are strongly discussed as new branching hormones in<br />

plants and fungi [5] we decided to test if trisporoids have an influence on plant<br />

cells. Amazingly the β-C18-ketone strongly inhibited root hair development of<br />

Arabidopsis thaliana, Solanum nigrum and Zea maize in a nanomolar range.<br />

The inhibition is linked to disruption of ROS-production by the root-hairassociated<br />

NADPH-oxidase and caused a collapse of the actin cytoskeleton.[6]<br />

[1] Sutter R P et al.(1996) Fung. Gen. Biol. 20, 268-279.<br />

[2] Schachtschabel D et al. (2005) Phytochemistry 66, 1358-1365<br />

[3] Schachtschabel D Boland W (2007) J. Org. Chem. 72, 1366-1372<br />

[4] Schachtschabel D et al. (2008) ChemBioChem 9, 3004-3012<br />

[5] Schachtschabel D, Boland W (2008) ChemBioChem, in press<br />

[6] Schlicht M et al.(2008) Plant J. 55,709-717<br />

KD 06<br />

Development of experimental tools for the investigation of<br />

the barley–pow<strong>der</strong>y mildew pathosystem<br />

G. Hensel *1<br />

1 Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Molecular<br />

Cell Biology, Gatersleben, Germany<br />

Fungal diseases are a major cause of yield losses in crop species grown<br />

worldwide. In or<strong>der</strong> to further our knowledge on the interaction between plants<br />

and fungal pathogens and to eventually improve crop plants, valuable<br />

experimental tools have been developed to facilitate detailed studies of barley–<br />

pow<strong>der</strong>y mildew interactions. A transient-induced gene silencing system<br />

(TIGS) was established using biolistic transfer of inverted-repeat RNAi<br />

constructs into epi<strong>der</strong>mal cells of detached leaf sections followed by<br />

inoculation with Blumeria graminis. This method enables us to screen large<br />

numbers of candidate genes. Interestingly, TIGS of some Blumeria graminis<br />

house-keeping genes in planta disrupts the development of this biotrophic<br />

fungus suggesting a usefulness of host-triggered RNAi in pathogens for plant<br />

protection. To verify the data of those transiently operating expression<br />

modulations stable barley transformation was conducted with selected<br />

constructs. To this end, the modular IPKb binary vectors were developed. This<br />

set includes GATEWAY compatible vectors for constitutive as well as<br />

epi<strong>der</strong>mis-specific overexpression or RNAi knock-down. Furthermore, highly<br />

efficient Agrobacterium-mediated gene transfer protocols to immature embryos<br />

or embryogenic pollen cultures have been developed. Beside the experimental<br />

model cvs. „Golden Promise“ and „Igri“ a number of further barley lines (e.g.<br />

cvs. „Ingrid“ and „Optic“) which are of particular interest for research on<br />

fungal pathosystems proved amenable to stable transformation. Together, the<br />

presented tools constitute a powerful experimental platform for the<br />

investigation of plant–microbe interactions in barley.<br />

KE 01<br />

Mitochondrial quality control systems: role in fungal<br />

development and lifespan control<br />

H.D. Osiewacz *1<br />

1 Department of Biosciences and Cluster of Excellence Macromolecular<br />

Complexes, Johann Wolfgang Goethe University, Frankfurt, Germany<br />

Mitochondria are cell organelles with a number of fundamental functions. Best<br />

known is their role in oxygenic energy transduction providing adenosine<br />

triphosphate (ATP) for energy consuming cellular processes. During the<br />

generation of ATP at the respiratory chain reactive oxygen species (ROS) are<br />

generated which are involved in cellular signaling but also lead to molecular<br />

damage and consequently to impaired functions, disease, and death.<br />

Fortunately, all biological systems contain a number of pathways designed to<br />

deal with this harmful situation. In addition to ROS scavenging systems (e.g.,<br />

scavenging enzymes), DNA and protein repair pathways, proteolytic systems,<br />

processes separating highly damaged parts of mitochondria and the subsequent<br />

degradation of these parts by ‘mitophagy’, and fusion of damaged with<br />

undamaged mitochondria are involved in keeping a functional population of<br />

mitochondria. In multicellular systems apoptotic removal of cells with severely<br />

damaged mitochondria represents another level of protection that acts at the<br />

organism level. In contrast, in unicellular organisms (e.g., Saccharomyces<br />

cerevisiae) and in filamentous fungi (e.g., Podospora anserina) apoptosis is the<br />

ultimate end point in the life cycle of the individuum.<br />

In the lecture I will provide results of investigations aimed at elucidating the<br />

impact of mitochondrial quality control systems on development and lifespan.<br />

The focus will be on the proteolytic system operating in mitochondria of the<br />

filamentous ascomycete P. anserina. Recent investigations revealed that<br />

genetic modulation of this system significantly affects lifespan. The data will be<br />

integrated into the network of quality control pathways of this fungus.<br />

KE 02<br />

A mitochondrial β-class carbonic anhydrase is involved in<br />

sexual reproduction of the filamentous fungus Sordaria<br />

macrospora<br />

S. Elleuche *1 , S. Pöggeler 1<br />

1 Abteilung Genetik eukaryotischer Mikroorganismen, Georg-August<br />

Universität, Göttingen, Germany<br />

Carbonic anhydrases (CAs) are ubiquitous enzymes, which catalyze the<br />

reversible hydration of carbon dioxide. CAs from human, prokaryotes, plants<br />

and fungi exhibit only a low level of sequence identity and can be divided into<br />

different classes. In eukaryotes, the subcellular distribution of CAs is highly<br />

diverse. Mammalian CAs belong to the α-class and can be found in the cytosol,<br />

in mitochondria, the plasma membrane or they can be secreted. Fewer CAs are<br />

encoded by fungal genomes. One β-CA gene has been investigated in the yeasts<br />

Saccharomyces cerevisiae and Candida albicans, whereas two β-CAs were<br />

described from the basidiomycete Cryptococcus neoformans. Interestingly,<br />

many prokaryotes as well as some ascomycetes contain CA genes from at least<br />

two different classes.<br />

In this study, we characterized three β-CAs from the filamentous ascomycete<br />

Sordaria macrospora. To investigate the role of the CAs, we deleted each gene<br />

and performed localization experiments. It turned out that the CAS2-protein<br />

localizes to mitochondria and is required for ascospore germination and<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

vegetative growth. CAS1 and CAS3 are localized to the cytosol and Δcas1,<br />

Δcas3 as well as the Δcas1/3 double deletion strains exhibit a wild-type like<br />

phenotype. A severe defect in fruiting body development was observed in a<br />

Δcas1/2 double mutant, indicating that the absence of cas1 increases the<br />

physiological defect in a Δcas2-background. Interestingly, the growth defect<br />

but not the impairment in germination efficiency of Δcas1/2 could be<br />

complemented by providing unspecific CA activity from overexpressed cas1,<br />

cas3 or from a non-mitochondrial cas2 variant. A detailed characterization of<br />

single and double knockout mutants will be presented.<br />

KE 03<br />

Cross species yeast two-hybrid analyses unravel cellular<br />

networks of fungal development<br />

S. Bloemendal *1 , I. Engh 1 , S. Seiler 2 , U. Kück 1<br />

1<br />

Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

2<br />

Abt. Molekulare Mikrobiologie und Genetik, Institut für Mikrobiologie und<br />

Genetik, Georg-August-Universität Göttingen, Göttingen, Germany<br />

Cellular differentiation processes are fundamental for eukaryotic organisms. An<br />

example is the formation of fruiting bodies in filamentous fungi as a<br />

multicellular differentiation process. The filamentous fungus Sordaria<br />

macrospora represents an excellent model system for cell differentiation during<br />

fruiting body development [1].<br />

For Sordaria macrospora, several proteins involved in this developmental<br />

process have been identified, including the proteins PRO22 and PRO40 [2, 3].<br />

Mutants with a point mutation in the ORF of pro22 and pro40, respectively, are<br />

unable to form mature fruiting bodies. For sexual development in filamentous<br />

fungi, hyphal fusion plays a critical role. Fluorescence microscopy using<br />

nuclear labeled strains revealed that mutants pro22 and pro40 are restricted in<br />

their ability to fuse, suggesting a role for PRO22 and PRO40 in hyphal fusion<br />

[3].<br />

For further analyses of these mutants, we want to identify interaction partners<br />

of the proteins PRO22 and PRO40. Due to the close relationship between S.<br />

macrospora and Neurospora crassa, a cDNA library of N. crassa was used for<br />

cross species yeast two-hybrid analyses to identify putative interaction partners.<br />

Our aim is to verify these putative interaction partners in vitro and in vivo and<br />

to identify a network involved in fruiting body formation.<br />

[1] Kück U et al. (<strong>2009</strong>) In: Anke T (ed) The Mycota XV, Springer-Verlag,<br />

Heidelberg<br />

[2] Engh I et al. (2007) Eukaryot Cell 6:831-843<br />

[3] Rech C et al. (2007) Curr Genet 52:259-266<br />

KE 04<br />

4-Dihydrotrisporin dehydrogenase from Mucor mucedo, an<br />

enzyme of the sex hormone pathway<br />

J. Wetzel *1 , O. Scheibner 2 , A. Burmester 1 , C. Schimek 1 , J. Woestemeyer 1<br />

1 Institute of Microbiology, Friedrich-Schiller-University, Jena, Germany<br />

2 ThermoFisher, ThermoFisher Scientific, Dreieich, Germany<br />

Zygomycetes use retinoid-like beta-carotene <strong>der</strong>ivatives, the trisporoids, as<br />

signals for partner recognition and internal regulation events during sexual<br />

development. Trisporoid synthesis involves oxidation of a hydroxyl group at<br />

the C4 atom in the ionone ring moiety of the molecule. In the (-) mating type,<br />

this reaction occurs at the conversion of 4-dihydromethyl trisporate into methyl<br />

trisporate. The same reaction is necessary to convert 4-dihydrotrisporin into<br />

trisporin. Until now, it was not clear if these reactions are catalyzed by the same<br />

or by different enzymes.<br />

We identified a C4-oxidizing enzyme from Mucor mucedo that is clearly<br />

different from the previously characterized 4-dihydromethyltrisporate<br />

dehydrogenase (TDH). The Mr 26 000 enzyme prefers 4-dihydrotrisporin over<br />

4-dihydromethyl trisporate as substrate and was therefore named 4dihydrotrisporin-dehydrogenase<br />

(TNDH).<br />

Using reverse genetics we identified a single copy gene, TSP2, encoding a 240<br />

amino acid short-chain dehydrogenase. This protein exhibits no similarity to<br />

TDH, an aldo-keto reductase, indicating independent evolutionary origins of<br />

the two genes.<br />

Expression of TSP2 depends on developmental stage in both mating types. At<br />

the enzyme level, activity is found exclusively in the (-) mating type. However,<br />

renaturation of proteins after denaturing PAGE reveals the presence of the<br />

TSP2 gene product in both mating types. These findings indicate<br />

posttranslational inactivation of 4-dihydrotrisporin dehydrogenase in the (+)<br />

mating type of Mucor mucedo, similar to the situation proposed for 4dihydromethyltrisporate-dehydrogenase.<br />

[1] J. Wetzel et al. (<strong>2009</strong>), Eukaryotic Cell, in press.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KE 05<br />

The role of the bZIP transcription factor MeaB in the<br />

nitrogen metabolite repression of Fusarium fujikuroi<br />

D. Wagner *1 , A. Schmeinck 1 , B. Tudzynski 1<br />

1 Institut für Botanik, Westfälische Wilhelms-Universität, Münster, Germany<br />

The rice pathogen Fusarium fujikuroi produces a broad spectrum of secondary<br />

metabolites. The synthesis of two of them, gibberellin and bikaverin, is subject<br />

to the nitrogen metabolite repression. One of the key elements of this complex<br />

regulatory network is the GATA transcription factor AreA which activates the<br />

expression of the gibberellin biosynthesis genes. In Aspergillus nidulans the<br />

main antagonist of AreA is NMR. The expression of nmr is positively<br />

controlled by the bZIP transcription factor MeaB which is therefore an indirect<br />

antagonist of AreA. We examined the role of MeaB in the nitrogen metabolite<br />

repression of Fusarium fujikuroi.<br />

Knockout and overexpression mutants of meaB were created, and the impact on<br />

the expression of nitrogen repressed genes was studied by Northern analysis. It<br />

was shown that MeaB has also a mainly negative effect on the expression of<br />

these genes in F. fujikuroi, but that this effect is not as strong as in A. nidulans.<br />

Interestingly the repressing effect of MeaB is not mediated via the expression<br />

control of nmr as proved by promoter studies in the ΔmeaB-background.<br />

Additionally, we identified two transcript sizes of meaB that are part of a<br />

complex regulation system. This allows MeaB to migrate into the nucleus only<br />

un<strong>der</strong> high nitrogen concentrations as shown by MeaB-GFP-Fusion. The results<br />

indicate the role of MeaB as a fine tuning regulator in the nitrogen regulation<br />

network and highlight differences between F. fujikuroi and A. nidulans<br />

concerning this role.<br />

KE 06<br />

Formation of itaconic acid by the fungus Ustilago maydis<br />

MB215<br />

M. Panakova *1 , N. Maassen 1 , M. Zimmermann 1 , M. Bölker 2 , U. Klinner 1<br />

1 Biology IV (Microbiology and genetics), RWTH Aachen, Aachen, Germany<br />

2 FB Biology, Philipps-University Marburg, Marburg, Germany<br />

Itaconic acid (ITA) is an unsaturated dicarboxylic acid which is produced<br />

biotechnologically and used as a platform chemical [1]. In the currently favored<br />

biotechnological production process Aspergillus terreus is used. In this<br />

filamentous fungus ITA is formed obviously from the tricarbonic acid<br />

intermediate cis-aconitate by the cis-aconitate decarboxylase (CAD). The<br />

sequence of the CAD gene of A. terreus has been published quite recently [2].<br />

However, nothing is known about regulatory principles and other metabolic<br />

details of the IA production by this fungus.<br />

Ustilago maydis appears to be a candidate for an alternative producer of ITA.<br />

This basidiomycetous fungus, that exhibits a free living yeast-like<br />

nonpathogenic and a filamentous pathogenic form, produces high amounts of<br />

ITA un<strong>der</strong> certain conditions as nitrogen limitation, a pH value of about 6 and<br />

presence of high amounts of glucose in the medium. We found out, that the<br />

production of ITA strongly depends on the amount of oxygen available for cells<br />

during cultivation.<br />

We have isolated REMI induced mutants of strain MB215 which were no<br />

longer able to produce ITA. To obtain more detailed information about the<br />

peculiarities of the ITA metabolic pathway in U. maydis we are characterizing<br />

these mutants and perform targeted disruptions of genes which are putatively<br />

involved in the ITA synthesis.<br />

[1] Willke T, Vorlop KD (2001) Appl Microbiol Biotechnol 56:289-295.<br />

[2] Kanamasa et al. (2008) Appl Microbiol Biotechnol 80:223-229.<br />

KE 07<br />

Novel DyP-type peroxidases from the jelly fungus<br />

Auricularia auricula-judae<br />

C. Liers *1 , C. Bobeth 1 , R. Ullrich 1 , M. Hofrichter 1<br />

1 Environmental Biotechnology, International Graduate School Zittau, Zittau,<br />

Germany<br />

The jelly fungus Auricularia auricula-judae („Jew´s ear fungus“ or „Tree<br />

ear“) produces exceptionally high levels (up to 8,000 U l -1 ) of extracellular<br />

peroxidase during growth in liquid, plant-based media (e.g. tomato juice<br />

medium). Purification of two peroxidase isoforms required three steps<br />

including anion and cation exchange chromatography as well as<br />

chromatofocusing, and resulted in an overall enzyme yield of approx. 60% for<br />

both proteins. Spectroscopic and electrophoretic analyses of purified proteins<br />

(designated as AjP1 and AjP2) revealed their heme-nature and molecular<br />

masses of approximately 40 and 51 kDa as well as acidic isoelectric points of<br />

59

60<br />

4.0 and 4.1, respectively. The enzymes catalyze not only the conversion of<br />

typical peroxidase substrates, such as 2,6-dimethoxyphenol or guaiacol in the<br />

absence of manganese, but also the decolourization of the synthetic azo-dye<br />

Reactive Black 5. In the presence of natural mediators like phenolic acids or<br />

linoleic acid, both peroxidases were capable of oxidizing a non-phenolic lignin<br />

model compound. The N-terminal amino acid sequence of the 51 kDa sized<br />

protein (AjP2) shows no homology to any known peroxidase. The N-terminal<br />

sequence of the minor isoform (AjP1), however, matches well with the<br />

sequence of a fungal peroxidase from Marasmius scorodonius (93% identity).<br />

Our results indicate that the peroxidases of A. auricula-judae belong to the<br />

novel “DyP-type” peroxidase family, which is postulated as an independent<br />

family separate from the classical peroxidases with unique structural and<br />

catalytic features. To confirm this, the substrate spectrum of the enzymes is<br />

currently <strong>bei</strong>ng studied more in detail.<br />

KF 01<br />

Blue-green biotechnology<br />

T. Börner *1<br />

1 Biologie/Genetik, Humboldt-Universität, Berlin, Germany<br />

Cyanobacteria (blue-green algae) represent a highly divers monophyletic group<br />

of microorganisms found in virtually all habitats including very extreme ones.<br />

Characteristic for all cyanobacteria is their ability to perform oxygen-evolving<br />

photosynthesis. This report will provide an overview and discuss recent trends<br />

in the use of cyanobacteria in biotechnology.<br />

A few species serve as food in several developing countries whilst there is a<br />

market for cyanobacteria as food supplements ("health food") in the US and<br />

Europe. The capability of many cyanobacteria to fix nitrogen from the air forms<br />

the basis for the application of several species as "biofertilizers". Cyanobacteria<br />

synthesize many products that are of commercial interest like amino acids,<br />

carotenoids, phycocyanin, (exo)polysaccharids or restriction enzymes. More<br />

recently it became evident that cyanobacteria are a particular rich source of<br />

bioactive secondary metabolites including ribosomal and non-ribosomal<br />

peptides, polyketides and others. There is hope that cyanobacterial metabolites<br />

may help to fight HIV and support the therapy of cancer. Recent research<br />

focuses on cyanobacteria as source of "bioenergy". The potential to combine<br />

photosynthesis with the production of hydrogen, biodiesel or bioethanol has<br />

attracted many scientists from both basic and applied sciences.<br />

KF 02<br />

Posttranslational modification by the nonribosomal peptide<br />

microcystin affects protein stability in the toxic<br />

cyanobacterium Microcystis<br />

E. Dittmann *1 , Y. Zilliges 1 , J.C. Kehr 1 , S. Mikkat 2 , M. Hagemann 3 , N. Tandeau<br />

de Marsac 4<br />

1<br />

Institut für Biologie/Molekulare Ökologie, Humboldt-Universität, Berlin,<br />

Germany<br />

2<br />

Proteomics Core Facility, Universität Rostock, Rostock, Germany<br />

3<br />

Institut für Biologie, Universität Rostck, Rostock, Germany<br />

4<br />

Unité de Cyanobacteries, Institut Pasteur, Paris, Germany<br />

Microcystis is predominant in many freshwater habitates and frequently forms<br />

surface exposed blooms. Light intensities at the surface normally lead to<br />

oxidative stress and photoinhibition. Here we provide evidence that the<br />

widespread hepatotoxin microcystin could be related to the remarkable<br />

resistance of Microcystis against oxidative stress in the field. The cyclic<br />

heptapeptide microcystin specifically inhibits protein phosphatases of types 1<br />

and 2A in higher eukaryotes. In particular, it interacts with the catalytic<br />

domains of these enzymes and forms a covalent bond to a cysteine. Using<br />

highly specific antibodies we have discovered that within Microcystis,<br />

microcystin interacts with a number of targets. Using immunoprecipitation and<br />

immunoaffinity purification and subsequent trypsin fingerprint analysis we<br />

have identified the putative microcystin interaction partners. Interestingly,<br />

enzymes known to be un<strong>der</strong> a redox control, such as Rubisco and<br />

phosphoribulokinase of the Calvin Cycle and glutathione reductase, but also<br />

cell surface proteins were among the interaction partners. In vitro studies<br />

revealed that microcystin binds via its N-methyldehydroalanine moiety to<br />

cysteines of these proteins. The binding is strongly enhanced un<strong>der</strong> oxidative<br />

stress conditions in vivo. A proteomic study of a microcystin-deficient mutant<br />

revealed that most of the microcystin binding partners showed clear differences<br />

in their abundance compared to the wild type. We therefore assume that<br />

microcystin fulfils an important thiol buffer function in Microcystis.<br />

KF 03<br />

Structure function relationships in Chlorophyta type<br />

[FeFe]-hydrogenases<br />

M. Winkler *1 , P. Knörzer 1 , T. Happe 1<br />

1<br />

AG Photobiotechnologie / LS Biochemie <strong>der</strong> Pflanzen, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

In contrast to the interaction of PetF with other redox partners like e.g.<br />

ferredoxin-NADPH-oxidoreductase (FNR) (1-3) the electron transfer process in<br />

green algae to or from the photosynthetic hydrogenase is still a matter of<br />

speculation (4-6). Complex formation between both proteins is a precondition<br />

for un<strong>der</strong>standing light driven hydrogen production in green algae. Recently,<br />

we reported about the establishment of a new heterologous expression system<br />

for diverse [FeFe]-hydrogenases including HydA1 of C. reinhardtii (7).<br />

In the present study we utilized this system to characterize the electron transfer<br />

interaction between HydA1 and PetF by testing the kinetics of site directed<br />

mutagenesis variants from both proteins generated on the basis of a preceding<br />

in silico docking analysis.<br />

By combining the elements of the photosynthetic electron transfer chain<br />

including plastocyanin, photosystem I (PsI) and PetF together with isolated<br />

[FeFe]-Hydrogenase we successfully reconstructed the coupling of HydA1 to<br />

the photosynthetic electron transport chain resulting in light driven hydrogen<br />

production.<br />

The H-cluster is featuring three CO- and two CN-ligands. For more information<br />

about the interaction between the CN-ligands and the amino acid backbone,<br />

side chains of interest were modified by site directed mutagenesis.<br />

[1] Morales, R., et al. (2000) EMBO Rep 1, 271-276<br />

[2] Kurisu, G., et al. (2001) Nat Struct Biol 8, 117-121<br />

[3] Palma, P. N., et al. (2005) FEBS Lett 579, 4585-4590<br />

[4] Florin, L., Tsokoglou, A., and Happe, T. (2001) J Biol Chem 276, 6125-<br />

6132<br />

[5] Winkler, M., Heil, B., and Happe, T. (2002) Biochim Biophys Acta 1576,<br />

330-334<br />

[6] Happe, T., and Kaminski, A. (2002) Eur J Biochem 269, 1022-1032<br />

[7] Girbal, L., von Abendroth, G., et al.(2005) Appl Environ Microbiol 71,<br />

2777-2781<br />

KF 04<br />

Construction of a continuous photobiological reactor<br />

system for cyanobacterial H2-production<br />

J.H. Kwon *1 , N. Waschewski 1 , G. Bernát 1 , M. Broekmans 1 , S. Rexroth 1 , M.<br />

Rögner 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

Hydrogen is one of the most promising energy sources. However, many<br />

technical aspects involved in the renewable supply of hydrogen remain<br />

unsolved. Hydrogen production in cyanobacteria has been identified as a<br />

candidate for a renewable energy source with small ecological foot print.<br />

Cyanobacterial systems are able to produce hydrogen using sunlight as energy<br />

source and atmospheric CO2 as substrate for biomass production along with O2<br />

as important by-product. For the industrial application of cyanobacterial<br />

hydrogen production, high investment and running cost, as well as low<br />

metabolic rates for hydrogen display the main obstacles.<br />

To address these issues, we apply three strategies: the metabolic engineering of<br />

cyanobacterial cells for improved H2-production, the construction of a lab-scale<br />

photobioreactor for the optimization of process parameters, and the<br />

development of a continuous cultivation system for stable and sustainable<br />

growth un<strong>der</strong> production conditions. For the reactor module, we constructed a<br />

flat-bed photobioreactor based on polymeric materials (coop. KSD, Hattingen)<br />

reducing the necessary investment costs to 10% of comparable systems. A<br />

steady-state fermentation process in this reactor has been realized un<strong>der</strong><br />

turbidostatic, as well as chemostatic conditions. The transition from batch to<br />

continuous culture enables efficient control of the metabolic state, which is a<br />

prerequisite for a reliable optimization of all process parameters. For<br />

improvement of the cyanobacterial metabolism for hydrogen production, we<br />

produced and characterized mutants involving especially reduced antenna size<br />

and partially uncoupled electron transport. These mutants displayed higher<br />

electron transport rates than the wildtype.<br />

Due to the insights resulting from this optimization process, the development of<br />

a designed micro-algae and the construction of an optimized reactor for<br />

hydrogen production appears to provide a high potential for technical<br />

application.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KF 05<br />

Analysis of the anaerobic response in Chlamydomonas<br />

reinhardtii regarding the impact of putative regulatory<br />

proteins.<br />

I. Hirschmann *1 , M. Terashima 1 , D. Petroutsos 1 , D. Krawietz 2 , T. Happe 2 , M.<br />

Hippler 1<br />

1 Institute of Plant Biochemistry and Biotechnology, Westfälische Wilhelms-<br />

Universität Münster, Münster, Germany<br />

2 Institute of Plant Biology and Biochemistry, Ruhr-Universität-Bochum,<br />

Bochum, Germany<br />

Using comparative proteomic approaches the anaerobic response in the wild<br />

type of C. reinhardtii was analysed (see Poster Mia Terashima) and led to the<br />

identification of proteins with a putative key role in the anaerobic adaptation<br />

process. Several RNAi mutant strains were created and provide an opportunity<br />

to get further insights in the regulatory pathways, which results to the vast<br />

metabolic changes un<strong>der</strong> anaerobiosis [1]. After cultivating various C.<br />

reinhardtii strains un<strong>der</strong> aerobic and anaerobic (4 hours of argon bubbling)<br />

conditions, the whole cell proteomes were compared using stable isotopic<br />

labelling of amino acids in cell cultures (SILAC [2]). The direct comparative<br />

quantification using any mutant strain and the 13 C6 Arginine-labeled wild type<br />

(Arg auxotrophic) strain facilitates in general the indirect comparision of not<br />

Arg auxotrophic strains un<strong>der</strong> altering conditions. The variation of the<br />

anaerobic adaptation process depending on the diminution of putative<br />

regulatory proteins will be discussed on this poster.<br />

[1] Mus et al. (2007) Anaerobic Acclimation in Chlamydomonas reinhardtii.<br />

Anoxic gene expression, hydrogenase induction, and metabolic pathways. The<br />

Journal of Biological Chemistry 282: 25475-25486<br />

[2] Naumann et al. (2005) N-terminal Processing of Lhca3 Is a Key Step in<br />

Remodeling of the Photosystem I-Light-harvesting Complex Un<strong>der</strong> Iron<br />

Deficiency in Chlamydomonas reinhardtii. The Journal of Biological<br />

Chemistry 280: 20431-20441<br />

KF 06<br />

Organelle transcriptomics and translatomics of<br />

Chlamydomonas reinhardtii<br />

A. Idoine *1 , R. Bock *1 , J. Rupprecht 1<br />

1 AG Bock / GoFORSYS, Max Planck Institute of Molecular Plant Physiology,<br />

Potsdam - Golm, Germany<br />

GoFORSYS is a research project supported by the Fe<strong>der</strong>al Ministry of<br />

Education and Research, which aims to perform a comprehensive systems<br />

analysis of the expression and regulation of photosynthesis in response to<br />

selected environmental factors in the model algae, Chlamydomonas reinhardtii<br />

(http://www.goforsys.de).<br />

C. reinhardtii is a very useful model organism for studying photosynthesis.<br />

This is because it is a single-celled microorganism, which removes many of the<br />

confounding effects of tissue- and developmental-specific regulation in higher<br />

plants. Its amenability to laboratory work, the ease of its genetic transformation<br />

and the availability of its full genome sequences, also adds to its appeal. In<br />

addition to the correlations to be drawn to higher plants, C. reinhardtii reveals<br />

an interesting and unique range of physiological pathways un<strong>der</strong> anaerobiosis.<br />

As part of the GoFORSYS project, the transcription and translation of<br />

organelle-encoded genes will be analysed. To this end, an oligonucleotide<br />

microarray has been designed which covers all genes present in the chloroplast<br />

and mitochondrial genomes. Analysis of steady-state mRNA accumulation<br />

(transcriptomics) will be combined with polysome isolation (translatomics) to<br />

give a comprehensive overview of transcript levels and their translational<br />

regulation un<strong>der</strong> a range of physiological conditions, including the effect of the<br />

circadian rhythm and anaerobiosis.<br />

These data will be combined with other microarray experiments, qRT-PCR,<br />

proteomics, and metabolomics to create an in depth un<strong>der</strong>standing of this<br />

important model system un<strong>der</strong> the investigated conditions. It will then be<br />

possible to use this information for applications such as improving<br />

photobiological hydrogen production, and it will be able to be extrapolated to<br />

higher plants due to the high conservation of the central processes of<br />

photosynthesis.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KG 01<br />

New approaches to the intracellular lifestyle of Salmonella<br />

enterica in eukaryotic cells<br />

M. Hensel *1<br />

1 Infektionsbiologische Abteilung, Universitätsklinikum Erlangen, Erlangen,<br />

Germany<br />

Salmonella enterica is an important gastrointestinal pathogen with a facultative<br />

intracellular lifestyle inside a membrane-bound compartment termed<br />

Salmonella-containing vacuole (SCV). Although major virulence determinants<br />

that enable the intracellular survival and replication of Salmonella have been<br />

identified, the cellular and molecular mechanisms how Salmonella manipulate<br />

their host cells are still enigmatic. A key factor for adaptation of Salmonella to<br />

intracellular life is the type III secretion system encoded by Salmonella<br />

Pathogenicity Island 2 that translocates a set of effector proteins across the SCV<br />

membrane. The effectors alter the maturation of the SCV, leading to formation<br />

of the compartment that is protected against killing and that permits replication<br />

of the bacteria. The analyses of alterations of the endosomal membrane system<br />

induced by intracellular Salmonella are of particular interest. Most previous<br />

observations were based on observations of single time points by<br />

immunofluorescence. Recently, new concepts for the intracellular life were<br />

developed based on advanced techniques of microscopy. Live cell imaging<br />

allowed time-resolved investigations of the SCV biogenesis and vesicular<br />

transport in infected cells. In addition, high resolution electron microscopy<br />

(EM) and EM tomography of Salmonella-infected host cells led to novel<br />

insights into the organization of the SCV and the manipulation of host cell<br />

membrane compartments by Salmonella.<br />

KG 02<br />

Staphylococcus aureus gene expression and -regulation<br />

during persistent colonization of the human nares<br />

M. Burian *1 , C. Wolz 1 , C. Goerke 1<br />

1 Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Tübingen,<br />

Tübingen, Germany<br />

Staphylococcus aureus asymptomatically colonizes the nose of one-fifth of the<br />

human population. Nasal carriage is associated with an increased risk of<br />

infection. Little is known about the adaptive gene expression of S. aureus to the<br />

nasal environment. We performed direct transcript analysis from nose swabs<br />

using quantitative RT-PCR and compared the transcription profile during<br />

colonization to that of the in vitro grown isolates. Nose swabs were obtained<br />

from four persistent S. aureus carriers and in a follow-up analysis 12 months<br />

later again from two of these carriers. Genes from the following categories<br />

were analyzed: i.) global virulence regulators, ii.) toxins, iii.) adhesins, iv.) cell<br />

wall remodeling enzymes, v.) DNA repair/metabolic regulators and vi.)<br />

immune modulators.<br />

The major global virulence regulators agr, sae, sigB and apsXRS were not<br />

active in the human nose, whereas the regulatory system for cell wall<br />

metabolism walK/R is highly active. Toxins which are implicated to play a role<br />

in invasive infection were not expressed in the nose. In contrast, factors<br />

important for tissue adherence like clumping factor B and the iron-regulated<br />

surface determinant A were highly expressed. For the permanent colonization<br />

of the human nose the action of immune-modulatory molecules and<br />

modification of the bacterial cell envelope seem to be important.<br />

In summary, we could show that the adaptation of S. aureus to its primary<br />

reservoir is a multifactorial process which greatly differs from the expression<br />

profile in vitro.<br />

KG 03<br />

Dynamics of type IV pili of Neisseria gonorrhoeae during<br />

infection<br />

D. Opitz *1 , M. Clausen 1 , B. Maier 1<br />

1 Institut für Allgemeine Zoologie und Genetik, Westfälische Wilhelms-<br />

Universität Münster, Münster, Germany<br />

Type IV pili are important bacterial virulence factors that mediate attachment to<br />

mammalian host cells and elicit downstream signals. When adhered to abiotic<br />

surfaces, the human pathogen Neisseria gonorrhoeae generates force by<br />

retracting these polymeric cell appendages. We recently found that single pili<br />

generate stalling forces that exceed 100pN, but it is unclear whether bacteria<br />

generate force once they adhere to their human host cells. We observed that pili<br />

retracted very actively during infection of human epithelial cells. Bacteria<br />

generated consi<strong>der</strong>able force during infection but the maximum force was<br />

reduced from (120±40)pN on abiotic surfaces to (70±20)pN on epithelial cells.<br />

The reduction of the maximum retraction force is most likely due to an elastic<br />

effect. Therefore we can not completely exclude that bacteria generate even<br />

61

62<br />

higher forces during infection. In previous studies we have shown that the<br />

distribution of retraction velocities in an abiotic environment is bimodal [1].<br />

During the infection the high velocity mode persisted at higher forces as<br />

compared to an abiotic environment. This increase in average velocity is<br />

consistent with an up-regulation of the putative motor protein PilT due to<br />

interaction with epithelial cells. Velocity and maximum force of pilus retraction<br />

were independent of the infection period within 1h and 24h post infection. Thus<br />

the force generated by type IV pili during infection is high enough to induce<br />

cytoskeletal rearrangements in the host cell.<br />

[1] Clausen, M.; Koomey, M. and Maier, B. Dynamics of type IV pili is<br />

controlled by switching between multiple states. Biophys. J., in print<br />

KG 04<br />

Transport of a Bradyrhizobium japonicum secreted protein<br />

into nodule cells and its impact on symbiosis<br />

S. Zehner *1 , M. Wenzel 1 , L. Friedrich 1 , M. Göttfert 1<br />

1 Institut für Genetik, Technische Universität Dresden, Dresden, Germany<br />

Bradyrhizobium japonicum is a slow growing soil bacterium and is able to fix<br />

nitrogen in root nodules of legumes. B. japonicum encodes a type III secretion<br />

system (T3SS) and secretes a number of proteins (Süß et al. 2006, Hempel et<br />

al. in press). The T3SS is supposed to transport proteins from the bacterial<br />

cytosol into the host cell. The role of the T3SS in the establishment of<br />

symbiosis is host dependent (Krause et al. 2002). We show the expression of<br />

the type III secretion effector protein NopE1 of B. japonicum in symbiosis.<br />

Using adenylate cyclase reporter of Bordetella pertussis (Sory & Cornelis<br />

1994) revealed that NopE1 is transported into the cytosol of nodule cells. The<br />

result also indicates that the signal for targeting the rhizobial protein into plant<br />

cells is located in the amino-terminal region of the secreted protein. The impact<br />

of the secreted protein NopE1 on symbiosis was studied on different host<br />

plants. Nodulation capacity on Vigna radiata was strongly affected in the<br />

presence of this protein.<br />

[1] Süß, C., Hempel, J., Zehner, S., Krause, A., Patschkowski, T., Göttfert, M.<br />

2006. J. Biotechnol. 126, 69-77.<br />

[2] Hempel, J., Zehner, S., Göttfert, M., Patschkowski, T. <strong>2009</strong> J. Biotechnol.<br />

in press<br />

[3] Krause, A., Doerfel, A., Göttfert, M. 2002. Mol. Plant-Microbe Interact. 12,<br />

1228-1235.<br />

[4] Sory, M.P., Cornelis, G.R. 1994. Mol. Microbiol. 14:583-594.<br />

KG 05<br />

Concomitant Fermentation and Denitrification along the<br />

Earthworm Alimentary Canal<br />

P.K. Wüst *1 , M.A. Horn 1 , H.L. Drake 1<br />

1<br />

Department of Ecological Microbiology, University of Bayreuth, Bayreuth,<br />

Germany<br />

The in situ conditions of the earthworm gut have been proposed to selectively<br />

stimulate ingested soil microorganisms. Thus, potential links between in situ<br />

conditions and anaerobic microbial activities during gut passage of Lumbricus<br />

terrestris were evaluated. H2 and N2O were emitted in vivo, and in situ<br />

microsensor analyses revealed H2 and N2O as concomitant products in the<br />

anoxic gut center. H2 concentrations were highest in foregut and midgut,<br />

whereas N2O concentrations were highest in crop/gizzard and hindgut. Thus,<br />

H2-producing fermentations were maximal in the foregut and midgut whereas<br />

denitrification was maximal in crop/gizzard and hindgut. Total carbon and total<br />

nitrogen were highest in foregut and decreased from anterior to posterior of the<br />

gut, and nitrite, ammonium, and iron(II) were highest in the crop/gizzard and<br />

decreased from anterior to posterior of the alimentary canal. Concentrations of<br />

polymeric and soluble saccharides decreased from the anterior to the posterior<br />

end of the alimentary canal. Maximal concentrations of organic acids occurred<br />

in the midgut, indicating that fermentation is ongoing during gut passage.<br />

These findings suggest that (a) ingested microorganisms are subject to changing<br />

in situ conditions during gut passage, (b) fermentation and denitrification occur<br />

concomitantly along the alimentary canal (b) the alimentary canal of the<br />

earthworm is a hotspot in aerated soils for carbon- and nitrogen-driven<br />

anaerobiosis, and (d) earthworms are a mobile source of reductant (i.e., emitted<br />

H2) for microbiota in aerated soils.<br />

KG 06<br />

Anti-inflammatory activity of a B. bifidum strain in Rag1 -/-<br />

colitic mice<br />

J. Preising *1 , J.H. Niess 2 , C.U. Riedel 1<br />

1 Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany<br />

2 Innere Medizin I, Uniklinik Ulm, Ulm, Germany<br />

Bifidobacteria have several beneficial effects for their host. Strain-dependent<br />

anti-inflammatory activity of various strains and species of bifidobacteria was<br />

previously shown by our group in in vitro experiments using LPS-challenged<br />

HT-29 cells. Here we analyse the anti-inflammatory effects of a strain of<br />

B. bifidum with good adhesive properties to intestinal epithelial cells in the<br />

Rag1 -/- mouse model of murine colitis. The transfer of CD4+ T-cell populations<br />

from wild type C57BL/6J into congenic Rag1 -/- mice leads to the development<br />

of colitis mediated by T-helper type 1 cells due to the lack of mature regulatory<br />

T-cells in these mice.<br />

In a placebo-controlled set up one group of Rag1 -/- mice(Rag1 tm1Mom ; n = 4)<br />

received one oral dose of our probiotic<br />

B. bifidum (2x10 9 cfu per animal) in PBS followed immediately by transfer of<br />

CD4+ T-cells. Two control groups received placebo of which one group also<br />

was transferred with CD4+ T-cells to induce colitis. Feeding with the probiotic<br />

and placebo was continued three times a week and weight was recorded for 34<br />

days when all animals were sacrificed. The anti-inflammatory effect of feeding<br />

the B. bifidum strain was assessed by measuring weight and length of dissected<br />

colons, histology scores of colonic tissue samples, and qRT-PCR for various<br />

pro-inflammatory cytokines. Additionally, the composition of the bacterial flora<br />

of all mice was analysed by fluorescence in situ hybridisation combined with<br />

flow cytometry.<br />

KH 01<br />

Analysis of the microtubule cytoskeleton in Aspergillus<br />

nidulans using fluorescent proteins<br />

R. Fischer *1 , N. Zekert 1<br />

1 Microbiology, University of Karlsruhe, Karlsruhe, Germany<br />

The advent of fluorescent proteins in microbiology went along with many new<br />

developments in light and fluorescent microscopy enabling cell biological<br />

studies in living bacterial and lower eukaryotic cells. One example for high<br />

resolution fluorecence microscopy is the analysis of the microtubule<br />

cytoskeleton in the filamentous fungus Aspergillus nidulans. In filamentous<br />

fungi microtubules are required for polarized growth for the delivery of cellend<br />

marker proteins, which in turn organize the actin cytoskeleton at the hyphal<br />

tip (Takeshita et al., 2008). In addition, microtubules are necessary for<br />

intracellular transport processes. In A. nidulans we found that at least two<br />

different microtubule populations exist. This discovery came from studies of an<br />

Unc-104 related kinesin motor protein, which preferentially moved along<br />

detyrosinated microtubules and transports vesicles. These microtubules are<br />

more stable than the tyrosinated ones and even remained intact during mitosis<br />

when other cytoplasmic microtubules were degraded (Zekert & Fischer, 2008).<br />

[1] Takeshita, N., Higashitsuji, Y., Konzack, S. & Fischer, R. (2008). Apical<br />

sterol-rich membranes are essential for localizing cell end markers that<br />

determine growth directionality in the filamentous fungus Aspergillus nidulans.<br />

Mol Biol Cell 19, 339-351.<br />

[2] Zekert, N. & Fischer, R. (2008). The Aspergillus nidulans kinesin-3 UncA<br />

motor moves vesicles along a subpopulation of microtubules. Mol Biol Cell in<br />

press.<br />

KH 02<br />

Live cell imaging in filamentous fungi<br />

I. Engh *1 , M. Nowrousian 1 , U. Kück 1<br />

1 General and Molecular Botany, Ruhr-University Bochum, Bochum, Germany<br />

The filamentous fungus Sordaria macrospora is a model system for eukaryotic<br />

cell differentiation. The vegetative state of its lifecycle is characterized by a<br />

mycelium consisting of interconnected hyphae. During sexual development,<br />

however, complex fruiting bodies are formed that consist of a number of<br />

different cell types forming different tissues. To gain insight into mechanisms<br />

and morphological changes leading to fruiting body formation, we have<br />

established several microscopic techniques for S. macrospora.<br />

First, we have tested standard fluorescent proteins like EGFP, EYFP and<br />

DsRed and novel fluorescent proteins like mKalama1 and EBFP2, tdTomato<br />

and mCherry. To obtain brighter fluorescence, we have further employed a<br />

codon-adapted mRFP1 protein. Bimolecular fluorescence complementation has<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

ecently been developed for filamentous fungi, and first results using BiFC in S.<br />

macrospora will be presented.<br />

Second, we have targeted fluorescent proteins to different organelles by fusing<br />

organellar target sequences or organelle-localized proteins to fluorescent<br />

proteins. Examples are ER-targeted DsRed and EGFP or histone fusion<br />

constructs with EYFP and ECFP. These enabled us to dissect organelle<br />

morphology in different developmental mutants of S. macrospora, and to<br />

analyze hyphal fusion frequency between different strains. Furthermore, we can<br />

now perform co-localization studies with labeled developmental proteins and<br />

organellar markers.<br />

Third, we have established a live cell imaging system for S. macrospora. The<br />

system is based on an upright microscope and utilizes a heating plate with small<br />

Petri dishes. With this live cell imaging method, we have been able to follow<br />

spore germination and formation of fruiting body initials in S. macrospora.<br />

KH 03<br />

Electron cryotomography of Campylobacter jejuni<br />

A. Müller *1 , A. McDowall 2 , G.J. Jensen 2 , W.M. Clemons, Jr. 1<br />

1 Department of Chemistry and Chemical Engineering, Caltech, Pasadena,<br />

United States<br />

2 Department of Biology, Caltech, Pasadena, United States<br />

Campylobacter jejuni is a leading cause of gastroenteritis and the main trigger<br />

for the autoimmune disease Guillain Barré Syndrome (GBS).<br />

Here we present C. jejuni´s ultrastructure, which we obtained by electron<br />

cryotomography of intact, frozen-hydrated cells. Our data reveals several novel<br />

features.<br />

C. jejuni are spiral bacteria that rely on their motility to colonize the mucus<br />

lining of their host’s gastrointestinal tract. Most of them have one flagellar<br />

motor at each pole. The structure making up the machinery is unusually large.<br />

The motors span the periplasm and are embedded in a crater-like structure of<br />

the outer membrane. The cytoplasmic region close to the poles features<br />

chemoreceptor grids that form a polar cap. In many cases we see a storage<br />

vesicle in or close to this region. Surprisingly, ribosomes are excluded from<br />

these polar regions.<br />

KH 04<br />

In vivo localization of the DNA translocator in Acinetobacter<br />

baylyi ADP1<br />

A. Desch* 1, B. Averhoff1,<br />

1Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,<br />

Goethe University Frankfurt/Main, Frankfurt, Germany<br />

Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium which is<br />

ubiquitous in natural enviroments and notable for its high competence of<br />

natural transformation. Gene disruption and mutant studies led to the<br />

identification of 16 distinct genes essential for natural transformation in<br />

Acinetobacter baylyi ADP1. The conserved proteins of the Acinetobacter DNA<br />

translocator can be assigned to two dinstinct groups: I. type IV pili (tfp)- and<br />

type II protein transport-related proteins (pseudopilins, prepilin-processing<br />

lea<strong>der</strong> peptidases, secretins, biogenesis factors) and II. widely conserved<br />

proteins in DNA transport systems (polytopic inner membrane proteins, DNAbinding<br />

proteins). Despite the similarities tfp and natural transformation were<br />

found to be functionally unrelated in A. baylyi. To get insights into the function<br />

of distinct competence proteins DNA binding studies were performed, which<br />

revealed that the competence protein ComEA exhibits DNA binding activity.<br />

Furthermore we have addressed the in vivo localization of ComEA by<br />

generating functional ComEA-GFP fusions. Expression of the ComEA-GFP<br />

fusions in A. baylyi un<strong>der</strong> the control of the comEA promoter led to the<br />

detection of ComEA in the cell periphery. The cells showed no evidence of<br />

polar localization of ComEA, such as most of the cells exhibited distinct lateral<br />

foci. Extended analyses are currently un<strong>der</strong>way address the effect of the growth<br />

phase-dependent natural transformation frequencies on the localization of the A.<br />

baylyi DNA translocator.<br />

KH 05<br />

Fluoresent Proteins for Anaerobic Applications<br />

M. Puls *1 , J. Potzkei 2 , F. Circolone 2 , T. Eggert 1 , T. Drepper 2<br />

1<br />

evocatal GmbH, Düsseldorf, Germany<br />

2<br />

Insitut für molekulare Enzymtechnologie, Heinrich-Heine-Universität,<br />

Düsseldorf, Germany<br />

Fluorescent proteins are most effective tools for the visualization of cellular<br />

processes in cell biology and biotechnology and have thus become<br />

indispensable research tools in these fields during the last years. Nevertheless,<br />

all commercially available fluorescent proteins to date have the severe<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

limitation that they stringently require oxygen in or<strong>der</strong> to constitute their<br />

fluorophoric group. We recently cloned and developed a novel type of<br />

fluorescent marker proteins containing a flavin-mononucleotide-based<br />

fluorophore that is not dependent on the presence of oxygen anymore. It shows<br />

bright fluorescence even in strictly anaerobic environment and thus opens up<br />

new vistas for many fields of research dealing with conditions of limited<br />

oxygen such as the study of anaerobic metabolism, exploration of biofilms or<br />

infection pathways of anaerobic pathogens.<br />

KH 06<br />

GFP based monitoring tools for isolation of new mobile<br />

genetic elements and assessment of conjugative plasmid<br />

transfer in biofilms<br />

K. Schiwon *1 , K. Arends 1 , E. Grohmann 1<br />

1<br />

Umweltmikrobiologie/Genetik, Technische Universität Berlin, Berlin,<br />

Germany<br />

The vast majority of bacteria in the environment live in complex microbial<br />

communities, known as biofilms. Surface-associated bacteria were also isolated<br />

from container systems, like the International Space Station (ISS) and the<br />

CONCORDIA Station in Antarctica. To study intragramic and intergramic<br />

plasmid transfer among bacterial ISS and CONCORDIA isolates we developed<br />

gfp-tagged monitoring tools based on the type IV secretion-like transfer system<br />

of the broad-host-range conjugative plasmid pIP501. The mobilisable plasmid<br />

contains oriTpIP501, the pIP501 relaxase and gfp. The monitoring tool for<br />

conjugative transfer contains the complete pIP501 transfer region and the gfp<br />

gene. The transfer genes are un<strong>der</strong> the control of the nisA promoter. To<br />

distinguish between donors and transconjugants we developed a plasmid with<br />

red fluorescence to label the donor bacteria.<br />

Plasmid mobilization and transfer in Gram-positive bacteria is monitored by<br />

fluorescence microscopy, antibiotic resistance test and gene specific PCR. To<br />

mimic natural conditions, plasmid transfer in biofilms from bacterial ISS and<br />

CONCORDIA isolates is <strong>bei</strong>ng investigated and mobilization frequencies<br />

determined. Single, two and multi species biofilms were formed by Bacillus<br />

thuringiensis, Staphylococcus aureus, Staphylococcus epi<strong>der</strong>midis and<br />

Enterococcus faecalis in biofilm reactors. Triparental mating experiments in<br />

biofilm reactors were carried out with Enterococcus faecalis and<br />

Staphylococcus aureus as donors and E. faecalis as recipient. We achieved<br />

mobilization rates of 3x10 -8 for S. aureus and 6x10 -7 for E. faecalis. The final<br />

goal is to carry out long term plasmid transfer studies in mixed biofilm<br />

communities to quantify plasmid transfer un<strong>der</strong> quasi-natural conditions by<br />

FACS.<br />

KI 01<br />

The mitochondrial translation machinery: From protein<br />

synthesis to membrane integration<br />

J. Herrmann *1 , M. Prestele 1 , S. Funes 1 , H. Bauerschmitt 1 , M. Ott 1<br />

1 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany<br />

The mitochondrial proteome represents a mosaic of different genetic origin:<br />

While most genes of mitochondrial proteins reside in the nucleus, a small set of<br />

proteins is mitochondrially encoded and synthesized by mitochondrial<br />

ribosomes. Despite their small number, these components are of outstanding<br />

relevance for the cell as they form the membrane-embedded reaction centers of<br />

the respiratory chain complexes and the ATPase. In or<strong>der</strong> to form these<br />

complex enzymes, the synthesis, membrane integration and assembly of their<br />

subunits must occur in a concerted co-translational reaction. Protein synthesis<br />

and membrane integration are kinetically and physically coupled by tethering of<br />

mitochondrial ribosomes to the inner membrane and by the recruitment of the<br />

insertion component Oxa1 to polypeptide exit tunnel on the large ribosomal<br />

subunit. However, we still know very little about the molecular processes<br />

which allow the coordination of the assembly of the different subunits.<br />

Recently we identified a component of the large ribosomal subunit, Mrpl36,<br />

which plays a critical role in the biogenesis of respiratory chain complexes, in<br />

particular of cytochrome oxidase. Mrpl36 consists of two domains: The Nterminal<br />

part of Mrpl36 anchors the protein to ribosomes and is crucial for<br />

protein synthesis per se. In contrast, the C-terminal domain of Mrpl36 is<br />

dispensable for protein synthesis but determines the fate of the synthesized<br />

proteins. In the absence of this latter domain, translation products fail to be<br />

assembled into respiratory chain complexes and are rapidly degraded. This<br />

observation is surprising as it indicates that mitochondrial ribosomes are not<br />

only critical for the production of mitochondrial translation products but also<br />

determine later steps in the assembly of respiratory chain complexes.<br />

63

64<br />

KI 02<br />

Translation elongation factor EF-Tu is part of the bacterial<br />

cytoskeleton<br />

H.J. Defeu Soufo 1 , C. Reimold 1 , U. Linne 2 , J. Gescher 1 , P. Graumann *1<br />

1 Biologie, Mikrobiologie, Universität Freiburg, Freiburg, Germany<br />

2 Chemie, Universität Marburg, Marburg, Germany<br />

EF-Tu has for long been proposed to play a role as a cytoskeletal element in<br />

bacteria, based on its ability to form filaments in vitro. We show that EF-Tu<br />

interacts with MreB, an actin-like cytoskeletal element present in many<br />

bacteria, in vitro and in vivo, and increases the ATPase activity of MreB.<br />

Consistent with this, EF-Tu leads to depolymerization of MreB filaments in<br />

vitro and in vivo, showing that it is an important modulator of MreB activity.<br />

EF-Tu localizes in a helical pattern in B. subtilis, and co-localizes with MreB,<br />

but in contrast to MreB, EF-Tu structures are rather static. Localization of<br />

MreB and of EF-Tu is interdependent, showing that indeed, EF-Tu is part of the<br />

cytoskeleton in bacteria. Lowering the levels of EF-Tu has a minor effect on<br />

translation, but a strong effect on cell shape and on the localization of MreB,<br />

supporting the idea that EF-tu plays a dual role in translation and in cell shape<br />

maintenance.<br />

KI 03<br />

Structural basis of N-end rule substrate recognition in<br />

Escherichia coli by the ClpAP adaptor protein ClpS<br />

V.J. Schuenemann *1 , S.M. Kralik 2 , R. Albrecht 1 , S.K. Spall 2 , K.N. Truscott 2 ,<br />

D.A. Dougan 2 , K. Zeth 1<br />

1 Department Protein Evolution, Max Planck Institute for Developmental<br />

Biology, Tuebingen, Germany<br />

2 Department for Biochemistry, La Trobe University, Melbourne, Australia<br />

In Escherichia coli the ClpAP protease, in concert with the adaptor protein<br />

ClpS, is responsible for the degradation of proteins bearing an N-terminal<br />

destabilizing amino acid (N-degron). Here we have determined the 3dimensional<br />

structures of ClpS in complex with three peptides, each bearing a<br />

different destabilising residue (Leu, Phe or Trp) at its N-terminus. All peptides,<br />

regardless of the identity of their N-terminal residue, are bound in a surface<br />

pocket on ClpS in a stereo-specific manner. Several highly conserved residues<br />

in this binding pocket interact directly with the backbone of the N-degron<br />

peptide and hence are critical for binding of all N-degrons. In contrast, two<br />

hydrophobic residues define the volume of the binding pocket and influence the<br />

specificity of ClpS. Collectively our data suggest that ClpS has been optimised<br />

for the binding and delivery of N-degrons bearing an N-terminal Phe or Leu.<br />

KI 04<br />

Spatial control of division site selection in Bacillus subtilis<br />

S. van Baarle *1 , M. Bramkamp 1<br />

1 Institut für Biochemie, Universität zu Köln, Koeln, Germany<br />

In bacteria, cell division is initiated when the tubulin homologue FtsZ<br />

polymerizes to form a ring-like structure, called the Z-ring, at midcell. In<br />

Bacillus subtilis, the placement of the Z-ring is regulated by two inhibitory<br />

systems. The nucleoid occlusion system prevents Z-ring formation over the<br />

nucleoid through the action of the DNA-binding protein Noc. The Min system<br />

suppresses division at the poles and consists of MinC, MinD and DivIVA,<br />

which are localized to the cell poles and the late septa. MinD is a membraneassociated<br />

protein which recruits MinC, the actual inhibitor of FtsZ<br />

polymerization, to the membrane. DivIVA is the topological factor for this<br />

system and in its absence, MinCD is no longer localized to the cell poles.<br />

However, a direct interaction between DivIVA and MinCD has never been<br />

shown. Recently, we identified a new component of the Min system (MinJ).<br />

This protein localizes to the cell poles and to septum in a DivIVA-dependent<br />

manner. In turn, localization of MinCD depends on the presence of MinJ.<br />

Bacterial two-hybrid assays showed that MinJ interacts with DivIVA and<br />

MinD, while both MinC and MinD do not interact with DivIVA. These data<br />

indicate that MinJ is a novel protein of the Min system, linking DivIVA to<br />

MinCD. Furthermore, analysis of minJ and divIVA mutants showed that their<br />

block in division occurs downstream of FtsZ. In conclusion, our data challenges<br />

the traditional view of the role of the Min system in cell division.<br />

KI 05<br />

Binding of SeqA to the Escherichia coli chromosome<br />

T. Waldminghaus *1 , K. Skarstad 1<br />

1 Institute for Cancer Research, Rikshospitalet-Radiumhospitalet-HF, Oslo,<br />

Norway<br />

The SeqA protein of Escherichia coli is involved in regulation of replication<br />

initiation and is also proposed to act in organization and segregation of<br />

daughter chromosomes. SeqA binds specifically to hemimethylated GATCsites<br />

that are produced during DNA-replication. The DNA remains<br />

hemimethylated until the Dam methyltransferase methylates the DNA fully.<br />

SeqA dependent regulation of replication initiation is based on its binding to<br />

recently replicated origins of replication. This prevents re-initiation of the new<br />

origins and also protects the origins from remethylation by Dam. While the role<br />

of SeqA in regulation of replication initiation has been investigated in detail its<br />

role in chromosome organization and segregation is poorly un<strong>der</strong>stood. There<br />

are about 20.000 GATC sites on the E. coli chromosome and these are potential<br />

SeqA binding sites. We analyzed binding of SeqA to all these sites using<br />

chromatin immunoprecipitation coupled with microarray analysis (ChIP on<br />

Chip). In addition we analyzed the methylation status of various chromosomal<br />

regions. SeqA was found to bind with different preferences to GATC sites<br />

depending on their grouping and spacing on the chromosomal DNA.<br />

Implications for chromosome organization and segregation are discussed.<br />

KI 06<br />

Proteins in the contact site of the two hyperthermophilic<br />

archaea, Ignicoccus hospitalis and Nanoarchaeum equitans<br />

T. Burghardt 1 , U. Küper 1 , F. Siedler 2 , R. Wirth 1 , H. Huber 1 , R. Rachel<br />

1 Dept of Microbiology, University of Regensburg, Regensburg, Germany<br />

2 Dept Membrane Biochemistry, MPI for Biochemistry, Martinsried, Germany<br />

The two Archaea Ignicoccus hospitalis and Nanoarchaeum equitans form a<br />

unique intimate association, the character of which is not yet fully un<strong>der</strong>stood.<br />

Ultrastructural investigations showed that I. hospitalis and N. equitans cells can<br />

be in direct contact via their surfaces [1]. Live-dead staining confirmed the tight<br />

physiological dependency between the two microorganisms [2]. Using cryopreparation<br />

methods, cells were shown to be preserved in a „close-to-native“<br />

state [3]. This permitted a more differentiated view of the contact site. At least<br />

two modes were observed: 1. Cell surfaces are close to each other but not<br />

always in direct contact, and some „fibrous material“ is found in the gap; in<br />

this case, the cytoplasmic membrane of I. hospitalis cells is involved in<br />

building up the contact. 2. Cell surfaces are in direct contact, and periplasmic<br />

vesicles of the I. hospitalis cell are involved in the formation of the contact site,<br />

while the cytoplasmic membrane does not contribute.<br />

For a deeper un<strong>der</strong>standing of the contact site, detailed biochemical knowledge<br />

of its composition is necessary. Therefore, an approach was launched aiming to<br />

isolate the contact site. Detergent-solubilized membrane proteins of a co-culture<br />

of I. hospitalis and N. equitans were purified using size exclusion<br />

chromatography. We obtained a fraction highly enriched in two proteins which<br />

are already known to be the major constituents of the cell envelopes, namely<br />

Ihomp1, the major outer membrane protein of I. hospitalis [4], and the S-layer<br />

protein of N. equitans. Most interestingly, this fraction also contains further, yet<br />

unknown membrane or cell envelope proteins [5], which are candidate<br />

molecules involved in the transport of metabolites between both<br />

microorganisms.<br />

[1] Huber et. al. 2002 Nature 417:63<br />

[2] Jahn et. al. 2008 J Bact 190:1743<br />

[3] Junglas et. al. 2008 Arch Microbiol 190:395<br />

[4] Burghardt et. al.2007 Mol Microbiol 63:166<br />

[5] Burghardt et. al. 2008 Arch Microbiol 190:379<br />

KI 07<br />

Mechanisms of subcellular DivIVA targeting in Bacillus<br />

subtilis<br />

S. Halbedel *1 , R. Breitling 2 , L.W. Hamoen 1<br />

1 Center for Bacterial Cell Biology, Institute for Cell and Molecular<br />

Biosciences, Newcastle University, Newcastle upon Tyne, United Kingdom<br />

2 Jena Bioscience GmbH, Jena, Germany<br />

DivIVA is a highly conserved cytoplasmic protein and functions in different<br />

morphogenetic and developmental processes in Gram-positive bacteria. In these<br />

organisms, DivIVA localizes to the site of cell division and to the cell poles.<br />

This localisation pattern is an important prerequisite for the biologic function of<br />

DivIVA also in Bacillus subtilis. In this organism, proper DivIVA localisation<br />

* 1<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

is essential for the correct placement of the cell division septum at midcell<br />

during vegetative growth. But it is also required for the transfer of one<br />

chromosome copy into the prespore compartment during sporulation. Genetic<br />

evidence has indicated that the N-terminal domain of DivIVA is important for<br />

DivIVA targeting to the poles and the division sites. Despite of this, little is<br />

known about the mechanisms of subcellular DivIVA targeting. We have<br />

adressed this question using different in vitro and in vivo approaches. Here, we<br />

will present data demonstrating that DivIVA associates with the cytoplasmic<br />

membrane. Membrane binding is presumably just one step in a more complex<br />

targeting pathway. So, we have hypothesized that any protein that is involved in<br />

this targeting process in vivo, should directly bind to DivIVA at a certain point<br />

during the cell cycle. We therefore used formaldehyde based cross-linking and<br />

subsequent affinity purification of cross-linked protein complexes to identify<br />

such interaction partners. We will present different genetic and cytological<br />

experiments that were used to test the contribution of individual interaction<br />

partners to the localisation and activity of DivIVA.<br />

KJ 01<br />

Communities of Shewanella oneidensis MR-1: what can we<br />

learn?<br />

K. Thormann *1<br />

1 Ökophysiologie, MPI für terrestrische Mikrobiologie, Marburg, Germany<br />

The perception that the majority of microorganisms in nature reside in surfaceassociated<br />

communities is almost as old as the field of microbiology itself.<br />

However, during the last two decades the interest in microbial communities,<br />

now often also referred to as biofilms, has increased enormously. Community<br />

formation enables tight cell-cell- and cell-surface-interactions, creating a<br />

heterogeneous environment. Microbes in biofilms exhibit drastically elevated<br />

resistance towards all kinds of environmental perturbations, including biocides<br />

and antibiotics, or predatory assaults. Despite extensive research on a number<br />

of different bacteria we are only beginning to un<strong>der</strong>stand the processes<br />

un<strong>der</strong>lying the structure and dynamics of bacterial communities.<br />

Biofilm formation occurs in several steps, from initial attachment to the rise of<br />

three-dimensional structures. This is enabled by production and/or excretion of<br />

extracellular polymeric substances, one hallmark during community<br />

development. Referring to studies on the metal-ion reducing bacterium<br />

Shewanella oneidensis MR-1, different aspects of biofilm formation, regulation<br />

and dynamics will be introduced and discussed.<br />

KJ 02<br />

Biofilm formation in Salmonella typhimurium: expression<br />

analysis of CsgD at the single cell level<br />

N. Grantcharova 1 , U. Römling *1<br />

1 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet,<br />

Stockholm, Germany<br />

Bacterial colonization and survival in different ecological niches often involves<br />

growth on surfaces and the formation of biofilm. The aggregation of the cells<br />

among each other as well as the adherence which enables surface colonisation<br />

is thereby mediated by the production of specific adhesions and extracellular<br />

structures like curli fimbriae or cellulose. In Salmonella enterica serovar<br />

Typhimurium the development of the multicellular behaviour is positively<br />

regulated by the transcription factor CsgD which itself is regulated by<br />

environmental signals, regulatory proteins and c-di-GMP signaling. So far little<br />

is known about the regulation and expression of CsgD within the biofilm, the<br />

interaction of CsgD with other proteins or its localization.<br />

We have constructed a translational CsgD-GFP fusion protein expressed from<br />

the native genomic locus of csgD un<strong>der</strong> its natural promoter. Fluorescence<br />

microscopy was used to study the expression of the CsgD-GFP at the single cell<br />

level un<strong>der</strong> a variety of environmental conditions in terms of media<br />

composition, temperature and oxygen availability. Preliminary results indicate<br />

the establishment of a differential pattern of csgD expression and a<br />

corresponding pattern of task distribution within the bacterial culture. Thus, the<br />

subpopulations of cells involved in forming biofilm structures, express higher<br />

amounts of csgD.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KJ 03<br />

Life Above Water for Uropathogenic Escherichia coli<br />

C. Hung *1 , J. Pinkner 1 , S. Hultgren 1<br />

1 Molecular Microbiology, Washington University School of Medicine, St.<br />

Louis, United States<br />

Bacteria living in an organized community within protective matrices fair better<br />

than their planktonic counterparts in surviving a harsh environment. Recently,<br />

bacterial biofilms have been consi<strong>der</strong>ed to play an important factor in human<br />

diseases, ranging from chronic wound to urinary tract infections (UTIs). In a<br />

diabetic murine UTI model, uropathogenic Escherichia coli (UPEC) was<br />

demonstrated to form organized structures within the infected kidney tubules.<br />

In vitro studies also showed that UPEC poses the ability to form biofilm un<strong>der</strong><br />

different environmental conditions.<br />

Methods: Prototypic UPEC, UTI89, and its isogenic mutants were grown in<br />

YESCA medium statically at 30°C. The ultra-structures of UPEC pellicles were<br />

examined by various microscopic techniques including confocal laser scanning,<br />

immunofluorescence, transmission electron and scanning electron<br />

microscopies.<br />

Results: Growth of the prototypic clinical UPEC isolate, UTI89, in YESCA<br />

medium at 30°C resulted in formation of a floating biofilm, termed pellicle.<br />

Antibody staining, transcriptional fusion reporter assay and isogenic mutant<br />

analyses indicated that curli plays a critical role in the development of UTI89<br />

pellicles. In addition, mutational studies also implicated cellulose and flagella<br />

to be integral parts of the pellicle development. Microscopic examination of<br />

UTI89 pellicles revealed complex structures in this type of bacterial biofilm.<br />

Analyses with other clinical UPEC isolates indicated that pellicle biofilm<br />

formation phenotype, although not ubiquitous, is also a characteristic of many<br />

UPEC isolates.<br />

Conclusions: Biofilm formation is a critical part of UPEC pathogenesis. This<br />

study showed the contribution of curli, cellulose and flagella in the<br />

development of UPEC in vitro biofilm.<br />

KJ 04<br />

3D Structure of the Archaea Ignicoccus hospitalis and<br />

Nanoarchaeum equitans, as determined by serial section<br />

electron microscopy<br />

T. Heimerl 1 , N. Wasserburger 1 , C. Meyer 1 , T. Burghardt 1 , B. Junglas 1 , H.<br />

Huber 2 , R. Wirth 2 , R. Rachel *1<br />

1 Centre for Electron Microscopy, and Inst. for Microbiology, University of<br />

Regensburg, Regensburg, Germany<br />

2 Institute for Microbiology, University of Regensburg, Regensburg, Germany<br />

The hyperthermophilic Archaeon Ignicoccus hospitalis has a unique CO2<br />

fixation pathway [1] and is the only known host for the smallest Archaeon,<br />

Nanoarchaeum equitans [2]. These two Archaea form an intimate association;<br />

it is not clear yet to which extent N. equitans cells have damaging effect to their<br />

host cells [3]. N. equitans is not able to synthesize lipids, amino acids, and<br />

nucleotides, according to physiological and genome studies [4]. Therefore, the<br />

interaction of these cells with I. hospitalis is of high interest, in or<strong>der</strong> to<br />

un<strong>der</strong>stand, how cells specifically recognize each other and how metabolite<br />

transport is organized. Labelling studies showed that archaeal lipids [5] and<br />

amino acids are transported from I. hospitalis to N. equitans [3].<br />

In or<strong>der</strong> to achieve the best preservation of the delicate contact site, we used<br />

cultivation in capillary tubes, cryo-fixation, freeze-substitution and resin<br />

embedding; serial sections were visualized by transmission electron<br />

microscopy, and data aligned and visualized [6]. In about 75% of the cells, the<br />

cytoplasm of I. hospitalis cells is involved in the interaction; in about 25%,<br />

periplasmic vesicles appear to be unloaded at the contact site, possibly releasing<br />

´cargo´ to the N. equitans cell. Immuno-localisation confirms that the Ihomp1<br />

protein of the I. hospitalis outer membrane [7] and the S-layer of N. equitans<br />

are present at and involved in the contact site. Biochemical studies helped to<br />

identify further proteins which might be relevant for cell-cell interaction and/or<br />

metabolite transport, like components of ABC transporters [8]. They are in the<br />

focus of ongoing studies on the contact site.<br />

[1] Huber et al. 2008 PNAS 105:7851<br />

[2] Huber et al. 2002 Nature 417:63<br />

[3] Jahn et al. 2008 J Bact 190:1743<br />

[4] Waters et al. 2003 PNAS 100:12984<br />

[5] Jahn et al. 2004 Arch Microb 182:404<br />

[6] Junglas B et al. 2008 Arch Microb 190:395<br />

[7] Burghardt et al. 2007 Mol Microb 63:166<br />

[8] Burghardt et al. 2008 Arch Microb 190:379<br />

65

66<br />

KJ 05<br />

Comparison of methods for simultaneous identification of<br />

bacterial species and determination of metabolic activity by<br />

protein-based stable isotope probing (Protein-SIP)<br />

experiments<br />

M. Taubert *1 , F. Schmidt 1,3 , N. Jehmlich 1 , M. von Bergen 1 , H.H. Richnow 3 , C.<br />

Vogt 3<br />

1<br />

Department of Proteomics, Helmholtz Centre for Environmental Research -<br />

UFZ, Leipzig, Germany<br />

2<br />

Interfaculty Institute for Genetics and Functional Genomics, University of<br />

Greifswald, Greifswald, Germany<br />

3<br />

Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental<br />

Research - UFZ, Leipzig, Germany<br />

By employing protein-based stable isotope probing (Protein-SIP) in metabolic<br />

labelling experiments with stable isotope labelled substrates, a concept for<br />

analyzing carbon and nitrogen flux in microbial communities was developed.<br />

For identification of microbial species, intact protein profiling (IPP) was used,<br />

whereas the assessment of their metabolic activity had been achieved by<br />

shotgun mass mapping (SMM). Microbial cultures were grown on substrates<br />

containing 13 C or 15 N. For the identification of species we tested both the IPP<br />

and SMM approach. Mass spectra (MALDI-MS) were taken from mixtures of<br />

either intact proteins or of peptides from tryptic digestion for generating<br />

species-specific peak patterns. In case of SMM, fragmentation of peptides was<br />

additionally used to obtain sequence information for species identification.<br />

Mass spectra of peptide sequences allowed calculation of the amount of 13 C or<br />

15 N incorporation within peptides for determining metabolic activity of the<br />

specific species. Comparison of IPP and SMM revealed a higher robustness of<br />

species identification by SMM. In addition, the assessment of incorporation<br />

levels of 13 C and 15 N into peptides revealed a lower uncertainty (0.4-6.3 atom<br />

%) compared to IPP (1.0-8.9 atom %). Allowing determination of metabolic<br />

activity and function of specific species, Protein-SIP has proved as a valuable<br />

tool for analysis of carbon and nitrogen flux within microbial communities.<br />

KJ 06<br />

Sulfate reducing bacteria associated with ANME2aggregates<br />

L. Schreiber *1 , A. Meyerdierks 1 , K. Knittel 1 , B. Fuchs 1 , R. Amann 1<br />

1<br />

Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen,<br />

Germany<br />

Microbial consortia mediating the anaerobic oxidation of methane (AOM) with<br />

sulfate have been shown to consist of anaerobic methanotrophic (ANME)<br />

archaea and sulfate reducing bacteria (SRB). Phylogenetically, ANME are not<br />

monophyletic and form different groups within the methanogenic archaea.<br />

Depending on the ANME group (ANME-1, ANME-2 or ANME-3), the<br />

associated bacteria have been identified to be closely related to the<br />

deltaproteobacterial genera Desulfosarcina, Desulfococcus, or Desulfobulbus.<br />

In our study we focused on the sulfate reducing partner of ANME-2 which was<br />

proposed to belong to the Desulfosarcina-related SEEP/SRB-1 group. The<br />

group comprises 16S rRNA sequences predominantly found at methane seeps.<br />

Applying phylogenetic analysis, we defined six subgroups (A-F) within the<br />

SEEP/SRB-1 group and designed a set of 16S rRNA-targeted oligonucleotide<br />

probes covering the subgroups. These probes were used for fluorescence in situ<br />

hybridization (FISH) studies on ANME-2 enrichment cultures from the<br />

sediment above gas hydrates at Hydrate Ridge (Cascadia margin, NE Pacific)<br />

and from sediment at the Isis mud volcano (Eastern Mediterranean). We<br />

observed that in both enrichments the SRB which were in direct contact with<br />

ANME-2 cells in shell type consortia, belong to only one specific SEEP/SRB-1<br />

subgroup. Ongoing FISH-studies on environmental samples from different<br />

AOM hot spots will reveal whether the association between this group and<br />

ANME-2 is of a general nature, and how abundant SEEP/SRB-1 bacteria of the<br />

different subgroups are at these sites.<br />

KJ 07<br />

Chemotaxis toward inorganic phosphate – an adaptive<br />

strategy of marine bacteria with implications for nutrient<br />

cycling in the ocean<br />

A. Hütz *1 , M. Mayer 1 , K. Schubert 1 , J. Overmann 1<br />

1 Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-<br />

Universität München, München, Germany<br />

Very low in situ phosphate concentrations characterize the Eastern<br />

Mediterranean Sea – one of the most oligotrophic habitats on Earth.<br />

Chemotaxis toward inorganic phosphate would constitute one strategy to cope<br />

with low concentrations of the growth-limiting nutrient since nutrient "hot<br />

spots" can be exploited. During Meteor cruise M71/3 chemotaxis experiments<br />

revealed that marine bacteria significantly accumulated in capillaries containing<br />

phosphate. The response toward phosphate was in general much faster than<br />

toward organic carbon substrates. Phylogenetic analyses of the chemotactically<br />

enriched bacterioplankton showed a dominance of the genera Alteromonas,<br />

Pseudoalteromonas, Vibrio, Alcanivorax and Thalassospira. Of these<br />

Thalassospira exhibited an exclusive response toward inorganic phosphate and<br />

thus was selected as a model system for the study of this so far largely<br />

unknown type of chemotaxis. A corresponding isolate of Thalassospira from<br />

the Eastern Mediterranean Sea as well as the described species Thalassospira<br />

profundimaris and Thalassospira lucentensis showed chemotaxis toward<br />

phosphate also in pure cultures. Phosphate starved bacteria accumulated in<br />

capillaries containing phosphate within 1.5 hours and were able to detect<br />

phosphate concentrations lower than 1 µM. Although Thalassospira sp.<br />

represents only up to 0.5% of the total bacterioplankton community in the water<br />

column of the Eastern Mediterranean Sea, its chemotactic behavior possibly<br />

leads to an acceleration of nutrient cycling in the open ocean and explains the<br />

competitive success of non ultraoligotrophic bacteria in this extremely nutrient<br />

limited environment.<br />

KK 01<br />

The SILVA project for comprehensive, quality checked and<br />

aligned ribosomal RNA databases<br />

E. Prüße *1 , C. Quast 1 , K. Dietrich 1 , K. Knittel 2 , B. Fuchs 2 , W. Ludwig 3 , J.<br />

Peplies 4 , F.O. Glöckner 1<br />

1<br />

Microbial Genomics Group, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

2<br />

Department of Molecular Ecology, Max Planck Institute for Marine<br />

Microbiology, Bremen, Germany<br />

3<br />

Department of Microbiology, Technische Universität München, München,<br />

Germany<br />

4<br />

Research and Development, Ribocon GmbH, Bremen, Germany<br />

Sequencing ribosomal RNA (rRNA) genes is currently the method of choice for<br />

phylogenetic reconstruction, nucleic acid based detection and quantification of<br />

microbial diversity. The exponential increase of publicly available rRNA<br />

sequence data has ren<strong>der</strong>ed the maintenance of rRNA databases difficult over<br />

the last years. To cope with the deluge of data, the SILVA system (from Latin<br />

silva, forest) was implemented to provide a comprehensive web resource for<br />

up-to-date, quality controlled databases of aligned rRNA sequences from the<br />

Bacteria, Archaea and Eukarya domains.<br />

All databases are fully compatible with the worldwide used ARB software<br />

package for sequence analysis and probe design. A novel incremental, dynamic<br />

profile sequence aligner (SINA) was developed for rapid and accurate sequence<br />

alignment. The system is able to align more than 1,000,000 SSU sequences<br />

overnight using our manually curated, high quality seed alignment as the<br />

reference. All sequences are checked for anomalies using the Pintail software<br />

and carry a rich set of process information. An intuitive quality ranking allows<br />

the user to get a rapid overview about the sequence and alignment quality. The<br />

databases are designed as a central comprehensive resource by integrating<br />

multiple taxonomic classifications taken from RDP II and greengenes and the<br />

latest validly described nomenclature provided by the German Collection of<br />

Microorganisms and Cell Cultures (DSMZ). Information, such as whether a<br />

sequence was <strong>der</strong>ived from a cultivated organism, a typestrain, or belongs to a<br />

genome project has been integrated from the straininfo.net bioportal, the Living<br />

Tree project and EMBL.<br />

As of release 96, October 2008, SILVA hosts a total of 886,153 sequences split<br />

into 756,668 small subunit and 129,485 large subunit rRNAs. The databases<br />

can be accessed at www.arb-silva.de.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KK 02<br />

Methanogenic Community in Terrestrial and Submarine<br />

Permafrost of the Siberian Laptev Sea Region<br />

K. Koch *1 , D. Wagner 1<br />

1<br />

Research Department Potsdam, Alfred Wegener Institute for Polar and Marine<br />

Research, Potsdam, Germany<br />

Global warming contributes to the degradation of permafrost-affected soils and<br />

perennially frozen sediments in Arctic Polar Regions. The subsequent emission<br />

of methane from thawing carbon rich permafrost sediments has been<br />

acknowledged as a significant contribution to the global carbon cycle. In or<strong>der</strong><br />

to un<strong>der</strong>stand the effect of increasing temperature on the carbon dynamics in<br />

climate-sensitive arctic permafrost environments, the characterization of<br />

methanogenic communities was conducted with submarine permafrost,<br />

characterized by a mean temperature of -2°C, in comparison to its terrestrial<br />

counterpart with a mean temperature of -12°C. Methane concentrations varied<br />

along the submarine permafrost sediments between 0 and 284 nmol CH4 g -1 ,<br />

and along the terrestrial permafrost deposits between 0.4 and 40 nmol CH4 g -1 .<br />

Archaeal 16S rRNA clone libraries were constructed for samples taken at three<br />

depths from both the terrestrial and submarine permafrost. Sequencing and<br />

phylogenetic analyses of representative clones showed that most of the rRNAs<br />

were closely related to Methanosarcinales. For quantitative analysis of bacteria<br />

and methanogenic archaea the TaqMan gene expression assay for real time<br />

PCR was designed. Additionally, to the real-time PCR quantifications, direct<br />

cell counts and fluorescent in situ hybridization (FISH) were applied. The<br />

observed differences in the methanogenic community structure in submarine<br />

permafrost sediments in comparison to its terrestrial counterpart may result<br />

from the up to 10°C higher in situ temperature of submarine permafrost.<br />

Therefore, submarine permafrost is consi<strong>der</strong>ed as an ideal natural laboratory to<br />

study the impact of changing environmental conditions, particularly increasing<br />

permafrost temperatures, on the structure and function of microbial<br />

communities in climate-sensitive permafrost habitats.<br />

KK 03<br />

Deep sequencing reveals exceptional diversity and<br />

alternative lifestyles of marine bacterial sponge symbionts<br />

F. Behnam *1 , M. Horn 1 , M. Taylor 2 , S. Luecker 1 , S. Whalan 3 , T. Rattei 4 , N.<br />

Webster 3 , M. Wagner 1<br />

1<br />

Department of Microbial Ecology, University of Vienna, Vienna, Austria<br />

2<br />

School of Biological Sciences, University of Auckland, Auckland, New Zealand<br />

3<br />

Microbiology and Symbiosis, Australian Institute of Marine Science,<br />

Townsville, Australia<br />

4<br />

Department of Genome Oriented Bioinformatics, Technische universität<br />

München, Munich, Germany<br />

Marine sponges (phylum Porifera) contain complex bacterial communities of<br />

consi<strong>der</strong>able ecological and biotechnological importance, with many of these<br />

organisms postulated to be specific to sponge hosts. Testing this hypothesis in<br />

light of the recent discovery of the rare microbial biosphere, three Australian<br />

sponges were investigated by massively parallel 16S rRNA gene tag<br />

pyrosequencing. More than 250,000 sponge-<strong>der</strong>ived sequence tags were<br />

assigned to 28 bacterial phyla and revealed up to 2996 operational taxonomic<br />

units (95% sequence similarity) per sponge species. While 48% of the 33<br />

detected, previously described "sponge-specific" cluster were found exclusively<br />

in adults and larvae -implying strict vertical transmission - the remaining taxa,<br />

including "Poribacteria", were also found at very low abundance among the<br />

135,000 tags retrieved from surrounding seawater. Thus, members of the rare<br />

seawater biosphere may serve as seed organisms for widely occurring symbiont<br />

populations in sponges and their host association might have evolved much<br />

more recently than previously thought. In or<strong>der</strong> to look for phylogenetic<br />

novelty within the tag sequences which could not be assigned to known phyla<br />

(similarity to curated SILVA database was below 75%) specific primers were<br />

designed and applied together with a conserved bacterial forward primer. All<br />

amplified 16S rRNA gene fragments (~1000-bp) had no closer relatives in the<br />

database used for the assignment and some of the retrieved sequences showed<br />

similarities of below 85% to all publically available 16S rRNA gene sequences.<br />

These findings demonstrate that at least some of the non-assignable tag<br />

sequences might be indicative for members of new bacterial phyla.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KK 04<br />

Microbial diversity in Arctic polygonal tundra soils<br />

S. Liebner *1 , T. Stührmann 2 , J. Har<strong>der</strong> 3 , J. Zeyer 1 , D. Wagner 4<br />

1<br />

Institute for Biogeochemistry and Pollutant Dynamics, Group of<br />

Environmental Microbiology, ETH Zurich, Zurich, Switzerland<br />

2<br />

Group of Molecular Ecology, MPI for Marine Microbiology, Bremen,<br />

Germany<br />

3<br />

Group of Microbiology, MPI for Marine Microbiology, Bremen, Germany<br />

4<br />

Research Department Potsdam, Alfred Wegener Institute for Polar and Marine<br />

Research, Potsdam, Germany<br />

Together with alpine tundra, Arctic tundra covers 7.4 % of the northern<br />

hemisphere’s land area. Arctic, permafrost-affected tundra exposed to seasonal<br />

freezing and thawing thereby tends to form polygonal patterns. These polygons<br />

display small-scale heterogeneities regarding water and energy regime,<br />

morphology, methane and oxygen concentrations. Little research has been done<br />

on what shapes microbial diversity in Arctic polygonal tundra soils. Based on<br />

molecular techniques we, therefore, investigated how site specific gradients in<br />

these environments affect structure and diversity of microbial communities.<br />

In a low-centred polygon on Samoylov Island, Lena Delta, overall bacterial<br />

diversity was high exceeding that of boreal forest and tundra. It, however,<br />

decreased towards the permafrost table. The bacterial community structure of<br />

the aerobic near surface of a polygon rim thereby varied significantly from<br />

those near the permafrost table and a water-saturated polygon centre [1].<br />

Contrary to the entire bacterial community, aerobic methanotrophic<br />

Proteobacteria (AMP), a physiologically and phylogenetically narrow group of<br />

bacteria, were little diverse on the genus level but formed micro-diverse<br />

clusters closely related to known psychrophilic and psychrotrophic organisms<br />

(Mb. psychrophilus and Mb. tundripaludum) isolated from the Arctic. Despite<br />

vertically decreasing temperatures and oxygen concentrations and an increase<br />

in pore-water methane content, the diversity of AMP remained constant<br />

towards the permafrost table [2]. We suggest that the extreme gradients of<br />

Arctic polygonal tundra enable diverse but site-specific and specialized<br />

bacterial communities to be established.<br />

[1] Liebner et. al. (in press) Microb Ecol DOI: 10.1007/s00248-008-9411-x<br />

[2] Liebner et. al. (2008) Intern Microbiol 11: 195-202<br />

KK 05<br />

New insights into the microbial diversity in spacecraft<br />

assembly clean rooms and the impact on planetary<br />

protection<br />

M. Stieglmeier 1 , C. Moissl-Eichinger *1<br />

1 Lehrstuhl für Mikrobiologie, Universität Regensburg, Regensburg, Germany<br />

One main task of Planetary Protection programs of NASA and ESA is to<br />

prevent the forward contamination of extraterrestrial environments with Earth<br />

life, preserving the integrity of future life detection missions. The knowledge<br />

and profound study of the spacecraft assembly clean room flora is an important<br />

prerequesite to avoid contaminations as far as possible.<br />

Facing ESA’s ExoMars mission, a biodiversity study of European assembly<br />

facilities and the Herschel space observatory located therein was performed.<br />

Molecular screenings revealed the presence of a broad variety of mainly<br />

human-associated Bacteria, and also eury- and crenarchaeal signatures were<br />

detected in all clean rooms.<br />

Despite the desiccated, nutrient-bare conditions within these clean rooms many<br />

Bacteria were cultivated, most of them capable of tolerating extreme<br />

conditions: facultatively anaerobic, autotrophic and nitrogen fixing Bacteria<br />

were detected, and, for the first time, also strictly anaerobic Bacteria were<br />

isolated from spacecraft harbouring clean rooms. These capabilities play a<br />

significant role in Planetary Protection consi<strong>der</strong>ations, since extraterrestrial<br />

environments like Mars do not provide organic carbon and nitrogen, or enough<br />

oxygen for full aerobic microbial growth. Our multi assay cultivation approach<br />

was the basis for the detection of Bacteria, that have not been cultivated from<br />

these special environments before, and led also to the discovery of two novel<br />

species.<br />

Our isolates, as well as the detection of archaeal 16S rRNA genes shows the<br />

presence of an extraordinary diverse microbial community in spacecraft<br />

assembly clean rooms and our findings will impact futural studies of these<br />

environments and space agency’s Planetary Protection consi<strong>der</strong>ations.<br />

67

68<br />

KK 06<br />

Ultrastructure, tactic behaviour and potential for sulfate<br />

reduction of a novel multicellular magnetotactic prokaryote<br />

from North Sea sediments<br />

R. Wenter *1 , G. Wanner 2 , D. Schüler 1 , J. Overmann 1<br />

1 Department Biologie I, Bereich Mikrobiologie, Ludwig-Maximilians-<br />

Universität München, München, Germany<br />

2 Department Biologie I, Bereich Botanik, Ludwig-Maximilians-Universität<br />

München, München, Germany<br />

Multicellular magnetotactic prokaryotes (MMPs) represent highly organised,<br />

spherical and motile aggregates of 10-40 bacterial cells, each with its own<br />

magnetosomes and flagellation. They orient themselves within a magnetic field<br />

and exhibit magnetotaxis. MMPs are most closely affiliated with dissimilatory<br />

sulfate reducing Desulfosarcina variabilis of the family Desulfobacteraceae.<br />

These unique microorganisms may serve as future model system for studing<br />

bacterial multicellularity and interaction. In coastal tidal sand flats of the North<br />

Sea, MMPs were found for the first time outside North and South America.<br />

Electron microscopy revealed the presence of bullet-shaped magnetosomes<br />

which are aligned in several parallel chains and oriented in the same direction<br />

across the entire aggregate. Energy dispersive X-ray analysis showed that the<br />

magnetosome crystalls were composed of an iron sulfide mineral. This<br />

particular morphology, arrangement and composition of magnetosomes has not<br />

been observed previously. The 16S rRNA gene sequence of MMPs from the<br />

North Sea exhibited phylogenetic distance of more than 4% to all other<br />

bacteria. Fluorescence in situ hybridisation demonstrated that the entire<br />

population of MMPs and all individual cells of the same multicellular aggregate<br />

belonged to the same phylotype. Genes for dissimilatory sulfite reductase<br />

(dsrAB) and dissimilatory adenosine-5´-phosphate reductase (aprA) were<br />

detected in magnetotactically purified MMP samples, suggesting that MMPs<br />

are capable of sulfate reduction. Chemotaxis assays with 41 test substances<br />

yielded strong responses towards acetate and propionate, which are typical<br />

growth substrates for sulfate-reducing bacteria. These newly-discovered<br />

properties of the novel MMP confer in a selective advantage over competing<br />

sulfate-reducers in stratified marine sediments.<br />

KK 07<br />

Assessment of the yeast diversity in soils un<strong>der</strong> different<br />

land use management<br />

A. Yurkov *1 , M. Kemler 1 , D. Begerow 1<br />

1 AG Geobotanik, Ruhr University Bochum, Bochum, Germany<br />

Sustainable agriculture and forestry are completely dependent on the soil they<br />

are growing on. The acquisition of nutrients by plants thereby heavily relies on<br />

a microbial network in the soil containing bacteria, animals and fungi. By<br />

decomposing plant residues fungi play an important role in the carbon cycle.<br />

Yeasts represent a formal group of fungi designated by the presence of<br />

unicellular in their life cycle. Studies in the last decade have shown that the<br />

contribution of yeasts to soil processes is greater than has been expected. Also,<br />

the diversity of that group of fungi has significantly increased due to molecular<br />

techniques in species delimitation. Yet it is supposed that less than 10% of the<br />

estimated yeast species are discovered.<br />

Studies addressing soil yeast diversity and ecology in Middle Europe are very<br />

scrappy. Therefore, we have studied yeast fungi in various soils (within the<br />

scope of the „German biodiversity exploratories“) by combining conventional<br />

plating techniques and culture independent methods. The culture-based<br />

approach was evaluated statistically to provide quantitative analysis of the soil<br />

yeast community and species diversity was estimated additionally by direct<br />

DNA extraction and subsequent cloning. Thereby we were able to culture more<br />

than 30 different yeast species. The species regularly observed in soil are likely<br />

to be the autochthonous soil microflora. Some of these yeasts are rarely<br />

reported in the early studies either due to misidentification or association with<br />

the soils of studied territory. Additionally, several new taxa have been isolated.<br />

In total about 70% of the isolates were able to assimilate lignino-cellulosic<br />

compounds. That implies involvement of yeasts in carbon cycle via conversion<br />

of woody compounds into soil organic matter. Significant changes of the yeast<br />

communities’ structures, species evenness in particular, along the land use<br />

gradient have been observed. A common notable characteristic of managed<br />

territories is a higher abundance of ascomycetous yeasts. Our results suggest<br />

yeast fungi to be promising tool displaying human impact on the soil microbial<br />

community.<br />

KL 01<br />

The Gram-positive sugar toppings - teichoic acids and<br />

related cell wall glycopolymers in staphylococcal physiology<br />

and host interaction<br />

A. Peschel *1<br />

1<br />

Cellular and Molecular Microbiology, University of Tübingen, Tübingen,<br />

Germany<br />

Staphylococcus aureus and most other Gram-positive bacteria contain<br />

characteristic membrane- or peptidoglycan-attached carbohydrate-based<br />

polymers in their cell walls whose structure, function, and biosynthesis is only<br />

superficially un<strong>der</strong>stood. The composition of these cell wall glycopolymers<br />

(CWG) is highly variable and often species and strain specific. CWGs have<br />

been implicated in adherence to host cells, activation of innate or adaptive<br />

immune responses and susceptibility to antibiotics and antimicrobial host<br />

defenses. Accordingly, the peptidoglycan-anchored wall teichoic acid (WTA)<br />

plays a crucial role in S. aureus nasal colonization, in endovascular infections,<br />

and in biofilm formation. CWGs are also thought to be important in bacterial<br />

physiology by controlling autolytic enzymes, storing divalent cations, or<br />

attaching surface proteins. Recent studies have yielded a growing picture of the<br />

biosynthetic pathways of WTA and the membrane anchored lipoteichoic acid<br />

(LTA). Accordingly, CWG represent a very important topic for research that<br />

may help to identify candidate targets for new antimicrobials, vaccines, and<br />

diagnostics.<br />

KL 02<br />

The role of wall teichoic acid of S. aureus in resistance<br />

against antimicrobial fatty acids from the human skin<br />

T. Kohler *1 , C. Weidenmaier 1 , A. Peschel 1<br />

1 Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Tübingen,<br />

Tübingen, Germany<br />

S. aureus is a well known cutaneous pathogen and nose colonizer in human. It<br />

was shown, that the colonization with S. aureus is a major risk factor for an<br />

endogenous infection with the own strain. Therefore it is crucial to un<strong>der</strong>stand<br />

the different factors which are involved in the process of colonization. The<br />

human skin is the first barrier against bacteria. And furthermore the innate<br />

immunity is well equipped against bacterial intru<strong>der</strong>s: Physically with highly<br />

crosslinked keratin and chemically with antimicrobial peptides and fatty acids.<br />

Staphylococcus aureus colonizes affected skin in nearly all patients with atopic<br />

<strong>der</strong>matitis and this could be related to a altered fatty acid composition on the<br />

skin as shown before.<br />

The hydrophobicity of the cell wall of S. aureus seems to play an important role<br />

in resistance against antimicrobial fatty acids and monoacylglycerols from the<br />

human skin. Here we show, that the loss of wall teichoic acid leads to an<br />

increase in hydrophobicity and therefore to more sensitivity against<br />

antimicrobial fatty acids and furthermore to an increase in binding of<br />

fluorescent labelled fatty acids.<br />

KL 03<br />

In contrast to human isolates, animal isolates of S.<br />

saprophyticus subsp. saprophyticus are not internalized into<br />

human urinary blad<strong>der</strong> carcinoma cell line 5637<br />

F. Szabados *1 , S. Rodepeter 1 , B. Heck 1 , A. Albrecht 1 , B. Kleine 1 , M. Kaase 1 , S.<br />

Gatermann 1<br />

1 Department of Medical Microbiology, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

Introduction: Uropathogenic E. coli (UPEC) and S. saprophyticus may cause<br />

acute and recurrent urinary tract infections (UTI). Internalization is described in<br />

uropathogenic E.coli as an important pathogenicity factor of acute and recurrent<br />

infections. Internalization into the urothelial carcinoma cell line 5637 was<br />

described for both uropathogens recently. For S. saprophyticus an animal<br />

reservior was discussed in the disease propagation. We therefore investigated<br />

the ability of human and animal isolates of S. saprophyticus spp. saprophyticus<br />

for internalization into the urinary blad<strong>der</strong> carcinoma cell line 5637 using a<br />

FACS-based method.<br />

Methods: S. saprophyticus ATCC 15305, S. saprophyticus wildtype strain<br />

7108,S. aureus Cowan I, S. carnosus TM300 were used as controls in a FACSinternalization-assay<br />

as described previously. Several animal (n=8) and human<br />

clinical isolates of S. saprophyticus spp. saprophyticus (n=11) were tested in<br />

this study.The mean fluoresence of S. saprophyticus spp. saprophyticus was<br />

normalized to the mean fluorescence of noninvasive S. carnosus TM300.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Results: We could show that human isolates of S. saprophyticus spp.<br />

saprophyticus in contrast to animal isolates were internalized into urinary<br />

blad<strong>der</strong> carcinoma cell line 5637. Human isolates have a significant higher (<br />

5,6-fold, p< 0.01 ) internalization compared to animal isolates.<br />

Conclusion: Recurrent urinary tract infections due to S. saprophyticus spp.<br />

saprophyticus could be explained by internalization of the uropathogen into the<br />

uroepithelial cell. The lack of internalization of animal isolates suggests a<br />

revision of the infection propagation model in S. saprophyticus spp.<br />

saprophyticus, where animals are described as an important reservoire.<br />

KL 04<br />

The type IV DNA secretion system of Neisseria gonorrhoeae<br />

E. Pachulec 1 , S. Jain 1 , W. Salgado-Pabón 2 , J. Dillard 2 , M. Valevich 3 , E.M.<br />

Heller 3 , C. Van <strong>der</strong> Does *3<br />

1 Molecular Microbiology, University of Groningen, Groningen, Netherlands<br />

2 Medical Microbiology and Immunology, University of Wisconsin, Madison,<br />

United States<br />

3 Ecophysiology, Max-Planck-Institute for terrestrial Microbiology, Marburg,<br />

Germany<br />

In Gram negative bacteria, substrates secreted into the medium have to cross<br />

both the inner and the outer membrane. The versatile family of type IV<br />

transporters, involved in one of the specialized pathways for secretion across<br />

the outer membrane has been extensively studied in recent years (eg, the Fplasmid<br />

conjugation in Escherichia coli and the VirD4/B system of<br />

Agrobacterium tumefaciens). The assembly of the complex, the mechanism of<br />

transport and the energy usage at molecular level is still not well defined. This<br />

system consists of a large complex of 12 to 25 proteins which spans both<br />

membranes and extends a pilus structure which attaches to the recipient cell. In<br />

the case of DNA conjugation systems, a protein, the relaxase, is covalently<br />

coupled to the DNA. This protein-DNA complex is transferred via the type IV<br />

machinery to the recipient cell. Recently, a gonococcal genetic island (GGI) has<br />

been identified in the human pathogen Neisseria gonorrhoeae which has<br />

similarity to the F plasmid conjugation system. This type IV secretion system<br />

was found to secrete DNA directly into the medium. The secreted DNA is then<br />

taken up We aim to decipher the mechanism of substrate transport by the type<br />

IV secretion machinery of N. gonorhoeae.<br />

An extensive mutagenesis study on all the genes encoded within the GGI<br />

revealed remarkable features of this type IV DNA secretiuon machinery. We<br />

will also the mechanism of the assembly the pilus, a structure which consists of<br />

remarkable circular proteins, and the function of the pilus in the transport of<br />

DNA and proteins.<br />

KL 05<br />

The genomic mutS-rpoS region of extraintestinal pathogenic<br />

E. coli (ExPEC): a minimal predictor of phylogenetic<br />

background and in vivo pathogenicity<br />

C. Ewers *1 , T. Homeier 1 , L.H. Wieler 1<br />

1 Institute for Microbiology and Epizootics, Freie Universität Berlin, Berlin,<br />

Germany<br />

The chromosomal mutS-rpoS region in Escherichia coli is subjected to genetic<br />

exchange during the evolution of pathogenic lineages, and high levels of<br />

variation in this genomic region have been suggested to enforce the emergence<br />

of E. coli pathogens.<br />

The characterization of this genomic region in 442 extraintestinal pathogenic E.<br />

coli (ExPEC), and 186 commensal as well as environmental E. coli strains of<br />

human and animal source by PCR revealed a great variability of the upstream<br />

fhlA-mutS and the downstream o454-nlpD region un<strong>der</strong>lining the high genetic<br />

and phylogenetic diversity within these groups of strains. One distinct pattern<br />

could be designated as "extraintestinal pathogenic mutS-rpoS pattern", as<br />

irrespective of their host origin and clinical history E. coli strains exhibiting this<br />

pattern possessed high numbers of ExPEC-related virulence genes and in<br />

addition were highly pathogenic in a chicken infection model in contrast to<br />

strains harbouring other mutS-rpoS patterns. Thus, our data give strong<br />

evidence for a direct link between the mosaic structure of this genomic region<br />

not only with the virulence gene profile and phylogenetic background of a<br />

strain but, what is of utmost importance, with its in vivo pathogenicity. The<br />

mutS-rpoSregion could therefore be of great value in identifying highly<br />

extraintestinal virulent strains among the mixed population of E. coli promising<br />

to be the basis of a future typing tool for ExPEC and the identification of their<br />

reservoir.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KL 06<br />

Dissection of the invasion process of Candida albicans<br />

B. Wächtler 1 , F. Dalle 2 , B. Hube *1<br />

1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for<br />

Natural Product Research and Infection Biology -Hans Knöll Institute (HKI),<br />

Jena, Germany<br />

2 Microbiologie Médicale et Moléculaire (EA562), Faculté de Médecine,<br />

Hôpital du Bocage, Dijon, France<br />

Candida albicans is the most common opportunistic fungal pathogen of<br />

humans which may also cause systemic infections in immunocompromised<br />

patients. The mechanisms by which C. albicans can invade epithelial barriers in<br />

or<strong>der</strong> to penetrate into deeper tissue and disseminate within the host is only<br />

partially un<strong>der</strong>stood.<br />

It has been shown that C. albicans is capable of adhering, invading and<br />

damaging oral epithelial cells during oral infections. Two different invasion<br />

mechanisms have been proposed: induced endocytosis and active penetration.<br />

Binding of distinct cell surface components of C. albicans to host cell surface<br />

receptors leads to host actin rearrangements and subsequent uptake of the<br />

fungus. Active penetration includes physical forces of hyphae and the<br />

production of secreted aspartic proteases (Saps). Although filamentation seems<br />

to be necessary for C. albicans to invade oral epithelial cells, activity from the<br />

fungus is dispensable for induced endocytosis because killed fungi are still<br />

endocytosed. However, damage of epithelial cells by killed hyphae is<br />

dramatically reduced.<br />

To elucidate which fungal factors contribute to adhesion, invasion and damage,<br />

we quantified the cellular invasion of different mutants with defects in<br />

morphology, signalling pathways, surface proteins associated with hyphal<br />

formation, or secreted proteases. To elucidate the relative contribution of active<br />

penetration versus induced endocytosis, we either blocked induced endocytosis<br />

or killed the fungus to quantify the remaining invasion potential.<br />

Our data show that the invasion process can be dissected into adhesion,<br />

invasion and dissemination and that certain fungal factors are important for<br />

distinct stages. Furthermore, while the contribution of induced endocytosis to<br />

invasion decreases over time, the contribution of active penetration increases.<br />

KM 01<br />

The regulatory potential of non-coding RNAs in<br />

photosynthetic processes<br />

D. Dienst 1 , I. Axmann 2 , S. Legewie 2 , J. Georg 3 , W.R. Hess 3 , A. Wilde *4<br />

1<br />

Institute of Biology, Humboldt-University Berlin,, Berlin, Germany<br />

2<br />

Institute of Theoretical Biology, Humboldt-University Berlin,, Berlin, Germany<br />

3<br />

Experimental Bioinformatics and Genetics, University of Freiburg, Freiburg,<br />

Germany<br />

4<br />

Institute of Microbiology and Molecular Biology, Justus-Liebig-University<br />

Giessen, Giessen, Germany<br />

Bioinformatics and experimental studies revealed the existence of a large<br />

number of putative regulatory RNAs in cyanobacteria. However, for the vast<br />

majority of these putative regulators functions are still unknown. In<br />

Enterobacteria the function of a number of small non-coding RNAs (ncRNA)<br />

depends on the RNA chaperone Hfq. Most cyanobacteria contain the gene for<br />

the RNA chaperon Hfq. Mutants lacking Hfq do not show any growth defects,<br />

suggesting that photosynthesis is not affected by this mutation. But hfq mutant<br />

cells were not naturally transformable anymore and exhibited a non-phototactic<br />

phenotype compared to the wild type.<br />

Further analysis suggests that the cis-encoded antisense RNA IsrR regulates<br />

photosynthetic processes independently of Hfq. Cyanobacteria respond to iron<br />

deficiency by expressing the protein IsiA (iron stress induced protein A). IsiA<br />

forms a giant antenna ring around photosystem I, thus enhancing light<br />

absorption by the PSI trimer un<strong>der</strong> iron limitation. We show this process to be<br />

controlled by IsrR, a cis-encoded antisense RNA transcribed from the isiA noncoding<br />

strand.<br />

Artificial overexpression of IsrR un<strong>der</strong> iron stress causes a strongly diminished<br />

amount of IsiA-photosystem-I-supercomplexes, whereas IsrR depletion results<br />

in premature expression of IsiA. The coupled degradation of IsrR/isiA-mRNA<br />

duplexes appears as a reversible switch to respond to environmental changes.<br />

IsrR is the first known RNA regulating a photosynthesis component. However,<br />

our bioinformatic and experimental studies revealed a number of yet non<br />

described ncRNAs potentially controlling photosynthetic processes.<br />

69

70<br />

KM 02<br />

Transient PSII subcomplexes in Thermosynechococcus<br />

elongatus<br />

M. Nowaczyk *1 , N. Grasse 1 , J. San<strong>der</strong> 1 , P. Striebeck 1 , K. Leischner 1 , M.<br />

Rögner 1<br />

1 Biochemie <strong>der</strong> Pflanzen, Ruhr Universität Bochum, Bochum, Germany<br />

The large membrane protein complex Photosystem II (PSII) is part of the<br />

photosynthetic electron transfer chain in cyanobacteria, algae and vascular<br />

plants and performs one of the key reactions on our planet - the light driven<br />

oxidation of water. In-depth biochemical and mass spectrometry analysis of<br />

His-tagged PSII complexes from T. elongatus revealed the presence of at least<br />

four different PSII subpopulations in our preparation (Nowaczyk et al. 2006,<br />

The Plant Cell).<br />

One inactive monomeric subfraction is characterized by the loss of the extrinsic<br />

subunits – instead Psb27 is bound to donor site of PSII. This transient factor<br />

was shown to carry a specific lipid modification which mediates the interaction<br />

with the complex during assembly. Moreover, 15 N pulse label experiments led<br />

to the conclusion that the PSII/Psb27 subcomplex is involved in the repair cycle<br />

of PSII – a unique mechanism for the selective exchange of the D1 key-subunit.<br />

Analysis of PSII complexes isolated from PSII deletion mutants and cells<br />

grown un<strong>der</strong> various growth conditions revealed the presence of previously<br />

uncharacterized transient PSII subcomplexes. One example is a new dimeric<br />

PSII-Psb27 complex which accumulates un<strong>der</strong> low temperature growth<br />

conditions. Detailed analysis of this complex gave new insights into the<br />

function of the Psb27 protein.<br />

KM 03<br />

Segregation of Membrane Domains in Gloeobacter violaceus<br />

S. Rexroth *1 , C. Mullineaux 2 , M. Rögner 1 , F. Koenig 3<br />

1 Biochemie <strong>der</strong> Pflanzen, Ruhr-Universität Bochum, Bochum, Germany<br />

2 School of Biological and Chemical Sciences, Queen Mary, University of<br />

London, London, United Kingdom<br />

3 Molekulare Pflanzenphysiologie, Universität Bremen, Bremen, Germany<br />

The unusual cyanobacterium Gloeobacter violaceus lacking the internal<br />

thylakoid membrane system found in other cyanobacteria was subjected to<br />

biochemical membrane fractionation, as well as in vivo imaging using confocal<br />

microscopy.<br />

With both approaches, the plasma membrane could be shown to be segregated<br />

in two distinct domains. Applying sucrose gradient centrifugation subsequent<br />

to French press treatment, two membrane fractions could be separated out with<br />

green and orange colour. These two membrane fractions were subjected to<br />

analysis on proteome, pigment and lipid level and were shown to display<br />

properties typical of the thylakoid and plasma membrane fractions of normal<br />

cyanobacteria. Applying confocal microscopy, patches with vast differences in<br />

chlorophyll autofluorescence intensities displaying the segregation into two<br />

membrane phases were observed in living cells.<br />

Applying proteome analyses to the two membrane fractions, 136 proteins were<br />

identified with a proportion of 44 % membrane integral proteins. For both<br />

fractions a distinct protein composition was detected with 80 and 29 proteins<br />

exclusively found in the green and orange membrane fractions, respectively.<br />

The green fraction contains a high chlorophyll to carotenoid ratio and the vast<br />

majority of bio-energetically active proteins, as it is usually observed for<br />

thylakoid membranes of typical cyanobacteria; the orange fraction might play a<br />

role in biogenesis of membrane protein complexes or in the assembly of cell<br />

wall components.<br />

KM 04<br />

Intracellular redoxpotential and photosynthetic membrane<br />

production in Rhodospirillum rubrum: Correlation with<br />

culture-redox, substrate concentrations and oxygen supply<br />

A.B. Carius *1 , M. Saeger 1 , R.P. Bollin 1 , H. Grammel 1<br />

1 Redox Phenomena in Photosynthetic Bacteria, Max-Planck-Institute for<br />

Dynamics of Complex Technical Systems Magdeburg, Magdeburg, Germany<br />

The facultative anaerobic nonsulfur purple bacterium Rhodospirillum rubrum<br />

shows photosynthetic intracellular membrane (pm) production in the dark, if it<br />

is grown un<strong>der</strong> semiaerobic conditions with succinate as substrate. The pmproduction<br />

can be improved by adding fructose to the culture medium.<br />

Therefore, the pm-absorption at 880nm (bacteriochlorophyll) in correllation to<br />

the cell density (optical density at 660nm) can be used as an admeasurement for<br />

semiaerobic conditions.<br />

The biotechnological production of intracellular pm could be used for<br />

carotenoid production, bacteriochlorophyll production or heterologous<br />

expression of membrane proteins. Since high cell density cultivation of<br />

R.rubrum has now been established in our group, but not yet semiaerobic high<br />

cell density, it has now become more mportant to characterize the semiaerobic<br />

effects more precisely.<br />

We have observed, that 1mM reduced glutathione added to the culture medium<br />

could replace the „fructose-effect“ and also improve it, whereas other reducing<br />

agents such as dithiothreitol (DTT) brought about the op- posite effect.<br />

Together with fructose, glutathione even was able to induce pm production<br />

un<strong>der</strong> aerobic conditions. These results indicate, that the intracellular redox<br />

state is very important for the regulation of the pm- production.<br />

In this work, we want to clarify, which factor influences the membrane<br />

production the most and how the intracellular redox potential responds to<br />

substrate availability, to the extracellular potential and oxygen concentrations.<br />

Several cultivations of R.rubrum with different culture conditions were<br />

compared concerning their NADH/NAD and NADPH/NADP ratios,<br />

glutathione content and photosynthetic membrane production.<br />

KM 05<br />

Electron bifurcation catalyzed by the MvhADG-HdrABC<br />

complex from Methanothermobacter marburgensis<br />

A.K. Kaster *1 , K. Parey 2 , R.K. Thauer 1<br />

1 Max Planck Institute for Terrestrial Microbiology, Biochemistry, Marburg,<br />

Germany<br />

2 Max Planck Institute for Biophysics, Molecular Membrane Biology, Frankfurt,<br />

Germany<br />

Most methanogenic archaea can grow at the expense of CO2 reduction with H2<br />

to methane. In this metabolism reduction of the heterodisulfide CoM-S-S-CoB<br />

of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) with H2 is one of three<br />

exergonic steps. In methanogens that contain cytochromes the reaction involves<br />

the two membrane associated enzyme complexes VhoACG and HdrDE, of<br />

which the subunits VhoC and HdrE are b type cytochromes. Electron transfer<br />

from H2 to CoM-S-S-CoB proceeds via methanophenazine and is coupled with<br />

the built up of an electrochemical proton potential. In methanogens without<br />

cytochromes CoM-S-S-CoB reduction with H2 is catalyzed via the cytoplasmic<br />

enzyme complex MvhADG-HdrABC, of which the subunit HdrA harbors an<br />

iron-sulfur-flavoprotein. We report here that the complex purified from<br />

Methanothermobacter marburgensis catalyzes the heterodisulfide dependent<br />

reduction of ferredoxin with H2. Apparently the free energy associated with<br />

CoM-S-S-CoB reduction (E o´ = -140 mV) with H2 (E o´ = -414 mV) is used to<br />

drive the en<strong>der</strong>gonic reduction of ferredoxin (E = -500 mV) with H2, the most<br />

likely stoichiometry <strong>bei</strong>ng 2 H2 + CoM-S-S-CoB + Fd → HS-CoB + HS-CoM<br />

+ Fdred 2- + 2 H + . It is proposed that the FAD-containing subunit HdrA is the site<br />

of electron bifurcation [1, 2].<br />

[1] Herrmann, G., Yayamani, E., Mai, G. and Buckel, W.; Energy conservation<br />

via electron-transferring flavoprotein in anaerobic bacteria. J. Bacteriol. 190,<br />

784-791 (2008)<br />

[2] Thauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W., and Hed<strong>der</strong>ich, R.;<br />

Methanogenic archaea: ecologically relevant differences in energy<br />

conservation. Nature Reviews Microbiology 6, 579-591 (2008)<br />

KM 06<br />

Functional role of a conserved aspartic acid residue in the<br />

motor of the Na + -driven flagellum from Vibrio cholerae<br />

T. Vorburger 1 , A. Stein 1 , G. Kaim 2 , J. Steuber *1<br />

1 Department of Biochemistry, University of Zurich, Zurich, Switzerland<br />

2 Gene Analytics, Bonstetten (D), Bonstetten, Germany<br />

The flagellar motor consists of a rotor and a stator and couples the flux of<br />

cations (H + or Na + ) to the generation of the torque necessary to drive rotation of<br />

the flagellar filament. The inner membrane proteins PomA and PomB are stator<br />

components of the Na + -driven flagellar motor from Vibrio cholerae. Affinitytagged<br />

variants of PomA and PomB were co-expressed in trans in the nonmotile<br />

V. cholerae pomAB deletion strain to study the role of the conserved<br />

D23 in the transmembrane helix of PomB. At pH 8.0 and un<strong>der</strong> saturating Na +<br />

concentrations, the D23E variant restored motility to 84% of that observed with<br />

wild type PomB, whereas the D23N variant resulted in a non-motile phenotype,<br />

indicating that a carboxylic group at position 23 in PomB is of functional<br />

importance for flagellar rotation. Motility tests at decreasing pH un<strong>der</strong><br />

otherwise identical conditions revealed a pronounced decline of flagellar<br />

function with a motor complex containing the PomB-D23E variant. This<br />

indicates that the protonation state of the glutamate residue at position 23<br />

determines the performance of the flagellar motor, most likely by altering the<br />

affinity of Na + to PomB. The conserved aspartate residue in the transmembrane<br />

helix of PomB and its H + -dependent homologs is proposed to act as a ligand for<br />

the coupling cation in the flagellar motor.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KN 01<br />

Localization and quantification of the flavin cofactors of the<br />

Na + -translocating NADH:quinone oxidoreductase from<br />

Vibrio cholerae<br />

M.S. Casutt 1 , T. Huber 1 , J. Steuber *1<br />

1 Biochemisches Institut, Universität Zürich, Zürich, Switzerland<br />

The Na + -translocating NADH:quinone oxidoreductase (Na + -NQR) from Vibrio<br />

cholerae is a respiratory membrane protein complex that couples the exergonic<br />

oxidation of NADH with quinone to the transport of Na + across the membrane.<br />

Na + -NQR is composed of six subunits (NqrA to NqrF) and contains one Fe-S<br />

centre, ubiquinone-8, one FAD, and two covalently bound FMNs as cofactors.<br />

Preparations of purified Na + -NQR additionally contained riboflavin. The<br />

question whether riboflavin is an additional flavin cofactor or whether it is an<br />

artefact originating from the hydrolysis of FAD or FMNs is discussed<br />

controversially [1,2].<br />

We present a new method for the quantification of the covalently linked FMNs<br />

allowing the determination of the total flavin content. Riboflavin is confirmed<br />

as <strong>bei</strong>ng an intrinsic cofactor of the Na + -NQR [1], and is demonstrated to be<br />

localized on the membrane-bound NqrB subunit.<br />

[1] M. Tao, M. S. Casutt, G. Fritz, J. Steuber, Oxidant-induced formation of a<br />

neutral flavosemiquinone in the Na+-translocating NADH:quinone<br />

oxidoreductase (Na+-NQR) from Vibrio cholerae, Biochim. Biophys. Acta<br />

1777 (2008) 696-702.<br />

[2] A. V. Bogachev, Y. V. Bertsova, D. A. Bloch, M. I. Verkhovsky,<br />

Thermodynamic properties of the redox centres of Na+-translocating<br />

NADH:quinone oxidoreductase, Biochemistry 45 (2006) 3421-3428.<br />

KN 02<br />

Ach1p from Saccharomyces cerevisiae and the connection<br />

to acetate metabolism<br />

C. Fleck *1 , M. Brock 1<br />

1 Leibniz Institute for Natural Product Research and Infection Biology e.V.,<br />

Hans-Knöll-Institute (HKI), Jena, Germany<br />

Ach1p was investigated for the first time approximately 20 years ago as acetyl-<br />

CoA hydrolase. However, the physiological impact of such an „energy<br />

wasting“ enzyme was unclear. To solve this question, we re-investigated the<br />

enzyme due its high identity to a recently characterised CoA-transferase from<br />

Aspergillus nidulans.<br />

The enzyme was homologously overexpressed in a Δach1 background and<br />

purified to homogeneity. Ach1p revealed significant CoA-transferase activity<br />

with acetate, succinate, propionate and the corresponding CoA-esters as<br />

substrates but displayed only minor acetyl-CoA-hydrolase activity. This data<br />

implicate that Ach1p acts as a CoA-transferase rather than a CoA-hydrolase.<br />

Investigations on an ach1 deletion mutant showed a concentration-dependent<br />

growth defect on acetate but no phenotype was observed on other acetyl-CoA<br />

generating carbon sources like ethanol, pyruvate or lactate. The reduced growth<br />

on acetate was softened when the cells were already adapted to acetate but the<br />

growth speed of wild type cells was never reached. Even more, growth on<br />

glucose leads to the secretion of acetate due to an "overflow metabolism", but<br />

only the wild type was able to consume the acetate efficiently after glucose was<br />

exhausted. Therefore, our data implicate an importance of Ach1p especially for<br />

the utilisation of acetate. Acetic acid can enter the mitochondria, when<br />

accumulating within the cytoplasm. The main function of Ach1p seems to be<br />

the detoxification of mitochondrial acetate by transferring the CoASH-moiety<br />

from succinyl-CoA to acetate.<br />

Due to the widespread distribution of the enzyme in the fungal kingdom,<br />

detoxification of mitochondrial acetate seems to represent a major problem<br />

during growth on acetate.<br />

KN 03<br />

The Ethylmalonyl-CoA Pathway – a Story of New<br />

Reactions and Substrates<br />

T.J. Erb *1 , B.E. Alber 2 , G. Fuchs 1<br />

1<br />

Institut für Biologie II, Mikrobiologie, Albert-Ludwigs Universität Freiburg,<br />

Freiburg i. Br., Germany<br />

2<br />

Department of Microbiology, The Ohio State University, Columbus (OH),<br />

United States<br />

Many organic substrates are metabolized via acetyl-coenzyme A (CoA) and<br />

enter the central carbon metabolism on the level of this compound. We recently<br />

described the outlines of the ethylmalonyl-CoA pathway, a new acetyl-CoA<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

assimilation strategy that operates in a number of well known bacteria such as<br />

Rhodobacter sphaeroides, Methylobacterium extorquens and various<br />

streptomycetes [1,2].<br />

Here, we present the complete reaction sequence of the ethymalonyl-CoA<br />

pathway with focus on the key enzymes of the central reaction steps: Two<br />

molecules of acetyl-CoA are transformed to crotonyl-CoA that is reductively<br />

carboxylated by ATP- and biotin-independent crotonyl-CoA<br />

carboxylase/reductase to ethylmalonyl-CoA, the characteristic intermediate of<br />

this pathway [1]. Ethylmalonyl-CoA is in turn converted to methylsuccinyl-<br />

CoA by coenzyme B12 dependent ethylmalonyl-CoA mutase [2] and<br />

subsequently oxidized by methylsuccinyl-CoA dehydrogenase, a FADdependent<br />

enzyme, to mesaconyl-CoA. After its conversion to β-methylmalyl-<br />

CoA, the C5-carbon skeleton is cleaved into glyoxylate and propionyl-CoA that<br />

are transformed to malate and succinyl-CoA, respectively.<br />

The enzymes of the ethylmalonyl-CoA pathway are similar to well studied<br />

proteins, but some of them show new enzymatic functions compared to the<br />

originally described representatives. Examples are the reductive carboxylation<br />

instead of reduction, or thioester hydrolysis instead of Claisen condensation.<br />

Others are highly specific for their C5-substrates and represent therefore new<br />

enzymatic subfamilies (e.g. ethylmalonyl-CoA mutase, methylsuccinyl-CoA<br />

dehydrogenase). The basis of the reaction and substrate specificity will be<br />

discussed.<br />

[1] Erb TJ, Berg IA, Brecht V, Müller M,Fuchs G, Alber BE (2007) PNAS,<br />

104:10631-6.<br />

[2] Erb TJ, Rétey J, Fuchs G, Alber BE (2008) JBC, 283:32283-93.<br />

KN 04<br />

Studies of aconitase in Corynebacterium glutamicum<br />

M. Baumgart *1 , M. Bott 1<br />

1 Institut für Biotechnologie 1, Forschungszentrum Jülich, Jülich, Germany<br />

The Gram-positive soil bacterium Corynebacterium glutamicum is used<br />

industrially for production of amino acids such as L-glutamate and L-lysine.<br />

The tricarboxylic acid cycle (TCA cycle) is a central metabolic pathway which,<br />

besides its function in energy generation, provides the biosynthetic precursors<br />

for glutamate and lysine, namely 2-oxoglutarate and oxaloacetate. In or<strong>der</strong> to<br />

improve production strains of C. glutamicum, it is necessary to have a detailed<br />

knowledge of the enzymes and regulatory mechanisms involved in the TCA<br />

cycle. Aconitase (Acn) was shown to be extensively regulated at the<br />

transcriptional level by the regulators AcnR, RipA and RamA, but the enzyme<br />

itself has not been characterised so far. It is not only part of the TCA cycle, but<br />

also involved in the glyoxylate cycle and the methylcitrate cycle. Acn was<br />

heterologously overproduced and purified to determine kinetic constants for all<br />

three substrates (citrate, cis-aconitate and isocitrate) as well as the pH and<br />

temperature optimum. In addition, an acn deletion mutant was characterized<br />

regarding its growth behaviour and global gene expression using DNA<br />

microarrays and proteome analysis. Surprisingly, we discovered that deletion of<br />

acn and also of icd (encoding isocitrate dehydrogenase) was accompanied by<br />

secondary mutations leading to an inactive or missing citrate synthase. This<br />

indicates that accumulation of citrate or isocitrate in the cytoplasm is toxic for<br />

the cell, a hypothesis that is currently tested experimentally.<br />

KN 05<br />

Physiological insights into hydrogen-based lifestyle<br />

Y. Kohlmann *1 , A. Pohlmann 1 , A. Otto 2 , D. Becher 2 , M. Hecker 2 , B. Friedrich 1<br />

1<br />

Institut für Biologie/ Mikrobiologie, Humboldt-Universität zu Berlin, Berlin,<br />

Germany<br />

2<br />

Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald,<br />

Greifswald, Germany<br />

Ralstonia eutropha is a strictly respiratory β-proteobacterium capable of<br />

generating energy from oxidation of molecular hydrogen. Un<strong>der</strong> an atmosphere<br />

of hydrogen, oxygen and carbon dioxide, R. eutropha thrives<br />

lithoautotrophically fixing carbon dioxide via the Calvin-Benson-Bassham<br />

cycle. Alternatively, the bacterium grows organoheterotrophically using a wide<br />

variety of organic substrates. This investigation compares hydrogen-based<br />

autotrophic to succinate-driven heterotrophic metabolism in reference to the<br />

genomic data deciphered previously [1].<br />

Shotgun proteome analysis provided a comprehensive protein expression<br />

pattern of R. eutropha H16 cells grown either heterotrophically on the fast<br />

growth-promoting substrate succinate or autotrophically with hydrogen and<br />

carbon dioxide as sole sources of energy and carbon, respectively. To obtain<br />

quantitative data, cell cultivation was coupled with metabolic labelling using<br />

the stable isotope 15 N. Furthermore, proteins were identified by the simple but<br />

powerful approach of GeLC-MS/MS. This technique gave access not only to<br />

71

72<br />

soluble but also to membrane proteins involved in processes like transport,<br />

signalling or energy metabolism. Pathway reconstruction based on the<br />

proteome of soluble and membrane fraction led to identification of central<br />

metabolic pathways, including nucleotide biosynthesis, glycolysis, TCA cycle,<br />

fatty acid and amino acid metabolism. Comparative analysis of the protein<br />

pattern of lithoautotrophically and heterotrophically grown cells yielded distinct<br />

molecular signatures illustrated by a huge number of proteins <strong>bei</strong>ng upregulated<br />

or even exclusively formed un<strong>der</strong> lithoautotrophic growth conditions. This<br />

demonstrates that hydrogen-based lifestyle relies on an extensive protein<br />

repertoire adapting the organism to the alternative nature of energy and carbon<br />

source.<br />

[1] Pohlmann A., et al., Nat. Biotechnol. 2006 Oct; 24(10):1257-62.<br />

KN 06<br />

Quantifying the Thiol Redox Proteome<br />

L.I. Leichert *1 , F. Gehrke 2 , H.V. Gudiseva 2 , M. Ilbert 2 , A.K. Walker 3 , J.R.<br />

Strahler 3 , U. Jakob 2<br />

1<br />

MPC - AG Redox-Proteomics, Ruhr-Universität Bochum, Bochum, Germany<br />

2<br />

Dept. of Molecular, Cellular and Developmental Biology, University of<br />

Michigan, Ann Arbor, Michigan, United States<br />

3<br />

Michigan Proteome Consortium, University of Michigan, Ann Arbor,<br />

Michigan, United States<br />

One of the main in vivo targets of ROS in the cell is the thiol group of<br />

cysteines. We have developed a mass spectrometry based thiol trapping<br />

technique termed OxICAT, which allows the precise quantification of oxidative<br />

cysteine modifications in hundreds of different proteins in a single experiment.<br />

We used OxICAT to identify target proteins of hydrogen peroxide (H2O2) and<br />

sodium hypochlorite (NaOCl) stress in Escherichia coli and to define the<br />

cysteine(s) affected un<strong>der</strong> these stress conditions. To our surprise a large<br />

number of proteins appears not to be affected indiscriminately but rather<br />

specifically either by H2O2 or NaOCl. These results indicate that individual<br />

ROS target distinct proteins in vivo. One third of the redox-sensitive proteins<br />

we identified are conditionally essential in E. coli, a finding that might explain<br />

the antibacterial effect of oxidative stress. Another subset of redox-sensitive<br />

proteins identified with our technique protects E. coli against oxidative stress.<br />

These experiments demonstrate that OxICAT, which can be used in a variety of<br />

different cell types and organisms, is a very powerful tool to identify, quantify<br />

and monitor ROS induced thiol modifications.<br />

KO 01<br />

Impact of stringent response on survival and usp gene<br />

expression in Pseudomonas aeruginosa<br />

N. Boes 1 , A. Steen 1 , K. Schreiber 2 , M. Scheer 2 , M. Schobert *1<br />

1 Institute of Microbiology, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

2 Bioinformatics and Biochemistry, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

The opportunistic human pathogen Pseudomonas aeruginosa, can cause acute<br />

and chronic infections and is characterized by its high antibiotic tolerance.<br />

During persistent infection P. aeruginosa forms biofilms in an oxygen limited<br />

to anaerobic environment and is exposed to energy starvation. Un<strong>der</strong> these<br />

conditions a set of five universal stress proteins (UspK, UspL, UspM, UspN<br />

and UspO) is produced. Mutant studies showed that these proteins are<br />

important for survival and persistence. The corresponding genes are un<strong>der</strong><br />

control of the oxygen-sensing regulator Anr and the stringent response. The<br />

stringent response is one of the global regulatory networks in bacteria,<br />

providing a rapid adaptation to a variety of growth inhibiting stress conditions.<br />

The regulatory components of the stringent response are the guanosine<br />

nucleotides ppGpp and its precursor pppGpp, which is produced by RelA and<br />

SpoT.<br />

We investigated the RelA/SpoT-dependent regulation of the five usp genes in<br />

stationary phase in more detail using reporter gene fusions and identification of<br />

32 P-labelled ppGpp via thin layer chromatography. Unexpectedly, alkaline pH<br />

was found to elicit a SpoT-controlled stringent response. Transcriptome<br />

analysis identified the members of the stringent response regulon and<br />

phenotypic characterization of a P. aeruginosa relA/spoT mutant strain<br />

indicated an important role for stringent response during biofilm growth.<br />

KO 02<br />

Acyl-homoserine lactone mediated regulation and LuxR<br />

receptors in the insect pathogen Photorhabdus luminescens<br />

I. Hitkova 1 , S. Linnerbauer 1 , K. Jung 1 , R. Heermann *1<br />

1 Mikrobiologie, LMU München, Martinsried/München, Germany<br />

Photorhabdus luminescens is a Gram-negative enterobacterium, which forms<br />

an entomopathogenic symbiosis with soil nematodes belonging to the species<br />

Heterorhabditis bacteriophora, and in turn is highly pathogenic towards<br />

insects. Regulation of mutualism and pathogenicity is frequently controlled by<br />

acyl-homoserine lactones (AHLs) via LuxI/LuxR quorum sensing systems in<br />

Gram-negative bacteria. Remarkably, P. luminescens has two potential AHL-<br />

LuxR receptors, but no LuxI homologue what makes them incapable to produce<br />

own AHLs. To obtain evidence for an AHL-dependent regulation in P.<br />

luminescens, we investigated the proteome of wild-type cells exposed to a mix<br />

of ten different AHLs, and of two mutants, each lacking one of the two AHL-<br />

LuxR receptors. The presence of AHLs altered the proteome with respect to<br />

several toxins, putative adhesion factors, regulators, and metabolic enzymes. In<br />

the mutants, the production of proteins putatively involved in amino acid<br />

metabolism, protein biosynthesis, membrane permeability, and in virulence was<br />

influenced. These results demonstrate that P. luminescens uses AHLs for<br />

regulation. This unorthodox AHL signaling reveals that this species does not<br />

communicate via AHLs, but rather spies out the environment for signals<br />

produced by potential competitors to adapt properly within the surrounding<br />

community.<br />

KO 03<br />

Structural basis for the regulatory function of the CpxP<br />

adaptor protein of Escherichia coli<br />

X. Zhou 1 , R. Fleischer 1 , N. Krauß 2 , P. Scheerer 3 , S. Hunke *1<br />

1 Cpx Envelope Stress, Humboldt University Berlin, Berlin, Germany<br />

2 Schoolof Biological and Chemical Sciences, Queen Mary, University of<br />

London, London, United Kingdom<br />

3 IMPB, Charité - University Medicine Berlin, Berlin, Germany<br />

The cell envelope of Gram-negative bacteria is directly subjected to permanent<br />

changes in the environment. Mechanisms that preserve the integrity of this<br />

compartment are regulated by the Cpx stress response pathway [1]. The Cpx<br />

system consists of the histidine kinase CpxA, the response regulator CpxR and<br />

the periplasmic CpxP protein. Sensing envelope perturbation by an unknown<br />

feature the sensor CpxA transmits a signal via a phosphorelay to CpxR, which<br />

in response acts as a transcription regulator of genes, whose products are<br />

mainly folding catalysts and proteases of the envelope [1]. The Cpx stress<br />

response is controlled by feedback inhibition [1]. Recently, we have shown that<br />

CpxP acts at the initiation point of signal transduction by reducing CpxA<br />

autophosphorylation activities in the reconstituted CpxRA system [2].<br />

Interestingly, it was shown that CpxP acts as an adaptor protein for leading<br />

misfolded pili subunits to the DegP protease for degradation [3].<br />

Here, we will present the structural basis for regulatory function of the CpxP<br />

adaptor protein. The high-resolution crystal structure of the truncated CpxP<br />

protein was solved as a dimer in the asymmetric unit at 1.45 Å resolution. The<br />

monomer displays a new folding motif which we called a "three-finger motif".<br />

Interestingly, the dimer reveals a stable α-helical domain swapping and is<br />

stabilized by four salt bridges and a His-His-sandwich. We constructed the<br />

respective CpxP mutants and analyzed their in vivo inhibitory functions,<br />

stabilities and dimer formation. Our data indicate that CpxP is only able to<br />

inhibit the Cpx-signalling cascade as a functional domain swapped dimer.<br />

[1]Ruiz, N. and Silhavy, T.J. (2005) Curr. Opin. Microbiol. 8, 122-126.<br />

[2]Fleischer, R., Herrmann, R., Jung, K. and Hunke, S. (2007) J. Biol. Chem.<br />

282, 8583-8593.<br />

[3]Isaac, D.D., Pinkner, J.S., Hultgren, S.J. and Silhavy, T.J. (2005) Proc. Natl.<br />

Acad. Sci. U.S.A. 102, 17775-17779.<br />

KO 04<br />

Analysis of ECF sigma factor regulation through regulated<br />

intramembrane proteolysis in Bacillus subtilis<br />

J. Heinrich 1 , K. Schäfer 2 , K. Hein 3 , T. Wiegert *2<br />

1 Lehrstuhl für Genetik, Universität Bayreuth, Bayreuth, Germany<br />

The Gram-positive model bacterium Bacillus subtilis encodes seven alternative<br />

sigma factors of the ECF family that control genes related to extracytoplasmic<br />

function. The activity of alternative sigma factors has to be regulated in a way<br />

that they become active and bind to the RNA polymerase core enzyme only<br />

upon certain stress signals. In many cases, the ECF sigma factor is sequestered<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

y a transmembrane anti-sigma factor. For the E. coli SigmaE/RseA and the B.<br />

subtilis SigmaW/RsiW system it has been shown that certain cell envelope<br />

stresses trigger degradation of the anti-sigma factor in a mechanism called<br />

regulated intramembrane proteolysis (RIP), resulting in the release of the<br />

corresponding sigma factor for interaction with RNAP. RsiW is degraded in a<br />

concerted action of at least three proteases. We could identify PrsW, which<br />

belongs to a new family of membrane embedded metalloproteases of unknown<br />

function, <strong>bei</strong>ng involved in the first committed step of RsiW proteolysis [1].<br />

The site-1 proteolytic product of RsiW then becomes a substrate for the<br />

intramembrane cleaving protease RasP [2]. Our work mainly focuses on the<br />

mechanism of RsiW degradation through PrsW and RasP, and on the question<br />

whether other B. subtilis sigma/anti-sigma factor pairs are controlled by RIP in<br />

a similar manner. New results on the action of PrsW and on the regulation of<br />

SigX/RsiX will be presented.<br />

[1] Heinrich, J. and T. Wiegert. (2006). YpdC determines site-1 degradation in<br />

regulated intramembrane proteolysis of the RsiW anti-sigma factor of Bacillus<br />

subtilis. Mol Microbiol 62:566-579.<br />

[2] Schöbel, S., Zellmeier, S., Schumann, W., and Wiegert, T. (2004). The<br />

Bacillus subtilis SigmaW anti-sigma factor RsiW is degraded by<br />

intramembrane proteolysis through YluC. Mol Microbiol 52, 1091-1105.<br />

KO 05<br />

Light-dependent gene regulation in Rhodobacter<br />

sphaeroides - Investigating the interplay between the<br />

AppA/PpsR- and PrrB/PrrA-system<br />

S. Metz *1 , G. Klug 1<br />

1 Institut für Mikro- und Molekularbiologie, Justus Liebig Universität Gießen,<br />

Gießen, Germany<br />

Rhodobacter sphaeroides, a gram-negative α-proteobacterium, can grow either<br />

by aerobic respiration, anaerobic respiration, fermentation or anoxygenic<br />

photosynthesis. This broad spectrum of possible energy generating mechanisms<br />

requires an extensive regulation. We are interested in the control of the puf-<br />

(light harvesting complex I (LHCI), reaction centre (RC) and the puc- (light<br />

harvesting complex II (LHCII) operon, that encode proteins required for<br />

formation of the photosynthetic apparatus in Rhodobacter sphaeroides.<br />

Regulation of these two operons has been investigated intensively and revealed<br />

a complex network of regulating factors. Both operons are on the one hand<br />

controlled by the PrrB (sensor kinase)/PrrA (response regulator) two<br />

component system that leads to an increase in transcription un<strong>der</strong> low oxygen<br />

and high light conditions through a cbb3 oxidase mediated signal. On the other<br />

hand the AppA (antirepressor)/PpsR (repressor) system greatly influences the<br />

expression of photosynthesis genes. PpsR acts as a strong repressor by directly<br />

binding the DNA, while AppA releases this repression. AppA itself reacts to<br />

both blue light and redox signals.<br />

By using Northern Blot analysis and semi-quantitative RT-PCR we could show<br />

a relation between the PrrB/PrrA system and the AppA/PpsR system.<br />

Photosynthetic gene expression un<strong>der</strong> semi-aerobic conditions does not solely<br />

depend on the release of PpsR repression – but also a PrrA mediated increase of<br />

transcription. Additionally, by in vivo analysis of AppA base-exchange<br />

mutants, we could show that the PrrA dependent activation greatly differs in its<br />

light sensitivity in contrast to the AppA dependent repression.<br />

KO 06<br />

Nitrogen control in Corynebacterium glutamicum: impact on<br />

glutamine metabolism<br />

N. Rehm *1 , E. Hiery 1 , T. Georgi 2 , M. Bott 2 , A. Burkovski 1<br />

1 Lehrstuhl für Mikrobiologie, Friedrich-Alexan<strong>der</strong>-Universität Erlangen-<br />

Nürnberg, Erlangen, Germany<br />

2 Institut für Biotechnologie I, Forschungszentrum Jülich, Jülich, Germany<br />

In the Gram positive soil bacterium Corynebacterium glutamicum assimilation<br />

of nitrogen sources is transcriptionally regulated by the repressor AmtR [1,2].<br />

Ammonium, the preferred nitrogen source of this bacterium, is mainly<br />

metabolized by the glutamate dehydrognase. Un<strong>der</strong> conditions of high<br />

ammonium concentrations AmtR represses the transcription of at least 33<br />

genes. In response to limiting amounts of ammonium transcription of these<br />

genes is initiated and the nitrogen starvation response is induced. Ammonium is<br />

then preferentially metabolized by the AmtR-controlled glutamine<br />

synthetase/glutamate synthase system. Besides ammonium, glutamine supports<br />

fast growth of C. glutamicum when used as nitrogen source. Glutamine can also<br />

be assimilated as carbon source although growth is decelerated in this case. In<br />

or<strong>der</strong> to examine metabolism of glutamine in C. glutamicum DNA microarray<br />

and subsequent mutant analyses were carried out. Unexpectedly, the nitrogen<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

starvation response was induced in wild type cells grown with glutamine as<br />

nitrogen source. The function of different enzymes such as glutaminase,<br />

glutamate dehydrogenase, glutamate synthase and glutamine synthetase was<br />

investigated using transcriptome, proteome and metabolome analyses. It could<br />

be shown that glutamate synthase is crucial for the assimilation of glutamine as<br />

nitrogen source. Moreover, this enzyme is responsible for internal glutamate<br />

accumulation when glutamine serves as nitrogen source, whereas upon growth<br />

with ammonium this task is primarily accomplished by the glutamate<br />

dehydrogenase.<br />

[1] Beckers, G., Strösser, J., Hildebrandt, U., Kalinowski, J., Farwick, M.,<br />

Krämer, R. and Burkovski, A. (2005). Mol. Microbiol. 58, 580-595.<br />

[2] Hänßler, E. and Burkovski, A. (2008). In Burkovski, A. (ed.)<br />

Corynebacteria: genomics and molecular biology. Caister Academic Press,<br />

Norfolk, UK, 183-201.<br />

KP 01<br />

Molecular aspects of ergot alkaloid biosynthesis<br />

P. Tudzynski *1<br />

1 Institut für Botanik, Westf.Wilhelms Universität Münster, Münster, Germany<br />

Ergot alkaloids and their <strong>der</strong>ivatives have been traditionally used as therapeutic<br />

agents in migrain, blood pressure regulation and help in child birth and<br />

abortion. Their production in submerse culture is a long established<br />

biotechnological process. Ergot alkaloids are produced mainly by members of<br />

the genus Claviceps, with C. purpurea as best investigated species, with respect<br />

to biochemistry of alkaloid biosynthesis and recently also molecular genetics.<br />

Genes encoding enzymes involved in alkaloid biosynthesis have been shown to<br />

form a cluster; recent data on functional analyses of cluster genes will be<br />

presented. Especially the analyses of the non-ribosomal-peptide synthetases<br />

(NRPS) involved in the synthesis of the peptide moiety of ergopeptines open<br />

interesting perspectives for the synthesis of new ergot alkaloids; on the other<br />

hand defined mutants can be generated producing interesting intermediates or<br />

only single peptide alkaloids (instead of the alkaloid mixtures usually produced<br />

by industrial strains). The various Claviceps species differ with respect to their<br />

host specificity and their alkaloid content; comparison of the ergot alkaloid<br />

clusters in these species (and of clavine alkaloid clusters in other genera) yields<br />

interesting insights into the evolution of cluster structure.<br />

KP 02<br />

Molecular analysis of the velvet complex in Penicillium<br />

chrysogenum and its role in regulating secondary<br />

metabolism and morphogenesis<br />

B. Hoff *1 , I. Zadra 2 , H. Kürnsteiner 2 , U. Kück 1<br />

1 Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

2 Development Anti Infective Microbiology, Sandoz GmbH, Kundl, Austria<br />

The filamentous fungus Penicillium chrysogenum is the main industrial<br />

producer of the pharmaceutical relevant beta-lactam antibiotic penicillin. All<br />

three biosynthesis genes are found in a single cluster and the expression of<br />

these genes is known to be controlled by a complex network of global<br />

regulators. Recently, the so called velvet complex was isolated and functionally<br />

identified in Aspergillus nidulans. The main components of this complex<br />

compromising the velvet protein, a velvet-like protein VelB and the<br />

methyltransferase LaeA regulate both secondary metabolism and conidiospore<br />

development.<br />

In a first attempt, we have unambiguously identified homologues of all three<br />

genes in P. chrysogenum and generated deletion strains for further functional<br />

characterization using a Pcku70 deletion strain with an improved gene targeting<br />

efficiency. Using expression and HPLC analyses, we have determined that the<br />

proteins characterized control penicillin biosynthesis in P. chrysogenum.<br />

Furthermore, detailed microscopic investigations such as bimolecular<br />

fluorescence complementation together with results from array analyses have<br />

shown that all components are important regulators of cellular differentiation in<br />

the industrially used fungus P. chrysogenum. All these findings extend options<br />

for industrial strain improvement programs.<br />

73

74<br />

KP 03<br />

Volatiles of Serratia odorifera: Emission profile analysis and<br />

growth promotion/inhibition of Arabidipsis thaliana<br />

M. Kai *1 , B. Piechulla 1<br />

1 Department of Biochemistry, University of Rostock, Rostock, Germany<br />

Bacteria are able to produce a wealth of volatiles. The reasons for such<br />

emissions are presently not well un<strong>der</strong>stood. The volatiles may e.g., act as<br />

signals for inter and intraorganismic and/or, cell-to-cell communication, be<br />

released as dispensable carbon, or deliver bioactive compounds into the closer<br />

or distant surrounding to effect organisms in the same habitat. Indeed, in cocultivation<br />

bioassays, volatiles from various bacteria, including Serratia<br />

odorifera 4Rx13, inhibit the growth of Rhizoctonia solani and of many other<br />

phytopathogenic fungi.<br />

The volatile profile of S. odorifera was now investigated in more detail. Up to<br />

60 different volatiles are emitted by this bacterial isolate, e.g.,<br />

dimethyldisulfide, dimethyltrisulfide, 2-phenylethanol and „Sodorifen“. The<br />

volatile profiles vary depending on the growth phase, e.g. 24-48 hours after<br />

inoculation the highest number of volatiles is released, and characteristic<br />

volatile patterns are distinguished.<br />

An interesting observation was made when Arabidopsis thaliana was cocultivated<br />

with S. odorifera in an open or closed system. Growth promotion<br />

was observed in the closed system, while in the open system the growth was<br />

inhibited. The GCMS volatile analysis revealed an increased volatile<br />

accumulation in the closed system (maximum of 40% increase), which however<br />

turned out not to be responsible for the observed growth promotion of A.<br />

thaliana. In the intention to elucidate the different effects in closed and open<br />

systems, individual volatile compounds were separately investigated and tested,<br />

and respective results will be presented.<br />

KP 04<br />

Overproduction of riboflavin is not needed for mycelial<br />

growth of Ashbya gossypii but leads to protection of its<br />

hyaline spores against UV-light<br />

S. Nieland *1 , K.P. Stahmann 1<br />

1 Technische Mikrobiologie, Fachbereich Bio-, Chemie- und Verfahrenstechnik,<br />

Fachhochschule Lausitz, Senftenberg, Germany<br />

Riboflavin, a yellow pigment, is needed in tiny amounts e.g. as FAD precursor.<br />

For humans 2 mg per day is sufficient. If more is taken up excretion occurs<br />

staining the urine yellow. The hemiascomycete Ashbya gossypii can produce up<br />

to 100 mg per g biomass, five or<strong>der</strong>s of magnitudes more than can be explained<br />

by its role as coenzyme precursor. Accumulation in the vacuoles leading to<br />

yellow mycelium suggests a role as pigment. In chemostatic cultivation<br />

overproduction for riboflavin is down-regulated at constant dilution rate [Appl<br />

Microbiol Biotechnol 76:569]. Three RIB genes showed minimal RT-PCR<br />

signals compared with ACT1. The same was true for expression of a reporter<br />

gene fused with the RIB3 promoter. Starvation induced RIB3, RIB4, and RIB5<br />

expression. Then riboflavin concentration increased and sporulation occurred.<br />

The spores of A. gossypii were found to be hyaline. Not a single of 10 3 spores<br />

survived 254 nm irradiation for 300 seconds. In contrast more than 65 % of<br />

Aspergillus nidulans conidia survived the same treatment and complete<br />

inactivation was reached after 1800 seconds. The proportion of surviving<br />

spores increased when 0.8 mM riboflavin was added to the spore suspension.<br />

These experiments show that over-produced riboflavin is not a primary but a<br />

secondary metabolite because it is not needed for growth. Photoprotection of<br />

the UV sensitive spores is a possible function. Whether this is true in planta,<br />

e.g. in the leaves of Gossypium hirsutum, the name giving host of A. gossypii, is<br />

not known.<br />

KP 05<br />

Antagonistic potential of Streptomyces dendra sp. nov<br />

associated with marine sponge Dendrilla nigra<br />

J. Selvin *1<br />

1 Dept of Microbiology, Bharathidasan University, Tiruchirappalli, India<br />

We have extensively explored the chemical ecology of marine sponge Dendrilla<br />

nigra. It was found that the sponge D. nigra contained potential bioactives<br />

including antibacterial, antifungal, cytotoxic, insecticidal, anticoagulant, antifouling<br />

and anti-predation properties. But the recollection of substantial<br />

quantity of source organism for the isolation of active principles was<br />

unsuccessful. Therefore efforts were taken for the isolation of potential<br />

producers from D. nigra as a sustainable source of supply for producing<br />

bioactives and exploring the chemical ecology of host sponge. Novel isolation<br />

media was developed based on the biochemical composition of host sponge,<br />

habitat ecology and phylogenetic relatedness of Candidatus bacterial<br />

symbionts. In the present study, a „regulatory-inductive approach“ was<br />

adopted for the development of novel cultivation media. The strain MSI051<br />

was the first isolate obtained in the efforts for culturing the Candidatus<br />

heterotrophic bacteria found associated with the marine sponge D. nigra. Based<br />

on the biochemical characteristics and phylogenetic analysis, the strain MSI051<br />

was named as Streptomyces dendra sp. nov., since the isolate was a<br />

endosymbiont of D. nigra. In or<strong>der</strong> to explore the functional role of MSI051 in<br />

the host sponge D. nigra, the phospholipase A2 (PLA2) activity was<br />

determined. The isolate MSI051 was a potential antagonistic producer which<br />

showed broad spectrum antibacterial activity. The polyketide synthase gene<br />

type II in the MSI051 ultimately increased the scope for combinatorial<br />

biosynthesis and genetic engineering for the production of new antibiotics.<br />

Thus, the new strain MSI051 associated with the sponge D. nigra would be a<br />

sustainable source of supply for the discovery novel drug leads and exploring<br />

the chemical ecology of host sponge.<br />

KP 06<br />

Triggering Cryptic Natural Product Biosynthesis in<br />

Microorganisms<br />

K. Scherlach * 1 , S. Bergmann 1 , J. Schümann 1 , A. Funk 1 , V. Schroeckh 1 , A.A.<br />

Brakhage 1 , C. Hertweck 1<br />

1<br />

Leibniz Institute for Natural Product Research and Infection Biology (HKI),<br />

Jena, Germany<br />

No abstract submitted<br />

KQ 01<br />

How small RNAs target multiple mRNAs to rewire gene<br />

expression post transcription<br />

J. Vogel *1<br />

1<br />

RNA Biology Group, Max Planck Institute for Infection Biology, Berlin,<br />

Germany<br />

Small noncoding RNAs (sRNAs) have increasingly been identified as posttranscriptional<br />

regulators of multiple transcriptionally uncoupled genes. How<br />

single sRNAs can select multiple mRNAs for repression or activation is yet<br />

poorly un<strong>der</strong>stood. In the canonical pathway of bacterial mRNA repression,<br />

sRNAs base-pair with the target’s Shine-Dalgarno sequence (SD) or AUG start<br />

codon to inhibit ribosome entry and thus translational initiation. However, ever<br />

more sRNAs fail prediction of SD or AUG pairing, suggesting that alternative<br />

pathways of target recognition and translational control exist.<br />

I will present data on two Salmonella sRNAs, GcvB and RybB, each of which<br />

regulate a large number of functionally related yet structurally diverse mRNAs.<br />

GcvB is a ~200 nt sRNA expressed in fast growing cells; it functions to limit<br />

the synthesis of ABC transporters of oligopeptides and amino acids. GcvB<br />

selects its targets by means of a deeply conserved G/U-rich stretch that can<br />

make lose antisense contacts with C/A-rich translational enhancer elements<br />

located in the 5’ UTRs of ABC transporter mRNAs [1].<br />

RybB is a ~80 nt sRNA activated by the envelope stress sigma factor, Sigma E,<br />

when unfolded outer membrane proteins (OMPs) accumulate in the periplasm.<br />

To prevent further folding stress, RybB accelerates the decay of all major porin<br />

and many minor OMP mRNAs [2-3]. We have discovered that a short<br />

conserved „seed“ region at the RybB 5’ end mediates short antisense<br />

interactions with all target omp mRNAs. Similar to GcvB, RybB interacts with<br />

few of its targets at the canonical SD or start codon position.<br />

Our results suggest that conserved „regulator domains“ and the targeting of<br />

non-canonical mRNA regions facilitate multiple mRNA regulation by bacterial<br />

sRNAs.<br />

[1] Sharma et al. 2007 Genes & Development (21): 2804-2817<br />

[2] Papenfort et al. 2006 Molecular Microbiology (62): 1674-1688<br />

[3] Bouvier et al. 2008 Molecular Cell, in press<br />

KQ 02<br />

Small non-coding RNAs in Rhodobacter sphaeroides and the<br />

singlet oxygen stress response<br />

B. Berghoff *1 , J. Glaeser 1 , C. Sharma 2 , J. Vogel 2 , G. Klug 1<br />

1 Institute for Microbiology and Molecular Biology, Justus-Liebig-University,<br />

Giessen, Germany<br />

2 RNA Biology, Max Planck Institute for Infection Biology, Berlin, Germany<br />

Photooxidative stress occurs when singlet oxygen ( 1 O2) is generated by the<br />

light-mediated energy transfer from a photosensitizer to molecular oxygen. In<br />

photosynthetic organisms chlorophyll molecules can act as natural<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

photosensitizers. As a consequence, every photosynthetic organism has to deal<br />

with photooxidative stress when light and oxygen are present simultaneously.<br />

In the photosynthetic Alphaproteobacterium Rhodobacter sphaeroides it is<br />

known that the alternative sigma factors RpoE and RpoHII play major<br />

regulatory roles in the photooxidative stress response but there is also strong<br />

evidence for an important role of small non-coding RNAs (sRNAs). In this<br />

study a genome wide search for sRNAs in R. sphaeroides was performed.<br />

Cultures of R. sphaeroides were either treated with superoxide radicals (O2 - ) or<br />

1 O2 to identify sRNAs which are related to both oxidative and photooxidative<br />

stress. Total RNA samples were enriched for primary transcripts and analysed<br />

by high-throughput pyrosequencing. Most abundant sRNAs were then verified<br />

by northern blot analysis. We observed that one sRNA (RSs0019) was induced<br />

by 1 O2. This sRNA also strongly depends on RpoE. Another set of three sRNAs<br />

(RSs0682a/b/c) is most likely cotranscribed from a putative RpoHI/RpoHII<br />

specific promoter. The most abundant of these three, RSs0682a, is induced<br />

un<strong>der</strong> oxidative and photooxidative stress conditions as well as un<strong>der</strong> heat<br />

shock. The processed form of RSs2461 shows slight induction un<strong>der</strong> different<br />

oxidative stress conditions and RSs0680 is specifically processed upon<br />

exposure to 1 O2. In conclusion, our data provide evidence for several sRNAs<br />

with a putative role in the photooxidative stress response of R. sphaeroides.<br />

KQ 03<br />

YmoA activates expression of the virulence regulator gene<br />

rovA in Yersinia pseudotuberculosis through the Csr<br />

regulatory system<br />

K. Böhme *1 , A.K. Heroven 1 , P. Dersch 1<br />

1 Department of Molecular Infection Biology, Helmholtz Centre for Infection<br />

Research, Braunschweig, Germany<br />

The enteropathogen Yersinia pseudotuberculosis is responsible for a variety of<br />

gut-associated diseases. The bacteria pass the intestinal tract and penetrate the<br />

epithelial cell layer through the M-cells to colonize the un<strong>der</strong>lying Peyer´s<br />

patches. This infection phase is primarily mediated by the outer membrane<br />

protein invasin. The expression of this virulence factor is tightly regulated in<br />

response to environmental signals by the global regulatory protein RovA. We<br />

found that the nucleoid-associated protein YmoA, a member of the Hha protein<br />

family, to be an important component of the regulatory network affecting RovA<br />

synthesis. YmoA is responsible for increased rovA expression. The protein<br />

level of the LysR-regulator RovM, which represses rovA expression, is<br />

downregulated. Moreover, we observed that YmoA affects the components of<br />

the carbon storage regulator (Csr) system, the regulatory RNA CsrC and the<br />

RNA binding protein CsrA. Experimental data indicate that YmoA is essential<br />

for CsrC synthesis. In a ymoA mutant the CsrC RNA level is strongly reduced.<br />

This results in a CsrA-mediated activation of RovM repressing the transcription<br />

of rovA and thus RovA-controlled invasin synthesis. Our results show that<br />

YmoA represents a key regulator, controlling expression of virulence factors<br />

such as invasin, which are crucial for the early steps of a Y. pseudotuberculosis<br />

infection.<br />

KQ 04<br />

CRISPR - a novel prokaryotic immune system that<br />

provides acquired resistance against phages<br />

N. Heidrich *1 , A. Raine 1 , G.H. Wagner 1<br />

1 Dep. of Cell and Molecular Biology, Uppsala University, Uppsala, Germany<br />

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are a<br />

ubiquitous feature of most bacterial and archeal genomes. Together with a set<br />

of so-called cas (CRIPSR-associated) genes the CRISPR system provides<br />

immunity against bacteriophages. Bioinformatics analyses of CRISPR and cas<br />

genes led to the hypothesis that this prokaryotic defence system may function<br />

analogous to RNA interference (RNAi). Recently, it has been demonstrated in<br />

E. coli that CRISPR sequences are transcribed into a multiunit RNA precursor<br />

that is cleaved into CRISPR monomers by a Cas protein complex termed<br />

Cascade. The CRISPR monomers, containing spacer segments (homologous to<br />

phage sequences) between processed repeats of CRISPR are supposed to<br />

mediate silencing of incoming phages nucleic acids. However, apart from this<br />

fascinating observation, the mechanism of CRISPR action remains entirely<br />

unknown.<br />

To un<strong>der</strong>stand the mechanism that provides bacterial immunity against foreign<br />

genetic elements we are investigating CRISPR elements in E. coli. We<br />

established a plasmid-based read-out system that monitors CRISPR-cas activity<br />

and shows that CRISPR transcripts ultimately can target both sense and<br />

antisense sequences. This intriguing observation might indicate a doublestranded<br />

effector intermediate or suggests that CRISPR RNAs target phage<br />

DNA. Biochemical tests on affinity-tagged, overproduced Cas proteins and<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

their putatively associated RNAs are expected to give information about their<br />

roles in immunity and may answer the question of whether RNAi, or a related<br />

mechanism, does indeed apply to prokaryotes.<br />

KQ 05<br />

The ibpAB operon of Escherichia coli – regulation after heat<br />

shock<br />

L. Gaubig *1 , F. Narberhaus 1<br />

1 LS für Biologie <strong>der</strong> Mikroorganismen, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

The ibpAB operon of E. coli encodes two small heat shock proteins. On<br />

transcriptional level the operon is regulated by a sigma32 promotor (1).<br />

Regulation on posttranscriptional level, at least for ibpA, is realized by an RNA<br />

thermometer. There is also a predicted RNA thermometer in the intergenic<br />

region between ibpA and ibpB. RNA thermometers are regulatory elements,<br />

which are located in the 5´ untranslated regions of many small heat shock genes<br />

in α- and γ- proteobacteria. RNA thermometers directly sense temperature as an<br />

environmental parameter. They influence expression of the downstream gene<br />

via structural changes around the SD sequence and thereby control its<br />

accessibility for ribosomes [2]. At physiological temperatures, translation is<br />

blocked because the ribosome binding site is masked by imperfect base pairing.<br />

At elevated temperatures, melting of the structure permits translation initiation<br />

[3].<br />

At different timepoints after heat shock a total of four transcripts of the ibpAB<br />

operon were detected. Here we present results on the heat shock regulation of<br />

the operon with an emphasis on the posttranscriptional level.<br />

[1] Allen S P, Polazzi J O, Gierse J K, Easton A M. (1992). Two novel heat<br />

shock genes encoding proteins produced in response to heterologous protein<br />

expression in E. coli. J Bacteriol 174: 6938-6947<br />

[2] Narberhaus F, Waldminghaus T, Chowdhury S. (2006). RNA thermometers.<br />

FEMS Microbiol Rev 30: 3-16<br />

[3] Chowdhury S, Maris C, Allain F, Narberhaus F. (2006). Molecular basis for<br />

temperature sensing by an RNA thermometer. EMBO J 25: 2487–2497<br />

KQ 06<br />

The influence of RNase E and RNase J on maturation and<br />

stability of non-coding RNAs in S. meliloti<br />

E. Evguenieva-Hackenberg *1<br />

1 Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität,<br />

Gießen, Germany<br />

The important function of small non-coding RNAs (ncRNAs) in the regulation<br />

of many processes in bacterial cells was recognised during the last years. In this<br />

work, the characterisation of several ncRNAs in S. meliloti is presented. The<br />

expression profiles of the highly conserved signal recognition particle (SRP)<br />

RNA and 6S RNA as well as of novel ncRNAs with unknown function were<br />

studied un<strong>der</strong> different growth conditions and un<strong>der</strong> stress. Additionally, the<br />

maturation, the steady state amount and the stability of the ncRNAs in RNase<br />

E- and RNase J-deficient S. meliloti strains were analysed. It will be shown that<br />

in contrast to the well studied model organisms E. coli and B. subtilis, which<br />

possess either RNase E or two RNase J homologs with important regulatory<br />

functions, respectively, S. meliloti harbours the two non-orthologous<br />

endoribonucleases RNases E and J. These RNases exhibit overlapping but not<br />

identical functions in the cell.<br />

[1] Hölscher M., Baumgardt K., Voss B., Hess W. and Evguenieva-<br />

Hackenberg, E. Small RNAs with regulatory functions in Sinorhizobium and<br />

Rhizobium. Poster at <strong>VAAM</strong> 2007.<br />

[2] Pobigaylo N, Wetter D, Szymczak S, Schiller U, Kurtz S, Meyer F,<br />

Nattkemper TW, Becker A. (2006) Appl. Environ. Microbiol. 72:4329-37.<br />

[3] Evguenieva-Hackenberg, E. and Klug, G. (<strong>2009</strong>) Progress in Nucleic acids<br />

Research and Molecular Biology (in press).<br />

KR 01<br />

Transcriptomics in the Ustilago-maize pathosystem<br />

M. Vranes 1 , K. Heimel 1 , R. Wahl 1 , A. Zahiri 1 , G. Döhlemann 2 , R. Kahmann 2 , J.<br />

Kämper *1<br />

1 Institut für Angewandte Biowissenschaften, Abteilung Genetik, Universität<br />

Karlsruhe, Karlsruhe, Germany<br />

2 Organismische Interaktionen, Max-Planck-Institut für Terrestrische<br />

Mikrobiologie, Marburg, Germany<br />

The basidiomycetous fungus Ustilago maydis is a ubiquitous pathogen on<br />

maize, one of the world’s most important cereal crops. As a biotrophic fungus,<br />

U. maydis depends on its host plant for proliferation. The fungus does not use<br />

75

76<br />

aggressive virulence strategies, and infected plant cells remain alive.<br />

Apparently, the fungus must have evolved strategies that cope with the plant<br />

defence program, ensure the proliferation of fungal cells, but also secure the<br />

survival of the host plant.<br />

We have used comparative transcriptomics of both fungal and plant cells to get<br />

insight into the infection process. Our data reveal a complex cascade of fungal<br />

transcription factors that coordinate cell cycle and filamentous growth. Specific<br />

regulators orchestrate a complex repertoire of secreted proteins thought to<br />

establish the biotrophic interaction, which includes suppression of plant defence<br />

and reprogramming of metabolic resources within the plant. Gene expression-<br />

and metabolome-analysis of the host plant during the infection process revealed<br />

that U. maydis is recognized initially and elicits plant defense reactions. With<br />

the establishment of the biotrophic interaction these responses are repressed by<br />

the fungus. Our data indicate further that U. maydis interferes with normal leaf<br />

development by preventing the transition from sink to source tissue. Our studies<br />

provide novel insights into the complexity of a biotrophic interaction.<br />

KR 02<br />

Quorum signal integration and subpopulation signalling in<br />

sporulating B. subtilis communities<br />

I. Bischofs *1 , J. Hug 2 , A. Liu 3 , D. Wolf 1 , A. Arkin 3<br />

1 Physical Biosciences Division, Lawrence Berkeley Lab, Berkeley, United<br />

States<br />

2 Department of Electrical Engineering, UC Berkeley, Berkeley, United States<br />

3 Department of Bioengineering, UC Berkeley, Berkeley, United States<br />

A common form of quorum sensing in gram positive bacteria is mediated by<br />

peptides which act as phosphatase regulators (Phr) of receptor aspartyl<br />

phosphatases (Raps). In B. subtilis several Phr signals are integrated in<br />

sporulation phosphorelay signal transduction. Based on a theoretical model we<br />

demonstrate that the phosphorelay can perform a sensitive division operation of<br />

inductive kinase encoded signals by instructive quorum modulated Rap signals,<br />

indicative of cells computing a „food per cell“ estimate. In addition, we show<br />

experimentally that the rapA‐phrA operon is heterogeneously induced in<br />

sporulating microcolonies. Cells delaying sporulation sustain PhrA expression<br />

during periods of active growth, while cells committing to sporulation do not<br />

induce or downregulate PhrA. Together with the model these findings suggest<br />

that the phosphorelay normalizes environmental signals by the size of the<br />

subpopulation actively competing for nutrients as signaled by PhrA. Hence we<br />

speculate that the various Phrs could facilitate subpopulation communication in<br />

isogenic communities to control cell differentiation by interpreting<br />

(environmental) signals based on the spatio‐temporal community structure.<br />

KR 03<br />

SulfoSYS - Sulfolobus Systems Biology: towards a Silicon<br />

Cell Model for the central carbohydrate metabolism of the<br />

Archaeon Sulfolobus solfataricus un<strong>der</strong> temperature<br />

variation<br />

M. Zaparty *1 , B. Siebers 1 , . SulfoSYS consortium 2<br />

1 Department of Chemistry, Biofilm Centre, Molecular Enzyme Technology and<br />

Biochemistry, University of Duisburg-Essen, Duisburg, Germany<br />

2 ., ., Germany<br />

Sulfolobus Systems Biology (SulfoSYS; 1) focuses on the study of the central<br />

carbohydrate metabolism (CCM) of Sulfolobus solfataricus and its regulation<br />

un<strong>der</strong> temperature variation at the systems level. In Archaea carbohydrates are<br />

metabolized by modifications of the classical pathways known from Bacteria or<br />

Eukarya [for review see 2], e.g. the unusual branched Entner-Doudoroff (ED)<br />

pathway [3], which is utilized for glucose degradation also in S. solfataricus.<br />

Although the complexity and modifications of archaeal glycolytic pathways are<br />

well established, knowledge about their regulation as well as energetics is<br />

rather scarce. The archaeal model organism of choice, S. solfataricus [4], is a<br />

thermoacidophilic Crenarchaeon that optimally grows at 80°C (60-92°C) and<br />

pH 2-4. The organism is a strict aerobe and grows heterotrophically on various<br />

carbon sources, amino acids and peptides [5].<br />

In general, life at high temperature requires very efficient adaptation to<br />

temperature changes, which is most difficult to deal with for organisms and it is<br />

unclear how biological networks can withstand and respond to such changes.<br />

The integrative project combines genomic, transcriptomic, proteomic,<br />

metabolomic, as well as kinetic and biochemical information. The final goal of<br />

SulfoSYS is the construction of a silicon cell model for this part of the living<br />

cell that will enable computation of the CCM network. Here we report on one<br />

of the first archaeal Systems Biology projects.<br />

[1] Albers S.-V. et al. (<strong>2009</strong>) Biochem. Soc. Trans., in press<br />

[2] Siebers B. and Schönheit P. (2005) Curr. Opin. Microbiol. 8, 695-705<br />

[3] Ahmed et al. (2005) Biochem. J. 390, 529-540<br />

[4] Zillig W. et al. (1980) Arch. Microbiol. 125, 259-269<br />

[5] Grogan D.W. (1998) J. Bacteriol. 171, 6710–6719<br />

KR 04<br />

Origin and primary function of the luciferase reaction in<br />

bacteria<br />

B.A. Hense *1 , J. Müller 2 , C. Kuttler 2<br />

1<br />

Institute of Biomathematics and Biometry, Hemholtz Zentrum Munich,<br />

Neuherberg/Munich, Germany<br />

2<br />

Centre for Mathematical Sciences, Technical University Munich,<br />

Garching/Munich, Germany<br />

Several bacteria species produce light by an O2 and energy consuming reaction<br />

catalyzed by luciferases. As these species often live in symbiosis with<br />

eukaryotes, the function of luminescence has been connected with fitness<br />

benefits of the host. However, the evolutionary origin and even the recent<br />

primary function is often still unclear. One hypothesis assumes that the original<br />

function of the luciferase reaction was to decrease the amount of oxygen and<br />

thus of its toxic reactive <strong>der</strong>ivates. This antioxidative activity has developed<br />

times as a reaction to the incipient increase of O2 concentration due to<br />

photosynthetic activity. According to this hypothesis, bacterial luminescence<br />

nowadays only survived with altered functions, as high recent O2 concentration<br />

made costly O2 detoxification inefficient. Interestingly, in most luminescent<br />

bacteria the luciferase reaction is controlled by autoinducers, following a<br />

strategy usually referred as quorum sensing. We introduced a mathematical<br />

model for the autoinducer-luciferase system based on data of Vibrio fischeri.<br />

Main results are: 1.) It is possible to estimate the reachable O2 concentration, to<br />

which each cell is exposed, by autoinducers. 2.) When cells live in aggregates<br />

(microcolonies), only a limited number of cells suffer from high energetic costs<br />

of the luminescence reaction. 3.) The fraction of cells with high energetic<br />

burdens declines with increasing colony size. Thus, activating luciferase<br />

reaction in the aggregate size dependent manner by autoinducers may enable a<br />

cost efficient function of the reaction for O2 elimination even at current<br />

environmental conditions.<br />

KR 05<br />

Proteomic analysis of the response of the human-pathogenic<br />

fungus Aspergillus fumigatus to hypoxia<br />

O. Kniemeyer *1 , M. Vödisch 1 , K. Scherlach 2 , R. Winkler 2 , C. Hertweck 2 , U.<br />

Horn 3 , A.A. Brakhage 1<br />

1 Molecular and Applied Microbiology, Leibniz Institute for Natural Product<br />

Research and Infection Biology (HKI), Friedrich Schiller University Jena,<br />

Jena, Germany<br />

2 Biomolecular Chemistry, Leibniz Institute for Natural Product Research and<br />

Infection Biology (HKI), Jena, Germany<br />

3 Bio Pilot Plant, Leibniz Institute for Natural Product Research and Infection<br />

Biology (HKI), Jena, Germany<br />

Aspergillus fumigatus is a ubiquitous, pathogenic filamentous fungus requiring<br />

atmospheric levels of oxygen for optimal growth. Upon inhalation, A.<br />

fumigatus spores germinate, un<strong>der</strong>go hyphal growth and spread in the lungs<br />

causing deadly invasive aspergillosis in immunocompromised patients.<br />

Surprisingly, little is known about the mechanisms by which the pathogen<br />

adapts to the low-oxygen (hypoxic) microenvironments in infected, necrotic<br />

tissue. Just recently it was shown that hypoxia adaptation is an important<br />

virulence attribute of human-pathogenic fungi. To identify novel hypoxiasensing<br />

and adapting pathways we have performed proteomic analyses of an A.<br />

fumigatus strain in response to low oxygen partial pressure by using an oxygencontrolled<br />

chemostat. Proteins involved in glycolysis, amino acid biosynthesis,<br />

stress response and respiration including the pyruvate dehydrogenase showed<br />

an increased spot volume ratio un<strong>der</strong> hypoxic conditions. In addition, molecular<br />

oxygen incorporating monooxygenases of the sterol and ubiquinone<br />

biosynthesis pathways were up-regulated un<strong>der</strong> hypoxic growth conditions as<br />

well. In contrast, proteins involved in sulphate assimilation and acetate<br />

activation were down-regulated. Strikingly, proteins encoded by genes<br />

organised in secondary metabolite clusters, showed also an increased level of<br />

expression un<strong>der</strong> hypoxic growth conditions. This finding was confirmed on<br />

the transcriptional level by Northern blot analysis. Possible roles of the<br />

differently expressed proteins will be discussed.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KR 06<br />

Comparison of composition and adaptation of the<br />

Corynebacterium glutamicum proteome un<strong>der</strong> different<br />

physiological conditions<br />

A. Poetsch *1 , D. Schluesener 1 , F. Fischer 1 , U. Haussmann 1 , C. Troetschel 1 , D.<br />

Wolters 2 , S.J. Liu 3<br />

1 Plant Biochemistry, Ruhr University Bochum, Bochum, Germany<br />

2 Analytical Chemistry, Ruhr University Bochum, Bochum, Germany<br />

3 Institute for Microbiology, Chinese Academy of Sciences, Beijing, China<br />

Corynebacterium glutamicum is one of the most important industrial<br />

microorganisms, used mainly for amino acid production. Proteomics has<br />

matured to a state that allows the comprehensive coverage of cytosolic,<br />

secreted, and recently even integral membrane proteins [1]. Furthermore,<br />

technologies enabling relative and absolute protein quantification have become<br />

available. We have applied these technologies to un<strong>der</strong>stand the physiological<br />

adaptation of C. glutamicum to a variety of conditions: L-lysine production,<br />

alternative carbon sources, and stress conditions (e.g. hyperosomolarity).<br />

Results of these studies will be compared and special attention will be paid to<br />

the composition to the membrane proteome. One focus will be the most<br />

abundant components in the membrane, which was estimated by analysis of the<br />

mass spectrometry data (spectral counting). For example, high abundances<br />

were found for members of the respiratory chain, substrate uptake, and protein<br />

secretion. A second focus will be similarities and differences in the proteome<br />

composition between the analyzed conditions. Here, concerning substrate<br />

uptake, extremely strong induction of new substrate uptake systems were found<br />

for growth on the alternative carbon sources citrate and benzoate, while the<br />

abundance of sugar uptake systems only slightly decreased. Furthermore, an<br />

increase of members of the respiratory chain was observed on alternative<br />

carbon sources. Also for proteins involved in osmoregulation such as MscL,<br />

differences were observed un<strong>der</strong> the compared growth conditions, which<br />

un<strong>der</strong>score the power of current proteomics technologies to concomitantly<br />

disclose occurring adaptation processes on a global scale.<br />

[1] Fischer et al., Mol Cell Proteomics, 5.3, 444-453 (2006).<br />

KS 01<br />

Mitochondrial protein biogenesis<br />

P. Rehling *1<br />

1<br />

Department of Biochemistry II, Faculty of Medicine, University of Goettingen,<br />

Goettingen, Germany<br />

Mitochondria are known as the powerhouse of eukaryotic cells since they<br />

produce the majority of cellular ATP. This chemical energy carrier is<br />

synthesized by the F1Fo-ATPase in cooperation with the respiratory chain<br />

protein complexes of the inner mitochondrial membrane. The five respiratory<br />

chain complexes are multi-subunit membrane protein complexes mainly<br />

composed of nuclear encoded proteins that are synthesized in the cytosol and<br />

imported into mitochondria. In fact, the majority of mitochondrial proteins are<br />

imported into mitochondria post-translationally from the cytosol and only small<br />

sets of proteins (eight in yeast and thirteen in humans) are encoded by<br />

mitochondria DNA.<br />

Multi-protein translocases in the membranes recognize and transport precursor<br />

proteins into mitochondria [1]. The outer membrane translocase (TOM<br />

complex) transports proteins across the outer membrane. Two translocase<br />

complexes in the inner membrane (TIM complexes) accept precursor proteins<br />

from the TOM complex and mediate further transport steps across and into the<br />

inner membrane. The twin-pore carrier translocase (TIM22 complex) is<br />

specialized to insert multi-spanning membrane proteins into the inner<br />

membrane [2]. In contrast, proteins with N-terminal presequences use the<br />

presequence translocase (TIM23 complex) for transport [3,4]. After insertion of<br />

imported proteins into the inner mitochondrial membrane by the translocase<br />

complexes their assembly into multi subunit protein complexes can take place.<br />

For this, individual subunits need to associate with each and do so most likely<br />

in a defined or<strong>der</strong>. Assembly factors that act as chaperones in this process<br />

appear to play an important role for this coordinated process [5].<br />

[1] Rehling, P. et al. (2004). Nature Rev. Mol. Cell Biol. 5: 519-530<br />

[2] Rehling, P. et al. (2003). Science 299: 1747-1751<br />

[3] Chacinska, A. et al. (2005). Cell 120: 817-829<br />

[4] Meinecke, M. et al. (2006). Science 312, 1523-1526<br />

[5] Mick et al. (2007). EMBO J. 26 , 4347-4358<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

KS 02<br />

Oxidative Protein Folding in the Intermembrane Space of<br />

Mitochondria<br />

M. Bien 1 , S. Longen 1 , N. Mesecke 1 , K. Bihlmaier 1 , J.M. Herrmann 1 , J. Riemer *1<br />

1 Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany<br />

For a long time, the endoplasmic reticulum (ER) was consi<strong>der</strong>ed to be the only<br />

compartment of the eukaryotic cell in which proteins are folded by dedicated<br />

enzymes in an oxidation-driven process. However, it became recently evident<br />

that eukaryotic cells harbor another oxidizing compartment, the small lumen<br />

between the outer and inner membranes of mitochondria - the intermembrane<br />

space (IMS). In mitochondria, protein oxidation can be used to drive protein<br />

translocation from the cytosol across the outer membrane. Moreover, redox<br />

reactions have been implied in many IMS-connected processes like apoptosis,<br />

aging and the regulation of the respiratory chain.<br />

The IMS machinery that drives oxidative protein folding consists of three<br />

highly conserved components: the import receptor Mia40 and the sulfhydryl<br />

oxidase Erv1 as well as the zinc-binding protein Hot13. Mia40 contains an<br />

essential redox-active disulfide bond in a cysteine-proline-cysteine signature<br />

which forms mixed disulfides with cysteine residues of newly imported<br />

polypeptide chains. These cysteine residues are initially kept reduced by Zn<br />

ions that are removed by Hot13 before interaction of substrate and Mia40. The<br />

Mia40-dependent oxidation of incoming proteins locks them in a stably folded<br />

state in which they are unable to traverse the outer membrane, thereby leading<br />

to a directed net transport of Mia40 substrates into the IMS of mitochondria.<br />

Mia40 is re-oxidized by the flavoprotein Erv1. Erv1 contains two essential<br />

redox-active cysteine-x-x-cysteine pairs which shuffle electrons from Mia40 to<br />

the FAD cofactor. In or<strong>der</strong> to be reoxidized, Erv1 efficiently interacts with<br />

cytochrome c which passes electrons from Erv1 on to cytochrome c oxidase<br />

and molecular oxygen giving rise to the production of water. Here, we present<br />

the reconstitution of the complete Mia40-Erv1 pathway. We thereby provide<br />

detailed insight into the molecular mechanism of this machinery and the flow of<br />

electrons from substrates via Mia40 and Erv1 to the respiratory chain.<br />

KS 03<br />

Peroxisomal matrix protein import: Critical steps of the<br />

receptor cycle<br />

C. Cizmowski *1 , E. Hambruch 1 , W. Stanley 2 , M. Willmanns 2 , W. Schliebs 1 ,<br />

H.W. Platta 1 , W. Girzalsky 1 , R. Erdmann 1<br />

1 Institute of Physiological Chemistry, Department for Systems Biochemistry,<br />

Ruhr-University Bochum, Bochum, Germany<br />

2 EMBL, Hamburg Outstation, Hamburg, Germany<br />

Peroxisomal proteins carrying a peroxisome targeting signal type 1 (PTS1) are<br />

recognized in the cytosol by the cycling import receptor Pex5p. The binding of<br />

cargo is accompanied by conformational changes within the TPR-domain of the<br />

receptor (I). The receptor-cargo complex docks at the peroxisomal membrane<br />

where it binds to several peroxins. After dissociation of the receptor-cargo<br />

complex, Pex5p is released into the cytosol to perform another round of import.<br />

During the receptor cycle Pex5p is tightly bound to the peroxisomal membrane.<br />

Although no transmembrane regions are predictable Pex5p displays typical<br />

features of an integral membrane protein. In fact, we could demonstrate that<br />

both human and yeast PTS1 receptor possess intrinsic lipid-binding activity.<br />

Purified soluble PTS1- receptors are capable of inserting spontaneously into<br />

artificial and cellular phospholipid membranes. Liposome binding and<br />

subsequent flotation analysis have been used as a tool to map the lipidinteracting<br />

sites of Pex5p (II). Recycling of Pex5p from the peroxisomal<br />

membrane back to the cytosol strongly depends from the activity of the AAA<br />

ATPases Pex1p and Pex6p. This was demonstrated by an in vitro export assay.<br />

Dislocation of Pex5p was achieved when membrane fractions were mixed with<br />

cytosolic fractions containing AAA-peroxins in the presence of an ATPregeneration<br />

system. When one of the AAA-peroxins or the ATP-regeneration<br />

system was omitted, Pex5p completely remained in the organellar pellet<br />

fraction (IV).<br />

KS 04<br />

YggB (MscS) of Corynebacterium glutamicum:<br />

Mechanosensitive channel and/or glutamate exporter<br />

K. Boerngen *1 , N. Moeker 1 , S. Morbach 1 , R. Kraemer 1<br />

1 Institute of Biochemistry, University of Cologne, Koeln, Germany<br />

Corynebacterium glutamicum is a Gram-positive, apathogenic soil bacterium<br />

with exceptional importance for the industrial production of various amino<br />

acids, especially L-glutamate. Although C. glutamicum has been used for<br />

glutamate production for decades the export mechanism of this amino acid is<br />

77

78<br />

still unknown. Recently, evidence was provided that the small<br />

mechanosensitive channel protein YggB (MscS) of C. glutamicum is linked to<br />

glutamate excretion un<strong>der</strong> glutamate production conditions in biotechnological<br />

applications [1]. C. glutamicum YggB was identified as mechanosensitive<br />

channel which is required for solute efflux in response to hypoosmotic<br />

conditions [2, 3]. It is homologous to MscS from E. coli (286 AA) concerning<br />

its N-terminal part, but, in addition, carries a long C-terminal domain of<br />

approximately 250 amino acids including a fourth transmembrane segment.<br />

Based on the membrane topology, different C-terminal truncations of C.<br />

glutamicum YggB were created and analyzed with respect to their function as<br />

mechanosensitive channel on the one hand and concerning a possible new role<br />

in export of glutamate, on the other. The expression of a truncated form of the<br />

YggB protein was shown to have a dramatic impact on the function of the<br />

protein affecting also the cell integrity. Glutamate excretion occurred<br />

spontaneously after the truncation of parts of the C-terminal domain.<br />

Furthermore, cells expressing various YggB constructs seem to have a<br />

decreased capability to cope with osmotic stress conditions.<br />

[1] Nakamura et al. (2007) Appl Environ Microbiol. 73, 4491-4498<br />

[2] Ruffert et al. (1999) J. Bacteriol. 181, 1673-1676<br />

[3] Nottebrock et al. (2003) FEMS Microb. Lett. 218, 305-309<br />

KS 05<br />

Two copper translocating ATPases of the thermoacidophilic<br />

archaeon Sulfolobus solfataricus<br />

C. Völlmecke 1 , G. Schröter 1 , S. Drees 1 , M. Zoltner 1 , S. Albers 2 , A. Driessen 2 ,<br />

M. Lübben *1<br />

1 LS Biophysik, Ruhr-Universität Bochum, Bochum, Germany<br />

2 Dept of Molecular Microbiology, University of Groningen, Groningen,<br />

Netherlands<br />

The thermoacidophile Sulfolobus solfataricus grows aerobically at pH 2-3 and<br />

at temperatures up to 90 deg in mineral media supplemented with complex<br />

carbon sources. In or<strong>der</strong> to sustain heavy metal homeostasis in this<br />

environment, P-type transport ATPases, also named CPX-ATPase due to<br />

conserved amino acid motifs, are required. CopA and CopB were the only<br />

CPX-ATPases that could be identified by genome sequencing of S. solfataricus.<br />

Both ATPases have been heterologously expressed in Escherichia coli, have<br />

been purified to homogeneity and subjected to polyclonal antibody formation.<br />

Both enzymes showed significant ATP hydrolytic activity that could by<br />

stimulated severalfold by copper or silver ions, which strongly supports their<br />

role in copper transport in vivo. However, the accurate biological tasks of CopA<br />

and CopB have to be elucidated. When Sulfolobus was grown in subtoxic<br />

concentrations of copper, CopA biosynthesis could be strongly induced. This<br />

behavior qualifies CopA as an active exporter. Un<strong>der</strong> these growth conditions<br />

however, no changes in the expression level of CopB were observed, which<br />

suggests that this ATPase does not work as copper resistance factor. In or<strong>der</strong> to<br />

characterize the molecular function of CopB, its encoding gene was disrupted<br />

in Sulfolobus. The phenotypic characterization of the copB mutant is reported.<br />

KS 06<br />

Tat-dependent transport of protein substrates with long<br />

unstructured linker peptides between the signal peptide and<br />

a folded domain<br />

U. Lindenstrauß 1 , T. Brüser *1<br />

1<br />

Institute of Biology / Microbiology, University of Halle-Wittenberg, Halle,<br />

Germany<br />

The Tat system serves to translocate folded and often cofactor-containing<br />

proteins across biological membranes. Unstructured proteins of up to 20-30<br />

kDa in size are also accepted by the Tat system, but only if they are polar on<br />

their surface. Using the iron-sulfur cofactor-containing model Tat-substrate<br />

HiPIP, we now demonstrate that the bacterial Tat system can translocate small<br />

globular proteins even when long unstructured linker peptides are sandwiched<br />

between the signal peptide and the N-terminus of the mature domain. The ironsulfur<br />

cofactor was fully assembled in the transported protein, which<br />

demonstrates that HiPIP was folded during translocation. A variation of linker<br />

lengths and C-terminal domains indicated that the Tat compatibility is limited<br />

by the volume of the transported protein. The unexpected tolerance towards<br />

unstructured linker peptides challenges our current un<strong>der</strong>standing of the Tat<br />

mechanism.<br />

KT 01<br />

Corynebacterium glutamicum as platform for production of<br />

fine chemicals:<br />

carbon control and access to new carbon substrates<br />

V.F. Wendisch *1<br />

1 Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms<br />

University Muenster, Muenster, Germany<br />

Amino acid production by Corynebacterium glutamicum amounts to about 2.5<br />

million tons per year and, thus, is a proven large-scale biotechnological process.<br />

The traditional product spectrum has recently been widened by metabolic<br />

engineering approaches, e.g. for production of 3-aminopropionic acid or<br />

ethanol. The generally recognized as safe C. glutamicum has been shown to be<br />

robust against a variety of inhibitory compounds and to be able to efficiently<br />

co-utilize different carbon source mixtures. The characterization of genetic<br />

control mechanisms of carbon metabolism, which are distinct from those of the<br />

model bacteria E. coli and B. substilis, enabled strain development for<br />

improved carbon substrate utilization. In addition, pathways for access to new<br />

carbon sources have been engineered, e.g. for efficient use of glycerol, which<br />

arises in large quantities in the biodiesel process as major by-product of plant<br />

seed oil transesterification with methanol. Progress and future challenges to<br />

establish C. glutamicum as platform for the production of fine chemicals will be<br />

discussed.<br />

[1] Engels V, Lindner SN & Wendisch VF (2008) J Bacteriol 190: 8033-8044.<br />

[2] Rittmann D, Lindner SN & Wendisch VF (2008) Appl Environ Microbiol<br />

74: 6216-6222.<br />

[3] Youn J-W, Jolkver E, Krämer R, Marin K & Wendisch VF (2008) J<br />

Bacteriol 190: 6458-6466.<br />

[4] Georgi T, Engels V & Wendisch VF (2008) J Bacteriol 190: 963-971.<br />

[5] Polen T, Schluesener D, Poetsch A, Bott M & Wendisch VF (2007) FEMS<br />

Microbiol Lett 273: 109–119.<br />

[6] Sindelar G & Wendisch VF (2007) Appl Microbiol Biotechnol 76: 677-689.<br />

[7] Engels V & Wendisch VF (2007) J Bacteriol 189: 2955-2966.<br />

KT 02<br />

Solvent-tolerant Pseudomonas: platform organisms for<br />

whole-cell biocatalysis?<br />

L.M. Blank *1 , J. Ruehl 1 , B. Ebert 1 , B. Bühler 1 , A. Schmid 1<br />

1 Department of Bio- and Chemical Engineering, Chair of Chemical<br />

Biotechnology, TU Dortmund, Dortmund, Germany<br />

A key limitation of whole-cell redox biocatalysis for the production of valuable,<br />

specifically functionalized products is substrate/product toxicity, which can<br />

potentially be overcome by using solvent-tolerant microorganisms. Selected<br />

and adapted strains of Pseudomonas putida were reported to tolerate high<br />

concentrations of organic solvents and grew in the presence of a second octanol<br />

[1], toluene [1], and styrene [2] phase. Using (13)C tracer based metabolic flux<br />

analysis, we investigated in solvent-tolerant P. putida strains the<br />

interrelationship of butanol or octanol tolerance and energy metabolism and<br />

quantified the NAD(P)H regeneration rate in the presence of these toxic<br />

solvents. The harsh growth conditions increased the energy demand of the<br />

solvent-tolerant P. putida strains. We show that solvent-tolerant P. putida have<br />

the remarkable ability to compensate for high energy demands by boosting their<br />

energy metabolism to levels up to an or<strong>der</strong> of magnitude higher than those<br />

observed during unlimited growth. According to the driven by demand concept,<br />

the NAD(P)H regeneration rate was increased up to eightfold by two<br />

mechanisms: (a) an increase in glucose uptake rate without secretion of<br />

metabolic side products, and (b) reduced biomass formation. This points to a<br />

high energy and redox cofactor demand for cell maintenance, which limits for<br />

example the potential for redox biocatalysis in the presence of octanol. An<br />

estimated upper bound for the NAD(P)H regeneration rate available for redox<br />

biocatalysis suggests that cofactor availability does not limit however<br />

biocatalysis un<strong>der</strong> optimized conditions, for example, in the absence of toxic<br />

solvent. The results are discussed in the context of the applicability of these<br />

extremophiles as hosts for industrial biotechnology.<br />

[1] Blank LM et al.: Febs J 2008, 275(20):5173-5190<br />

[2] Park JB et al.: Biotechnol Bioeng 2007, 98(6):1219-1229<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

KT 03<br />

Expression of a novel bacterial tyrosinase in Escherichia<br />

coli<br />

M. Fairhead 1 , K. Grie<strong>der</strong> 1 , L. Thöny-Meyer *1<br />

1 Laboratory of Biomaterials, Swiss Fe<strong>der</strong>al Laboratories for Materials Testing<br />

and Research, St. Gallen, Switzerland<br />

Tyrosinase (EC 1.14.18.) is a type 3 copper oxidase enzyme that converts<br />

tyrosine to DOPA to dopaquinone, which is a precursor in melanin<br />

biosynthesis. Tyrosinases have many potential biotechnological applications,<br />

one of them <strong>bei</strong>ng the ability to catalyze protein-protein or proteinpolysaccharide<br />

cross-linking.<br />

While fungal tyrosinases are made as zymogens which are processed by<br />

chymotrypsin-like serine proteases to their mature form, bacterial tyrosinases<br />

do not require proteolytic activation.<br />

We report on the successful expression of a tyrosinase-like gene from the<br />

aquatic bacterium Verrucomicrobium spinosum in Escherichia coli. The<br />

overproduced enzyme was able to form the black pigment melanin when<br />

tyrosine was added to the medium. The protein was purified to homogeneity<br />

and shown to contain 1.72 copper atoms per polypeptide, which is in agreement<br />

with a binuclear copper site. Both mono-and diphenol oxidase activites were<br />

present as shown by spectrophotometric assays for tyrosine hydroxylation.<br />

Specific activity, temperature and pH optimum will be presented. In addition,<br />

preliminary data on cross-linking experiments indicate that this enzyme may be<br />

used for immobilization of enzymes on surfaces or in aggregates.<br />

KT 04<br />

Conversion of polycyclic aromatic and heterocyclic<br />

hydrocarbons by extracellular fungal peroxygenases<br />

E. Aranda *1 , M. Kluge 2 , R. Ullrich 2 , M. Hofrichter 2 , G. Kayser 1<br />

1<br />

Unit of Environmental Process Engineering, International Graduate School<br />

Zittau, Zittau, Germany<br />

2<br />

Environmental Biotechnology, International Graduate School Zittau, Zittau,<br />

Germany<br />

For decades, the conversion of polycyclic aromatic hydrocarbons (PAHs) and<br />

heterocyclic compounds has been subject of microbiological and biochemical<br />

studies due to their recalcitrance, ecotoxicological effects and importance as<br />

precursors in pharmaceutical and fine-chemical synthesis. Various<br />

microorganisms including fungi and bacteria and their enzymatic systems were<br />

investigated, which led to the elucidation of different pathways and numerous<br />

metabolites including epoxides, hydroxylation and ring-cleavage products.<br />

Recently, we have discovered a new type of peroxide-consuming enzyme with<br />

exceptional catalytic properties – the aromatic peroxygenase (APO) – that<br />

oxygenates a huge number of aromatics including PAHs and heterocycles. This<br />

enzyme is an extracellular heme-thiolate protein produced by agaric<br />

mushrooms such as Agrocybe aegerita and Coprinellus radians and shows<br />

hybrid functions of peroxidases and cytochrome P450 monooxygenases. Here<br />

we report on new pathways and reactions for the conversion of polyaromatic<br />

compounds like dibenzothiophene, dibenzofuran, fluorene, phenanthrene and<br />

anthracene catalyzed by APO from Agrocybe aegerita and Coprinellus radians<br />

(abbreviated as AaP and CrP). AaP was able to hydroxylate the aromatic rings<br />

of all substrates tested at different positions as well as the heterocyclic sulfur in<br />

case of dibenzothiophene. In contrast, CrP showed a limited capacity for<br />

aromatic ring-hydroxylation and oxygenated dibenzothiophene just at the<br />

sulfur. Despite these differences, both enzymes oxygenated naphthalene<br />

regioselectively leading to naphthalene 1,2-oxide as initial metabolite that<br />

spontaneously hydrolyzed to form 1-naphthol and 2-naphthol as major and<br />

minor product, respectively. Recent experiments have indicated that also larger<br />

PAHs are subject of APO oxidation and the limits of oxygenation regarding<br />

size and structure of PAHs are currently un<strong>der</strong> investigation.<br />

KT 05<br />

Improving thermal stability of a cold-active lipase by<br />

directed evolution<br />

C. Elend *1 , A. Basner 1 , W.R. Streit 2 , G. Antranikian 1<br />

1<br />

Technische Mikrobiologie, Technische Universität Hamburg-Harburg,<br />

Hamburg, Germany<br />

2<br />

Biozentrum Klein Flottbek / Allgemeine Mikrobiologie und Biotechnologie,<br />

Universität Hamburg, Hamburg, Germany<br />

Lipases [EC 3.1.1.3] are currently one of the most prominent enzymes used in<br />

biotechnology. They often possess a wide substrate range, although they<br />

catalyse highly specific chemo-, regio- and enantioselective reactions without<br />

need of co-factors.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

A metagenome <strong>der</strong>ived cold-active, but heat-labile lipase LipCE was subjected<br />

to directed evolution to improve stability at elevated temperatures. The enzyme<br />

was introduced to two rounds of random mutagenesis using epPCR with an<br />

average mutation rate of 3 bp per 1000. A mutant library with 4230 active<br />

clones in E. coli was constructed in 96 well plates. The crude extracts in the<br />

plates were heat-treated for 45 min at 50°C and enhanced thermostability was<br />

assayed by measuring residual activity with pNP-laurate as a substrate.<br />

The most thermostable clone from the first round was subjected to another<br />

round of epPCR, yielding the thermostable mutant IIIB2/VIIF6. Sequence<br />

analysis revealed six mutations leading to four amino acid substitutions in the<br />

476 aa protein. Two of the four amino acid substitutions have taken place in the<br />

proximity of the active site. The mutant enzyme was purified and compared to<br />

the wild-type lipase. A significant increase in thermal stability was measured,<br />

the half-life at 50°C increased from 3 to 30 minutes without loss in specific<br />

enzyme activity. A slightly elevated conversion of smaller pNP-acylesters as<br />

well as pNP-myristate was observed. An impact on the enzymatic activity<br />

converning the pH- and temperature profile was not observed. The enzyme<br />

retained its high activity at low temperatures.<br />

KT 06<br />

Metabolic Engineering of Escherichia coli towards the<br />

Production of Tocotrienol<br />

S. Ghanegaonkar *1 , G. Sprenger 1 , C. Albermann 1<br />

1 Institute of Microbiology, Universität Stuttgart, Stuttgart, Germany<br />

Tocotrienols and tocopherols together form the family of Vitamin E.<br />

Tocotrienols consist of an aromatic polar head <strong>der</strong>ived from aromatic aminoacid<br />

pathway and an unsaturated isoprenoid lipophilic tail <strong>der</strong>ived from DXP<br />

pathway. In recent years tocotrienols have gained importance due to their<br />

neuroprotective, anticancer and antioxidant properties. Tocotrienols are<br />

exclusively synthesized in photosynthetic organisms. [1]<br />

Recently, the in-vivo biosynthesis of δ-tocotrienol in recombinant Escherichia<br />

coli cells has been reported using an expression plasmid carrying all the<br />

necessary genes. [2] Plasmid systems are suitable for high level protein<br />

expression but can result in decreased productivity with probable loss of<br />

plasmid during high cell density cultivation in bioreactor. Hence, a robust E.<br />

coli strain was constructed for the heterologous biosynthesis of the key<br />

intermediate of all the forms of tocotrienol i.e. MGGBQ (2-methyl-6geranylgeranyl-benzoquinol)<br />

by integrating the genes encoding for phydroxyphenyl-pyruvate<br />

dioxygenase, geranylgeranylpyrophosphate synthase,<br />

and homogentisate-phytyltransferase, into the chromosome of E. coli<br />

BW25113. This strain was cultivated in minimal medium with glucose or<br />

glycerol as the sole carbon and energy sources in absence of any selection<br />

marker. Cell growth of recombinant strain was similar to wild type without<br />

loss of its chromosomal integrated genes. After induction with IPTG the<br />

recombinant strain exclusively produced MGGBQ which is accumulated in the<br />

cell. Integration of an additional gene encoding for isopentenyl pyrophosphate<br />

isomerase from DXP pathway enhanced the MGGBQ production. Integration<br />

of the gene encoding for tocopherol/tocotrienol cyclase resulted in formation of<br />

δ-tocotrienol.<br />

[1] D. DellaPenna, et al. Annu. Rev. Plant Biol. 2006, 57, 711-738.<br />

[2] C. Albermann et al. Chembiochem. 2008, 9, 2524-2533.<br />

KT 07<br />

Monoseptic cultivation un<strong>der</strong> non-sterile conditions in 350<br />

litre scale<br />

K.P. Stahmann *1 , K. Schnitzlein 2 , M. Huddar 2 , S. Barig 1<br />

1<br />

Technische Mikrobiologie, Fachbereich Bio-, Chemie- und Verfahrenstechnik,<br />

Fachhochschule Lausitz, Senftenberg, Germany<br />

2<br />

Lehrstuhl für Chemische Reaktionstechnik, Brandenburgische Technische<br />

Universität Cottbus, Cottbus, Germany<br />

Applied Microbiology is on the way to replace chemical processes. A first<br />

example is the production of riboflavin by Bacillus subtilis or Ashbya gossypii.<br />

Both processes co-exist in industrial scale and are both economically and<br />

ecologically better than the chemical synthesis (Appl Microbiol Biotechnol<br />

53:509). But both processes and many more, e.g. L-amino acid or enzyme<br />

production are run with cost-intensive sterile technique. High investment<br />

expenses e.g. for steel vessels and tight systems as well as high running costs<br />

for energy allow only financially sound companies to start a new process. High<br />

selling prices inhibit e.g. replacement of chemical reactions by enzymatic<br />

catalysis in production plants. A cheap and robust process is needed.<br />

Since lipases are already successful in several industrial applications because<br />

they work in organic solvents a new production system was developed. Five<br />

conditions were found to generate selectivity for stable monoseptic one-week-<br />

79

80<br />

cultivations: mineral salts medium, nitrate as sole nitrogen source, plant<br />

triglycerides as sole carbon and energy source, pH 3-4 (Patent Application<br />

WO/2008/067882). Botrytis cinerea and five fungal isolates from soil or<br />

compost were found to grow un<strong>der</strong> these conditions. Best results concerning<br />

lipase production were obtained with Phialemonium spec. AW02. In a 500 litre<br />

rain water barrel filled with 350 litre culture medium, kept at 28°C, and aerated<br />

by 100 litre per minute 20 kU (para-nitrophenylpalmitate hydrolysis) were<br />

obtained after 7 days of cultivation. To keep the pH between 3 and 4 more than<br />

1 litre of 1M HCl had to be added.<br />

KU 01<br />

Protein splicing of fungal inteins<br />

S. Pöggeler *1 , S. Elleuche 1<br />

1 Institute of Microbiology & Genetics / Genetics of eukaryotic Microorganisms,<br />

Georg-August University Göttingen, Göttingen, Germany<br />

Inteins are protein-intervening sequences found inside the coding region of<br />

different host proteins and are translated in-frame with them. They can selfexcise<br />

through protein splicing, which ligates the host protein flanks, termed<br />

exteins, with a peptide bond. Large inteins comprise independent proteinsplicing<br />

and endonuclease domains whereas mini-inteins lack the central<br />

endonuclease domain. We identified mini-inteins in the PRP8 protein of species<br />

in the genus Penicillium and Eupenicillium. We demonstrated that these fungal<br />

PRP8 mini-inteins un<strong>der</strong>go autocatalytic protein splicing when heterologously<br />

expressed in E. coli, in a model host protein, and in a divided GFP model<br />

system. They are among the smallest known nuclear-encoded, active splicing<br />

protein elements. Moreover, we demonstrated that the PRP8 intein of<br />

Penicillium chrysogenum, the major producer of the β-lactam antibiotic<br />

penicillin, is capable of protein splicing in trans when expressed as a bicistronic<br />

operon in E. coli. To identify mini-intein domains that are essential for protein<br />

splicing, deletions were introduced at different sites of the 157-aa PRP8 miniintein<br />

of P. chrysogenum. The removal of eight and six amino at two different<br />

sites resulted in a functional eukaryotic mini-intein of only 143 aa and is the<br />

smallest functional eukaryotic intein engineered so far. In the future, the PRP8<br />

intein of P. chrysogenum may be potentially useful for engineering and<br />

manipulating proteins as well as various applications in protein chemistry.<br />

KU 02<br />

Nuclear gene targeting in Chlamydomonas as exemplified<br />

by disruption of the PHOT gene<br />

B. Zorin *1 , Y. Lu 2 , I. Sizova 3 , P. Hegemann 1<br />

1<br />

Experementelle Biophysik, Humboldt Universität zu Berlin, Berlin, Germany<br />

2<br />

Max-Planck-Institut, Molekulare Pflanzenphysiologie, Potsdam, Germany<br />

3<br />

Division of Radiation Biophysics, Petersburg Nuclear Physics Institute,<br />

Gatchina/St. Petersburg, Russia<br />

Chlamydomonas reinhardtii is the most powerful photosynthetic eukaryotic<br />

unicellular model organism. However, its potential is not fully exploitable since<br />

as in most green plants specific targeting of nuclear genes is not routinely<br />

possible. Recently, we have shown by repair of an introduced truncated model<br />

gene that transformation of Chlamydomonas with single stranded DNA greatly<br />

suppresses random integration of the DNA in the genome whereas homologous<br />

recombination (HR) is left unchanged. However, endogenous genes still could<br />

not be targeted. Here we present optimized transformation conditions that<br />

further improved HR and suppressed non-homologous DNA integration (NHI).<br />

The improved transformation strategy allowed us now to specifically inactivate<br />

in two different Chlamydomonas strains the nuclear PHOT gene, which<br />

encodes for the blue light photoreceptor phototropin (PHOT). The option to<br />

target mo<strong>der</strong>ately expressed Chlamydomonas nuclear genes with high<br />

efficiency now further improves the utility of this this alga for basic science and<br />

biotechnology.<br />

KU 03<br />

Progress in the formation of the DNA Bank Network<br />

H.P. Klenk *1 , G. Haszprunar 2 , J.W. Wägele 3 , B. Gemeinholzer 4<br />

1<br />

Department of Microbiology, German Collection of Microorganisms and Cell<br />

Cultures, Braunschweig, Germany<br />

2<br />

Bavarian State Collection of Zoology, München, Germany<br />

3<br />

Forschungsmuseum König, Bonn, Germany<br />

4<br />

Botanical Garden and Botanical Museum Berlin-Dahlem, Freie Universität<br />

Berlin, Berlin, Germany<br />

The formation of a DNA bank network was funded by the German Science<br />

Foundation (DFG) since Spring 2007. In the meantime the infrastructure for the<br />

network has been established at four locations: microorganisms at the German<br />

Collection of Microorganisms and Cell Cultures (DSMZ); botanical samples at<br />

the Botanical Garden and Botanical Museum Berlin-Dahlem (BGBM), and<br />

zoological samples at the Bavarian State Collection of Zoology (ZSM) and the<br />

Zoologisches Forschungsmuseum Alexan<strong>der</strong> König (ZFMK). More than<br />

12000 DNA samples have been accessed into the collection (including almost<br />

4000 microbial samples) and biological material for the extraction of over<br />

30000 additional samples is maintained at the four research collections.<br />

Evaluation of optimal long-term storage conditions for DNAs is in progress.<br />

The main focus of the network is to enhance taxonomic, systematic, genetic,<br />

conservation and evolutionary studies by providing: (1) at-cost availability of<br />

DNA material; (2) high quality, long-term storage of DNA material on which<br />

molecular studies have been performed; (3) complete on-line documentation of<br />

each sample, including the provenance of the original material, the place of<br />

voucher deposit, information about DNA quality and extraction methodology,<br />

digital images of vouchers and links to published molecular data if available.<br />

The DNA bank databases of all network partners are decentralised administered<br />

and accessible via a central Web portal (http://www.dnabank-network.org/),<br />

providing DNA samples of complementary collections of wildlife organisms.<br />

The design of the database system and shared Web portal facilitates both<br />

accesses to DNA samples as well as to associated specimen and DNA data.<br />

KU 04<br />

Initial steps for the characterization of biofilm formation by<br />

the bioleaching acidophilic bacterium Acidithiobacillus<br />

ferrooxidans following a microarray transcriptome analysis<br />

M. Vera *1 , T. Rohwer<strong>der</strong> 1 , V. Bonnefoy 2 , W. Sand 1<br />

1 Aquatic Biotechnology, Biofilm Centre, University of Duisburg-Essen,<br />

Duisburg, Germany<br />

2 Laboratoire de Chimie Bactérienne,, Institut de Microbiologie de la<br />

Mediterranee, CNRS, Marseille, France<br />

Bioleaching is the extraction of metals, such as copper or gold, from ore by<br />

microorganisms. To obtain their energy for growth, these microorganisms<br />

catalyze oxidation of ferrous iron and/or oxidation of reduced inorganic sulfur.<br />

Ferric iron and/or protons then chemically attack metal sulfides leading to<br />

soluble metal release, which are subsequently recovered. The most studied<br />

leaching microorganisms belong to the Acidithiobacillus and Leptospirillum<br />

genera.<br />

Bacterial attachment increases leaching activities since a special environment is<br />

formed between the bacterium and the metal sulfide surface. This process may<br />

depend on abiotic parameters such as the purity and the degree of<br />

crystallization of the metal sulfide as well as biotic ones such as the capacity of<br />

the bacteria for detecting favourable sites for attachment (chemotaxis) and for<br />

synthesizing a suitable cell envelope (EPS quantity and composition), which<br />

allows attachment. Therefore, planktonic and sessile cells should significantly<br />

differ their gene expression patterns. We have chosen Acidithiobacillus<br />

ferrooxidans ATCC 23270 because its genome sequence is complete and DNA<br />

microarrays are available.<br />

In or<strong>der</strong> to analyze the transcriptional differences between planktonic and<br />

sessile cells, one of the crucial steps is to obtain RNA of good quality. The high<br />

content of EPS of the biofilms is interfering with the RNA extraction and<br />

purification. These polymeric substances bind to nucleic acids, interfering with<br />

DNAse activity and inhibiting RT-PCR amplification. As the first step of this<br />

project we have improved the cultivation of A. ferrooxidans on pyrite (FeS2)<br />

and improved RNA extraction method from this sessile cell population.<br />

KU 05<br />

Darwin or Lamarck: Genetic analysis of a suppressor<br />

mutation in Bacillus subtilis<br />

S. Tholen *1 , K. Gunka 1 , J. Stülke 1<br />

1 Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

In Bacillus subtilis, like in all other organisms, glutamate is the central amino<br />

group donor for all nitrogen containing compounds of the cell. Furthermore<br />

glutamate can serve as a source of carbon by <strong>bei</strong>ng degraded to α-ketoglutarate<br />

and ammonium. This reaction is catalyzed by the glutamate dehydrogenase<br />

(GDH) [1]. The genome of B. subtilis encodes two GDHs but only the product<br />

of the rocG gene is enzymatically active in the strain 168. The second gene<br />

gudB encodes a cryptic GDH that is expressed constitutively but does not have<br />

enzymatic activity due to a duplication of three amino acids in the active site of<br />

the enzyme. By the precise excision of the corresponding 9 duplicated base<br />

pairs the gene product regains its activity [2]. This mutation, designated as<br />

gudB1, occurs with a high rate in a rocG mutant and complements the growth<br />

defect of this mutant on rich medium. In our studies we analyzed the frequency<br />

of the gudB reversion in a rocG mutant background and investigated the<br />

mechanism that is involved in the decryptification of the gudB gene. The<br />

excision of the 9 base pairs of the direct repeat occurs in a RecA independent<br />

manner. In a genetic screen we studied whether the high mutation rate depends<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

on selection or not. To elucidate the mechanism of this high-frequency<br />

mutagenesis we performed a transposon mutagenesis and analyzed mutants that<br />

show a drastically reduced mutation rate of the gudB gene.<br />

[1] Commichau et al. (2006) Curr Opin Microbiol, 9: 167-172.<br />

[2] Belitsky et al. (1998) J. Bacteriol. 180:6298-6305.<br />

KU 06<br />

Metagenomic-<strong>der</strong>ived quorum-quenching clones interfering<br />

with P.aeruginosa biofilm formation<br />

P. Bijtenhoorn *1 , C. Schipper 1 , C. Hornung 1 , M. Quitschau 2 , S. Grond 2 , W.<br />

Streit 1<br />

1 Mikrobiologie und Biotechnologie, University of Hamburg, Hamburg,<br />

Germany<br />

2 Institut für Organische und Biomolekulare Chemie, University of Göttingen,<br />

Göttingen, Germany<br />

Many opportunistic pathogenic bacteria regulate their virulence expression by<br />

quorum sensing. In Pseudomonas aeruginosa, quorum sensing-regulated gene<br />

expression contributes to the formation and maintenance of biofilms and their<br />

tolerance to conventional antimicrobials. Thus quorum sensing is a<br />

consequential target for new antimicrobial drugs which could block quorum<br />

sensing signal reception. As quorum sensing is not directly essential for growth<br />

of the bacteria, inhibition of quorum sensing does not put strong selective<br />

pressure on the bacteria for development of resistance as with antibiotics.<br />

Metagenomics offers the possibility to scan the uncultivated bacteria for new<br />

biocatalysts, among those also quorum sensing inhibiting biomolecules.<br />

We found two novel quorum-quenching ORFs, designated bpiB04 and bpiB05,<br />

by screening a genome bank with an Agrobacterium tumefaciens reporter<br />

strain. The ORFs were cloned into the broad-host-range vector pBBR1MCS-5<br />

and transferred into Pseudomonas aeruginosa PAO1 by electroporation, where<br />

they caused a decreased motility and biofilm formation. An N-3-oxo-octanoylhomoserin-lactone<br />

degradation assay together with HPLC-MS analyses proofs<br />

decomposition of these autoinducers. Current work focuses on a detailed<br />

biochemical characterisation of the Bpi proteins and their impact on P.<br />

aeruginosa biofilm formation.<br />

[1] Schipper et al., 2008, Metagenome-<strong>der</strong>ived clones encoding for two novel<br />

lactonase family proteins involved in biofilm inhibition in Pseudomonas<br />

aeruginosa Appl. Environ. Microbiol.<br />

KU 07<br />

Quantitative proteomics approach to the establishment of<br />

interaction networks of peroxisomal membrane proteins in<br />

Saccharomyces cerevisiae<br />

B. Reinartz 1 , S. Oeljeklaus 1 , I. Michels 1 , C. Stephan 1 , M. Eisenacher 1 , W.<br />

Schliebs 2 , R. Erdmann 2 , H.E. Meyer 1 , B. Warscheid *1<br />

1 Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany<br />

2 Department for Systems Biochemistry, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

The biogenesis of peroxisomes is mediated by the concerted action of several<br />

membrane-bound protein complexes. For a comprehensive characterization of<br />

the structural composition of distinct protein complexes and cross-talk between<br />

them we developed a global proteomics strategy using epitope tagging and<br />

affinity purification combined with metabolic labeling of yeast and quantitative<br />

high resolution mass spectrometry. Moreover, when combining this functional<br />

proteomics strategy with advanced bioinformatics data analysis and statistics it<br />

provides a most valuable tool for the establishment of entire protein interaction<br />

networks with high accuracy. In this work and as proof of principle, we applied<br />

this approach to the study of the interaction network of Pex14p, an integral<br />

member of the matrix protein import machinery in peroxisomes.<br />

Our investigation resulted in the definition of a Pex14p „core“ complex<br />

comprising the entire importomer as well as two further proteins Pex11p and<br />

Dyn2. This result illustrates the high potential of our functional proteomics<br />

strategy not only to reliably discriminate between true interacting proteins and<br />

co-purifying contaminants but also to identify new interacting partners of<br />

Pex14p. We next extended our approach to the identification of transient and/or<br />

weak interacting partners. As a result we report for the first time a number of<br />

proteins which transiently/weakly interact with Pex14p in a direct or indirect<br />

manner. This data provides a wealth of new information potentially leading to<br />

better un<strong>der</strong>standing the molecular mechanisms un<strong>der</strong>lying peroxisome<br />

biogenesis. In our ongoing studies we apply this strategy to establish a most<br />

comprehensive interaction network of membrane proteins involved in the<br />

proliferation of peroxisomes.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PA 01<br />

The effect of the oligosaccharides over the bacteriocins in<br />

or<strong>der</strong> to obtain a product as biocontroller of the human<br />

intestinal microflora<br />

E. Vamanu *1<br />

1 Biotehnol Centre Bucharest, USAMV Bucharest, Bucharest, Romania<br />

A probiotic product is currently essential for maintaining the normal intestinal<br />

microflora. This is due to the incidence of the colon cancer, high cholesterol,<br />

irritable colon, dysfunctions in the digestive tract and to the necessity of<br />

preventing and correcting the effects due to constipation. All these problems are<br />

frequently encountered to the groups of risk. An alternative increasingly used to<br />

the conventional products is the use of pobiotics, prebiotics, or symbiotics in<br />

or<strong>der</strong> to firstly prevent and then to correct and treat the negative effects<br />

occurring after these health problems.<br />

The aim of this study is to determine the effect of certain prebiotics over the<br />

synthesis of bacteriocins. The Lactobacillus paracasei CMGB16 strain<br />

producing bacteriocins was used. As sensitive strain, Escherichia coli was used.<br />

In the nutritive environment (MRS), the carbon source (glucose) was<br />

supplemented with inulin from chicory and Dahlia, raffinose and lactulose. The<br />

strains were cropped in these environments for 96 hours. The cells have been<br />

eliminated by centrifuging at 5,000 rpm for 10 minutes. The pH of the resulted<br />

supernatant was adjusted to the value of 5.5 with NaOH 0.2N. The inhibitory<br />

activity was determined by agar well diffusion method. The resistance to<br />

various inhibitory substances was also determined, such as: pepsin, trypsin,<br />

pronase E, subtilisin, catalase in concentration of 0.5mg/ml.<br />

A significant increase of the activity of the bacteriocin was noticed, when<br />

supplementing the cropping environment with inulin, lactulose, raffinose,<br />

within the time range 25 – 96 hours. The diameter of the inhibition area was of<br />

at least 2cm, visible in the case of using all prebiotics. The largest inhibitory<br />

area is visible after 24 and 48 hours of fermentation.<br />

On these lines, it may be concluded that the strain may be successfully used for<br />

obtaining a probiotic or prebiotic product for adjusting the problems due to the<br />

intestinal microflora dysfunction.<br />

PA 02<br />

New insights in the anaerobic butyrate metabolism of<br />

Syntrophomonas wolfei<br />

N. Müller *1 , B. Schink 1<br />

1 Mikrobielle Ökologie, Universität Konstanz, Konstanz, Germany<br />

Microbial fermentation of butyrate to acetate and molecular hydrogen is an<br />

en<strong>der</strong>gonic reaction un<strong>der</strong> standard conditions and therefore cannot support<br />

microbial growth. Only if the hydrogen partial pressure is kept low by<br />

methanogenic archaea or sulfate reducers the reaction can provide enough<br />

energy for ATP synthesis, even though only to a small extent. ATP is<br />

synthesized by substrate level phosphorylation in the acetate kinase reaction,<br />

but part of this ATP has to be reinvested into a reversed electron transport by<br />

which electrons arising in oxidation of butyryl-CoA to crotonyl-CoA can be<br />

released finally as molecular hydrogen. From cell free extracts of<br />

Syntrophomonas wolfei an NADH dehydrogenase complex was enriched which<br />

could play an essential role in its butyrate metabolism. The enzyme reacted<br />

with a variety of different electron acceptors including quinones and<br />

tetrazolium dyes as observed before with NADH dehydrogenases. The activity<br />

was inhibited significantly by trifluoperazine (TPZ), an antitubercular agent<br />

acting against NADH:menaquinone oxidoreductase of Mycobacterium<br />

tuberculosis. TPZ inhibited also the oxidation of butyrate with tetrazolium dyes<br />

by intact cells of Syntrophomonas wolfei. Therefore we assume that this<br />

enzyme is involved in reversed electron transport during the degradation of<br />

butyrate.<br />

PA 03<br />

Nitrite-oxidizing phototrophic bacteria<br />

J. Schott *1 , B.M. Griffin 2 , B. Schink 1<br />

1 Microbial Ecology, Universität Konstanz, Konstanz, Germany<br />

2 Genomic Biology, University of Illinois, Urbana, United States<br />

The nitrogen cycle is one of the major redox cycles in littoral sediments with<br />

nitrite as a key intermediate, product or substrate in almost all dissimilatory<br />

pathways, e.g. nitrification, ammonification, denitrification or anammoxreaction.<br />

Although anoxygenic phototrophic bacteria are frequently found in<br />

both littoral sediments and shallow waters and are known to oxidize various<br />

organic or inorganic compounds, nitrogen cycle compounds could not be found<br />

as electron donors for photosynthesis until recently (Griffin et al., 2007).<br />

81

82<br />

Based on enrichment cultures which oxidized nitrite stoichiometrically to<br />

nitrate, two types of dominating bacteria, an irregular rod shaped and a<br />

sphaerical shaped bacterium could be isolated. Thus the nitrite/nitrate couple<br />

with a redox potential of +430 mV at pH 7 is the highest know electron donor<br />

for anoxygenic photosynthesis so far.<br />

Strain KS1 was none-motile, surrounded by a slime capsule, gram-negative and<br />

had a size of 2-3 µm in diameter. It showed no vitamin dependence and 16SrRNA-gene-analysis<br />

revealed 98% similarity to several Thiocapsa strains.<br />

When the strain was starved for molybdenum, no nitrite oxidation was<br />

observed, whereas the full oxygen tension of air had no direct effect on nitrite<br />

oxidation. Strain KS1 closest relative Thiocapsa roseopersicina could also<br />

utilize nitrite as sole electron donor. Cells of strain LQ17 were irregular 1-4 µm<br />

long rods with 0.6-1 µm in diameter. 16S rRNA analysis revealed high<br />

similarity to Rhodopseudomonas strains.<br />

Both strains, KS1 and LQ17, contained bacteriochlorophyll a as major pigment<br />

and utilized a broad variety of organic and inorganic electron donors.<br />

PA 04<br />

The impact of chlorinated ethenes on the formation and<br />

subcellular localization of the tetrachloroethene reductive<br />

dehalogenase in Desulfitobacteria strains<br />

A. Reinhold *1 , M. Westermann 2 , T. Schubert 1 , G. Diekert 1<br />

1 Institute of Microbiology, Friedrich-Schiller-University, Jena, Germany<br />

2 Centre of Electron Microscopy, Friedrich-Schiller-University, Jena, Germany<br />

Earlier studies on the regulation of the tetrachloroethene (PCE) reductive<br />

dehalogenase in gram-negative Sulfurospirillum multivorans revealed two<br />

novel types of regulation mechanisms: a) The cellular localization depended on<br />

the presence of PCE [1]; b) Long-term subcultivation in the absence of PCE<br />

resulted in the loss of PCE dehalogenase activity (unpublished); the<br />

corresponding pceA gene then was inducible.<br />

Here we tested if both types of regulation apply to gram-positive strains of<br />

Desulfitobacterium hafniense (PCE-S and Y51). It was found that PCE also<br />

effected the cellular localization in D. hafniense and long-term subcultivation in<br />

the absence of PCE led to a loss of enzyme activity. This points to similar<br />

regulation mechanisms in these dechlorinating bacteria independent on the<br />

phylogenetic affiliation of the organisms. The molecular basis of these<br />

regulatory mechanisms is not yet known; however, preliminary experiments<br />

indicate differences in the long-term regulation between the gram-negative and<br />

the gram-positive bacteria.<br />

[1] John M, Schmitz RPH, Diekert G (2006) Arch Microbiol 186: 99-106<br />

PA 05<br />

Thermodynamic equilibria and substrate specificity of 2hydroxyacyl-CoA<br />

dehydratases<br />

A. Parthasarathy *1 , D.M. Smith 2 , W. Buckel 1<br />

1<br />

Fachbereich Biologie Mikrobiologie, Philipps-Universität, Marburg, Germany<br />

2<br />

Division of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute,<br />

Zagreb, Croatia<br />

2-Hydroxyglutaryl-CoA dehydratase (Hgd) is the key enzyme in the<br />

fermentation of glutamate in Clostridium symbiosum. It catalyses the reversible<br />

dehydration of (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA, which is<br />

chemically difficult due to the high pK = 40 of the β-proton. The iron-sulfur<br />

clusters of the dehydratase accept an electron from a cluster on the ATPhydrolyzing<br />

activating enzyme. This high-energy electron generates a radical<br />

on the substrate that reduces the pK by 26 units [1]. A chemical method<br />

developed to selectively synthesize (R)-2-hydroxyglutaryl-CoA enabled the<br />

accurate measurement of the equilibrium constant (K´ = 57; calculated 8.4).<br />

The analogous dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-<br />

CoA catalysed by the key enzyme of leucine fermentation in Clostridium<br />

difficile occurs irreversibly within the limits of detection (K´ > 1000; calculated<br />

1610).<br />

In contrast, the equilibrium of the dehydration of (R)-lactyl-CoA to acrylyl-<br />

CoA catalysed by the key enzyme of alanine fermentation in Clostridium<br />

propionicum strongly favours the hydroxy compound (K´ = 0.02; calculated<br />

0.02). Ab initio molecular orbital calculations rationalized this unexpected large<br />

substituent effect. In agreement with these measurements only with 2hydroxyisocaproyl-CoA<br />

dehydratase a product-based allylic ketyl radical has<br />

been observed by EPR spectroscopy [2].<br />

Interestingly, Hgd is a promiscuous enzyme accepting also 2hydroxyhexanedioyl-CoA<br />

(2-hydroxyadipyl-CoA), 2-hexene-4-oxodioyl-CoA,<br />

hexadienedioyl-CoA (muconyl-CoA), and acetylenedicarboxylate-CoA as<br />

substrates. The latter is hydrated to oxaloacetyl-CoA, which spontaneously<br />

hydrolyses to oxaloacetate.<br />

[1] Smith, D. M., Buckel, W. & Zipse, H. (2003) Angew. Chem. Int. Ed. Engl.<br />

42, 1867-1870.<br />

[2] Kim, J., Darley, D., Buckel, W. & Pierik, A. J. (2008) Nature 452, 239-242.<br />

PA 06<br />

Pseudomonas aeruginosa NirE: a SAM-dependent<br />

uroporphyrinogen III methyltransferase for heme d1<br />

biosynthesis<br />

S. Storbeck *1 , J. Walther 1 , J. Müller 1 , G. Layer 1<br />

1 Institute of Microbiology, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

Pseudomonas aeruginosa uses the anaerobic respiration process of<br />

denitrification as a powerful strategy for energy generation un<strong>der</strong> anaerobic<br />

growth conditions. The second step of denitrification is the reduction of NO2 - to<br />

NO catalyzed by dissimilatory nitrite reductases. Cytochrome cd1 nitrite<br />

reductase contains two different tetrapyrroles as essential prosthetic groups,<br />

heme c and heme d1. The biosynthesis of the isobacteriochlorin heme d1 is<br />

currently unknown; however, mutant studies revealed that proteins encoded by<br />

genes of the P. aeruginosa nir-operon (nirSMCFDLGHJEN) are involved in<br />

heme d1 biosynthesis. Based on amino acid sequence analysis the NirE protein<br />

was predicted to be a SAM-dependent uroporphyrinogen III methyltransferase<br />

catalyzing the methylation of uroporphyrinogen III to precorrin-2.<br />

Recombinant P. aeruginosa NirE was produced in E. coli and<br />

chromatographically purified. During protein production the reaction product of<br />

NirE accumulated in the cells as was shown by UV-VIS spectroscopy and<br />

HPLC analysis. Gelfiltration experiments indicate that P. aeruginosa NirE is a<br />

dimeric protein. Furthermore, an in vitro NirE activity assay was established.<br />

Using this assay we observe the conversion of uroporphyrinogen III into<br />

precorrin-2 catalyzed by NirE, proving its predicted catalytic activity.<br />

PA 07<br />

Towards the in-vitro methylation of metals and metalloids:<br />

Capability of corrinoid-dependent methyltransferases from<br />

Methanosarcina mazei to volatilise metal(loid)s<br />

F. Thomas *1 , B. Huber 1 , R. Diaz-Bone 2 , R. Hensel 1<br />

1 Mikrobiologie 1, Universität Duisburg-Essen, Essen, Germany<br />

2 Institut für Umweltanalytik, Universität Duisburg-Essen, Essen, Germany<br />

Formation of volatile <strong>der</strong>ivatives of metals and metalloids (in general<br />

methylated or hydrated <strong>der</strong>ivatives) by microorganisms are widespread in<br />

anaerobic habitats like sewage-sludge, geothermal vents as well as intestinal<br />

tracts of mammalian species including human. In most cases, these compounds<br />

represent methyl-<strong>der</strong>ivatives of metal(loid)s and exhibit a higher toxicity than<br />

their inorganic educts. As suggested from recent studies, methylation of<br />

metal(loid)s is an inherent feature of methanoarchaea. Nevertheless, the<br />

biochemical mechanisms of the synthesis of these <strong>der</strong>ivatives by<br />

methanoarchaea is still poorly un<strong>der</strong>stood.<br />

Here we present evidence that two methyltransferases from the<br />

methanoarchaeum Methanosarcina mazei are able to transform inorganic<br />

metal(loid)s into volatile <strong>der</strong>ivatives. Their enzymatic properties regarding<br />

substrate specifity are analysed and un<strong>der</strong>lying reaction mechanisms discussed.<br />

PA 08<br />

The Activating Enzyme of Acetobacterium dehalogenans –<br />

Identification of a new protein family of Reductive<br />

Activators of Corrinoid Enzymes (RACEs)<br />

S. Studenik *1 , A. Schilhabel 1 , A.J. Pierik 2 , G. Diekert 1<br />

1 Institute for Microbiology, Friedrich-Schiller-University Jena, Jena, Germany<br />

2 Institute of Cytobiology and Cytopathology, Philipps University Marburg,<br />

Marburg, Germany<br />

O-demethylases mediate the transfer of the methyl group of phenyl methyl<br />

ethers to tetrahydrofolate. They are key enzymes in the methylotrophic<br />

metabolism of strictly anaerobic homoacetogens, e. g. Acetobacterium<br />

dehalogenans. The O-demethylase complex consists of four distinct proteins,<br />

namely two methyltransferases, a corrinoid protein and an Activating Enzyme.<br />

The Activating Enzyme of A. dehalogenans is a 2Fe/2S protein and catalyzes<br />

the ATP-dependent „reactivation“ (i. e. reduction) of the corrinoid cofactor.<br />

The analysis of the genetic organization of the Activating Enzyme (AE)<br />

showed that a large number of putative AE-encoding genes are present in<br />

numerous genomes of Bacteria and Archaea. These genes are usually located<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

within or close to operons encoding B12-dependent methyltransferase systems.<br />

In most cases the function of the putative AE-encoding genes is unknown so<br />

far. The biochemical characterization of the AE of A. dehalogenans as well as<br />

the elucidation of the reaction mechanism of the ATP dependent reactivation of<br />

corrinoid enzymes allows the classification of a new enzyme family (RACE =<br />

Reductive Activators of Corrinoid Enzymes).<br />

PA 09<br />

Closing the gap. Electron transport chain from the inner<br />

membrane to insoluble iron in Shewanella oneidensis<br />

B. Schütz *1 , J. Gescher 1<br />

1 Institute for Biology II / Department of Microbiology, University Freiburg,<br />

Freiburg, Germany<br />

Coupling the reduction of ferric iron to energy generation is a form of<br />

respiration that is highly distributed amongst several known bacterial and<br />

archaeal genera. The γ-proteobacterium Shewanella oneidensis and the δproteobacterium<br />

Geobacter metallireducens became model organisms to study<br />

dissimilatory iron reduction un<strong>der</strong> neutrophilic conditions. Ferric iron forms<br />

highly insoluble minerals like ferrihydrite or hematite. Therefore, dissimilatory<br />

metal reducers are challenged to perform electron transfer reactions from the<br />

cytoplasmic membrane through the periplasm to the outer membrane to bring<br />

the electrons into contact with the terminal electron acceptor. We were<br />

interested in the electron transfer mechanism that is used by S. oneidensis to<br />

transport electrons from the cytoplasmic membrane to ferric iron. The c-type<br />

cytochrome proteins MtrA and FccA were identified as two abundant<br />

periplasmic cytochromes in a heme stain based screen. With in vitro and<br />

synthetic biology experiments we could show direct coupling of MtrA to<br />

CymA with an electron transfer rate very similar to the rate of CymA catalyzed<br />

FccA reduction. We conducted further experiments that show a direct electron<br />

transfer between MtrA and FccA in vitro. The results imply that FccA might<br />

not only be a periplasmic fumarate reductase but could be furthermore involved<br />

in the electron transport chain to ferric iron. Our experiments also show that a<br />

direct electron transfer between MtrA and OmcB is possible, thereby closing<br />

the gap between periplasmic and outer membrane electron transfer reactions.<br />

PA 10<br />

Does the unconventional octahaem c-type cytochrome<br />

MccA play a role in sulfur metabolism?<br />

A. Mager *1 , J. Simon 2<br />

1 Institut für Mikrobiologie und Genetik, Technische Universität Darmstadt,<br />

Darmstadt, Germany<br />

Recently, a novel octahaem cytochrome c (MccA) was overproduced and<br />

isolated from the periplasm of the Epsilonproteobacterium Wolinella<br />

succinogenes [1]. MccA (80 kDa) contains seven haem c groups attached via<br />

conventional CXXCH haem c binding motifs whereas the eighth haem c is<br />

bound by both cysteine residues of an unprecedented CX15CH signature<br />

sequence. The mcc gene cluster also encodes proteins that are likely to deliver<br />

electrons from the quinol pool to MccA as well as a dedicated cytochrome c<br />

haem lyase [1,2]. The presence of these accessory genes is conserved in mcc<br />

gene arrangements from several Campylobacter and Shewanella species.<br />

W. succinogenes cells grow by anaerobic respiration with polysulfide as<br />

terminal electron acceptor but do not convert thiosulfate or tetrathionate [3].<br />

Nonetheless, quantitative RT-PCR showed that the presence of 10 mM sodium<br />

thiosulfate led to a 20-fold up-regulation of mccA transcript levels. Similarly,<br />

thiosulfate was found to increase the mccA transcript amount in Shewanella<br />

oneidensis MR-1 [4] suggesting that MccA might have a function in the<br />

turnover of a sulfur compound.<br />

The presented work will focus on the regulation of W. succinogenes mccA<br />

transcription in response to several sulfur-containing substrates and the possible<br />

involvement of a novel two-component regulatory system whose components<br />

are encoded upstream of mccA.<br />

[1] R.S. Hartshorne et al. (2007) Mol. Microbiol. 64, 1049-1060<br />

[2] J. Simon and M. Kern (2008) Biochem. Soc. Trans. 36, 1011-1016<br />

[3] R. Hed<strong>der</strong>ich et al. (1999) FEMS Microbiol. Rev. 22, 353-381<br />

[4] A.S. Beliaev et al. (2005) J. Bacteriol. 187, 7138-7145<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PA 11<br />

Acetophenone Carboxylase, a novel carboxylase involved in<br />

anaerobic ethylbenzene degradation in Aromatoleum<br />

aromaticum EbN1<br />

K. Schühle *1 , J. Hei<strong>der</strong> 1<br />

1 Laboratorium für Mikrobiologie, Philipps-Universtität Marburg, Marburg,<br />

Germany<br />

The β-proteobacterium Aromatoleum aromaticum strain EbN1 degrades various<br />

aromatic compounds un<strong>der</strong> denitrifying conditions, including the aromatic<br />

hydrocarbon ethylbenzene. Initially, ethylbenzene is anaerobically oxidised to<br />

(S)-1-phenylethanol by the periplasmatic, molybdenum/iron-sulfur/heme b<br />

enzyme ethylbenzene dehydrogenase, which belongs to the DMSO reductase<br />

family of molybdenum enzymes. (S)-1-Phenylethanol is transported into the<br />

cytosol and further oxidised to acetophenone. Subsequently, acetophenone is<br />

carboxylated to benzoylacetate by a novel type of carboxylase in an ATPdependent,<br />

biotin-independent reaction.<br />

Acetophenone carboxylase has been biochemically characterised and properties<br />

of the enzyme will be shown. The enzyme shows some sequence similarity<br />

with acetone carboxylase, but differs in several crucial aspects of composition,<br />

cofactor dependence and reaction mechanism. The catalytic properties of the<br />

enzyme and its reaction mechanism was investigated.<br />

PA 12<br />

Tungsten-containing Benzoyl-CoA Reductase from the<br />

Strictly Anaerobic Geobacter metallireducens<br />

J.W. Kung *1 , M. Boll 1<br />

1 Institut für Biochemie, Universität Leipzig, Leipzig, Germany<br />

Benzoyl-CoA reductases (BCR) are key enzymes in the anaerobic degradation<br />

of aromatic compounds that catalyze the two-electron reduction of the aromatic<br />

ring to a cyclic dienoyl-CoA. Facultative anaerobes feature a 3x[4Fe-4S]<br />

clusters containing ATP-dependent BCR (1), whereas strictly anaerobic<br />

bacteria appear to use an ATP-independent BCR complex encoded by the<br />

benzoate-induced bamBCDEFGHI genes (2). Using an assay that followed the<br />

reversal of BCR reaction, the enzyme-, time- and electron-acceptor dependent<br />

rearomatization of dienoyl-CoA to benzoyl-CoA, the active site containing<br />

components of BCR from Geobacter metallireducens were purified and<br />

characterized. The dienoyl-CoA aromatizing activity was sensitive to dilution<br />

and oxygen exposure. The purified enzyme consisted of two gene products that<br />

were identified as BamB (75 kDa, annotated as aldehyde:ferredoxin<br />

oxidoreductase) and BamC (20 kDa, annotated as FeS module of hydrogenases)<br />

by MS-analysis. The molecular mass of 185 kDa suggested an α2β2<br />

composition. Metal analysis revealed 0.9 W, 8-10 Fe, 2.2 Ca and 1.0 Zn per αβ<br />

module. UV/vis spectroscopic analysis of the enzyme as isolated revealed a<br />

typical spectrum of oxidized FeS-clusters that could only be reduced by<br />

dienoyl-CoA but not by any other reductant.<br />

[1] Boll, M. (2005), J Mol Microbiol Biotechnol. 10(2-4):132-42<br />

[2] Wischgoll et al. (2005), Mol Microbiol. 58(5):1238-52<br />

PA 13<br />

Riboflavin as a prosthetic group in enzymes<br />

E. Jayamani *1 , C.D. Boiangiu 1 , T. Selmer 1 , W. Buckel 1<br />

1<br />

Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität,<br />

Marburg, Germany<br />

Riboflavin (vitamin B1) is consi<strong>der</strong>ed only as precursor of the prosthetic groups<br />

riboflavin-5´-phosphate (FMN) and FAD, which occur in several hundreds of<br />

enzymes. Recent studies, however, reported the presence of riboflavin in a<br />

small number of enzymes. Riboflavin was first discovered by R. Kuchta and R.<br />

Abeles (1985) in the radical enzyme lactyl-CoA dehydratase from Clostridium<br />

propionicum. Later riboflavin has been detected in the 2-hydroxyglutaryl-CoA<br />

dehydratases from Fusobacterium nucleatum (A. Klees, W. Buckel, 1992) and<br />

Acidaminococcus fermentans (U. Müller, W. Buckel, 1995), but not in that of<br />

Clostridium symbiosum (M. Hans, W. Buckel, 1999). Other 2-hydroxyacyl-<br />

CoA dehydratases are devoid of riboflavin (J. Kim, W. Buckel, 2004).<br />

Riboflavin has been also detected in NADH:quinone oxidoreductase (Nqr) and<br />

confirmed recently (B. Barquera, R. Gennis, 2002 and 2008, J. Steuber 2008).<br />

Here report the presence of riboflavin in the Nqr/Rnf-related ferredoxin-NAD+<br />

reductase from Clostridium tetanomorphum. The rnfCDGEAB operon has been<br />

almost completely sequenced and aligned with the sequences of C. tetani. The<br />

complex contains both non-covalently bound flavin as well as covalently bound<br />

flavin. The non-covalently bound flavin was identified as FMN and riboflavin<br />

in a 1:1 stochiometric ratio, each 0.3 mol/mol Rnf complex (180 kDa). The<br />

83

84<br />

subunits RnfG and RnfD contain covalently bound flavin linked via<br />

phosphodiester bonds. The iron was determined as 25 ± 1 mol per Rnf complex<br />

confirming the six deduced ferredoxin-like [4Fe-4S] clusters. Routinely, Rnf<br />

activity is measured with NADH and ferricyanide at 420 nm. In or<strong>der</strong> to<br />

measure NAD+ reduction with reduced ferredoxin catalysed by the Rnf<br />

complex, the electron carrier was purified from C. tetanomorphum and reduced<br />

in situ by Ti(III)citrate at pH 7.0.<br />

PA 14<br />

Heme d1 biosynthesis in Pseudomonas aeruginosa<br />

G. Layer *1 , S. Storbeck 1 , J. Walther 1<br />

1 Institut für Mikrobiologie, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

Anaerobic growth and survival of Pseudomonas aeruginosa is essential for<br />

biofilm formation and infection. Replacement of the electron acceptor oxygen<br />

by nitrate during anaerobic denitrification is a powerful strategy for anaerobic<br />

energy generation. In the second step of the denitrification process the<br />

dissimilatory nitrite reductase (cytochrome cd1) utilizes the prosthetic groups<br />

heme c and heme d1 for the reduction of nitrite to NO. Heme d1 is not a real<br />

heme, rather an isobacteriochlorin related to siroheme, vitamin B12 and<br />

coenzyme F430. The multistep biosynthesis of this unique cofactor is only<br />

poorly un<strong>der</strong>stood. Mutational analysis of different bacterial species possessing<br />

a cytochrome cd1 nitrite reductase led to the identification of multi open reading<br />

frame loci whose gene products seem to be involved in heme d1 biosynthesis. In<br />

P. aeruginosa the nir genes, including those for cytochrome cd1 itself, two ctype<br />

cytochromes and several uncharacterized proteins most likely involved in<br />

heme d1 biosynthesis, are organized in one large operon (nirSMCFDLGHJEN).<br />

The genes nirF, nirD, nirL, nirG, nirH, nirJ and nirE are all necessary for the<br />

biosynthesis of heme d1 as was shown by insertional mutagenesis and<br />

complementation analyses. Encoded proteins include a SAM-dependent<br />

uroporphyrinogen III methyltransferase (NirE), a Radical SAM protein (NirJ)<br />

and a potential dehydrogenase (NirF). In our group we investigate heme d1<br />

biosynthesis by (1) isolation and identification of biosynthetic intermediates<br />

accumulated in vivo and (2) by production, purification and biochemical<br />

characterization of recombinant Nir proteins. First results concerning the NirE<br />

protein and a novel potential biosynthetic intermediate will be presented.<br />

PA 15<br />

Towards the Identification of the Source of CO on the<br />

Maturation Pathway of the [NiFe]-Hydrogenases<br />

C. Pinske *1 , G. Sawers 1<br />

1<br />

Institute of Biology/ Microbiology, Martin-Luther-University Halle-<br />

Wittenberg, Halle/Saale, Germany<br />

Hydrogenases catalyze the reversible oxidation of molecular hydrogen to<br />

protons and electrons. Escherichia coli contains three anaerobically synthesized<br />

[NiFe]-hydrogenases whose bimetallic active sites contain nickel, iron, and the<br />

diatomic ligands cyanide and carbon monoxide. The diatomic ligands are<br />

attached to the iron atom. Whilst CN is <strong>der</strong>ived from carbamoylphosphate the<br />

metabolic source of CO is still unknown [A, B]. We aim to identify the origin<br />

and the route of transfer of the CO-ligand by constructing defined multiple<br />

knockout-mutants of genes for C1-metabolism. The chosen target genes interact<br />

in glycine, tetrahydrofolate, or purine metabolism and catalyze transfer or<br />

decarboxylating reactions. In practice construction of the mutants is achieved<br />

by repeated phage transduction and elimination of the selectable marker. The<br />

mutants created are screened systematically for their hydrogenase activity.<br />

During this procedure a mutant was isolated which was unable to produce H2gas<br />

during mixed acid fermentation and which exhibited an unusual phenotype<br />

not comparable with any of the previously isolated hyp-gene mutants. None of<br />

the large hydrogenase-subunits is processed in the mutant and the amount of<br />

hydrogenase 2 large-subunit is significantly reduced in synthesis and/or<br />

stability. Phenotypic complementation was not possible by formatesupplementation.<br />

Further FT-IR characterisation of the active site ligands will<br />

reveal the influence of this mutation on hydrogenase maturation.<br />

[1] Forzi et al., (2007) FEBS Lett, 581: p. 3317-21.<br />

[2] Lenz et al., (2007) FEBS Lett, 581: p. 3322-6.<br />

PA 16<br />

Metabolite Analysis of the Central Metabolic Pathways of<br />

Rhodospirillum rubrum for Biotechnological Applications<br />

C. Rudolf *1 , B. Klein 2 , M. Oldiges 2 , H. Grammel 1<br />

1 Redox phenomena in phototsynthetic bacteria, Max Planck Institute for<br />

Dynamics of Complex Technical Systems, Magdeburg, Germany<br />

2 Institute of Biotechnology 2, Forschungszentrum Jülich, Jülich, Germany<br />

The high potential of the facultative photosynthetic bacterium Rhodospirillum<br />

rubrum S1 to produce biopolyesters (PHB), carotenoids, succinate or molecular<br />

hydrogen is known for years. Many products originate from precursor<br />

metabolites of the central metabolism. Therefore, analysis and a better<br />

un<strong>der</strong>standing of the central metabolism is crucial for optimization<br />

biotechnological production processes. In this project we use experimental<br />

determination of metabolites in combination with metabolic network analysis<br />

for bioprocess development and strain optimization.<br />

To investigate the product formation, cultivations in stirred tank reactors (5 l)<br />

un<strong>der</strong> dark conditions with fructose as sole carbon source were performed.<br />

After an aerobic growth phase, the formation of the various products was<br />

induced by gassing with a nitrogen/carbon dioxide mixture (10 % CO2). For<br />

metabolome analysis the cells were quenched with cold methanol-buffer,<br />

extracted with chloroform-methanol-buffer and measured with LC-MS/MS.<br />

The potential of Rhodospirillum rubrum S1 for biotechnological applications<br />

could be demonstrated by production of hydrogen (42 ml/h), photosynthetic<br />

membranes (Abs880 nm/660 nm > 1.00), PHB (up to 10 % (w/w)) and succinate<br />

(YP/S = 0.71 mol succinate/mol fructose). A comparable cultivation with the<br />

lycopene producing strain Slyc18 yielded 3.50 mg lycopene / g cdw. We also<br />

show that commonly employed methods for metabolic quenching and<br />

extraction reveal a high loss of intracellular metabolites of the cells (up to 80<br />

%). Nevertheless, after switching to anaerobic conditions significant changes in<br />

metabolite pools of the citric acid cycle could be observed, whereas metabolite<br />

pools of glycolysis or pentose phosphate pathway remained constant.<br />

PA 17<br />

A novel screening-system detects regulative factors of the<br />

hydA1-promoter in Chlamydomonas reinhardtii<br />

M. Pape *1 , A. Hemschemeier 1 , T. Happe 1<br />

1 Biochemistry of Plants, Photobiotechnology, Ruhr-University Bochum,<br />

Bochum, Germany<br />

The photosynthetic green algae Chlamydomonas reinhardtii generates a<br />

complex hydrogen-metabolism un<strong>der</strong> anaerobic acclimation. Responsible for<br />

hydrogen-evolution is an O2-sensitive [FeFe]-Hydrogenase (HydA1), which is<br />

localized in the chloroplast and is coupled to the photosynthetic electrontransport<br />

chain "Melis A., Happe T., 2001". The expression of the hydA1 gene<br />

un<strong>der</strong> oxygen deprivation is presumably controlled on the transcriptional level<br />

"Happe T., Kaminski A., 2002; Stirnberg M., Happe T., 2004", but to date<br />

knowlegde about regulative mechanisms is limited.<br />

A novel screening-system makes it possible to detect regulative factors of the<br />

hydA1 expression. We therefore use the Chlamydomas strain MR9 which<br />

integrated a chimeric construct consisting of the hydA1-promoter fused to the<br />

reportergen arylsulfatase (ars2) in its genome. An active promoter results in<br />

expression of the periplasmic enzyme arylsulfatase. Ars-activity can be<br />

measured by cleavage of the artificial substrate X-SO4 (5-Bromo-4-chloro-3indolyl-sulfat)<br />

that causes a blue staining of the algae colony. A mutant library<br />

was constructed via insertional mutagenesis and transformants were tested<br />

un<strong>der</strong> anaerobic and aerobic conditions concerning their ars-activity to get<br />

information about a potentiell knock-out in repressive or activating regulative<br />

factors of the hydA1-promoter. First mutants with changed ars-activity could be<br />

obtained and new data will be presented.<br />

PA 18<br />

A genetic system for Castellaniella defragrans strain<br />

65Phen, a monoterpene-mineralizing denitrifying<br />

Betaproteobacterium<br />

A. Dikfidan 1 , F. Lüddeke *1 , J. Har<strong>der</strong> 1<br />

1 Department of Microbiology, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

Castellaniella defragrans degrades anaerobically natural monoterpenes, which<br />

are well known as essential oils and are climate relevant components emitted by<br />

trees. We started the development of a genetic system for C. defragrans strain<br />

65Phen metabolizing phellandrene to have a tool for molecular analysis of<br />

enzymes of the monoterpene degradation pathway.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Rifampicin was applied to obtain spontaneous mutants. Wild type strain and<br />

rifampicin resistant mutant strains exhibited the same physiology regarding<br />

their growth on several monoterpenes as carbon source un<strong>der</strong> anaerobic<br />

conditions. The mutation was relatively stable in rifampicin-free liquid media<br />

over five passages: 75 % of the bacteria retained their resistance.<br />

The broad-host-range vector pBBR1MCS-2 carries kanamycin resistance and<br />

was successfully used for conjugational plasmid transfer from E. coli S17-1 to<br />

C. defragrans 65Phen-RIF2. Two <strong>der</strong>ivatives of the suicide vector pK19mob<br />

(Km R ) served as basis for deletion mutagenesis by homologous recombination.<br />

Flanking regions of a geraniol dehydrogenase were amplified and inserted into<br />

pK19mobsacB for the generation of a plasmid with an in-frame deletion of<br />

geraniol dehydrogenase (pΔORF31). Transfer to strain 65Phen by conjugation<br />

yielded kanamycin resistant colonies.<br />

So far, we have established the conjugational transfer and a single cross-over<br />

integration of suicide vectors into the genome of C. defragrans. Future work<br />

will address the isolation of double cross-over deletion mutants to verify the<br />

function of the geraniol dehydrogenase in anaerobic monoterpene degradation.<br />

PA 19<br />

Membrane proteins involved in the anaerobic catabolism of<br />

aromatic compounds in Geobacter metallireducens<br />

S. Wischgoll *1 , D. Heintz 2 , K. Ullmann 3 , M. Boll 1<br />

1 Institut für Biochemie, Universität Leipzig, Leipzig, Germany<br />

2 Institut de Biologie Moléculaire des Plantes, Centre national de la recherche<br />

scientifique, Strasbourg, France<br />

3 Institut für Zelltherapie und Immunologie, Fraunhofer IZI, Leipzig, Germany<br />

A previous analysis of the soluble proteome of the aromatic compound<br />

degrading Geobacter metallireducens provided initial evidence that strictly<br />

anaerobic bacteria use a yet uncharacterized, ATP-independent enzyme for<br />

dearomatization of the central intermediate benzoyl-CoA. The corresponding<br />

putative BamB-I complex comprises soluble molybdenum, selenocysteine and<br />

Fe/S cluster containing components. In or<strong>der</strong> to identify anticipated but so far<br />

unknown membrane proteins involved in benzoate catabolism of G.<br />

metallireducens, the proteome of washed membrane fractions from cells grown<br />

on benzoate and acetate was quantitatively analyzed by spectral counting. A<br />

total of 931 proteins were identified by combining 1D-SDS-PAGE with LC-<br />

MSMS; 16 newly identified membrane associated proteins showed clearly<br />

increased expression levels in cells grown on benzoate. The newly identified<br />

proteins comprise components with similarities to modules of NiFe/heme b<br />

hydrogenases, cytochrome bd oxidases, dissimilatory nitrate reductases, and<br />

transporter related proteins. The transcriptional regulation of differentially<br />

produced proteins was analyzed by reverse transcription qPCR and revealed the<br />

induction of further genes putatively coding for energy-converting<br />

hydrogenases. In vitro activities of benzoate induced hydrogenase and nitrate<br />

reductase were determined. The results obtained provide evidence that most of<br />

the newly identified proteins are consi<strong>der</strong>ed to be involved in (i) electron<br />

transfer reactions entangled in anaerobic aromatic metabolsim including<br />

hydrogenase modules, (ii) protection of the BamB-I complex from oxygen<br />

damage, (iii) coregulation of molybdenum-cofactor containing enzymes, and<br />

(iv) uptake of aromatic growth substrates.<br />

PA 20<br />

Anaerobic O-Demethylation: Characterization and<br />

Mutagenesis of the methyl transferase Ivan of<br />

Acetobacterium dehalogenans<br />

S. Kreher *1 , S. Studenik 1 , G. Diekert 1<br />

1 Institute of Microbiology, Friedrich Schiller Universität Jena, Jena, Germany<br />

Anaerobic bacteria such as Acetobacterium dehalogenans are able to grow on a<br />

variety of phenyl methyl ethers. Key enzymes in the utilization of these<br />

substrates are the O-demethylases, which mediate the cleavage of the ether<br />

bond and the transfer of the methyl group to tetrahydrofolate. Here we focused<br />

on the vanillate O-demethylase. The first step of the ether cleavage is catalyzed<br />

by the zinc containing methyl transferase Ivan (MT Ivan) which transfers the<br />

methyl group to a super-reduced corrinoid protein. MT Ivan was heterologously<br />

expressed in E. coli and further characterized. The N-terminus of the enzyme<br />

was deleted to investigate the impact of this part of the peptide chain on the<br />

activity. The mutants obtained were characterized.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PA 21<br />

Cyclohexane-1,2-dione Hydrolase: a special enzyme in<br />

several ways<br />

S. Fraas *1 , J. Har<strong>der</strong> 2 , E. Warkentin 3 , U. Ermler 3 , P.M.H. Kroneck 1<br />

1<br />

Fachbereich Biologie, Universität Konstanz, Konstanz, Germany<br />

2<br />

Abteilung Mikrobiologie, Max-Planck-Institut für Marine Mikrobiologie,<br />

Bremen, Germany<br />

3<br />

Molekulare Membranbiologie, Max-Planck-Institut für Biophysik, Frankfurt,<br />

Germany<br />

Cyclohexane-1,2-dione Hydrolase (CDH) is the key enzyme in the anoxic<br />

degradation of the alicyclic compound cyclohexane-1,2-diol. It is the first and<br />

only α-ketolase known and carries ThDP, Mg 2+ and FAD as cofactors [1]. Its<br />

substrate cyclohexane-1,2-dione (CDO) originates from cyclohexane-1,2-diol.<br />

CDO un<strong>der</strong>goes a hydrolytic cleavage to 6-oxohexanoate which is further<br />

oxidized to adipate [1]. So far, among the ThDP dependent enzymes, only CDH<br />

will accept cyclic compounds as substrate. Structural information – deduced<br />

from high resolution X-ray crystallography – confirm the ThDP binding site as<br />

the catalytic center of CDH. Moreover, our results indicate a conformational<br />

change of CDH during catalysis. Un<strong>der</strong> our current experimental conditions,<br />

the enzyme crystallizes as a tetramer. Further crystallization experiments reveal<br />

that the ring-cleavage will be catalyzed by the CDH tetramer.<br />

Ultracentrifugation and gel filtration studies indicate the presence of a CDH<br />

dimer as the major species in solution. On the other hand, according to dynamic<br />

light scattering experiments dimers, trimers and tetramers appear to exist. Thus,<br />

the catalytically active form of CDH in solution remains unclear and requires<br />

further investigation.<br />

[1] Fraas, S., Steinbach, A.K., Tabbert, A., Har<strong>der</strong>, J., Ermler, U., Tittmann, K.,<br />

Meyer, A., Kroneck, P.M.H. (2008), J. Mol. Cat. B, in revision.<br />

[2] Har<strong>der</strong>, J. (1997) Arch. Microbiol. 168: 199-204<br />

PA 22<br />

A denitrifying Betaproteobacterium (strain HxN1) as a<br />

model organism for the study of anaerobic alkane<br />

degradation<br />

K. Webner *1 , I. Werner 2 , F. Widdel 1 , O. Grundmann 1<br />

1<br />

Abteilung Mikrobiologie, Max-Planck-Institut für marine Mikrobiologie,<br />

Bremen, Germany<br />

2<br />

Sanofi Aventis, Sanofi Aventis, Frankfurt, Germany<br />

Strain HxN1, a member of the Betaproteobacteria, can grow anaerobically with<br />

n-alkanes of chain lenghts from C6 to C8 and nitrate as electron acceptor that is<br />

reduced to N2. Strain HxN1 was chosen as a model organism for the study of<br />

anaerobic alkane degradation because it exhibits the fastest growth of the<br />

presently known anaerobic alkane degra<strong>der</strong>s and grows without cell attachment<br />

to the alkane phase. Metabolite analyses, measurements with cell extracts, the<br />

identification of alkane-specific proteins and un<strong>der</strong>lying genes as well as<br />

analogies to anaerobic toluene activation indicated that n-alkanes are added to<br />

fumarate via a radical mechanism yielding (1-methylalkyl)succinates. The<br />

alkane-activating enzyme, (methylalkyl)succinate synthase (Mas), consists of at<br />

least of three different subunits, but the exact composition and the enzyme is<br />

unknown. Also, there is still uncertainty about the enzyme (protein-activating<br />

enzyme) that introduces the radical. Until now heterologous expression of<br />

active proteins in Escherichia coli was not successful. A deeper un<strong>der</strong>standing<br />

of anaerobic alkane activation will therefore depend on biochemical and genetic<br />

studies directly with strain HxN1. To explore genetic accessibility,<br />

transformation of strain HxN1 was attempted. A broad-host-range vector<br />

mediating ampicillin resistance was introduced by electroporation. The<br />

transformation efficiency was > 6 x 10 5 transformants per µg DNA. This may<br />

open the possibility for directed mutagenesis and deletion of individual mas<br />

genes and associated ones.<br />

PA 23<br />

Catalytic mechanism of the dissimilatory sulfite reductase<br />

from Archaeglobus fulgidus<br />

K. Parey *1 , U. Ermler 1 , P. Kroneck 2<br />

1 Molekulare Membranbiologie, Max Planck Institut für Biophysik,<br />

Frankfurt/Main, Germany<br />

2 Fachbereich Biologie, Universität Konstanz, Konstanz, Germany<br />

Dissimilatory sulfite reductase (dSiR) catalyzes the reduction of sulfite to<br />

sulfide, a key step within the global biogeochemical sulfur cycle: HSO3 - + 6e - +<br />

6H + -> HS - + 3H2O.<br />

It also converts nitrite to ammonia, again a multi-electron, multi-proton transfer<br />

reaction.<br />

85

86<br />

The enzyme from various sulfate-reducing bacteria has been described as<br />

α2β2γmδn-multimer, with a molecular mass of approximately 200 kDa (α ~ 50<br />

kDa, β ~ 45 kDa, β ~ 11 kDa, δ ~ 8 kDa) [1]. Dissimilatory sulfite reductase<br />

from the strict anaerobe A. fulgidus was purified and crystallized un<strong>der</strong> the<br />

exclusion of dioxygen, applying the hanging drop vapor diffusion method [2,3].<br />

Crystals of A. fulgidus dSiR revealed an α2β2 heterotetrameric organization,<br />

with a size of 125 Å x 80 Å x 60 Å. The characteristic trilobal architecture of<br />

the molecule shows the presence of four siroheme-[4Fe-4S] sites, and four extra<br />

[4Fe-4S] clusters. To obtain further insight into the mechanism of the reduction<br />

of sulfite, catalyzed by dSiR, crystallographic structures of the enzyme were<br />

solved in complex with the substrates sulfite and the proposed reaction product<br />

sulfide. Further HPLC analysis of products was performed to show differences<br />

and analogies in the reaction mechanism to that of the assimilatory sulfite<br />

reductase (SiRHP) from E. coli [4].<br />

[1] Fritz G, Einsle O, Rudolf M, Schiffer M, Kroneck PMH (2005) Key<br />

Bacterial Multi-Centered Metal Enzymes Involved in Nitrate and Sulfate<br />

Respiration. J Mol Microbiol Biotechnol 10:223-233<br />

[2] Schiffer, A. (2004) Dissertation, Universität Konstanz<br />

[3] Dahl C, Trüper HG (2001) Sulfite Reductase and APS Reductase from<br />

Archaeoglobus fulgidus. Methods Enzymol 331:472-441<br />

[4] Stroupe and Getzoff (2001) Sulfite reductase hemoprotein. In “Handbook of<br />

metalloproteins” (Messerschmidt A, Huber R, Poulos T, Wieghardt K, eds)<br />

PA 24<br />

Acetate metabolism in Methanosarcina mazei<br />

C. Krätzer *1 , U. Deppenmeier 1<br />

1 Institut für Mikrobiologie und Biotechnologie, Universität Bonn, Bonn,<br />

Germany<br />

The archaeal organism Methanosarcina mazei is a methanogenic Archaeon that<br />

is characterized by the ability to generate methane as the major end product of<br />

metabolism. M. mazei has a limited substrate spectrum and can utilize acetate in<br />

the so called acetoclastic pathway, as well as CO2 + H2 and methylated C1compounds.<br />

The biochemistry of methanogenesis has gained much attention in<br />

the past decades, but the components of the membrane electron transport chain<br />

involved in acetate utilization are still unclear. Acetate is cleaved by the CO<br />

dehydrogenase/acetyl-CoA synthase that donates electrons to ferredoxin. The<br />

proteins that are responsible for transferring electrons from ferredoxin to the<br />

respiratory chain are still a matter of debate. Genome wide transcription<br />

analysis has shown that the genes encoding the so called Fcc complex are<br />

highly upregulated with acetate as sole substrate. Therefore, we propose that<br />

the Fcc complex plays a major role in the acetoclastic pathway and is involved<br />

in energy conservation in M. mazei. To address this question, a mutant strain<br />

lacking one of the key subunits of the Fcc complex was generated and<br />

analyzed. Furthermore, several proteins of the Fcc complex have been<br />

overproduced in Escherichia coli to provide a basis for an in depth<br />

characterization of the complex and to elucidate electron flow from ferredoxin.<br />

The characteristics and the proposed function of these proteins will be<br />

presented.<br />

PA 25<br />

How different is different? Carbon isotope fractionation in<br />

the two branches of the acetyl-CoA pathway.<br />

M. Blaser *1 , R. Conrad 1<br />

1<br />

Biogeochemie, Max-Planck-Institut für terrestrische Mikrobiologie, Marburg,<br />

Germany<br />

In methanogenic environments homoacetogenic bacteria contribute to the<br />

degradation of organic matter by a concerted action with fermenting, hydrolytic<br />

and methanogenic bacteria. To distinguish the different degradation pathways,<br />

the isotopic composition of substrates and products can be used to determine<br />

the fractionation factors of the individual processes. One of the major<br />

contributions of homoacetogenic bacteria is the reduction of CO2 and H2 to<br />

form acetate via the acetyl-CoA pathway. During this process two mole CO2<br />

are reduced to deliver the carboxyl- and methyl- group of acetate. It seems<br />

likely that the isotopic signature of both groups differ. To investigate this, the<br />

acetate formed by three different homoacetogenic cultures (A. woodii, T. kivui,<br />

S. ovata) was purified and pyrolysed. The obtained methane (former methylgroup)<br />

was analyzed using a GC-IRMS. First interpretations suggested that,<br />

even though the overall fractionation of the pathway is similar in all three<br />

strains, there is a species specific differentiation of the two branches.<br />

PA 26<br />

The effect of energy limitation on the carbon isotope<br />

fractionation factor of homoacetogenic bacteria<br />

A. Rafiei *1 , M. Blaser 1 , R. Conrad 1<br />

1<br />

Biogeochemie, Max-Planck-Institut für terrestrische Mikrobiologie, Marburg,<br />

Germany<br />

Anoxic soil is a complex system containing a variety of diverse microorganisms.<br />

This diversity leads to competition and by this to carbon and energy<br />

limitation. One of the communities present in soil are homoacetogenic bacteria.<br />

These micro-organisms utilise CO2 and H2 as sole carbon and energy source<br />

and produce acetate via the acetyl-CoA pathway. In or<strong>der</strong> to find out how these<br />

bacteria may affect the isotopic composition of acetate in soil, we analysed two<br />

homoacetogenic cultures (Acetobacterium woodii and Thermoanaerobacter<br />

kivui), grown in a minimal medium. The effect of energy limitation on the<br />

carbon isotope fractionation factor was evaluated by restricting the amount of<br />

H2. The concentration of CO2 was kept constant at 20 percent, while the<br />

hydrogen concentration varied between 80 and 0 percent. The concentration<br />

and carbon isotope composition of CO2 and acetate was measured using<br />

chromatographic separation and isotope ratio mass spectrometer (IRMS)<br />

quantification. It appears that the limitation of hydrogen has a consi<strong>der</strong>able<br />

effect on fractionation.<br />

PA 27<br />

Heterologous production of multihaem c-type cytochromes<br />

in Wolinella succinogenes<br />

M. Kern *1 , J. Simon 1<br />

1 Department of Microbiology and Genetics, TU Darmstadt, Darmstadt,<br />

Germany<br />

The Epsilonproteobacterium Wolinella succinogenes has a high capacity to<br />

produce c-type cytochromes using the so-called biogenesis system II for<br />

covalent haem attachment. Moreover, this organism is nonhazardous, easy to<br />

grow, and tools for genetic engineering have been developed. In particular, W.<br />

succinogenes appears to be a suitable host for production of c-type cytochromes<br />

from other Epsilonproteobacteria like pathogenic Campylobacter species.<br />

A genetic strategy is presented that aimed at the efficient overproduction of<br />

strep-tagged pentahaem cytochrome c nitrite reductase (NrfA) from<br />

Campylobacter jejuni. A codon-optimised variant of the C. jejuni nrfA gene<br />

was used to replace genuine nrfA on the genome of W. succinogenes, thereby<br />

retaining the nrf promoter and the nucleotide stretch encoding the NrfA signal<br />

peptide. Using this approach, highly active C. jejuni NrfA was obtained that<br />

could be purified by affinity chromatography. NrfA from Campylobacter<br />

species is predicted to contain five haem c groups ligated by conventional<br />

CXXCH haem c binding motifs whereas the active site haem group of W.<br />

succinogenes is known to be bound via a CXXCK motif. It has been shown<br />

previously that the lysine residue acts as an axial haem ligand and that the<br />

replacement of this lysine by histidine substantially impaired NrfA activity<br />

[Pisa et al. (2002) Mol. Microbiol. 43, 763-770].<br />

The predicted properties and functions of several more, as yet poorly<br />

characterized mutihaem c-type cytochromes from various<br />

Epsilonproteobacteria will be discussed that are potential candidates for<br />

heterologous cytochrome c production in W. succinogenes.<br />

PA 27<br />

Anaerobic degradation of long-chain alkanes by strain<br />

HdN1<br />

J. Zedelius *1 , R. Rabus 2 , M.M.M. Kuypers 1 , F. Schreiber 1 , F. Widdel 1<br />

1<br />

Mikrobiologie, Max-Planck-Institut für Marine Mikrobiologie, Bremen,<br />

Germany<br />

2<br />

Institut für Chemie und Biologie des Meeres (ICBM), Carl von Ossietzky<br />

Universität Oldenburg, Oldenburg, Germany<br />

The degradation of hydrocarbons by anaerobic microorganisms involves<br />

unusual activation reactions with no similarity to those in aerobic bacteria<br />

which employ oxygenases. To our present knowledge, n-alkanes in denitrifying<br />

Betaproteobacteria and sulfate-reducing Deltaproteobacteria are anaerobically<br />

activated via a glycyl radical-catalyzed addition to fumarate yielding alkylsubstituted<br />

succinates. Strain HdN1, a member of the Gammaproteobacteria,<br />

grows anaerobically with n alkanes from C14 to C20, using nitrate as electron<br />

acceptor that is reduced to N2 (Ehrenreich et al., 2000). In this strain, alkylsubstituted<br />

succinates were not detectable, and a genomic analysis did not<br />

reveal genes encoding the characteristic glycyl radical enzyme for anaerobic<br />

hydrocarbon activation. This suggests that strain HdN1 does not make use of<br />

the otherwise common anaerobic alkane activation mechanism with fumarate as<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

a co substrate. First comparative proteomic analyses of cells grown for ten<br />

generations with n-hexadecane and alternative substrates indeed indicated some<br />

alkane-specific proteins that are not similar to glycyl radical enzymes for<br />

anaerobic hydrocarbon activation.<br />

[1] Ehrenreich, P., A. Behrends, et al. (2000). "Anaerobic oxidation of alkanes<br />

by newly isolated denitrifying bacteria (vol 173, pg 58, 2000)." Archives of<br />

Microbiology 173(3): 232-232<br />

PA 28<br />

Anaerobic degradation of cinnamic and hydrocinnamic<br />

acid by "Aromatoleum aromaticum" strain EbN1<br />

K. Trautwein *1 , D. Lange 2 , R. Rabus 1<br />

1 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl<br />

von Ossietzky University, Oldenburg, Germany<br />

2 Microbiology, Max-Planck-Institute for Marine Microbiology, Bremen,<br />

Germany<br />

"Aromatoleum aromaticum" strain EbN1 is able to degrade a wide variety of<br />

aromatic compounds un<strong>der</strong> nitrate-reducing conditions, including toxic organic<br />

solvents, petroleum hydrocarbons and plant-<strong>der</strong>ived aromatic compounds.<br />

Cinnamic and hydrocinnamic acid (trans-3-phenylacrylic and 3phenylpropionic<br />

acid, respectively) represent building blocks of lignin and are<br />

substrates for anaerobic growth of strain EbN1. Substrate-shift experiments<br />

with benzoate-adapted cells demonstrated that the capability to anaerobically<br />

degrade cinnamic acid (70 h lag phase) and hydrocinnamic acid (130 h lag<br />

phase) are inducible.<br />

Differential protein profiling (2D DIGE) of benzoate- vs. cinnamic or<br />

hydrocinnamic acid-grown cells revealed the specific up-regulation of several<br />

protein spots (up to 44-fold), most likely involved in the β-oxidation of<br />

cinnamic and hydrocinnamic acid to benzoyl-CoA. The genome of strain EbN1<br />

encodes a large number of enzymes catalyzing β-oxidation reactions, which<br />

could thus far not be assigned to defined substrate specificities. Further<br />

degradation of benzoyl-CoA is assumed to follow the known pathway, since the<br />

respective enzymes did not display differences in abundance un<strong>der</strong> all three<br />

tested substrate conditions.<br />

PA 29<br />

Nitrosative stress defence in Wolinella succinogenes<br />

J. Volz *1 , C. Winkler 1 , M. Kern 2 , J. Simon 2<br />

1 Institute of Molecular Biosciences, Goethe University, Frankfurt, Germany<br />

2 Department of Microbiology and Genetics, Technische Universität Darmstadt,<br />

Darmstadt, Germany<br />

Nitric oxide, hydroxylamine and its congeners exert toxic effects on microbes,<br />

and many bacteria have evolved numerous mechanisms for coping with<br />

nitrosative stress. The genomic inventory of Epsilonproteobacteria, such as<br />

Campylobacter jejuni and Wolinella succinogenes, appears rather limited with<br />

respect to proteins that are potentially involved in nitrosative stress defence.<br />

Growth experiments and disc diffusion assays were performed using W.<br />

succinogenes wild-type cells and several gene deletion mutants in the presence<br />

of nitrite, hydroxylamine and various NO-releasing compounds. We present<br />

evidence that cytochrome c nitrite reductase (NrfA) is involved in the<br />

detoxification of nitric oxide and hydroxylamine. Moreover, a putative<br />

flavodiiron protein (Fdp) appears to mitigate nitric oxide stress in the cytoplasm<br />

whereas the hybrid cluster protein (Hcp) seems not to be involved in<br />

detoxifying nitric oxide or hydroxylamine.<br />

The W. succinogenes genome encodes three Dnr-type transcriptional regulators<br />

of the Fnr-Crp-superfamily that may mediate nitric oxide sensing and induction<br />

of nitrosative stress defence proteins. Putative Dnr-binding boxes (consensus<br />

motif TTGAT-N4-ATCAA) were found upstream of the transcriptional start<br />

sites of several genes including the nap, nrf and nos loci that encode the<br />

respiratory electron transport systems for reduction of nitrate, nitrite or nitrous<br />

oxide, respectively. Two dnr deletion mutants of W. succinogenes have been<br />

constructed and their physiological properties will be presented.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PA 30<br />

Anaerobic toluene metabolism in denitrifying and sulfate<br />

reducing bacteria: Enzymes involved in toluene<br />

degradation<br />

M. Hilberg *1 , S. Kölzer 1<br />

1<br />

Laboratorium für Mikrobiologie, Philipps-Universität Marburg, Marburg,<br />

Germany<br />

Anaerobic degradation of toluene is initiated by an unusual addition reaction of<br />

the toluene methyl group to the double bond of a fumarate cosubstrate to form<br />

the first intermediate (R)-benzylsuccinate. This reaction is catalysed by (R)benzylsuccinate<br />

synthase, a glycyl radical enzyme. All enzymes required for<br />

the degradation of toluene to (R)-benzylsuccinate are encoded in the tolueneinducible<br />

bss-operon. In addition to the genes coding for the (R)benzylsuccinate<br />

synthase (bssABC) there is another gene (bssD), which codes<br />

for a protein similar to activating enzymes needed for radical formation in other<br />

glycyl radical enzymes. The gene for the activating enzyme in T. aromatica<br />

was cloned and overexpressed in E.coli. We present initial data on the<br />

biochemical properties of this enzyme.<br />

The further catabolism of (R)-benzylsuccinate to benzoyl-CoA and succinyl-<br />

CoA follows a modified β-oxidation pathway. Enzymes of this pathway are<br />

arranged in similar operons in denitrifying, Fe(III)-reducing and sulfate<br />

reducing bacteria capable of degrading toluene. The genes and gene products of<br />

these bbs-operons are analyzed genetically and biochemically to establish the<br />

conservedness or differences in energy metabolism between different<br />

physiological groups of toluene degrading bacteria.<br />

PA 31<br />

The Role of Conspicuous Methyltransferases in CO<br />

Metabolism of Methanosarcina acetivorans<br />

E. Oelgeschläger 1 , M. Rother *1<br />

1 Institut für Molekulare Biowissenschaften, Molekulare Mikrobiologie und<br />

Bioenergetik, Goethe-Universität Frankfurt, Frankfurt am Main, Germany<br />

Methanosarcina acetivorans is one of the few members of the methanogenic<br />

archaea shown to use carbon monoxide (CO) as the sole source of energy for<br />

growth. Carboxidotrophic growth of this organism is peculiar as it involves<br />

formation of acetate, formate, and methylated thiols (methanethiol and<br />

dimethylsulfide) besides methane. Un<strong>der</strong> these conditions three conspicuous<br />

proteins homologous to both corrinoid proteins and methyltransferases are<br />

highly abundant. To address their role in CO metabolism of M. acetivorans, a<br />

set of single- and double-mutants, and the triple-mutant, was constructed by<br />

deletion/disruption of the encoding genes. All mutants could still grow on CO<br />

like the wild type, which demonstrates that the methyltransferase homologs are<br />

not essential un<strong>der</strong> this condition. Experiments with resting cells revealed that<br />

the capacity to form methane from CO was not compromised in any of the<br />

mutants, ruling out their involvement in the CO2 reduction pathway of<br />

methanogenesis. Strikingly, the single- and double mutants were affected to<br />

various degrees in their capacity to generate dimethylsulfide (DMS) from CO<br />

and to form methane from DMS while the triple-mutant could neither produce<br />

nor utilize DMS. These data demonstrate that the "fused" methyltransferases<br />

are involved in, and required for, methylsulfide metabolism of M. acetivorans.<br />

PA 32<br />

Bionic nano-cellulosomes: reversible immobilisation of<br />

Clostridium thermocellum hydrolytic enzymes on<br />

nanoparticles<br />

J. Krauss *1 , V.V. Zverlov 1 , W.H. Schwarz 1<br />

1 Department of Microbiology, TU Munich, Freising, Germany<br />

The anaerobic bacteria Clostridium thermocellum is able to form an<br />

extracellular multi-enzyme-complex, the cellulosome, for effective degradation<br />

of cristalline cellulose. We found a mutant which does not express the main<br />

structural component of the cellulosome, the CipA-protein, but does produce<br />

the complete set of cellulosomal enzymes. The ORF of scaffoldin encoding<br />

gene cipA of this strain is interrupted by an IS element (IS1447) at an early<br />

position.<br />

In this study recombinant parts of the scaffoldin-protein were produced in E.<br />

coli, so called miniscaffoldins. The miniscaffoldins consist of one or more type<br />

I cohesins, for enzyme interaction, optional with or without a carbohydrate<br />

binding module (CBM). The cohesin containing miniscaffoldins are able to<br />

bind hydrolytic enzymes from Clostridium thermocellum mutant SM901. Via a<br />

bifunctional linker miniscaffoldins are bound on the surface of nanoparticles<br />

caused by high affinity interactions. Hydrolytic activity of bound enzymes on<br />

87

88<br />

immobilized miniscaffoldins is tested on different substrates like soluble or<br />

amorphous or crystalline cellulose. The results were compared with those of<br />

free enzymes and native Clostridium thermocellum cellulosome. The enzyme<br />

activity assays of the obtained bioconjugates display an enhanced hydrolytic<br />

activity and synergistic effect behaviour on crystalline and amorphous cellulose<br />

in contrast with that of free enzymes.<br />

PA 33<br />

Phylogenetic characterization of aggregate-forming,<br />

gaseous alkane-degrading and sulphate-reducing<br />

enrichment cultures from marine hydrocarbon seeps<br />

U. Jaekel *1 , F. Widdel 1 , F. Musat 1<br />

1<br />

Microbiology, Max Planck Institute for Marine Microbiology, Bremen,<br />

Germany<br />

Short-chain non-methane alkanes (C2-C5) are constituents of natural gas and<br />

crude oil. Bacteria degrading these gases in the presence of oxygen are well<br />

known. Recently, the anaerobic degradation of short-chain non-methane<br />

alkanes by sulphate-reducing bacteria was reported. The isolated strain BuS5<br />

oxidizes propane and butane completely to CO2. However, the diversity of<br />

anaerobic propane and butane degra<strong>der</strong>s at deep-sea hydrocarbon seeps is<br />

unknown. In the present study two aggregate-forming, propane- and butanedegrading<br />

sulphate-reducing enrichment cultures were obtained from deep-sea<br />

hydrocarbon seeps and characterized using molecular techniques based on 16S<br />

rRNA. Clone libraries from both enrichment cultures revealed mostly<br />

phylotypes affiliated with the Desulfosarcina/Desulfococcus-cluster of the<br />

Deltaproteobacteria. Whole-cell hybridization with group-specific fluorescent<br />

probes indeed confirmed a dominance of such phylotypes in the aggregates.<br />

Whole-cell hybridization with more specifically designed probes showed that at<br />

least 80% of the cells in the aggregates were closely related to strain BuS5.<br />

These cell types are assumed to be mainly responsible for degradation of the<br />

hydrocarbon gases in the enrichment cultures and may also play a role in the<br />

anoxic surroundings of marine hydrocarbon seeps. The present findings shed<br />

further light on the relatedness and diversity of anaerobic propane- and butanedegrading<br />

bacteria and on the metabolic capabilities in the<br />

Desulfosarcina/Desulfococcus-cluster. Members of this cluster are frequently<br />

found in diverse marine habitats; however their environmental role is<br />

insufficiently un<strong>der</strong>stood.<br />

PA 34<br />

High reactivity of naturally formed iron oxide colloids<br />

J. Bosch *1 , A. Fritzsche 2 , K.U. Totsche 2 , R.U. Meckenstock 1<br />

1<br />

Institut of Groundwater Ecology, HelmholtzZentrum München, Neuherberg,<br />

Germany<br />

2<br />

Institut für Geowissenschaften, Friedrich-Schiller Universität Jena, Jena,<br />

Germany<br />

Microbial dissimilatory iron reduction is an important biogeochemical process.<br />

Recent research has clearly demonstrated the impact of iron particle size on the<br />

rates at which this process is occurring. Compared to bulk phases with identical<br />

mineral composition, particles in the nanometer range exhibited greatly<br />

enhanced reduction rates. So far, in virtually all studies synthetic iron oxide<br />

colloids were used. We present microbial reduction experiments performed on<br />

naturally formed, heterogenuous iron hydroxide colloids, exhibiting a range of<br />

diameters.<br />

Material from a top horizon of a floodplain soil was fed un<strong>der</strong> water-saturated<br />

conditions with a low molar ionic solution in soil columns. After <strong>bei</strong>ng<br />

mobilized and discharged from the soil material, ferrous iron was oxidised and<br />

precipitated un<strong>der</strong> oxic conditions as iron oxide colloids in the soil leachate.<br />

Due to changes in interparticular stability, the hydrodynamic diameter of these<br />

particles ranged from >300 to 100 nm. The colloids were subsequently added to<br />

anoxic, dense cell suspensions of Geobacter sulfurreducens. This experimental<br />

set-up has been previously successfully used for testing synthetic iron<br />

hydroxides. Natural colloidal iron oxides were rapidly and almost completely<br />

reduced. Reduction rates were comparable to those of synthetic iron oxide<br />

(nano)particles, thus confirming studies basing on synthetic iron phases. Our<br />

results support the hypothesis that microbial iron hydroxide nanoparticle<br />

reduction in the environment is a process of significance for the<br />

biogeochemical turnover of iron.<br />

PA 35<br />

2-Naphthoyl-CoA reductase from the sulfate-reducing<br />

enrichment culture N 47<br />

F. Raulf 1 , J. Johannes *1 , R.U. Meckenstock 1<br />

1<br />

Institut für Grundwasserökologie, Helmholtz-Zentrum München, Neuherberg,<br />

Germany<br />

The bicyclic aromatic hydrocarbon naphthalene is an important constituent of<br />

petroleum and coal tar. Due to its high chemical stability and toxicity<br />

naphthalene is consi<strong>der</strong>ed to be of major environmental concern. As<br />

groundwater environments are often anoxic the anaerobic biodegradation of<br />

naphthalene is of special interest and a few sulfate-reducing microorganisms<br />

have been cultivated.<br />

The sulfate-reducing enrichment culture N 47 isolated from a contaminated<br />

aquifer near Stuttgart is proposed to initially activate naphthalene by<br />

methylation to 2-methylnaphthalene. This intermediate is then metabolized by<br />

fumarate addition to the methyl group, oxidation of the methyl carbon atom and<br />

subsequent thiolytic cleavage of naphthyl-2-oxomethyl-succinyl-CoA to 2naphthoyl-CoA<br />

(N-CoA) and succinyl-CoA [1]. The further breakdown of N-<br />

CoA is proposed to proceed by sequential reduction of the aromatic ring<br />

system. This proposal is supported by recent genomic data where genes similar<br />

to ATP dependent benzoyl-CoA reductases were found and assigned to a N-<br />

CoA reductase [2].<br />

We found the activity of a N-CoA reductase in crude extracts of N 47 cells<br />

grown on naphthalene as sole source of energy and cell carbon. Here we<br />

present initial data of the reaction regarding substrate utilization and products<br />

formed.<br />

[1] Safinowski and Meckenstock (2006): Environmental Microbiology 8(2),<br />

347–352<br />

[2] Selesi D, unpublished data<br />

PA 36<br />

Reconstruction of Central Carbohydrate metabolism in the<br />

Hyperthermophilic Archaeon Thermococcus kodakaraensis<br />

K. Matsubara *1 , B. Siebers 1<br />

1 Department of Chemistry, Biofilm Centre, Molecular Enzyme Technology and<br />

Biochemistry, University of Duisburg-Essen, Duisburg, Germany<br />

A gene disruption is a powerful tool to examine the function of a target gene or<br />

its product in vivo. The development of respective tools in hyperthermophiles<br />

was hampered by the heat instability of common antibiotics. However an<br />

effective novel system has been developed for hyperthermophilic archaeon<br />

Thermococcus kodakaraensis [1, 2]. This method is composed of two steps:<br />

First, the disruption vector harbouring flanking regions of the target gene and<br />

the orotidine-5-phosphate decarboxylase (pyrF) is introduced to the host mutant<br />

of T. kodakaraensis (the KU216; ΔpyrF). In the next step, selection of<br />

disruptants is achieved by pop-out of the region including the pyrF and the<br />

target gene, and is mediated by 5-fluoroorotate addition to the medium. One of<br />

the great advantages of this method is that by removing the marker gene pyrF,<br />

subsequent additional disruptions are possible.<br />

This strategy is currently used in the laboratory to study the complexity of<br />

central carbohydrate metabolism and its regulation in T. kodakaraensis. It has<br />

already been shown that T. kodakaraensis utilizes the Embden-Meyerhof<br />

pathway of glycolysis. Nevertheless, T. kodakaraensis harbours harbours gene<br />

encoding homologs involved in the Entner-Doudoroff (ED) pathway, known as<br />

another popular glycolytic pathway in archaea. To unravel the role of EDhomologs<br />

in T. kodakaraensis, disruptants of the genes were generated and<br />

analyzed in respect to their phenotypes.<br />

[1] Sato T, Fukui T. et al. (2005). Appl. Environ. Microbiol, 71(7), 3889-3899<br />

[2] Sato T, Fukui T. et al. (2003). J. Bacteriol, 185(1), 210-220<br />

PA 37<br />

Combining proteomic and stable isotope fractionation<br />

analyses to elucidate the anaerobic benzene degradation<br />

pathway by iron- and sulfate-reducing microorganisms<br />

N. Abu Laban *1 , D. Selesi 1 , R.U. Meckenstock 1<br />

1 Institute of Groundwater Ecology, Helmholtz Zentrum München- German<br />

Research Center for Environmental Health, Neuherberg, Germany<br />

Despite its high chemical stability, the aromatic hydrocarbon benzene is known<br />

to be biodegradable with various electron acceptors un<strong>der</strong> anaerobic conditions.<br />

However, our un<strong>der</strong>standing of the initial activation reaction and the<br />

responsible microorganisms is limited. In the present study, we performed a<br />

comparative analysis of the whole proteome of an iron-reducing enrichment<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

culture grown on benzene or benzoate, and a sulfate-reducing enrichment<br />

culture-grown on benzene only. The mass spectrometry-based comparative<br />

analysis of the iron-reducing culture revealed that 58 specific proteins were<br />

only induced in benzene-grown cells. These proteins were related to energy and<br />

nucleotide metabolism, membrane transport, heat shock, and cell structure. 20<br />

peptides were only related to proteins with unknown functions in the database<br />

and might constitute enzyme candidates for the unknown benzene activation<br />

reaction. We could not identify enzymes of anaerobic toluene or phenol<br />

degradation which might have indicated a methylation or hydroxylation as<br />

initial activation mechanism. These findings support our recent metabolitebased<br />

analysis [1, 2] where carboxylation was favored as initial biochemical<br />

mechanism of anaerobic benzene degradation.<br />

In or<strong>der</strong> to elucidate if carboxylation might be an universal activation<br />

mechanism of anaerobic benzene degradation, we additionally analyzed<br />

hydrogen isotope fractionation of the two benzene-degrading cultures. Similar<br />

hydrogen enrichment factors (εH) -56.2 ‰ and -53.4 ‰ were observed for the<br />

iron- and sulfate-reducing cultures indicating a common mechanism for the first<br />

enzyme reaction in anaerobic benzene degradation.<br />

[1] Kunapuli et al. (2008) Environ Microbiol 10 (7):1703-1712,<br />

[2] Abu Laban et al. (2008) FEMS Microbial Ecol, submitted<br />

PA 38<br />

Anaerobic degradation of short-chain and cyclic alkanes by<br />

sulfate-reducing enrichment cultures<br />

F. Musat *1 , U. Jaekel 1 , J. Zedelius 1<br />

1<br />

Department of Microbiology, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

Saturated aliphatic and alicyclic hydrocarbons are common constituents of<br />

natural gas and crude oil. Whereas their aerobic biodegradation has been known<br />

for a century, their anaerobic biodegradation has been recognized and studied<br />

relatively recently. Most of this research has focused on n-alkanes with chains<br />

with six and more carbon atoms (≥C6H14). Relatively little is currently known<br />

about the anaerobic degradation of cyclic alkanes, in particular the<br />

unsubstituted ones (cyclopentane, cyclohexane). In the present study, we<br />

investigated cyclohexane degradation with sulfate as electron acceptor in<br />

marine enrichment cultures. Cyclohexane was completely degraded to CO2,<br />

with stoichiometric reduction of sulfate to sulfide. The enrichment culture was<br />

dominated by free-living cells affiliating with the Desulfosarcina cluster of the<br />

Deltaproteobacteria, as demonstrated by whole-cell hybridization with<br />

specifically design oligonucleotide probes. Phylotypes of the Desulfosarcina<br />

cluster are frequently observed as naturally abundant phylotypes in natural<br />

marine sediments, whereas little is known about their function. The present<br />

results as well as the recent finding of sulfate-reducing propane and butane<br />

degra<strong>der</strong>s belonging to this cluster 1 indicate that it may include several<br />

metabolically specialized degra<strong>der</strong>s of hydrocarbons.<br />

[1] Kniemeyer, O., F. Musat, S. M. Sievert, K. Knittel, H. Wilkes, M.<br />

Blumenberg, W. Michaelis, A. Classen, C. Bolm, S. B. Joye, F. Widdel (2007)<br />

Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing<br />

bacteria. Nature 449: 898 – 901.<br />

PA 39<br />

Anaerobic naphthalene degradation by an iron-reducing<br />

enrichment culture<br />

D. Selesi *1 , Y. Wang 1 , J. Johannes 1 , R.U. Meckenstock 1<br />

1 Institute of Groundwater Ecology, Helmholtz Zentrum München - German<br />

Research Center for Environmental Health, Neuherberg, Germany<br />

The awareness of the public health risks associated with polycyclic aromatic<br />

hydrocarbons has directed research to the microbiological and biochemical<br />

un<strong>der</strong>standing of anaerobic PAH degradation. In the present study, we report a<br />

microbial culture enriched from a tar-oil contaminated site that oxidized<br />

naphthalene as sole source of carbon with iron(III) as electron acceptor. The<br />

community analysis based on 16S rRNA gene sequences revealed one<br />

dominant phylotype poorly affiliated with the Gram-positive genus Moorella.<br />

The next closest related, cultivated organism was Moorella<br />

perchloratireducens, a methanol-oxidizing, perchlorate-reducing bacterium<br />

(89% sequence similarity). The dominant organism in the culture was likely to<br />

be responsible for naphthalene oxidation and, thus, Gram-positive bacteria were<br />

shown to be involved in PAH degradation, for the first time.<br />

To elucidate the initial biochemical reaction of anaerobic naphthalene<br />

degradation metabolites were analysed in culture supernatants. 2-naphthoic acid<br />

was the only detectable intermediate compound. Moreover, the enrichment<br />

culture was able to grow on 2-naphthoic acid but not on 2-methylnaphthalene<br />

as sole source of carbon.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PA 40<br />

The bamA gene: a general functional marker for anaerobic<br />

aromatic metabolism<br />

K. Kuntze *1 , C. Vogt 2 , H.H. Richnow 2 , M. Boll 1<br />

1 Institute of Biochemistry, University of Leipzig, Leipzig, Germany<br />

2 Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental<br />

Research - UFZ, Leipzig, Germany<br />

In all anaerobic bacteria, most aromatic growth substrates (e.g. toluene, phenol,<br />

cresols, xylenes, ethylbenzenes, benzoate analogues, etc.) are channelled to the<br />

central intermediate benzoyl-CoA [1]. This compound is converted to<br />

cyclohex-1,5-diene-1-carboxyl-CoA by dearomatizing benzoyl-CoA<br />

reductases, which greatly differ in facultative and obligate anaerobes. The<br />

following steps are highly similar in all aromatic compounds degrading<br />

anaerobes: (i) addition of water by a dienoyl-CoA hydratase, (ii) oxidation of<br />

the hydroxyl group to 6-oxocyclohexenoyl-CoA (6-OCH) by an alcohol<br />

dehydrogenase, and finally (iii) hydrolytic ring cleavage and the addition of a<br />

water molecule to the double bond by a hydrolase yielding 6-OH-pimelyl-CoA,<br />

referred to as BamA. We established an assay with degenerated primers<br />

targeting the bamA gene from all anaerobic bacteria [2]. It provides a new and<br />

widely applicable tool for the detection of all types of anaerobic bacteria<br />

capable of degrading a wide variety of aromatic compounds. With the bamA<br />

gene probe benzene contaminated aquifers were analyzed using in situ<br />

microcosms (BACTRAP), loaded with benzene and incubated for five months<br />

at two different sites. The combined application of both, BamA and 16S-RNA<br />

analysis identified a Geobacter species as the only dominating species at one<br />

site, whereas at another site a species only distantly related to Azoarcus sp.<br />

dominated, most possibly representing a novel species or genus.<br />

[1] Boll, M 2005 J. Mol. Microbiol. Biotechnol. 10:132-142<br />

[2] Kuntze K, Shinoda Y, Moutakki H, McInerney MJ, Vogt C, Richnow HH,<br />

Boll M 2008 Environ Microbiol. 10(6):1547-56<br />

PA 41<br />

COSMIC: Systems Biology of Clostridium acetobutylicum –<br />

analysis of an oscillation phenomenon<br />

K. Schwarz *1 , W.M. de Vos 1 , J. van <strong>der</strong> Oost 1 , S.W.M. Kengen 1<br />

1 Laboratory of Microbiology, Wageningen University, Wageningen,<br />

Netherlands<br />

The COSMIC (Clostridium acetobutylicum Systems Microbiology) project is<br />

one of the collaborative EU promoted research initiatives on "Systems Biology<br />

of Microorganisms" and aims at the analyses and modelling of key regulatory<br />

and metabolic events occurring during the transition between the acidogenic<br />

and the solventogenic phase of the C. acetobutylicum metabolism.<br />

To guarantee reproducible data C. acetobutylicum was grown in a substrate<br />

limited chemostat allowing the direct control of the metabolic shift. Getting into<br />

"steady state" un<strong>der</strong> solvent producing conditions the culture exhibited an<br />

oscillation in its redox potential. Currently, research focuses on the analysis of<br />

this synchronized metabolic behaviour as it may reveal general regulatory<br />

patterns that play a key role un<strong>der</strong> solventogenic conditions. It could be shown,<br />

that the oscillation of the culture’s redox potential is effected by the metabolic<br />

status of the bacterium, the growth rate and the substrate limitation. Biomass<br />

and fermentation products were not affected and remained constant. However,<br />

both, physiological and microarray analyses suggest an involvement of the<br />

sulphur metabolism.<br />

PA 42<br />

Enzymology of caffeate respiration in Acetobacterium<br />

woodii<br />

E. Biegel *1 , S. Vitt 1 , S. Schmidt 1 , V. Müller 1<br />

1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,<br />

Goethe University Frankfurt, Germany, Frankfurt, Germany<br />

Ferredoxin is a central electron carrier in caffeate respiration in A. woodii. It is<br />

reduced by hydrogen or organic substrates and reoxidized by a membranebound<br />

ferredoxin:NAD + -oxidoreductase of so far unknown identity. Here, we<br />

will present genetic, immunological and biochemical evidence for a membranebound<br />

Rnf complex that is speculated to couple electron transfer from<br />

ferredoxin to NAD + with electrogenic Na + transport across the cytoplasmic<br />

membrane. NADH + H + produced by the Rnf complex is reoxidized by the<br />

previously identified electron transfer flavoproteins (Etfs) [1]. Here, we have<br />

isolated the etf genes and sequenced the up and downstream region. We will<br />

present evidence that the etf genes built an operon together with an acyl-CoA<br />

dehydrogenase and an acyl-CoA synthetase, indicating that caffeate is activated<br />

89

90<br />

to caffeyl-CoA prior to its reduction by the presumptive caffeyl-CoA<br />

reductase/Etf complex [2].<br />

[1] Imkamp, F., Biegel, E., Jayamani, E., Buckel, W. and Müller, V. 2007.<br />

Dissection of caffeate respiratory chain in the acetogen Acetobacterium woodii:<br />

identification of an Rnf-type NADH dehydrogenase as a potential coupling site.<br />

J. Bacteriol. 189:8145-8153<br />

[2] Herrmann, G., Jayamani, E., Mai, G. and Buckel, W. 2008. Energy<br />

conservation via electron-transferring flavoprotein in anaerobic bacteria. J.<br />

Bacteriol. 190:784-791<br />

PA 43<br />

Analysis of a Chlamydomonas reinhardtii pyruvate formate<br />

lyase mutant strain indicates a regulative connection<br />

between fermentative and hydrogen metabolism<br />

D. Krawietz 1 , G. Philipps *1 , A. Hemschemeier 1 , J.J. Higuera Sobrino 2 , T.<br />

Happe 1<br />

1 Biochemistry of Plants, Ruhr-University Bochum, Bochum, Germany<br />

2 Bioquímica y Biología Molecular, Universidad de Córdoba, Córdoba, Spain<br />

The unicellular green alga Chlamydomonas reinhardtii has a complex<br />

anaerobic metabolism, which is marked by fermentation and hydrogen<br />

production. A mutant library was generated by random insertional mutagenesis<br />

to screen for mutants with defects in anaerobic metabolism. A mutant with a<br />

single integration of the resistance cassette in the pyruvate formate lyase (Pfl1)<br />

gene has been identified and was chosen for further analyses. The absence of<br />

this key enzyme of fermentation in the mutant results in an increased<br />

production of ethanol and D-lactate compared to the wild type. Intriguingly, the<br />

lack of Pfl1 results also in a reduction of hydrogenase activity and hydrogenase<br />

gene expression. The wild type phenotype could be restored by<br />

complementation with the pfl1 gene. In this work the genetic and physiological<br />

results will be presented to characterize the impact of formate production on<br />

hydrogen metabolism for the green alga during anaerobiosis.<br />

PA 44<br />

Anaerobic toluene metabolism in Thauera aromatica :<br />

enzymes involved in β-oxidation of (E)-Phenylitaconyl-CoA<br />

M.L. Lippert *1 , J. Hei<strong>der</strong> 1<br />

1 Laboratorium für Mikrobiologie, Philipps-Universität Marburg, Marburg,<br />

Germany<br />

Anaerobic catabolism of toluene is initiated by addition of the methyl group of<br />

toluene to the double bond of a fumarate cosubstrate, yielding the first<br />

intermediate (R)-benzylsuccinate. This reaction is catalysed by (R)benzylsuccinate<br />

synthase, a glycyl radical enzyme. The further pathway of<br />

anaerobic toluene catabolism follows a β-oxidation pathway of (R)benzylsuccinate<br />

to benzoyl-CoA, the common intermediate in anaerobic<br />

aromatic metabolism, and succinyl-CoA. All enzymes required for this pathway<br />

are encoded in the bbs operon (beta-oxidation of (R)-benzylsuccinate). Two of<br />

the genes code for the subunits of a (R)-benzylsuccinate:succinyl-CoA<br />

transferase activating (R)-benzylsuccinate to the CoA-thioester which is<br />

oxidised to (E)-phenylitaconyl-CoA via (R)-benzylsuccinyl-CoA<br />

dehydrogenase, the bbsG gene product. Both enzymes have previously been<br />

studied in detail.<br />

Further enzymes involved in β-oxidation of (E)-phenylitaconyl-CoA are<br />

encoded by bbsH ((E)-phenylitaconyl-CoA hydratase), bbsCD (2-<br />

(hydroxymethylphenyl)-succinyl-CoA dehydrogenase) and bbsAB<br />

(benzoylsuccinyl-CoA-thiolase). They have been overproduced in E. coli and<br />

purified to homogeneity. We present the characterisation of these enzymes and<br />

biochemical reconstruction of the specific oxidation pathway of (E)phenylitaconyl-CoA<br />

to benzoyl-CoA and succinyl-CoA.<br />

PB 01<br />

Isolation and characterization of a novel, highly unusual<br />

Ignicoccus species<br />

M. Lange *1 , C. Meyer 2 , R. Rachel 2 , U. Kueper 1 , K. Beblo 3 , S. Sievert 4 , H.<br />

Harald 1<br />

1 Institute for Microbiology, University of Regensburg, Regensburg, Germany<br />

2 Centre of Electron Microscopy, University of Regensburg, Regensburg,<br />

Germany<br />

3<br />

Institute for Aerospace Medicine, German Aerospace Center, Cologne,<br />

Germany<br />

4<br />

Laboratory for Biogeochemistry, Woods Hole Oceanographic Institution,<br />

Woods Hole, United States<br />

A novel species of the archaeal genus Ignicoccus (strain MEX 13A) was<br />

isolated from a black smoker chimney wall of a deep sea hydrothermal vent<br />

system located in the South Pacific (depth 2507 m). Comparison of its 16S<br />

rRNA gene sequence showed a close relationship to Ignicoccus hospitalis<br />

(around 98 % sequence similarity). The coccoid cells usually exhibit cell<br />

diameters between 1.5 and 3 µm. However, un<strong>der</strong> special growth conditions<br />

giant cells with diameters up to 15 µm occur. The isolate grows optimally<br />

around 90°C at NaCl concentrations from 1.4 to 2.0% NaCl. Similar to all<br />

known Ignicoccus species, strain MEX 13A is a chemolithoautotroph gaining<br />

energy by the reduction of elemental sulfur using molecular hydrogen as<br />

electron donor. In contrast, isolate MEX 13A is unique in the ability to use<br />

nitrate as alternative electron acceptor, resulting in the production of ammonia<br />

and / or nitrite. Further structural, physiological and molecular similarities and<br />

differences to the described Ignicoccus species will be presented. An open<br />

question is at the moment, whether the new isolate (like I. hospitalis) can serve<br />

as host organism for Nanoarchaeum equitans.<br />

PB 02<br />

The ‘fibres’ of Ignicoccus hospitalis: Studies on a new type<br />

of cell appendage<br />

C. Meyer *1 , D. Müller 2 , H. Huber 3 , R. Wirth 3 , R. Rachel 1<br />

1 Centre for Electron Microscopy, University of Regensburg, Regensburg,<br />

Germany<br />

2 Dept. for Pathology, University Hospital Regensburg, Regensburg, Germany<br />

3 Institute for Microbiology, University of Regensburg, Regensburg, Germany<br />

Ignicoccus hospitalis, a hyperthermophilic Crenarchaea exhibiting a distinctive<br />

outer membrane, grows as a fastidious anaerobe, producing H2S in a<br />

chemolithoautotrophical way. It is described to possess flagella whereas no<br />

motility was detected in later analyses [1, 2]. By examining these cell<br />

appendages in greater detail, we found a new and unique structure, differing<br />

from the classical flagella in constitution, biochemistry and function.<br />

As a first step the cell appendages of I. hospitalis were enriched from<br />

supernatant by PEG/NaCl induced precipitation, CsCl-gradient centrifugation<br />

and dialysis. Electron microscopy showed long filaments with a diameter of 14<br />

nm which were built of one main 33 kDa protein, for which no similar protein<br />

could be detected in non-redundant protein databases. Antibodies against that<br />

protein were raised in rabbits and tested by Western Blot analysis and different<br />

immuno-labelling methods. They turned out to be highly specific and enabled<br />

us to study the intracellular distribution of the protein, which could be found in<br />

the cytoplasmic and the outer membrane as well as in the periplasmic vesicles.<br />

Ultrathin sections of high pressure frozen cells show that these cell appendages<br />

are anchored in the cytoplasmic membrane; supported by observations on<br />

structures found in lysed cells. Here a definite assembly between the filaments<br />

and different membrane types could be detected and specified by immunolabelling.<br />

At present different constructs of the protein are cloned in E. coli to elucidate<br />

the function of the first transmembrane helix, a structure conserved in some<br />

proteins found in selected Archaea.<br />

[1] Huber H. et al., 2000, IJSEM 50:2093<br />

[2] Paper W. et al., 2007, IJSEM 57:803<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PB 03<br />

S-layer - Or how to deal with „Heavy Metal“<br />

A. Klingl *1 , K. Etzel 2 , A. Mutter 2 , W. Depmeier 2 , G. Schmalz 3 , M. Thomm 1 , H.<br />

Huber 1 , R. Rachel 1<br />

1 Microbiology and Electron Microscopy, University of Regensburg,<br />

Regensburg, Germany<br />

2 Institute of Geosciences, University of Kiel, Kiel, Germany<br />

3 Department of Dentistry and Periodontology, University Hospital of<br />

Regensburg, Regensburg, Germany<br />

For a deeper un<strong>der</strong>standing of the interaction of two mesophilic Acidithiobacilli<br />

and two thermophilic Sulfolobus strains with pyrite surfaces during the<br />

leaching process [1-3] we used light and electron microscopy. In or<strong>der</strong> to<br />

preserve the ultrastructure of the cells and their interaction site with the mineral<br />

for electron microscopy, gentle cryo-preparation methods were employed.<br />

Transmission and scanning electron micrographs showed the interaction in all<br />

strains to be mediated by S-layers, two-dimensionally crystalline surface<br />

proteins. Apparently, these macromolecules are equivalent to the EPS<br />

substance observed in other studies [4]. The S-layer completely covers the cell<br />

and is also in direct contact with the mineral surface when cells are attached to<br />

pyrite. This could be confirmed via immuno localization of the surface proteins,<br />

confirming the even distribution of the proteins on the cell surface. S-layer<br />

sheets were isolated by detergent extraction, for both, immunizing rabbits and<br />

investigating their fine structure by TEM after negative staining, using<br />

correlation averaging [5]. For both strains of Acidithiobacilli, we could identify<br />

lattices with rare p2 symmetry, with almost identical lattice dimensions. For<br />

both archaeal strains, the proteinaceous surface layer exhibited p3 symmetry, as<br />

previously observed for closely related microorganisms of the Sulfolobales [6].<br />

PB 04<br />

PCR-Based DGGE and FISH Analysis of Methanogens in<br />

an Anaerobic Bioreactor for Treating Palm Oil Mill<br />

Effluent<br />

M. Tabatabaei *1 , M.R. Zakaria 1 , R. Abdul Rahim 2 , A.D.G. Wright 3 , Y. Shirai 4 ,<br />

N. Abdullah 5 , K. Sakai 6 , S. Ikeno 4 , M. Mori 6 , N. Kazunori 6 , A. Sulaiman 7 , M.A.<br />

Hassan 1<br />

1 Department of Bioprocess Technology, Universiti Putra Malaysia, Serdang,<br />

Malaysia<br />

2<br />

Department of Cell and Molecular Biology, Universiti Putra Malaysia,<br />

Serdand, Malaysia<br />

3<br />

CSIRO Livestock Industries, Queensland Bioscience Precinct, St. Lucia,<br />

Australia<br />

4 Graduate School of Life Science and Systems Engineering, Kyushu Institute of<br />

Technology, Kitakyushu, Japan<br />

5 Department of Microbiology, Universiti Putra Malaysia, serdang, Malaysia<br />

6 Department of Plant Resources, Kyushu University, Fukuoka, Japan<br />

7 Department of Food and Process Engineering, Universiti Putra Malaysia,<br />

serdang, Malaysia<br />

16S rRNA-targeted fluorescent in situ hybridization combined with PCRcloning,<br />

light microscopy using Gram stains, scanning electron microscopy and<br />

denatured gradient gel electrophoresis were used to reveal the distribution of<br />

methanogens within an anaerobic closed digester tank fed with palm oil mill<br />

effluent. For specific detection of methanogens, 16S ribosomal RNA (rRNA)cloning<br />

analysis was conducted followed by RFLP (restriction fragment length<br />

polymorphism) for presumptive identification of methanogens. To cover the<br />

drawbacks of the PCR-cloning study, the organization of the microorganisms<br />

was visualized in the activated sludge sample by using fluorescent<br />

oligonucleotide probes specific to several different methanogens, and a probe<br />

for bacteria. In situ hybridization with methanogen- and bacterial probes and<br />

denatured gradient gel electrophoresis within activated sludge clearly<br />

confirmed the presence of Methanosaetasp. and Methanosarcina sp. cells.<br />

Methanosaeta concilii was found to be the dominant species in the bioreactor<br />

for treating palm oil mill effluent (POME). These results revealed the presence<br />

of possibly new strain of Methanosaeta in the bioreactor called Methanosaeta<br />

concilii SamaliEB (Gene bank accession Number: EU580025). In addition,<br />

fluorescent hybridization pictured the close association between the<br />

methanogens and bacteria and that the number of methanogens was greater than<br />

the number of bacteria.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PB 05<br />

Methanogenesis in Holocene Permafrost Deposits of the<br />

Lena Delta<br />

D. Wagner *1 , M. Schloter 2 , A. Lipski 3<br />

1<br />

Research Unit Potsdam, Alfred Wegener Institute for Polar and Marine<br />

Research, Potsdam, Germany<br />

2<br />

Institute of Soil Ecology, Helmholtz Zentrum München, Neuherberg, Germany<br />

3<br />

Department of Microbiology, University of Osnabrueck, Osnabrueck,<br />

Germany<br />

Permafrost environments within the Siberian Arctic are natural sources of the<br />

climate relevant trace gas methane. In or<strong>der</strong> to improve our un<strong>der</strong>standing of<br />

the present and future carbon dynamics in high latitudes, we studied the<br />

methane concentration, the quantity and quality of organic matter, and the<br />

activity and biomass of the methanogenic community in permafrost deposits.<br />

For these investigations a permafrost core of Holocene age was drilled in the<br />

Lena Delta (72°22’N, 126°28’E). The organic carbon of the permafrost<br />

sediments varied between 0.6% and 4.9% and was characterized by an<br />

increasing humification index with permafrost depth. A high methane<br />

concentration was found in the upper 4m of the deposits, which correlates well<br />

with the methanogenic activity and archaeal biomass (expressed as PLEL<br />

concentration). Even the incubation of core material at -3 and -6°C with and<br />

without substrates showed a significant methane production (range: 0.04–0.78<br />

nmol CH4 h -1 g -1 ). The results indicated that the methane in Holocene<br />

permafrost deposits of the Lena Delta originated from mo<strong>der</strong>n methanogenesis<br />

by cold-adapted methanogenic archaea. Microbial generated methane in<br />

permafrost sediments is so far an un<strong>der</strong>estimated factor for the future climate<br />

development.<br />

PB 06<br />

The swimming behaviour of Archaea: a video-based<br />

comparative study<br />

B. Herzog *1 , R. Wirth 1<br />

1 Institute of Microbiology, University of Regensburg, Regensburg, Germany<br />

In Bacteria a great variety of motility structures occur, whereas in the case of<br />

Archaea only flagella are known. These flagella differ significantly from<br />

bacterial ones and very little is known about their assembly or the mechanism<br />

that leads to motion.<br />

Here we report data comparing the swimming behaviour of various<br />

representatives of the domain Archaea, namely Halobacterium salinarum,<br />

Methanococcus voltae, Methanococcus maripaludis, Methanocaldococcus<br />

jannaschii, Methanothermobacter thermoautotrophicus, Sulfolobus<br />

solfataricus, Pyrococcus furiosus and Ignicoccus hospitalis. For comparative<br />

analyses Escherichia coli and Proteus mirabilis were used as default for their<br />

motility patterns <strong>bei</strong>ng well studied.<br />

Our analysis system consists of a very light sensitive (16bit CCD) camera<br />

connected to an Olympus BX50 phase-contrast microscope with dark field, UV<br />

and heating equipment. This allows experiments to be performed un<strong>der</strong> nearly<br />

optimal growth conditions (e.g. 95°C and anaerobic atmosphere). The<br />

information obtained (as video or image-sequence) was evaluated using<br />

specific tracking and picture processing software. The swimming trails of the<br />

above mentioned organisms were analysed with respect to their directionality,<br />

their average speed and range of motion.<br />

Data will be presented indicating e.g. that H. salinarum is a very slow<br />

swimming Archaeon, whilst M. jannaschii represents an exceptionally fast<br />

swimming organism. In addition we will show that observations of the<br />

swimming behaviour un<strong>der</strong> "sub-optimal" conditions (e.g. aerobiosis and low<br />

temperature) lead to completely different results. Further analyses will ask for<br />

the potential alteration of archaeal swimming due to chemotaxis or aerotaxis.<br />

PB 07<br />

Purification of Pyrococcus furiosus flagella and approaches<br />

for cloning of the major flagellin<br />

S. Schopf *1 , R. Rachel 2 , R. Wirth 1<br />

1 Institute of Microbiology, University of Regensburg, Regensburg, Germany<br />

2 Institute of Microbiology and Electron Microscopy, University of Regensburg,<br />

Regensburg, Germany<br />

Flagella of the archaeon Pyrococcus furiosus have serveral functions: First of<br />

all they are putative motility tools used for swimming. But cells are also able to<br />

adhere to various surfaces via their flagella, resulting in a biofilm-like growth.<br />

Moreover, flagella can aggregate into highly or<strong>der</strong>ed "cable-like" cell-cell<br />

connections, probably used for genetic transfer.<br />

91

92<br />

So far only little is known about the structure of the flagella and their<br />

membrane anchor. In flagella preparations three flagellins were found, with<br />

FlaB0 constituting the major flagellin. Glykosylated, it has a molecular weight<br />

of about 30 kDa. The two other proteins are detectable only by<br />

immunolabeling; therefore it is assumed that they play a minor role for flagella<br />

structure.<br />

For further detailed studies the preparation of well structured flagella posessing<br />

potential anchoring structures is a fundamental prerequisite. Therefore, a new<br />

preparation protocol was established, based on the solubilization of membrane<br />

proteins in or<strong>der</strong> to obtain free flagella (not attached to the membrane and cell<br />

wall), followed by several purification steps. Flagella prepared that way can be<br />

further analyzed by various techniques, like electron microscopy or native gel<br />

electrophoresis to study the formation of complexes.<br />

Furthermore the generation of antibodies against the three flagellins is an<br />

important aim of our work; with the help of immunolabeling technique we<br />

migth be able to clarify the proteins´ location in the flagella. Various<br />

approaches to express recombinant flagellins for immunization in rabbit have<br />

been made and will be reported.<br />

PB 08<br />

Characterization of missing enzymes involved in the 3hydroxypropionate/4-hydroxybutyrate<br />

cycle of autotrophic<br />

carbon dioxide fixation in Metallosphaera sedula<br />

D. Kockelkorn *1 , I.A. Berg 1 , G. Fuchs 1<br />

1 Institut für Biologie / Mikrobiologie, Albert-Ludwigs-Universität Freiburg,<br />

Freiburg, Germany<br />

Autotrophic members of the archaeal or<strong>der</strong> Sulfolobales assimilate CO2 via a<br />

novel 3-hydroxypropionate/4-hydroxybutyrate cycle, with acetyl-<br />

CoA/propionyl-CoA carboxylase as the key carboxylating enzyme [1]. In this<br />

cycle, as studied in Metallosphaera sedula, one acetyl-CoA and two<br />

bicarbonate molecules are reductively converted via 3-hydroxypropionate to<br />

succinyl-CoA. Succinyl-CoA is further reduced to 4-hydroxybutyrate and<br />

converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA<br />

dehydratase. The genes for the key enzymes of the cycle are also present in the<br />

genomes of other autotrophic members of the Sulfolobales and in marine<br />

Cenarchaeles.<br />

Three enzymes of this cycle, for which no unambiguous candidate gene could<br />

be identified in the genome of M. sedula, were purified. These enzymes are<br />

malonate semialdehyde reductase, succinate semialdehyde reductase and a<br />

bifunctional crotonyl CoA hydratase/(S)-3-hydroxybutyryl-CoA<br />

dehydrogenase. The corresponding genes were deduced from internal peptide<br />

sequences, overexpressed in Escherichia coli, and the recombinant enzymes<br />

were characterized. Corresponding genes were present in all sequenced<br />

Sulfolobales genomes. Interestingly, the gene for crotonyl-CoA hydratase/(S)-<br />

3-hydroxybutyryl-CoA dehydrogenase was also found in the sequenced<br />

representatives or<strong>der</strong>s Desulfurococcales and Thermoproteales. These Archaea<br />

use a dicarboxylate/4-hydroxybutyrate CO2 fixation cycle [2], in which<br />

succinyl-CoA is converted to two acetyl-CoA via 4 hydroxybutyrate and<br />

crotonyl-CoA, as in Sulfolobales.<br />

[1] Berg et al. (2007) Science 318 (5857):1782-1786.<br />

[2] Huber et al. (2008) PNAS 105 (22):7851–7856<br />

PB 09<br />

Desiccation tolerance of methanogenic archaea from<br />

Siberian permafrost on a Martian soil simulant<br />

E. Rickert *1 , D. Wagner 1<br />

1 Alfred Wegener Institut for Polar and Marine Research, Research Unit<br />

Potsdam, Potsdam, Germany<br />

Mars is consi<strong>der</strong>ed as one of the most similar planets to Earth in our solar<br />

system, although it is presently characterized by an extreme cold and dry<br />

climate. If primitive life developed on Mars and survived un<strong>der</strong> the harsh<br />

conditions on present Mars, it will be possibly found in the protected<br />

subsurface of the planet.<br />

Due to the arid conditions on Mars, a possible niche for microorganisms could<br />

be the subsurface permafrost environments. As it was shown in one of our<br />

previous studies, methanogenic archaea from Siberian permafrost are<br />

remarkably resistant to desiccation as well as to simulated Martian thermophysical<br />

conditions. It was reported that pure cultures of methanogenic archaea<br />

survived a desiccation period of at least 25 days.<br />

The aim of this study was to investigate the growth and the desiccation<br />

tolerance of methanogenic archaea from permafrost on a Martian soil simulant.<br />

During the experiments methanogens from permafrost and non-permafrost<br />

habitats - latter used as reference organisms - were grown and desiccated on the<br />

soil simulant. By the increase of methane concentration the growth rate was<br />

determined, measured by gas chromatography. The survival rates after<br />

desiccation were calculated by fluorescence in situ hybridization (FISH).<br />

Our study presents new insights into the ability of archaea from Siberian<br />

permafrost to metabolize and survive during a prolonged desiccation period on<br />

a Mars soil simulant. The results indicate that these organisms could be used as<br />

model for putative microbial life on Mars.<br />

PB 10<br />

Biofilm formation un<strong>der</strong> hot conditions<br />

A. Koerdt *1 , K. Thormann 2 , S.V. Albers 1<br />

1 Independent Junior Research Group Molecular Biology of Archaea, Max<br />

Planck Institute for Terrestrial Microbiology, Marburg, Germany<br />

2 Department of Ecophysiology, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

The mechanism of biofilm formation and its importance for survival of<br />

microorganisms in their natural habitats has attracted more and more interest<br />

during the last few years. For bacteria and eukarya many of these systems have<br />

been described and characterized and confocal microscopy has played a vital<br />

role in this.<br />

For the archaeal domain only very few publications have addressed biofilm<br />

formation. We have chosen the crenarchaeal model organism Sulfolobus spp. to<br />

initiate studies on archaeal biofilms. Sulfolobus species are<br />

hyperthemoacidophiles growing optimally at 80°C and pH 3. Sulfolobus species<br />

are found all over the world in geothermically active environments like<br />

solfataric fields.<br />

We have developed assays to study biofilm formation at high temperatures. We<br />

studied the three species S. solfataricus, S. acidocaldarius and S. tokodaii in<br />

biofilm formation and motility assays. S. acidocaldarius easily formed biofilms<br />

in standing petri dishes in only two days whereas the other two strains needed<br />

longer to develop more advanced biofilm structures. Analysis of these<br />

structures with confocal microscope has been performed and its structures will<br />

be presented. Currently, factors which influence biofilm formation such as<br />

surface tension, O2 availability, temperature, and the addition of divalent<br />

cations are un<strong>der</strong> investigation. This is the first in depth analysis of biofilm<br />

formation in the domain of the archaea.<br />

PB 11<br />

Role of surface structures during attachment of the<br />

hyperthermophile Sulfolobus solfataricus to a variety of<br />

surfaces<br />

B. Zolghadr 1 , A. Kling 2 , A. Driessen 3 , R. Rachel 2 , S.V. Albers *1<br />

1<br />

Molecular Biology of Archaea, Max Plank Institute for terrestrial<br />

microbiology, Marburg, Germany<br />

2<br />

Lehrstuhl für Mikrobiologie & Archaeenzentrum <strong>der</strong> Universität Regensburg,<br />

Universität Regensburg, Regensburg, Germany<br />

3<br />

Molecular Microbiology, Rijks universiteit Groningen, Groningen,<br />

Netherlands<br />

Attachment of micro-organisms to surfaces is essential for their survival in their<br />

natural environment. However, in many laboratories micro-organisms are<br />

cultivated as planktonic species and many important aspects of their behaviour<br />

in their natural ecosystem are still unclear. The hyperthermophilic Sulfolobales<br />

strains are able to grow at extreme conditions of 80˚C and pH3 and are found in<br />

muddy acidic solfataric fields all over the world. In these habitats Sulfolobus<br />

spp. most likely stick to sand particles and other material present in these hot<br />

springs.<br />

At first attachment of Sulfolobus solfataricus P2 was tested to a variety of<br />

surfaces such as mica, glass and carbon coated electron microscopy grids. The<br />

materials were added to liquid cultures cultivated un<strong>der</strong> typical conditions with<br />

orbital shaking and attachment was observed after two days by analysis with<br />

electron microscopy or raster electron microscopy. S. solfataricus also showed<br />

the abundant formation of extracellular substances which composition is<br />

currently is un<strong>der</strong> investigation.<br />

In S. solfataricus several cell surface structures have been described, such as<br />

flagella, UV induced pili and the bindosome. For all these systems deletion<br />

strains have been characterized before and were now tested for their ability to<br />

attach to the afore mentioned materials. Although the wild type cells could very<br />

well attach to carbon coated electron microscopy grids, the flagella deletion<br />

mutant could not adhere any more showing the important role of flagella in this<br />

adhesion process. Also deletion mutants of the UV induced pili system were<br />

greatly impaired in attachment indicating that these pili might not only play an<br />

important role after UV stress, but also in adhesion to surfaces. Summarizing,<br />

this is the first study to show the involvement of cell surface structures in the<br />

archaeal domain by detailed analysis of deletion mutants.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PB 12<br />

New autotrophic CO2 fixation pathways in Crenarchaeota:<br />

Rules and seeming exceptions<br />

I. Berg *1 , D. Kockelkorn 1 , H. Ramos-Vera 1 , G. Fuchs 1 , A. Petri 1<br />

1<br />

Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg,<br />

Freiburg, Germany<br />

The common ancestor of life was proposed to be a chemoautotrophic<br />

hyperthermophile. Therefore, the studies of autotrophic carbon dioxide<br />

assimilation in extremophilic Archaea are important for our un<strong>der</strong>standing of<br />

the evolution of early life. Recently, two new pathways have been discovered in<br />

Crenarchaeota: the 3-hydroxypropionate/4-hydroxybutyrate cycle in<br />

Sulfolobales [1] and the dicarboxylate/4-hydroxybutyrate cycle in Ignicoccus<br />

hospitalis, belonging to the Desulfurococcales [2]. The latter pathway has also<br />

been identified in Thermoproteales. These pathways are different in the nature<br />

of carboxylating enzymes; more specifically, acetyl-CoA/propionylCoA<br />

carboxylase in the 3-hydroxypropionate/4-hydroxybutyrate cycle and pyruvate<br />

synthase and PEP carboxylase in the dicarboxylate/4-hydroxybutyrate cycle.<br />

The regeneration of acetyl-CoA, the primary CO2 acceptor, is similar in both<br />

pathways: succinyl-CoA, the CO2 fixation product, is reduced to 4hydroxybutyrate,<br />

which is further converted to two acetyl-CoA molecules with<br />

a participation of 4-hydroxybutyryl-CoA dehydratase, a key enzyme of the 4hydroxybutyrate<br />

part of the pathways. Autotrophic Crenarchaea studied so far<br />

use one of the two pathways. The usage of the aerotolerant biotin-dependent<br />

acetyl-CoA/propionyl-CoA carboxylase in the 3-hydroxypropionate/4hydroxybutyrate<br />

cycle and of the strictly anaerobic ferredoxin-dependent<br />

pyruvate synthase in the dicarboxylate/4-hydroxybutyrate cycle is determined<br />

by an aerobic or anaerobic mode of growth of the corresponding species. The<br />

presence of the 4-hydroxybutyryl-CoA dehydratase gene in different<br />

crenarchaeal or<strong>der</strong>s points to a possible ancestry of the acetyl-CoA regeneration<br />

through 4-hydroxybutyrate in Crenarchaeota.<br />

[1] Berg et al., 2007, Science 318, 1782-1786.<br />

[2] Huber et al., 2008, PNAS 105, 7851-7856.<br />

PB 13<br />

Autotrophic carbon dioxide assimilation in Thermoproteales<br />

revisited<br />

H. Ramos-Vera *1 , I. Berg 1 , G. Fuchs 1<br />

1 Mikrobiologie, Institut für Biologie II, Albert-Ludwigs-Universität Freiburg,<br />

Freiburg, Germany<br />

The dominant extant autotrophic pathway, the Calvin-Bassham-Benson cycle,<br />

is restricted to Bacteria and Eukarya and probably evolved late in the evolution.<br />

Within Archaea, two new pathways were recently described in Crenarchaeota.<br />

Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate cycle with acetyl-<br />

CoA/propionyl-CoA carboxylase as carboxylating enzyme (1). Ignicoccus<br />

hospitalis (Desulfurococcales) uses the dicarboxylate/4-hydroxybutyrate cycle,<br />

where pyruvate synthase and phosphoenolpyruvate carboxylase are responsible<br />

for CO2 fixation (2). In both cycles, the regeneration of acetyl-CoA from<br />

succinyl-CoA proceeds via 4-hydroxybutyrate. Thermoproteales would be an<br />

exception in Crenarchaeota, since the reductive tricarboxylic acid cycle was<br />

proposed for Thermoproteus neutrophilus (3). However, we could not confirm<br />

the presence of its key enzyme, ATP-citrate lyase. Moreover, 13 C-labeling<br />

studies are difficult to reconcile with a reductive carboxylation of succinyl-CoA<br />

to 2-oxoglutarate, but are in line with the operation of dicarboxylate/4hydroxybutyrate<br />

cycle. All enzymes of the dicarboxylate/4-hydroxybutyrate<br />

cycle were found in extracts of autotrophically grown T. neutrophilus cells, and<br />

the corresponding genes were identified in the genome. The key enzymes of the<br />

cycle were strongly up-regulated un<strong>der</strong> autotrophic growth conditions,<br />

indicating their involvement in autotrophic CO2 fixation. Moreover, T.<br />

neutrophilus incorporates [1- 14 C]4-hydroxybutyrate in all main cell<br />

constituents, pointing out its central position in the metabolism. We conclude<br />

that the dicarboxylate/4-hydroxybutyrate cycle is operating in CO2 fixation in<br />

Thermoproteales as well as in Desulfurococcales.<br />

[1] Berg et al., 2007, Science 318, 1782-6.<br />

[2] Huber et al., 2008, PNAS 105, 7851-6.<br />

[3] Strauss et al., 1992, Eur J Biochem 205, 853-66.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PB 14<br />

Is "Candidatus Nitrososphaera gargensis" a mixotrophic<br />

ammonia-oxidizing archaeon?<br />

R. Hatzenpichler *1 , N. Schuster 1 , N. Rychlik 2 , E. Spieck 2 , E. Lebedeva 3 , H.<br />

Daims 1 , M. Wagner 1<br />

1 Department of Microbial Ecology, University of Vienna, Vienna, Austria<br />

2 Biocenter Klein Flottbek, University of Hamburg, Hamburg, Germany<br />

3 Winogradsky Institute of Microbiology, Russian Academy of Sciences,<br />

Moscow, Russia<br />

The finding that some members of the Crenarchaeota, besides two well-known<br />

groups within the Beta- and Gammaproteobacteria, are capable of ammonia<br />

oxidation radically changed our perception of nitrification [1]. Since then, many<br />

habitats including numerous soil and marine systems, sediments and terrestrial<br />

hot springs, have been shown to be teeming with ammonia-oxidizing archaea<br />

(AOA). By analyzing a mo<strong>der</strong>ately thermophilic enrichment we recently<br />

demonstrated for "Candidatus Nitrososphaera gargensis" as the first member of<br />

the crenarchaeotal soil group (group CI.1b) the capability to oxidize ammonia<br />

[2]. Until now, this AOA is the only cultured representative of this globally<br />

distributed phylogenetic group. Furthermore, ammonia oxidation is the only<br />

reported physiology of this group. The autotrophic nature of "Candidatus<br />

Nitrososphaera gargensis" is in accordance with the physiology of the other<br />

three AOA which have been identified so far ("Candidatus Nitrosopumilus<br />

maritimus", "Candidatus Cenarchaeum symbiosum" and "Candidatus<br />

Nitrosocaldus yellowstonii"). However, recent studies revealed that at least a<br />

part of the crenarchaeotal community in mo<strong>der</strong>ate ecosystems has a<br />

heterotrophic or mixotrophic lifestyle [3-5]. Using a combination of<br />

fluorescence in situ hybridization (FISH) and single-cell microautoradiography<br />

(MAR) we analyzed the metabolic versatility of "Candidatus Nitrososphaera<br />

gargensis" by offering different heterotrophic substrates including amino acids<br />

in the presence or absence of ammonia.<br />

[1] Könneke, M. et al. (2005) Nature 437:543-6;<br />

[2] Hatzenpichler, R. et al. (2008) PNAS 105:2134-9;<br />

[3] Hallam, S. et al. (2006) PLoS Biol 4:e95;<br />

[4] Ingalls, A. E. et al. (2006) PNAS 103:6442-7;<br />

[5] Agogué, H. et al. (2008) Nature 456:788-92<br />

PB 15<br />

Development of a genetic system in Sulfolobus<br />

acidocaldarius<br />

M. Wagner *1 , S. Berkner 2 , G. Lipps 3 , S.V. Albers 1<br />

1 Molecular Biology of Archaea, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

2 Department of Biochemistry, University of Bayreuth, Bayreuth, Germany<br />

3 School of Life Sciences, University of Applied Sciences Northwestern<br />

Switzerland, Muttenz, Switzerland<br />

Sulfolobus acidocaldarius is a hyperthermophilic Crenarchaeon which lives in<br />

aerobic zones of terrestrial solfataric springs. It grows optimally at 80°C and<br />

pH 3 on carbon sources such as yeast extract and tryptone. Sulfolobus species<br />

belong to the best studied archaeal microorganisms. However, the lack of<br />

genetic tools such as deletion procedures and vector systems have long<br />

hampered detailed in vivo studies. A major problem is the absence of suitable<br />

selection markers. Because of the extreme environmental conditions common<br />

antibiotics such as ampicillin or kanamycin can not be used and it is only<br />

possible to get gene deletions via selection on auxotrophies. In this study<br />

Sulfolobus acidocaldarius strains with several and even multiple markers were<br />

prepared to facilitate a common and fast method to produce gene knockouts in<br />

this organism. The system is based on gene deletions in several amino acid<br />

pathways which were produced by markerless deletion in S. acidocaldarius<br />

strain DMS639. With this new set of auxotrophic mutants in this organism the<br />

foundation for obtaining deletions, gene replacements and complementations<br />

has been build.<br />

93

94<br />

PB 16<br />

Snap-shot genome analysis of a mo<strong>der</strong>ately thermophilic<br />

enrichment and genome reconstruction of the dominant<br />

ammonia-oxidising crenarchaeote<br />

N. Rychlik *1 , R. Hatzenpichler 2 , M. Schillhabel 3 , M. Wagner 2 , W. Streit 1 , E.<br />

Spieck 1<br />

1<br />

Abteilung allgemeine Mikrobiologie und Biotechnologie, Universität<br />

Hamburg, Hamburg, Germany<br />

2<br />

Department für Mikrobielle Ökologie, Universität Wien, Wien, Austria<br />

3<br />

Institut für klinische Molekularbiologie, Universitätsklinikum Schleswig-<br />

Holstein, Kiel, Germany<br />

During the last few years, the un<strong>der</strong>standing of the nitrogen cycle has changed<br />

dramatically. In 2005, it was discovered that Archaea can catalyse the first step<br />

of nitrification i.e. the oxidation of ammonia to nitrite [1]. The mo<strong>der</strong>ately<br />

thermophilic (46°C) ammonia oxidizing crenarchaeote "Candidatus<br />

Nitrososphara gargensis" [2] from a hot spring biofilm grows in a community<br />

together with different bacteria and was enriched to a final ratio of more than<br />

90 %. To clarify the composition of this consortium 16S rRNA-gene based<br />

analyses (clone library, DGGE) were performed and four bacteria were detected<br />

in addition to "Candidatus N. gargensis". The metagenome of the enrichment<br />

was sequenced by 454 pyrosequencing and ~ 200 mb sequence data were<br />

generated in or<strong>der</strong> to assemble and annotate the whole genome of N. gargensis.<br />

Therefore, more than 6.700 contigs were added to the pedant-database and the<br />

RAST server for annotation. Furthermore, a metagenomic cosmid library was<br />

constructed with E. coli Epi100 as heterologous host. Current work focuses on<br />

closing the gaps and binning of the obtained contigs. This will be the first<br />

assembled and annotated genome of an ammonia oxidising crenarchaeote of the<br />

cluster 1.I.b of the ubiquitary widespread soil group, which has not been<br />

cultivated as a single strain.<br />

[1] Könneke et al. (2005). Nature 437(7058): 543-6<br />

[2] Hatzenpichler et al. (2008). PNAS 105(6): 3134-9<br />

PB 17<br />

Gene conversion results in the harmonization of genome<br />

copies in the polyploid haloarchaeon Haloferax volcanii<br />

C. Lange *1 , J.A. Soppa 1<br />

1 Institut für Molekulare Biowissenschaften, Goethe-Universität, Frankfurt am<br />

Main, Germany<br />

The halophilic model archaeon Haloferax volcanii was recently shown to be<br />

polyploid [1]. It contains 18 genome copies during exponential growth and 10<br />

genome copies in the stationary growth phase [1]. As point mutants can easily<br />

be isolated and genetic engineering of the H. volcanii genome, e.g. construction<br />

of deletion mutants, easily results in cells with identical mutated genome<br />

copies, a mechanism for the harmonization of different alleles must exist.<br />

Therefore, we became interested in the mechanism, the kinetics and the<br />

direction of gene conversion in H. volcanii.<br />

Applying the pop-in/pop-out strategy [2], the trpA deletion mutant H53 [3] was<br />

used to construct a strain that carries two different genomes simultaneously<br />

within each cell, which contain either the leuB gene or the trpA gene at the leuB<br />

locus. Therefore, this strain is prototrophic for both amino acids. It could be<br />

shown that after the addition of either leucine or tryptophane the respective<br />

"unnecessary" allele is indeed lost. The velocity of gene conversion depends on<br />

the length of the DNA fragment that has to be newly synthesized. If selection<br />

was totally abrogated by adding both amino acids, genome harmonization<br />

consistently occurred in the direction of the leuB gene, which required less<br />

DNA synthesis. These data hint at a homologous recombination mechanism<br />

<strong>bei</strong>ng responsible for the gene conversion in H. volcanii. Further experiments<br />

will characterize genome copy harmonization in strains missing putative<br />

homologous recombination genes.<br />

[1] S. Breuert, T. Allers, G. Spohn and J. Soppa (2006) PLoS ONE 1:e92<br />

[2] T. Allers and H.-P. Ngo (2003) Biochem Soc Trans 31:706-710<br />

[3] T. Allers, H.-P. Ngo, M. Mevarech and R.G. Lloyd (2004) Appl Environ<br />

Microbiol 70:943-953<br />

PB 18<br />

Role of members of the aldehyde dehydrogenase<br />

superfamily in Sulfolobus solfataricus<br />

D. Esser *1 , T. Kouril 1 , M. Zaparty 1 , B. Siebers 1<br />

1 Department of Chemistry, Biofilm Centre, Molecular Enzyme Technology and<br />

Biochemistry, University of Duisburg-Essen, Duisburg, Germany<br />

Comparative biochemical studies on central carbohydrate metabolism revealed<br />

that Archaea utilize modifications of the classical EMP- and ED pathways,<br />

characterized by the presence of numerous novel enzymes [1]. Recent studies<br />

indicate that the branched ED pathway in S. solfataricus is promiscuous and<br />

represents an equivalent route for glucose and galactose catabolism [2]. In S.<br />

solfataricus two different GAP converting enzymes were characterized, the<br />

classical phosphorylating GAP dehydrogenase (GAPDH) and the nonphosphorylating<br />

GAPDH (GAPN) and a role of GAPN in metabolic<br />

thermoadaption has been suggested previously [3]. GAPN (Sso3194) is a<br />

member of the aldehyde dehydrogenase superfamily, interestingly in the<br />

genome of S. solfataricus four additional GAPN paralogs (Sso1629, Sso1842,<br />

Sso1218 & Sso3117) have been identified. So far only Sso3117 [4] has been<br />

show to posses 2,5-dioxopentanoate activity <strong>bei</strong>ng involved in the pentose<br />

phosphate metabolism. However, the physiological function of the three<br />

residual paralogs remained unclear.<br />

The three genes were cloned, the encoded proteins heterologously expressed in<br />

Escherichia coli, purified and analysed in respect to their catalytic properties in<br />

or<strong>der</strong> to elucidate their function in the carbohydrate metabolism of S.<br />

solfataricus. Detailed enzymatic studies for the three paralogs (Sso1218,<br />

Sso1842 & Sso1629) will be presented.<br />

[1] Siebers, B. and Schönheit, P., 2005, Curr Opin Microbiol 8:695-705<br />

[2] Lamble, H. J. et al., 2003, J Biol Chem 278 : 34066-34072<br />

[3] Ettema, T. J. G., et al., 2008, Extremophiles 12:75–88<br />

[4] Brouns, S. J. J., et al., 2006, J. Biol. Chem., Vol. 281, Issue 37, 27378-<br />

27388<br />

PB 19<br />

Multiprotein-bridging factor 1 (MBF1) in the archaeal<br />

transcription of the hyperthermophilic Crenarchaeon<br />

Thermoproteus tenax and the mesophilic Euryarchaeon<br />

Methanosarcina mazei.<br />

J. Marrero-Coto *1 , A. Ehrenhofer-Murray 2 , B. Siebers 1<br />

1 Department of Chemistry, Biofilm Centre, Molecular Enzyme Technology and<br />

Biochemistry, University of Duisburg-Essen, Duisburg, Germany<br />

2 Department for Genetics, University Duisburg-Essen, Essen, Germany<br />

Multiprotein-bridging factor 1 (MBF1) is a transcriptional activator that<br />

mediates transcriptional activation by bridging a sequence-specific activator<br />

and the TATA-box-binding protein (TBP). A homology search of the databases<br />

showed that the MBF1 sequence is highly conserved in Archaea and from<br />

Saccharomyces cerevisiae to human. Archaeal MBF1s (aMBF1) contain a Znribbon<br />

motif that is absent in their eukaryotic counterparts. Recent studies<br />

revealed diverse biological functions of MBF1 in yeast, insects, rats, plants and<br />

human [1, 2, 3, 4, 5]. MBF1 is present in all archaeal genomes, raising the<br />

question about its possible function. It has been described that yeast MBF1<br />

(yMBF1) mediates the GCN4-dependent transcriptional activation of the HIS3<br />

gene by bridging between GCN4 and TBP [1]. Interestingly, AtMBF1 paralogs<br />

from the plant Arabidopsis thaliana are able to complement the defects of an S.<br />

cerevisiae MBF1 deletion (mbf1Δ) [6]. In this study, it was tested whether<br />

MBF1 from Thermoproteus tenax (TtxMBF1) and Methanosarcina mazei<br />

(MmMBF1) are able to complement the function of the yeast MBF1 gene.<br />

[1] Takemaru K, Harashima S, Ueda H, Hirose S (1998) Mol Cell Biol 18:<br />

4971-4976<br />

[2] Liu Q, Nakashima-Kamimura N, Ikeo K, Hirose S, Gojobori T (2003)<br />

Development 130: 719-728<br />

[3] Jindra M, Gaziova I, Uhlirova M, Okabe M, Hiromi Y, Hirose S (2004)<br />

EMBO J 23: 3538-3547<br />

[4] Smith M, Johanson R, Rogers K, Coleman P, Slemmon R Mol Brain Res<br />

62: 12-24.<br />

[5] Mariotti M, De Benedictis L, Avon E, Maier J (2000) J Biol Chem 275:<br />

24047-24051<br />

[6] Tsuda K, Tsuji T, Hirose S, Yamazaki K (2004) Plant Cell Physiol 45: 225-<br />

231<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PB 20<br />

CRISPR in Thermoproteus tenax: Tracking the Cas<br />

complexes<br />

A. Plagens *1 , A. Hagemann 2 , B. Tjaden 1 , R. Hensel 1<br />

1 Department of Biology, Microbiology I, University of Duisburg-Essen, Essen,<br />

Germany<br />

2 Department of Chemistry, Biofilm Centre, Molecular Enzyme Technology and<br />

Biochemistry, University of Duisburg-Essen, Duisburg, Germany<br />

The clusters of regularly interspaced short palindromic repeats (CRISPR) are a<br />

novel feature of a variety of prokaryotic organisms, present in nearly all<br />

archaeal and 50% of all bacterial genomes. They consist of repeat sequences of<br />

24 to 48 nt, separated by slightly longer spacer sequences [1]. These clusters<br />

are accompanied by a set of conserved cas (CRISPR-associated) genes, e.g. cas<br />

genes 1 to 4. The encoded Cas proteins are involved in the propagation and<br />

function of CRISPR. It has been shown in E. coli that CRISPR, together with<br />

the Cas proteins, guide antiviral defence in prokaryotes with the specificity<br />

determined by sequence similarity between spacer and phage genome [2]. The<br />

detailed un<strong>der</strong>lying molecular mechanisms remained unknown yet.<br />

To get a closer insight into the functional impact of the CRISPR/Cas-system in<br />

Archaea the seven CRISPR clusters (TTX_1-7) and the ten relevant cas genes<br />

of the hyperthermophilic Crenarchaeote Thermoproteus tenax were subject to<br />

detailed analysis. The genes located between CRISPR cluster TTX_4 and 5<br />

show two typical operon-like genome structures with a putative transcriptional<br />

regulator (ORF TTX_1249) located in between. Stress induced upregulation of<br />

transcription could be verified by RT-PCR Southern Blots for both cas operons.<br />

The respective cas genes were heterologously expressed in E. coli; the entire<br />

Cas protein complexes 1 (TTX_1245-1248) and 2 (TTX_1250-1255) were<br />

purified and refolded from inclusion bodies and functionally analysed.<br />

[1] Jansen et al., Mol Microbiol 43(6), 2002<br />

[2] Brouns et al., Science 321, 2008<br />

PB 21<br />

Probing the reaction mechanism of the sulfur oxygenase<br />

reductase of Acidianus ambivalens<br />

A. Veith *1 , T. Urich 2 , K. Seyfarth 1 , A. Kletzin 1<br />

1 Institute of Microbiology and Genetics, Technical University of Darmstadt,<br />

Darmstadt, Germany<br />

2 Department of Genetics in Ecology, University of Vienna, Vienna, Austria<br />

The soluble sulfur oxygenase reductase (SOR) is the initial enzyme in the<br />

aerobic sulfur oxidation pathway of the archaeon Acidianus ambivalens<br />

catalyzing the oxygen-dependent sulfur disproportionation to sulfite and H2S.<br />

The globular, hollow holoenzyme is composed of 24 identical subunits. The<br />

active site comprises a mononuclear non-heme iron center with a low reduction<br />

potential and a cysteine persulfide.<br />

The active site is surrounded by a putative hydrogen bonding network probably<br />

contributing to the low reduction potential. Mutagenesis of two of the residues<br />

involved to alanine almost abolished activity, whereas a specific Glu-Asp<br />

exchange resulted in an increase of specific activity.<br />

Two consecutive pores provide substrate access to the active site, the first is<br />

formed by chimney-like structure in the outer shell while the others provides<br />

entrance to each of the active site pockets separately. A highly increased<br />

enzyme activity was obtained in deletion mutants of the chimney-like<br />

structures. Mutation of methionine residues M296/297 placed at the entrance to<br />

the active site pocket resulted in a significant decrease of enzyme activity with<br />

the exception of a M296V mutant thus showing the importance of the pore for<br />

activity.<br />

To investigate the question whether the active site iron un<strong>der</strong>goes a valence<br />

change during catalysis we exchanged it for other transition metals. The activity<br />

of Co 2+ -substituted SOR was similar to the wildtype Fe enzyme. An active<br />

Ga 3+ -substituted SOR showed that a valence change is not required and that<br />

oxygen is activated rather by the substrate than by the metal.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PB 22<br />

Insights into the NrpR regulon in Methanosarcina mazei<br />

Gö1<br />

K. Weidenbach 1 , C. Ehlers *1 , J. Kock 1 , R. Schmitz-Streit 1<br />

1 Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel,<br />

Germany<br />

The methanogenic archaeon Methanosarcina mazei strain Gö1 contains two<br />

homologues of NrpR, the transcriptional repressor of nitrogen assimilation<br />

genes recently discovered and characterized in Methanococcus maripaludis [1].<br />

Insertion of a puromycin-resistance conferring cassette into MM1085 encoding<br />

a single NrpR domain with an N-terminal helix–turn–helix domain (NrpRI)<br />

lead to a significant reduction of the lag-phase after a shift from nitrogen<br />

sufficiency to nitrogen limitation. Consistent with this finding, loss of NrpRI<br />

resulted in significantly increased transcript levels of genes involved in nitrogen<br />

fixation or nitrogen assimilation though growing un<strong>der</strong> nitrogen sufficiency as<br />

demonstrated by quantitative reverse transcriptional PCR analysis. Genomewide<br />

analysis using DNA-microarrays confirmed that transcript levels of 27<br />

ORFs were significantly elevated in the M. mazei MM1085::pac mutant un<strong>der</strong><br />

nitrogen sufficiency, including genes known to be up-regulated un<strong>der</strong> nitrogen<br />

limitation (e.g. nifH, glnA1, glnK1), and 17 additional genes involved in<br />

metabolism (4), encoding a flagella related protein (1) and genes encoding<br />

hypothetical proteins (12). Using cell extracts of Escherichia coli expressing<br />

MM1085 fused to the maltose binding protein (MBP–NrpRI) and employing<br />

promoter binding studies by DNA-affinity chromatography demonstrated that<br />

MBP–NrpRI binds specifically to the nifH-promoter. Deletion of various bases<br />

in the promoter region of nifH confirmed that the regulatory element ACC-N7-<br />

GGT is required for specific binding of NrpRI to the promoter.<br />

[1] Lie TJ, Leigh JA (2003) A novel repressor of nif and glnA expression in the<br />

methanogenic archaeon Methanococcus maripaludis. Mol Microbiol 47:235-<br />

246<br />

PB 23<br />

D-xylose degradation pathway in the halophilic archaeon<br />

Haloferax volcanii<br />

U. Johnsen *1 , M. Dambeck 2 , J. Soppa 2 , T. Fuhrer 3 , U. Sauer 3 , P. Schönheit 1<br />

1 Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität zu Kiel,<br />

Kiel, Germany<br />

2 Institut für Molekulare Biowissenschaften, Goethe-Universität Frankfurt,<br />

Frankfurt am Main, Germany<br />

3 Institut of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland<br />

The catabolic pathways of pentoses in archaea – with exception of D-arabinose<br />

in Sulfolobus solfataricus (1) – have not been analyzed in detail. Here we report<br />

the pathway of D-xylose degradation in the halophilic archaeon Haloferax<br />

volcanii. The analyses include (i) in vivo labelling experiments in growing<br />

cultures with 13 C-labelled xylose; (ii) DNA microarray experiments; (iii)<br />

characterization of enzymes, and (iv) analyses of in frame deletion mutants.<br />

The data indicate that D-xylose is oxidized to α-ketoglutarate, involving Dxylose<br />

dehydrogenase, xylonate dehydratase, 2-keto-3-deoxy-xylonate<br />

dehydratase and α-ketoglutarate semialdehyde dehydrogenase. This pathway<br />

differs from the classical D-xylose pathway in most bacteria involving the<br />

formation of xylulose-5-phosphate via xylose isomerase and xylulose kinase.<br />

[1] Stan J. J. Brouns et al. (2006) Journal of Biological Chemistry, 281, 27378-<br />

27388<br />

PB 24<br />

Cyclic 2,3-Disphosphoglycerate Synthetase: Thermosensor<br />

in Hyperthermophilic Methanoarchaea<br />

T. Knura 1 , B. Tjaden 1 , P. Moritz 1 , R. Hensel *1<br />

1 Mikrobiologie I, Universität Duisburg-Essen, Essen, Germany<br />

Some hyperthermophilic methanoarchaea (e.g. Methanothermus fervidus, M.<br />

sociabilis, Methanopyrus kandleri) respond changes of temperature with<br />

corresponding alterations of the intracellular concentration of the threefold<br />

negatively charged anion cyclic 2,3-diphosphoglycerate (cDPG) with highest<br />

concentrations of the molecule observed at supraoptimal growth temperature.<br />

The observation that this compound stabilizes intracellular enzymes of these<br />

organisms against thermo-inactivation supports its important thermoadaptive<br />

role. Key enzyme for the formation of cDPG is the cyclic 2,3diphosphoglycerate<br />

synthetase (cDPGS), which catalyses the reversible closure<br />

of the anhydride bond of the vicinal phosphate groups of the educt (non-cyclic<br />

2,3-diphosphoglycerate). Raising the growth temperature above the optimum<br />

95

96<br />

(up to 93 or 110°C, respectively)is followed by the formation of a cDPGS<br />

isoform with a significantly higher kcat for phosphoanhydride closure thus<br />

providing the basis for an immediate temperature adaptation through the<br />

intracellular concentration of cDPG. The structural and functional properties of<br />

the enzyme enabling its role as thermosensor and thermoadaptor at high<br />

temperature will be presented<br />

PB 25<br />

Elucidating the role of Sm-like proteins in Archaea<br />

S. Fischer *1 , J. Soppa 2 , T. Allers 3 , A. Marchfel<strong>der</strong> 1<br />

1 University of Ulm, Ulm, Germany<br />

2 Goethe University, Frankfurt, Germany<br />

3 University of Nottingham, Nottingham, United Kingdom<br />

In Eukarya Sm and Lsm (Sm-like) proteins are involved in a plethora of<br />

functions: they are essential components of small nuclear ribonucleoproteins<br />

(snRNPs) that function in pre-mRNA splicing. Additionally, Lsm RNPs are<br />

involved in mRNA degradation and maturation of precursor RNAs.<br />

The bacterial counterpart of eukaryotic Lsm proteins is the protein Hfq, holding<br />

a comparable multitude of functions. One of these is the interaction with<br />

sRNAs enabling thereby translational control and modulation of mRNA<br />

stability.<br />

Furthermore, Hfq acts as a translational regulator by interfering with mRNAribosome<br />

binding and was shown to modulate the activity of the key enzymes<br />

for mRNA decay. Lsm proteins have also been found in Archaea and using<br />

similarity searches we were able to identify the Haloferax volcanii Lsm protein,<br />

which we termed HvoLsm. In or<strong>der</strong> to clarify the biological functions of the<br />

archaeal Lsm protein, we constructed the deletion mutant ?hvolsm. The<br />

phenotype of this strain is currently <strong>bei</strong>ng analysed applying different growth<br />

conditions while proteome investigation is achieved using 2D gel<br />

electrophoresis.<br />

In addition we expressed the protein HvoLsm in E. coli to study its in vitro<br />

functions. We incubated the recombinant HvoLsm with sRNAs from Haloferax<br />

volcanii in gel shift assays, showing that the protein binds to sRNAs.<br />

PB 26<br />

Identity and abundance of ammonia-oxidising<br />

crenarchaeotes in the German Wadden Sea<br />

D. Wischer 1 , S. Standfest 1 , H. Cypionka 1 , M. Könneke *1 ,<br />

1 Institute for Chemistry and Biology of the Marine Environment (ICBM),<br />

University of Oldenburg, Oldenburg, Germany<br />

The cultivation of the mesophilic crenarchaeote Nitrosopumilus maritimus [1]<br />

and subsequent molecular studies have demonstrated the important role of<br />

ammonia-oxidising archaea (AOA) in the global nitrogen cycle. We had<br />

selectively enriched and isolated a novel AOA from a tidal channel of the<br />

German Wadden Sea (site Janssand). Strain Jan1 affiliates with the genus<br />

Nitrosopumilus and represents the first mesophilic AOA isolated from a natural<br />

environment. Here, we studied the distribution of AOA at site Janssand. MPN<br />

cultures from water and sediment were set up to estimate the abundance of both<br />

archaeal and bacterial nitrifyers. Cultures without amendment of antibiotics<br />

showed consumption of ammonia coupled to production of nitrate. In contrast,<br />

incomplete oxidation of ammonia to nitrite was found in media with antibiotics,<br />

indicating the inhibition of both ammonia-oxidising bacteria (AOB) and nitriteoxidising<br />

bacteria, and the presence of AOA. MPN cultures further indicated an<br />

up to 100-fold higher abundance of AOB compared to AOA in the surface<br />

sediment. PCR-based analysis confirmed the absence of bacteria and the<br />

presence of archaea in antibiotic-supplemented enrichments, while archaea<br />

were not detected at high dilutions of antibiotic-free enrichments. Cloning of<br />

archaeal 16S rRNA genes revealed the predominance of a single archaeal<br />

phylotype that affiliated with the genus Nitrosopumilus and showed a close<br />

relatedness to strain Jan1. The contribution of AOA to the archaeal community<br />

at site Janssand is currently <strong>bei</strong>ng investigated by cloning and CARD-FISH<br />

analysis of the upper, oxygenated sediment layer.<br />

[1] Könneke et al. (2005) Nature 437: 543–546.<br />

PC 01<br />

Separation and Characterization of Pumilacidin Isoforms<br />

Produced by Bacillus pumilus<br />

M. Gomaa *1 , P. Stolle 1 , G. Dräger 2 , G. Auling 1<br />

1 Institut für Mikrobiologie, Leibniz Universität Hannover, Hannover, Germany<br />

2 Institut für Organische Chemie, Leibniz Universität Hannover, Hannover,<br />

Germany<br />

A lipopeptide biosurfactant was produced by Bacillus pumilus IIBk9 in<br />

Casamino Acids medium. The culture filtrate of this strain lowered the surface<br />

tension to the very low value of 33.1 mN m -1 and displayed antifungal [1] and<br />

hemolytic activity as well. The crude lipopeptide was harvested after acidic<br />

precipitation. Subsequent extraction with dichloromethane and evaporation<br />

yielded a white pow<strong>der</strong>. High-resolution reversed phase HPLC on Nucleodur<br />

C18 column yielded 5 fractions which were characterized by MS-MS. This<br />

revealed isoforms of a cyclic lipopeptide (pumilacidin) built from variants of a<br />

heptapeptide and a beta-hydroxy fatty acid with chain lengths of 15 - 17 carbon<br />

atoms similar to a lipopeptide produced by B. pumilus M937-B1.[2] Since the<br />

latter strain is lost our work makes available lipoheptapeptide compounds with<br />

modified structures and different hydrophobicities which promise to have<br />

potential for biotechnological and pharmaceutical applications.<br />

[1] Fiss, M. (2001) Evaluierung von epiphytischen Antagonisten <strong>zur</strong><br />

biologischen Kontrolle des Apfelschorferregers Venturia inaequalis. Ph. D.<br />

thesis, University Hannover<br />

[2] Naruse et al. (1990) J. Antibiotics 43: 267 – 280.<br />

PC 02<br />

Plectasin, a fungal defensin antibiotic peptide, targets the<br />

bacterial cell wall precursor Lipid II<br />

T. Schnei<strong>der</strong> *1 , T. Kruse 2 , R. Wimmer 3 , I. Wiedemann 1 , V. Sass 1 , U. Pag 1 , A.<br />

Jansen 1 , A.K. Nielsen 2 , P.H. Mygind 2 , S. Neve 2 , B. Ravn 2 , L. de Maria 2 , H.G.<br />

Sahl 1 , H.H. Kristensen 2<br />

1 Institute for Medical Microbiology, Immunology and Parasitology –<br />

Pharmaceutical Microbiology Section, University of Bonn, Bonn, Germany<br />

2 Anti-Infective Discovery, Novozymes AS, Bagsvaerd, Denmark<br />

3 Department of Biotechnology, Chemistry and Environmental Engineering,<br />

Aalborg University, Aalborg, Denmark<br />

Antimicrobial peptides serve a vital role in first-line host defence and can be<br />

found throughout the animal and plant kingdom [1]. Although <strong>bei</strong>ng<br />

evolutionary ancient, AMPs still represent an effective weapon against Grampositive<br />

and Gram-negative bacteria, fungi and enveloped viruses killing very<br />

rapidly. Among the antimicrobial peptides, the defensins represent an important<br />

peptide family.<br />

The fungal defensin Plectasin is a 40 amino acid peptide produced by the<br />

saprophytic ascomycete Pseudoplectania nigrella [2]. Plectasin shares primary<br />

structural features with defensins from spi<strong>der</strong>s, scorpions, dragonflies and<br />

mussels and folds into a cysteine-stabilized alpha-beta-structure.<br />

Unlike conventional antibiotics which act via defined target molecules,<br />

antimicrobial defence peptides are assumed to act unspecifically by<br />

permeabilising the cell membrane.<br />

In contrast to this widely held view, we report for the first time the bacterial cell<br />

wall precursor Lipid II as a molecular target of a defensin.<br />

We used a series of cellular approaches and in vitro cell wall biosynthesis<br />

assays to narrow down the target pathway and the specific mechanism of<br />

action.<br />

[1] Zasloff M., Nature. 415(6870):389-95 (2002)<br />

[2] Mygind PH., Nature. 437(7061):975-80 (2005)<br />

PC 03<br />

Comparative analysis of transcriptional activities of<br />

heterologous promoters in A. friuliensis<br />

N. Wagner *1 , R. Biener 1 , D. Schwartz 1<br />

1 Fakultät Angwandte Naturwissenschaften, Hochschule Esslingen, Esslingen,<br />

Germany<br />

The rare actinomycete Actinoplanes friuliensis produces the lipopeptide<br />

antibiotic friulimicin. The complete friulimicin gene cluster consisting of 24<br />

open reading frames was characterised by sequence analysis [1]. In the<br />

friulimicin gene cluster four regulatory genes (regA,regB,regC,regD) have been<br />

identified. RegA seems to act as a main activator for the majority of friulimicin<br />

biosynthetic genes [2]. Genetic approaches to improve antibiotic production in<br />

A. friuliensis by overexpression of selected biosynthetic genes such as regA are<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

hampered by a lack of suitable replicative plasmids. Therefore strong<br />

heterologous promoters are of particular importance.<br />

So far analysis of transcription activities of such promoters in A. friuliensis and<br />

other Actinoplanes strains has not been described. Therefore the activities of<br />

several constitutive antibiotic resistance gene promoters as well as of phage<br />

promoters were compared by applying a reporter system in A. friuliensis and S.<br />

lividans. For that purpose a transcriptional fusion of the respective promoter<br />

regions with the promoterless egfp (enhanced green fluorescent protein) gene of<br />

the promoter probe vector pIJ8660 was carried out. Relative fluorescence of<br />

cell-free crude extracts was quantified for determination of promoter strength.<br />

In or<strong>der</strong> to directly compare the strength of heterologous and homologous<br />

promoters in A. friuliensis, the transcription rate of the friulimicin biosynthetic<br />

gene pstA in the wildtype and un<strong>der</strong> the control of PermE-up in a formerly<br />

described mutant [1] was determined by real-time-RT-PCR. By these<br />

experiments it was shown that the transcription rate of pstA is higher un<strong>der</strong> the<br />

control of the pathway specific promoter than un<strong>der</strong> the control of the PermEup<br />

promoter.<br />

[1] Müller et al.(2007): Antimicrob. Agents Chemother.51: 1028-1037<br />

[2] Nolden et al. (2008): J. Biotechnol. submitted<br />

PC 04<br />

Characterisation of inhibitory substances produced by<br />

Pseudoalteromonas citrea DSM 8771 T and<br />

Pseudoalteromonas aurantia DSM 6057 T<br />

J.E. Rau *1 , U. Fischer 1<br />

1 Zentrum für Umweltforschung und nachhaltige Technologien (UFT) und<br />

Fachbereich Biologie/Chemie, Abteilung Marine Mikrobiologie, Universität<br />

Bremen, Bremen, Germany<br />

The genus Pseudoalteromonas contains numerous marine species synthesizing<br />

biologically active substances which act upon a variety of target organisms.<br />

This seems to be a unique characteristic for this genus and may greatly benefit<br />

Pseudoalteromonas cells in their competition for nutrients and colonization of<br />

habitats. Species of the genus Pseudoalteromonas are generally found in<br />

association with marine eukaryotes and display antibacterial, antiviral,<br />

antitumor, antifouling, agarolytic, bacteriolytic, haemolytic, and algicidal<br />

activities. In this study, the inhibitory compounds produced by<br />

Pseudoalteromonas citrea DSM 8771 T and Pseudoalteromonas aurantia DSM<br />

6057 T have been investigated by FPLC using SEC, AIEX, and CIEX.<br />

Biologically active compounds of high molecular weight exhibiting<br />

antibacterial activity upon different target organisms have been detected in<br />

these two Pseudoalteromonas strains. Furthermore, inhibitory substances of<br />

low molecular weight have been examined as well. The possible structure of<br />

the inhibitory compounds has been studied by testing their effects on different<br />

enzymes such as proteases, glucanases, and oxidoreductases concerning the<br />

intensity of inhibition on the used target organisms.<br />

PC 05<br />

Bacillus licheniformis DSM13 produces an antimicrobial<br />

substance that is based on a putative lantibiotic gene cluster<br />

J. Dischinger *1 , M. Josten 1 , H.G. Sahl 1 , G. Bierbaum 1<br />

1 Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),<br />

University of Bonn, Bonn, Germany<br />

Lantibiotics are gene encoded antimicrobial peptides that are produced by a<br />

variety of Gram-positive bacteria. The lantibiotic prepeptides are ribosomally<br />

synthesized, contain an N-terminal lea<strong>der</strong> and a C-terminal propeptide sequence<br />

and do not show any antibiotic activity. During maturation the prepeptides<br />

un<strong>der</strong>go extensive posttranslational modifications including the introduction of<br />

the unique amino acids lanthionine and methyllanthione as well as the<br />

proteolytic removal of the lea<strong>der</strong>. The structural gene of the prepeptide (lanA)<br />

as well as the other genes which are involved in lantibiotic biosynthesis,<br />

modification (lanM, lanP), regulation (lanR, lanK), export (lanT(P)) and<br />

immunity (lanEFG) are organized in biosynthetic gene clusters.<br />

The existence of a putative two-peptide lantibiotic gene cluster, encoding<br />

lichenicidin, in Bacillus licheniformis DSM13 was first postulated by<br />

McClerren et al. (2007). This gene cluster comprises two structural genes<br />

(licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10<br />

ORFs that show sequence similarities to proteins involved in lantibiotic<br />

production.<br />

We were able to detect an antimicrobial activity in the culture supernantant as<br />

well as in an isopropanol wash extract of the cell pellet of Bacillus<br />

licheniformis DSM13. In agar well diffusion assays these antimicrobial<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

substances exhibited an activity against Gram-positive bacteria but were<br />

inactive against Gram-negatives and Candida albicans.<br />

A mutant Bacillus licheniformis DSM13 which harbours an insertion in the<br />

modification enzyme licM1 is characterized by a loss of the activity of the<br />

isopropanol extract. This indicates that the antimicrobial activity of the<br />

isopropanol extract is based on the putative lichenicidin gene cluster.<br />

PC 06<br />

Characterization of an antimicrobial substance produced<br />

by Bacillus cereus ATCC14579<br />

S. Feger *1 , J. Dischinger 1 , G. Bierbaum 1<br />

1 Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),<br />

University of Bonn, Bonn, Germany<br />

Lantibiotics are antimicrobial peptides that belong to the group of class I<br />

bacteriocins. The lantibiotic prepeptides are gene encoded and are ribosomally<br />

synthesized by a variety of Gram-positive bacteria. The prepeptides consist of<br />

an N-terminal lea<strong>der</strong> and C-terminal propeptide sequence. During maturation<br />

extensive posttranslational modifications occur at the propeptide sequence,<br />

resulting in the introduction of the non-proteinogenic amino acids lanthionine<br />

and methyllanthionine, respectively. The genes encoding the prepeptide (lanA)<br />

and the modification enzymes (lanB, lanC) are usually located in a gene<br />

cluster, that also contains genes for processing, transport (lanT(P)), immunity<br />

(lanEFG) and regulation (lanRK).<br />

Newly sequenced bacterial genomes often contain novel uncharacterized<br />

lantibiotic genes and by in silico analysis we found a number of such genes in<br />

Bacillus cereus ATCC14579. These include 4 identical prepetides (lanA), a<br />

putative dehydration enzyme (lanB) and an ABC-transporter that could be<br />

involved in immunity (lanEFG). Until now, no genes for cyclisation, transport<br />

and regulation have been identified.<br />

In agar well diffusion assays of isopropanol wash extracts of Bacillus cereus<br />

cells we were able to detect an antimicrobial substance with significant activity<br />

against a broad range of Gram-positive bacteria, including bacilli, enterococci,<br />

streptococci, staphylococci (even MRSA), whereas no activity against Gramnegative<br />

and Candida albicans was observable. Furthermore, stability assays<br />

were performed to determine the influence of temperature, pH and organic<br />

solvents and revealed a stable activity un<strong>der</strong> all tested conditions.<br />

In future experiments we want to show the relation between antimicrobial<br />

activity and the lantibiotic genes by detection of lanthionine and further amino<br />

acid composition analysis.<br />

PC 07<br />

Glycopeptide resistance in the producer strain<br />

Amycolatopsis balhimycina<br />

H.J. Frasch *1 , T. Schäberle 1 , E. Stegmann 1 , W. Wohlleben 1<br />

1 Microbiology/ Biotechnology, Eberhard Karls University, Tübingen, Germany<br />

Glycopeptides are the antibiotics of choice for treatment of severe infections<br />

caused by gram-positive bacteria when first-line antimicrobial agents have<br />

failed. The target of glycopeptides is the D-alanine-D-alanine (D-Ala-D-Ala)<br />

terminus of the side chain of the growing cell wall thereby cell wall<br />

biosynthesis is inhibited.<br />

Glycopeptide resistance is best studied in glycopeptide resistant enterococci.<br />

The transposon Tn1546 encodes for VanHAX, VanRS and VanY.<br />

VanHAX represents the essential enzymes for the replacement of the<br />

endstanding D-Ala with D-Lac in the peptidoglycan. VanRS is a bacterial twocomponent<br />

system which controls the expression of the vanHAX genes. VanY<br />

is a membrane associated D,D-carboxypeptidase contributing to high-level<br />

resistance.<br />

The strain Amycolatopsis balhimycina produces the vanyomycin-type antibiotic<br />

balhimycin. Several putative resistance genes were found in the balhimycin<br />

biosynthesis gene cluster, along with vanY and vanRS.<br />

A deletion mutant of vanR could be obtained but showed no phenotype<br />

concerning antibiotic production and resistance.<br />

Biochemical assays revealed that VanY is a carboxypeptidase, which can<br />

increase the resistance level in the presence of the vanHAX genes.<br />

In A. balhimycina the vanHAX genes are located outside the balhimycin<br />

biosynthesis cluster. RT-PCR analysis revealed that the vanHAX-genes are<br />

expressed as an operon. The expression of the A. balhimycina vanHAX genes in<br />

the glycopeptide sensitive strain S. coelicolor Müller un<strong>der</strong> the constitutive<br />

promoter permE* enabled the recombinant S. coelicolor Müller (vanHAX) to<br />

grow in the presence of balhimycin confirming the involvement of these genes<br />

in vancomycin resistance.<br />

Cell wall analysis of different mutants as well as cultivation of A. balhimycina<br />

un<strong>der</strong> different growth conditions indicated that a resistant cell wall is formed<br />

independently of balhimycin production.<br />

97

98<br />

PD 01<br />

Synthesis of aromatic beta-amino acids using novel cyclic<br />

amidases<br />

U. Bretschnei<strong>der</strong> *1 , C. Syldatk 1 , J. Rudat 1<br />

1 Institut für Bio- und Lebensmitteltechnik, Lehrstuhl für Technische Biologie,<br />

Universität Karlsruhe, Karlsruhe, Germany<br />

Optical active beta-amino acids are interesting building blocks for<br />

pharmaceuticals and fine chemicals. As their chemical synthesis is still<br />

inefficient and costly our approach is a modification of the well studied<br />

hydantoinase/carbamoylase system. It is industrially used to produce alphaamino<br />

acids from hydantoins. However, for beta-amino acid production<br />

dihydrouracils serve as substrates and a cyclic amidase with hydrolytic activity<br />

for these six-ring systems has to be applied.<br />

In a first step several aromatic dihydrouracils were prepared chemically from<br />

bulk chemicals. In whole cell biotransformation experiments with wild type<br />

strains showing cyclic amidase activity, these substrates were hydrolyzed to<br />

their corresponding N-carbamoyl beta-amino acids. A screening for<br />

carbamoylases able to catalyze the follow-up reaction to beta-amino acids is<br />

still in progress. Finally a whole cell biocatalytic system using the cyclic<br />

amidase/carbamoylase system for the efficient conversion of aromatic<br />

dihydrouracils to highly enantiopure aromatic beta-amino acids has to be<br />

established.<br />

PD 02<br />

New routes for the enantioselective synthesis of aromatic<br />

beta-amino acids<br />

J. Rudat *1 , U. Bretschnei<strong>der</strong> 1 , B. Brucher 1 , C. Syldatk 1<br />

1 Institut für Bio- und Lebensmitteltechnik, Bereich 2: Technische Biologie,<br />

Universität Karlsruhe (TH), Karlsruhe, Germany<br />

Beta-amino acids have unique pharmacological properties and their utility as<br />

building blocks of peptidomimetics, pharmaceutically important compounds<br />

and natural products is of growing interest.<br />

Several enzymatic methods have been developed for the preparation of various<br />

beta-amino acids, most of which exploit hydrolytic enzymes for the kinetic<br />

resolution of racemic mixtures, especially using acylases to stereoselectively<br />

cleave racemic N-acylated beta-amino acids.<br />

We are checking aromatically substituted dihydropyrimidines as novel<br />

substrates for cyclic amidases which have been shown to often exhibit some<br />

dihydropyrimidinase activity. Using whole cell biotransformations we obtained<br />

complete hydrolysis to the corresponding N-carbamoyl beta-amino acid with<br />

several strains with previously proven amidase activity for aromatically<br />

substituted hydantoins. We are now screening for certain carbamoylases to<br />

catalyse the follow-up hydrolysis of these compounds to beta-amino acids.<br />

In a second approach, we took the aromatic beta-amino acid of interest as<br />

screening substance, leading to the isolation of several bacterial strains with the<br />

ability to metabolise these unusual substrates as sole source of nitrogen by<br />

transferring the amino group to 2-oxoglutarate. First results indicate that this<br />

reaction is reversible which might lead to an additional route to aromatic betaamino<br />

acids using the corresponding beta-keto acid as substrate. We now<br />

started to purify one of these transaminases to find out whether it is a novel<br />

specific beta-transaminase or an alpha-transaminase also accepting certain betaamino<br />

acids as alternative substrates.<br />

PD 03<br />

Screening for novel aminotransferases for the production of<br />

enantiopure aromatic β-amino acids<br />

B. Brucher *1 , J. Rudat 1 , C. Syldatk 1<br />

1 Institut f. Bio- und Lebensmitteltechnik/ Bereich II: Technische Biologie,<br />

Universität Karlsruhe (TH), Karlsruhe, Germany<br />

There has been an increasing interest in β-amino acids in recent years due to the<br />

relative stability of their peptides [1] and their interesting pharmacological<br />

properties [2]. There is currently no single best method for the preparation of<br />

enantiomerically pure β-amino acids.<br />

Aminotransferases are widely applied in the synthesis of unnatural amino acids<br />

because of their high enantioselectivity and broad substrate specificity [3].<br />

In or<strong>der</strong> to find an efficient aminotransferase able to convert a 3-Oxo-3-arylpropionic<br />

acid to the corresponding β-amino acid we performed a selective<br />

enrichment culture with an aromatic β-amino acid as the sole nitrogen source.<br />

Several new strains possessing aminotransferase activity could be isolated. 16S<br />

rRNA sequencing showed that they belong to the genera Staphylococcus,<br />

Sphingomonas, Variovorax and Burkhol<strong>der</strong>ia. The aminotransferases are<br />

pyridoxal-5’-phosphate dependent and able to use pyruvate as well as α-ketoglutaric<br />

acid as amino acceptors.<br />

[1] Seebach D, Matthews JL (1997), Chem. Commun.: 21, 2015-2022.<br />

[2] Liljeblad A, Kanerva LT (2006), Tetrahedron: 25, 5831-5854.<br />

[3] Hwang BY, Cho BK, Yun H (2005), J. Mol. Catal. B- Enzymatic: 37, 47–<br />

55.<br />

PD 04<br />

An Asp-His catalytic dyad is the general acid/base catalyst<br />

of bacterial cell wall recycling β-N-acetylglucosaminidases<br />

S. Litzinger *1 , S. Fischer 1 , K. Die<strong>der</strong>ichs 1 , W. Welte 1 , C. Mayer 1<br />

1 Department of Biology, University of Konstanz, Konstanz, Germany<br />

β-N-acetylglucosaminidases like NagZ of E. coli are involved in the bacterial<br />

cell wall recycling by cleaving the glycosidic bond between Nacetylglucosamine<br />

(GlcNAc) and N-acetylmuramic acid (MurNAc) of<br />

peptidoglycan fragments. In contrast to NagZ, the β-N-acetylglucosaminidase<br />

3A of Bacillus subtilis (BsNag3A) is secreted and consists of two domains, a<br />

catalytic and a second domain of unknown function. Here we present the<br />

crystal structure of BsNag3A alone and with a competitive inhibitor bound to<br />

the active site (1.4 and 1.7 Å resolution, respectively). The first structure of a<br />

two-domain β-N-acetylglucosaminidase of family 3 glycosidases revealed an<br />

Asp-His dyad in the active site which is conserved in β-Nacetylglucosaminidases<br />

but absent in β-glucosidases of family 3. Structure and<br />

kinetic analyses of BsNag3A provide evidence for a mechanism in which<br />

His234 presumably acts as general acid/base catalyst, whereas Asp232<br />

coordinates His234 for proper function. The dyad is located on the N-terminal<br />

(β/α)8-barrel domain and His234 takes over the position of a conserved<br />

glutamate, which is the acid/base catalyst in β-glucosidases of family 3.<br />

Replacement of His234 or Asp232 with glycine reduces the rate of hydrolysis<br />

and ren<strong>der</strong>s the reaction invariant with pH in the range of 6-9. Furthermore,<br />

accumulation of the glycosyl-enzyme intermediate was identified by<br />

electrospray mass spectrometry in the His234Gly mutant. This is the first<br />

evidence of an Asp-His dyad involved in hydrolysis of β-glycosidic bonds<br />

which may function similar to the catalytic triad of serine proteases.<br />

PD 05<br />

DNA protection in starved bacteria by Dps and Dps-like<br />

proteins<br />

J. Ullmann *1 , B. Hernandez-Alvarez 1 , K. Zeth 1<br />

1<br />

Department Protein Evolution, Max Planck Institute for Developmental<br />

Biology, Tübingen, Germany<br />

Dps (DNA-binding protein from starved bacteria) and Dps-like proteins play an<br />

important role during starvation in bacteria via protecting the DNA against<br />

oxidative damage. The protection is carried out by iron uptake and proposed<br />

unspecific binding of DNA (in vitro binding is already shown). The<br />

dodecameric cave like structure of the proteins leads to the possibility to take<br />

up the iron ions into oligomeric ferritin shells as Fe 2+ , oxidized therein to Fe 3+<br />

which results in magnetite structures. The other purpose of the dodecameric<br />

structure is the DNA-binding which is proposed at the N-terminus Therefore<br />

the proteins are unregulated to 2% of the whole cell proteins during starvation<br />

phase.<br />

To further investigate DNA-binding we now accomplish EMSA<br />

(electrophoretic mobility shift assay) on agarose and polyacrylamide gels and<br />

try cocrystallisation with several DNA sequences. We also perform electron<br />

microscopy experiments to show the DNA-binding.<br />

PD 06<br />

Carbon and nitrogen isotope fractionation associated with<br />

bacterial hydrolysis of the herbicide atrazine<br />

A.H. Meyer *1 , M. Elsner 1<br />

1<br />

Institut für Grundwasserökologie, Helmholtz Zentrum München, Neuherberg,<br />

Germany<br />

Even after legislative prohibition in 1991 by the European Union, the pesticide<br />

atrazine and its metabolites are still frequently detected in surface and ground<br />

water exceeding the permitted drinking water concentration limit of 0,1 µg/L.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

However, despite much recent research on atrazine, its risk assessment in the<br />

environment is still a major challenge, because of the difficulty of establishing<br />

mass balances in the subsurface. To receive a better insight into the fate of<br />

atrazine in the environment we aimed at developing compound-specific stable<br />

isotope analysis (CSIA) for atrazine, a method that has become highly valuable<br />

for assessing degradation and initial pathways of other organic contaminants in<br />

subsurface environments. In such assessments, it is important to determine<br />

robust isotope fractionation factors beforehand. In ongoing work, we therefore<br />

measure carbon and nitrogen isotope fractionation patterns associated with biotransformation<br />

reactions of atrazine, to trace different degradation pathways. C<br />

and N isotope fractionation factors were determined in resting cell experiments<br />

for Pseudomonas sp. ADP, Chelatobacter heintzii and Arthrobacter aurescens<br />

TC1, strains that hydrolyse atrazine in the initial transformation reaction. First<br />

results are discussed with respect to the potential to use stable isotope<br />

fractionation for assessment of atrazine biodegradation occurring by different<br />

degradation-pathways.<br />

PE 01<br />

Toxicity of methionine-S-sulfoximine and its impact on<br />

ammonia synthesis in cyanobacteria<br />

F. Schlink *1<br />

1 Mikroorganismische Interaktionen, Uni Tübingen, Tübingen, Germany<br />

Ammonia is the most important form of combined nitrogen. All available forms<br />

of nitrogen (N2 NO3, urea…) are first transformed to ammonia before <strong>bei</strong>ng<br />

assimilated through incorporation into the carbon skelleton of 2-oxoglutarate<br />

via the GS-GOGAT-pathway. The essential enzyme of these reactions is the<br />

glutamine synthetase (GS), which catalyzes the reaction of glutamate and<br />

ammonia to glutamine. The GS can be inhibited by the herbicide methionine-<br />

S-sulfoximine (MSX), which is a structural analogue of glutamate, the substrate<br />

of GS. This inhibition is not reversible. The complete inhibition of the GS<br />

should first result in an intracellular accumulation of ammonia.<br />

One of our goals is to find out, whether the toxic effect of MSX to the cells in<br />

due to the accumulation of toxic concentrations of ammonia. Furthermore we<br />

are interested in the regulation of ammonia formation un<strong>der</strong> conditions of GS<br />

inhibition. Therefore, we work with a set of nitrogen regulatory mutants like the<br />

PII-mutant or PII modification mutants. These mutants are treated with MSX in<br />

different light qualities and some physiological parameters are determined, like<br />

the GS-activity, ammonia-, nitrite- and nitrate concentration in the medium.<br />

These tests are performed with different strains of Cyanobacteria, like the<br />

unicellular strains Synechococcus elongatus and Synechocystis 6803 and the<br />

filamentous Anabaena 7120.<br />

Preliminary data suggests that the effects on the viability are directly caused by<br />

MSX and not by intoxication with high concentrations of ammonia.<br />

PE 02<br />

A highly conserved gene cluster of unkown function is<br />

involved in PHA accumulation of Synechocystis sp.PCC<br />

6803<br />

M. Schlebusch *1 , K. Forchhammer 1<br />

1 Mikrobiologie / Organismische Interaktionen, Universität Tübingen, Tübingen,<br />

Germany<br />

Nitrogen frequently is a limiting nutrient in natural habitats. Therefore,<br />

cyanobacteria as well as other autotrophic organisms have developed multiple<br />

strategies to adapt to nitrogen deficiency. Transcriptomic analyses of the strain<br />

Synechococcus elongatus PCC 7942 un<strong>der</strong> nitrogen-deficient conditions<br />

revealed a highly induced gene cluster. The genes occur in the or<strong>der</strong> (1)<br />

hypothetical protein, (2) nitrilase, (3) radical S-adenosyl methionine (SAM)<br />

superfamily member, (4) acetyltransferase of the GNAT family, (5) AIR<br />

synthase. Upstream of the gene cluster lies a predicted flavoprotein which is<br />

located on the opposite strand. The flavoprotein and the gene cluster share a<br />

common binding site for the general nitrogen transcript factor NtcA.<br />

Bioinformatic analyses of the operon structure show, that homolog gene<br />

clusters occur in some cyanobacteria, in several beta- and gamma-<br />

proteobacteria as well as in some gram-positives.<br />

Knockout mutants in Synechococcus elongatus PCC 7942 were created for<br />

every gene of the operon, however, the mutants show no distinct phenotype. By<br />

contrast, in Synechocystis sp. PCC 6803 a knock out mutant of the first gene is<br />

unable to accumulate polyhydroxyalkanotes (PHA), a carbon and energy<br />

storage compound, which is not present in Synechococcus. We will present an<br />

extensive characterization of this mutant with respect to the accumulation of the<br />

PHA-granules, the expression of relevant mRNA’s, as well as several metabolic<br />

intermediates.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PE 04<br />

Chloroplast gene expression in Chlamydomonas reinhardtii:<br />

factors and mechanisms<br />

A. Bohne 1 , C. Schwarz 1 , F. Wang 1 , J. Nickelsen *1<br />

1 AG Molekulare Pflanzenwissenschaften, LMU München, Planegg, Germany<br />

The biogenesis of chloroplasts depends on a huge variety of different nucleusencoded<br />

factors which are imported by the organelle wherein they control<br />

almost all steps of plastid gene expression including transcription, RNA<br />

metabolism, translation as well as assembly of multisubunit-complexes. In the<br />

green alga Chlamydomonas reinhardtii, chloroplast synthesis of the D2 protein<br />

(PsbD), which is the rate-determining subunit for assembly of photosystem II,<br />

depends on a high molecular weight complex containing at least the RNA<br />

stability factor Nac2 and the translational regulator RBP40. This indicates that<br />

processes of 5´UTR-mediated RNA stabilization and translation initiation are<br />

tightly coupled in C. reinhardtii.<br />

Expression of the related psbA gene is also mediated by a high molecular<br />

weight complex. This complex contains at least one subunit of the plastid<br />

pyruvate dehydrogenase suggesting a possible relationship between chloroplast<br />

protein and lipid synthesis.<br />

PE 05<br />

Biochemical regulation of the<br />

Glucosylglycerolphosphatesynthase from Synechocystis sp.<br />

PCC 6803<br />

J. Novak *1 , M. Stirnberg 1 , R. Krämer 1 , K. Marin 1<br />

1 Institute for Biochemistry, University of Cologne, Cologne, Germany<br />

Upon high salt concentrations the mo<strong>der</strong>ately halotolerant cyanobacterium<br />

Synechocystis sp. PCC 6803 accumulates the compatible solute<br />

glucosylglycerol (GG) to balance the internal and external osmolalities. The<br />

activity of the key enzyme in GG synthesis, the<br />

glucosylglycerolphosphatesynthase (GgpS), is biochemically regulated in<br />

dependency of the external salt level. The mechanisms that un<strong>der</strong>lie regulation<br />

during the rapid response and steady-state acclimatization are by now unknown<br />

and the interaction with an inhibitory factor is assumed.<br />

During the last ten years several approaches were carried out to identify a<br />

putative interaction partner. The inhibitory factor is highly stable and triggers a<br />

conformational change of GgpS structure during limited proteolysis assays. We<br />

could show by SPINE analysis and protease treatment of the cell extract<br />

harboring the inhibitory factor that no protein-protein interaction or<br />

posttranslational modification is involved in regulation of GgpS activity.<br />

Focussing on the identification of a low molecular weight compound HPLC<br />

analysis yielded no clear results. By a systematic approach using an in vitro<br />

assay system we could finally identify the inhibitory factor. The interaction of<br />

the GgpS and its inhibitor is dose-dependently diminished at increasing NaCl<br />

concentrations. In conclusion we present a model for adjustment of the internal<br />

GG pool by the salt-dependent interaction of the GgpS enzyme with the newlyidentified<br />

inhibitor.<br />

PE 06<br />

Functional analysis of Raa4, a chloroplast splicing factor<br />

from Chlamydomonas reinhardtii<br />

A. Mishra *1 , S. Glanz 1 , K. Schmitt 1 , M. Tietze 1 , U. Kück 1<br />

1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr University, Bochum,<br />

Germany<br />

The expression of chloroplast genome is highly dependent on the large number<br />

of nucleus-encoded factors. These nuclear-encoded factors are believed to<br />

promote the maturation of chloroplast precursor RNAs and could be part of a<br />

putative chloroplast spliceosome. Trans-splicing of the psaA pre-mRNA of<br />

Chlamydomonas reinhardtii is most probably promoted by a multi subunit<br />

complex. At least 14 nucleus-encoded factors and a small chloroplast-encoded<br />

RNA (tscA) are required for the maturation of the psaA mRNA. We are<br />

interested in identification and functional characterization of components from<br />

the putative chloroplast spliceosome. In this regard by using forward genetic<br />

approach, we have generated trans-splicing mutants and rescued one of these<br />

mutants by genomic complementation. The affected gene, called Raa4, for<br />

RNA maturation of psaA 4, encodes a putative aminoacyl-tRNA synthetase.<br />

Here, we present the functional analysis of this protein by electrophoretic<br />

mobility shift assay using different organellar group II intron domains. For<br />

further characterization, detection of this protein as a part of the large molecular<br />

weight complex in the chloroplast has to be done by using sucrose density<br />

gradient centrifugation. In or<strong>der</strong> to investigate specific binding of the other<br />

chloroplast proteins, co-immunoprecipitation procedures will be used.<br />

99

100<br />

PE 07<br />

The novel anaerobically induced Ferredoxin of<br />

Chlamydomonas reinhardtii<br />

J. Jacobs *1 , C. Lambertz 1 , A. Hemschemeier 1 , T. Happe 1<br />

1 Plant Biochemistry/ Photobiotechnology, Ruhr University Bochum, Bochum,<br />

Germany<br />

* both authors contributed equally to this work<br />

The unicellular green alga Chlamydomonas reinhardtii has a complex<br />

anaerobic metabolism that is marked by the production of hydrogen gas and<br />

formate [1], catalysed by an [FeFe]-hydrogenase coupled to photosynthesis by<br />

the natural electron donor ferredoxin [2, 3] and by the bacterial-type pyruvate<br />

formate-lyase (PFL) [4,5], respectively.<br />

In addition to the gene encoding for the photosynthetic ferredoxin PetF, five<br />

further ferredoxin encoding genes can be found in C. reinhardtii. The analysis<br />

of the transcription pattern of these genes upon sulphur deprivation revealed a<br />

high upregulation of two of them. The strong increase of the fdx5 and fdx2<br />

transcript level indicates a vitale role of these novel proteins in the special<br />

physiology of anaerobic C. reinhardtii cultures.<br />

This study shows the first biochemical and genetic characterisation of Fdx5,<br />

which has been heterologously expressed in Escherichia coli and purified via<br />

StrepTag-chromatography. The purified protein was used for the production of<br />

antibodies, which allowed the detection of Fdx5 on protein level and the<br />

homologous purification of the native protein. Furthermore,the regulation of the<br />

fdx5 gene in response to O2 deprivation is analyzed by reporter gene assays.<br />

[1] T. Happe, A. Hemschemeier, M. Winkler and A. Kaminski, Trends Plant<br />

Sci, 2002, 7(6), 246-250<br />

[2] T. Happe, B. Mosler and J.D. Naber, Eur J Biochem , 1994, 222, 769-775<br />

[3] L. Florin, A. Tsokoglou and T. Happe, J. Biol. Chem., 1995, 276, 6125-<br />

6132<br />

[4] A. Hemschemeier and T. Happe, Biochem Soc Trans., 2005, 33, 39-41<br />

[5] A. Hemschemeier, J.Jacobs and T. Happe, Eukaryot. Cell, 2008, 7, 518-526<br />

PE 08<br />

The relationship between cyanobacteria and aerobic<br />

heterotrophic bacteria: Enemies and friends<br />

K. Kohls *1 , M. Schacht 2 , K. Palinska 2 , R.M.M. Abed 3<br />

1<br />

Max -Planck- Institut für Marine Mikrobiologie, Bremen, Bremen, Germany<br />

2<br />

Institute for Chemistry and Biology of the Marine Environment, Carl von<br />

Ossietzky University of Oldenburg, Oldenburg, Germany<br />

3<br />

College of Science-Biology Department, Sultan Qaboos University, Muscat,<br />

Oman<br />

Obtaining cyanobacterial axenic cultures has been (and still is) a major<br />

challenge due to associated aerobic heterotrophic bacteria (AHB). The identity<br />

of AHB and their relationship to cyanobacteria were investigated in unialgal<br />

cyanobacterial cultures isolated from hypersaline microbial mats. Denaturing<br />

gradient gel electrophoresis (DGGE) showed that most of the attached AHB<br />

belonged to the Bacteriodetes group but few were related to Alpha-,<br />

Gammaproteobacteria and Chloroflexus-like bacteria. Different cyanobacteria<br />

were found to be associated with different AHB, although some bacterial<br />

populations were in common. Cultivation of an axenic culture of Microcoleus<br />

chthonoplastes (PCC 7420) with and without AHB showed that the growth of<br />

the cyanobacterium was either stimulated or completely inhibited depending<br />

on the type of AHB added. AHB might stimulate the growth of cyanobacteria<br />

by using photosynthetically produced oxygen to consume cyanobacterial<br />

exudates or/and by providing cyanobacteria with necessary vitamins and other<br />

growth factors. Some of the substrates produced by AHB might inhibit the<br />

growth of certain cyanobacteria, however the identity of these substance is<br />

unknown. Flexibacter-related species were shown, using fluorescent in situ<br />

hybridization (FISH), to lyse cyanobacterial filaments and to grow on their<br />

fragments. In conclusion, the close association between cyanobacteria and<br />

AHB is highly specific and depends on the bacterial species as well as on the<br />

exchange of nutrients and organics. Further investigations will focus on the<br />

identity of these compounds in or<strong>der</strong> to gain a better un<strong>der</strong>standing of these<br />

relationships.<br />

PF 01<br />

Mutation in the amiC cluster of the filamentous<br />

cyanobacterium Nostoc punctiforme leads to filament<br />

dystrophy and lack of cell differentiation<br />

J. Lehner *1 , K. Forchhammer 1 , I. Maldener 1<br />

1 Mikrobiologie/Organismische Interaktionen, EK Universität Tübingen,<br />

Tübingen, Germany<br />

Nostoc is a complex filamentous cyanobacterium, which is able to form<br />

differentiated cells: heterocysts for N2 fixation, stress resistant akinetes and<br />

small mobile filaments, called hormogonia. It can be assumed that during cell<br />

differentiation massive changes of the cell wall structure take place. Recent<br />

studies, based on micro-array analysis, revealed that more than 120 genes<br />

involved in cell envelope formation are differentially expressed in the different<br />

cell types compared to vegetative cells.<br />

One of the genes, NpF1846, is a homolog to amiC, encoding N-acetylmuramyl-<br />

L-amidase, which functions in murein degradation in gram-negative bacteria. It<br />

has been shown that in gram- bacteria ami genes are necessary for the septum<br />

development and cell division. The amiC gene of N. punctiforme was<br />

inactivated by insertion of a neomycin cassette. Fully segregated mutants were<br />

viable, however, grew very slowly compared to the wild type and only in<br />

medium containing a combined nitrogen source. The mutant cells do not form<br />

filaments, but groups of cells in which diverse division planes lead to irregular<br />

aggregates. These cell clumps are not able to form neither heterocysts nor<br />

hormogonia. Fluorescence images and electron micrographs of ultra thin<br />

sections of the cells will be presented. The phenotype of the mutant suggests<br />

that amiC has an important function in filament morphogenesis in Nostoc,<br />

possibly <strong>bei</strong>ng involved in regulation of division plane formation.<br />

PF 02<br />

Assembly of the DNA uptake machinery in competent<br />

Bacillus subtilis cells<br />

M. Kaufenstein *1 , A.C. Zimmermann 1 , P.L. Graumann 1<br />

1 Institute of Biology, Microbiology, University of Freiburg, Freiburg, Germany<br />

Competent Bacillus subtilis cells are able to take up exogenous DNA and<br />

un<strong>der</strong>go transformation. Competence is induced by secreted peptide factors,<br />

which trigger a sophistical regulatory system. This system leads to the synthesis<br />

of the ComK master transcription regulator, which in turn activates all<br />

necessary competence proteins.<br />

Uptake of DNA requires the product of the ComG operon, which appears to<br />

form a pilus-like structure. This multiprotein assembly seems to be required to<br />

channel DNA through the cell wall to ComEA. ComEA acts as a receptor for<br />

double-stranded DNA and brings it to ComEC, which forms an aqueous<br />

channel to transport only single-stranded DNA across the membrane, while the<br />

other strand is degraded. Overall, about 15 proteins are involved in DNA<br />

uptake.<br />

Interestingly, fluorescence microscopical experiments have shown that several<br />

uptake proteins localized at a single cell pol, except for the protein ComEA,<br />

which is arranged throughout the whole membrane.<br />

We are interested in the question of how the DNA uptake machinery assembles<br />

at a single cell pole, and retains its localization. We study the dynamics of these<br />

proteins by time lapse microscopy and FRAP experiments. We show that<br />

several components of the machinery show different localization patterns and<br />

highly dynamic movements within the cell. Our data suggest that the DNA<br />

uptake machinery assembles and disassembles in a highly dynamic manner, and<br />

may consist of many short lived interactions rather than of a single static<br />

complex.<br />

We also investigate how the known proteins interact together and identify new<br />

protein-protein-interactions responsible for forming multicomplexes by FRET<br />

experiments, Blue Native Gel and TAP-tag experiments.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PF 03<br />

Calcineurin is involved in development of Dictyostelium<br />

discoideum<br />

S. Thewes *1 , K. Böckeler 2 , B. Weissenmayer 3 , R. Mutzel 1<br />

1 Institute for Biology – Microbiology, Freie Universität Berlin, Berlin,<br />

Germany<br />

2 Protein Phosphorylation Laboratory, Cancer Research UK London Research<br />

Institute, London, United Kingdom<br />

3 Conway Institute, University College Dublin, Dublin, Ireland<br />

Calcineurin is a protein phosphatase conserved from yeast to man regulating a<br />

plethora of cellular processes. It is composed of one catalytic (calcineurin A;<br />

CNA) and one regulatory (calcineurin B; CNB) subunit. Recently we have<br />

shown that silencing of calcineurin B in the social amoeba Dictyostelium<br />

discoideum results in delayed development, ectopic tip formation in<br />

culminating fruiting bodies and aberrant stalk cell morphology. Using a RNAinterference<br />

approach we show here that silencing of the catalytic subunit of<br />

calcineurin (CNA) in D. discoideum results in similar phenotypes compared<br />

with CNB-RNAi mutants. In CNA-RNAi mutants almost no expression of the<br />

calcineurin A mRNA and protein could be detected whereas expression of the<br />

calcineurin B protein was not affected. CNA-RNAi mutants show ectopic tip<br />

formation in culminating fruiting bodies and shorter stalks compared to the<br />

wildtype. These results show that the phosphatase activity of calcineurin is<br />

required for cell differentiation and morphogenesis of D. discoideum and that<br />

both subunits have to act together to achieve full activity.<br />

PF 04<br />

Characterisation of EspC, an histidine protein kinase in<br />

Myxococcus xanthus<br />

A. Schramm *1 , B. Lee 1 , P. Higgs 1<br />

1 Department of Ecophysiology, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

Upon starvation, Myxoccoccus xanthus starts a complex multicellular<br />

developmental program in which 10 5 cells migrate into mounds and then<br />

differentiate into environmentally resistant spores. Un<strong>der</strong> laboratory conditions,<br />

this process takes at least 72 hours and is spatially and temporally coordinated<br />

by a series of intra- and extracellular signals. Previous research has<br />

demonstrated that progression through this complex developmental program is<br />

coordinated by several histidine protein kinases that, when individually<br />

mutated, cause the cells to aggregate and sporulate earlier, forming smaller and<br />

more disorganized fruiting bodies.<br />

espA and espC, both encode orphan hybrid histdine protein kinases, and when<br />

deleted, share the same early developmental phenotype. Interestingly, the<br />

developmental phenotype of an espA espC double mutant is indistinguishable<br />

from the single mutants, suggesting that these two kinases may lie in the same<br />

signaling pathway.<br />

We have previously determined that in EspA the conserved phosphoaccepting<br />

histidine and aspartic acid residues in the kinase as well as the receiver<br />

domains, respectively, are essential for EspA function. Here we show that the<br />

conserved histidine residue is not necessary for EspC function, whereas the<br />

conserved aspartic acid residue in the receiver domain is essential for its<br />

function. We are currently exploring how EspA and EspC function together via<br />

phosphotransfer and protein interaction assays.<br />

PG 01<br />

Repeat induced point mutation and recombination in<br />

Aspergillus niger and Penicillium chrysogenum<br />

F. Kempken *1 , I. Braumann 1 , M. van den Berg 2<br />

1 Botanisches Institut, Christian-Albrechts-Universität zu Kiel, Kiel, Germany<br />

2 Anti-Infectives, DSM, Delft, Netherlands<br />

Transposons are mobile and mostly also repetitive sequences, which are found<br />

in all eukaryotic genomes. We have analyzed the transposon content in two<br />

fungal genomes, Aspergillus niger and Penicillium chryosogenum. In both<br />

fungi there is also strong evidence for a Repeat Induced Point mutation (RIP)like<br />

mechanism. We present data for RIP in multiple copies of the A. niger<br />

retrotransposon AniTa1 and the P. chrysogenum class II transposon PeTra2.<br />

RIP was identified in both elements, representing the first observation of RIP in<br />

these two fungi. For PeTra2 all sequences investigated seem to be effected by a<br />

mo<strong>der</strong>ate type of RIP in a varying extend. AniTa1 copies seem to be unRIPed<br />

with the exception of two copies in which 20% of all nucleotides have been<br />

altered due to RIP. These findings suggest a rather selective but very strong<br />

type of RIP in A. niger.<br />

Transposons are usually present in multiple copies in their hosts’ genomes.<br />

Recombination between two transposon copies can result in chromosomal<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

101<br />

rearrangements. Here, we describe a recombination event between two copies<br />

of the retrotransposon ANiTa1 within the genome of the fungus Aspergillus<br />

niger (strain CBS513.88). The observed chromosomal rearrangement appears<br />

to be strain-specific, as the corresponding genomic region in another strain,<br />

ATCC1015, shows a different organization. The striking genomic differences<br />

in ANiTa1 copy distribution leading to differences in the chromosomal structure<br />

between the two strains, ATTC1015 and CBS513.88, suggest that the activity<br />

of transposons may profoundly affect the evolution of different fungal strains.<br />

PG 02<br />

The complete genome sequence of the B12-producing<br />

Escherichia Blattae strain DSZM4481 isolated from a<br />

cockroach<br />

E. Brzuszkiewicz *1 , T. Waschkowitz 2 , J. Schuldes 1 , G. Gottschalk 1 , R. Daniel 2<br />

1 Goettingen Genomics Laboratory, Institute of Microbiology and Genetics,<br />

Georg-August University of Goettingen, Göttingen, Germany<br />

2 Department of Genomic and Applied Microbiology, Institute of Microbiology<br />

and Genetics, Georg-August-University of Goettingen, Goettingen, Germany<br />

3 Goettingen Genomics Laboratory, Institute of Microbiology and Genetics,<br />

Georg-August University of Goettingen, Goettingen, Germany<br />

The enteric bacterium Escherichia blattae DSMZ4481 has been isolated from<br />

the hind-gut of a cockroach. In contrast to its relative E. coli and other enteric<br />

bacteria, E. blattae is non-pathogenic for humans (safety level S1). In addition,<br />

we have shown that all molecular tools developed for E. coli such as cloning<br />

and expression systems are functional in E. blattae. In contrast to E. coli, E.<br />

blattae is able to synthesize B12 de novo. Thus, this organism is an interesting<br />

alternative to E. coli with respect to biotechnological applications.<br />

The complete genome sequence of E. blattae DSMZ4481 was determined and<br />

analyzed. The organism contains a single chromosome of 4,158,636 bp that<br />

encodes 3,947 ORFs. The G+C content of 56,51% is significantly higher than<br />

the one of E. coli (approximately 51%).<br />

In this report, we present the structure of the entire genome with special<br />

emphasis on genomic islands, inserted phages, and B12-dependent reactions. In<br />

or<strong>der</strong> to investigate the genetic organization, we compared the genome of E.<br />

blattae with the genomes of pathogenic and non-pathogenic enteric bacteria.<br />

One major difference of E. blattae to other enteric bacteria is the presence of 8<br />

rRNA operons. Comparative genomics revealed a mosaic structure of the E.<br />

blattae chromosome, indicating high genome plasticity and the occurrence of<br />

many horizontal gene transfer events.<br />

PG 03<br />

Adaptation of the Corynebacterium glutamicum membrane<br />

proteome to aromatic acids<br />

as alternative carbon sources<br />

U. Haußmann *1 , S.W. Qi 2 , D. Wolters 3 , M. Rögner 1 , S.J. Liu 2 , A. Poetsch 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

2 State Key Laboratory of Microbial Resources, Institute of Microbiology,<br />

Chinese Academy of Sciences, Beijing, China<br />

3 Analytical Chemistry, Ruhr-University Bochum, Bochum, Germany<br />

The ability of microorganisms to metabolize a variety of carbon sources is a<br />

key advantage during the constant competition for nutrition in natural<br />

environments, where aromatic compounds are found either as degradation<br />

intermediates of natural products (e.g. lignin) or industrial pollutants. Whereas<br />

the Corynebacterium glutamicum cytosolic proteomes have been characterized<br />

during growth on various aromatic compounds [1], only scarce information<br />

exists about the dynamic adaptation of the bacterial membrane protein<br />

composition in general and to aromatic carbon sources in particular.<br />

We are investigating the Corynebacterium glutamicum membrane proteome<br />

un<strong>der</strong> quantitative and qualitative aspects by applying gel-free and gel-based<br />

proteomics technologies. First, the adaptation of the membrane proteome to<br />

benzoate was analyzed using the gel-free MudPIT/SIMPLE technology [2],<br />

with a 15 N metabolically labeled internal standard [3] for relative protein<br />

quantification. During growth on benzoate co-expression of the two benzoate<br />

transporters BenE and BenK, changes in energy metabolism and a starvation<br />

response were observed compared to glucose-grown cells. Second, the<br />

AIEC/SDS-PAGE method [4] is applied to characterize the membrane<br />

proteome during growth on protocatechuate. This technology allows a<br />

combination of 15 N metabolic labeling and labeling with Cy fluorescent dyes<br />

for relative protein quantification.<br />

The comparison of the membrane proteome profiles of benzoate- and<br />

protocatechuate-grown cells will help to distinguish component-specific and<br />

more general adaptation processes.

102<br />

[1] Qi, SW et al. (2007) Proteomics 7:3775-3787<br />

[2] Fischer, F et al. (2006) Mol Cell Proteomics 5 (3):444-453<br />

[3] MacCoss, MJ et al. (2003) Anal Chem 75:6912-6921<br />

[4] Schlüsener, D et al. (2005) Proteomics 5:1317-1330<br />

PG 04<br />

Genome sequence of the polysaccharide-degrading,<br />

thermophilic anaerobe Spirochaeta thermophila<br />

A. Angelov *1 , S. Liebl 1 , H. Liesegang 2 , W. Liebl 1<br />

1 Department of Microbiology, Technische Universitaet Muenchen, Muenchen,<br />

Germany<br />

2 Institute of Microbiology and Genetics, Georg-August-Universitaet<br />

Goettingen, Goettingen, Germany<br />

The Spirochaetes form a distinct phylum within the phylogenetic tree of<br />

Bacteria. They have helically coiled cells, stain as Gram-negative and possess<br />

typical flagella, called axial filaments, which run lengthwise between the cell<br />

membrane and outer membrane. Although most of the species in the<br />

Spirochaetes phylum are free-living, genome sequences are available<br />

exclusively for members of the disease-causing genera Leptospira, Borrelia and<br />

Treponema. The genus Spirochaeta consists currently of 18 species, most of<br />

which are thermo- and alkaliphilic, anaerobic and are capable of degrading a<br />

great variety of polysaccharides. So far, no complete genome sequences are<br />

available for members of the genus. S. thermophila is the only extremely<br />

thermophilic representative of the genus (Topt = 65°C) and is able to use<br />

various sugar polymers as a sole carbon and energy source, e.g. starch,<br />

glycogen, pullulan, microcrystalline cellulose, lichenan, laminarin, chitin and<br />

xylan.<br />

We have determined the complete genome sequence of S. thermophila<br />

DSM6192 using a combination of shotgun sequencing (large insert fosmid and<br />

small insert plasmid library) and sequencing-by-synthesis (454 sequencing,<br />

Genome Sequencer FLX). The 2.47 Mb genome consists of a single<br />

chromosome. No extra-chromosomal elements could be detected. The<br />

determined GC-content of 61.9 deviates significantly from that of other<br />

Spirochaetes with available genome data, which have a GC-content ranging<br />

from 27.8 to 52.8, which possibly may be linked to the free-living and<br />

thermophilic lifestyle of S. thermophila. Initial ORF prediction and annotation<br />

led to the identification of a large number of genes encoding putative<br />

carbohydrate-active enzymes. By combining biochemical and in silico based<br />

methods we aim to investigate the molecular apparatus that performs the<br />

degradation and utilisation of complex carbohydrate polymers.<br />

PG 05<br />

Saccharomyces cerevisiae as a new screening host for largeinsert<br />

environmental libraries in function-based<br />

metagenomic analysis<br />

M. Taupp *1 , J. Stoepel 2 , J. Fuchs 1 , P. Hieter 2 , S.J. Hallam 1<br />

1 Department of Microbiology & Immunology, University of British Columbia,<br />

Vancouver, Canada<br />

2 Michael Smith Laboratories, Department of Medical Genetics, University of<br />

British Columbia, Vancouver, Canada<br />

Metagenomic analysis of the undiscovered microbial world comprises a<br />

virtually infinite and largely untapped pool of genetic diversity with wide<br />

ranging therapeutic and biotechnological potential. Function-based analysis of<br />

metagenomic libraries uncovered new enzymes and bioactive molecules with<br />

unique properties. However, screening efficiency is very low and hundreds of<br />

thousands of clones have to be screened in high-throughput assays. Screening<br />

efficiencies mainly depend on the applied screening host, which is in most<br />

cases E. coli. Besides several other prokaryotic screening hosts like<br />

Streptomyces, Pseudomonas, Rhizobium or Listeria, a eukaryotic host seems to<br />

be missing in or<strong>der</strong> to directly extract biological information from metagenomic<br />

libraries. Eukaryotic genes in metagenomic libraries are likely to be not<br />

discovered due to differences in bacterial and native expression systems, codon<br />

usage, missing chaperones, and posttranslational modifications. A host<br />

expression machinery more similar to a native one would be highly<br />

advantageous and Saccharomyces cerevisiae seems to be best suited regarding<br />

growth rates and stability and transformation efficiency of plasmids. To<br />

accomplish this, a pipeline for retrofitting existing E. coli fosmid libraries was<br />

established employing a constructed E. coli-Saccharomyces cerevisiae shuttle<br />

vector harbouring the conditional origin of replication oriR6K and the<br />

arabinose-inducible oriV as well as a single loxP-site for Cre/loxP-based<br />

retrofitting of existing metagenomic libraries. For a high-throughput retrofit, an<br />

in vivo recombinatorial system of large insert metagenomic libraries in the<br />

yeast host system was established applying an LEU2/URA3-γ-integration<br />

vector for counterselection. Both approaches open up the possibility to apply<br />

yeast in a wide range of metagenomic analysis.<br />

PG 06<br />

The cell wall and cell surface proteome of Rhodopirellula<br />

baltica SH1 T<br />

X.H. Cao *1 , B. Voigt 2 , K. Hempel 2 , D. Becher 2 , R. Sietmann 2 , F.O. Glöckner 3 ,<br />

R. Amann 3 , M. Hecker 2 , T. Schwe<strong>der</strong> 4<br />

1 Institute of Marine Biotechnology, EMAU Greifswald, Greifswald, Germany<br />

2 Institute for Microbiology, EMAU Greifswald, Greifswald, Germany<br />

3 Max Planck Institute for Marine Microbiology, Bremen & Jacobs University<br />

Bremen, Bremen, Germany<br />

4 Institute of Pharmacy, EMAU Greifswald, Greifswald, Germany<br />

Rhodopirellula baltica is a marine model of the fascinating and ubiquitous<br />

planctomycete phylum. These bacteria possess a proteinaceous cell wall<br />

consisting of unknown components and typical surface architectures including<br />

holdfasts, crateriforms, and appendage-like structures. We report here different<br />

proteomic approaches to reveal structural and physiological characteristics of<br />

cell wall and cell surface proteins. These approaches led to the identification of<br />

surface attached proteins which are mainly involved in the regulation of cell<br />

adhesion and in the degradation of sulfated compounds. Among the newly<br />

detected proteins were high molecular weight proteins involved in cell-cell<br />

attachment, including lipoproteins, fibrinogen-binding protein and the MAFp3<br />

aggregation factor. Interestingly, the identification of two sulfatases, an Nacetylglucosamine-6-phosphate<br />

deacetylase and a number of other<br />

carbohydrate degradation enzymes in these protein fractions support the<br />

hypothesis that R. baltica cells mineralize marine snow particles by an<br />

enzymatic system attached to their surface. Among the identified cell wall<br />

proteins, a planctomycete-typical YTV protein family displays an amino acid<br />

composition matching the previously chemically determined amino acid<br />

composition of the cell wall of R. baltica. The observation of YTV proteins in<br />

an SDS resistant cell wall protein fraction was confirmed by Westen blotting.<br />

Preliminary attempts were done to localize YTV proteins in R. baltica cells by<br />

transmission electron microscopy.<br />

PG 07<br />

Single-cell based analysis of the uncultivated giant rod<br />

Magnetobacterium bavaricum<br />

C. Jogler *1 , G. Wanner 2 , M. Niebler 1 , L. Wei 3 , M. Kube 4 , N. Petersen 5 , R.<br />

Amann 6 , R. Reinhardt 4 , D. Schüler 1<br />

1 Microbiology, LMU Munich, Munich, Germany<br />

2 Botany, LMU Munich, Munich, Germany<br />

3 Institute of Geology and Geophysics, University Beijing, Beijing, China<br />

4 Analytics and Computing, Max Planck Institut for Molecular Genetics, Berlin,<br />

Germany<br />

5 Department of Geo- and Environmental Science, LMU Munich, Munich,<br />

Germany<br />

6 Department of Molecular Ecology, Max Planck Institut for Marine<br />

Microbiology, Bremen, Germany<br />

The giant magnetotactic rod Magnetobacterium bavaricum that was previously<br />

discovered in sediments of the Lake Chiemsee (Germany) is unusual with<br />

respect to its deep-branching affiliation with the Nitrospira phylum, the up to<br />

1000 bullet-shaped magnetosome crystals, and its unique cell biology. Since<br />

all attempts to cultivate M. bavaricum failed so far, we relied on environmental<br />

samples to study the cell biology and the genetic basis of biomineralization.<br />

We developed a two step magnetic separation process, which allows the<br />

collection of M. bavaricum from environmental samples in sufficient<br />

homogeneity for comprehensive ultrastructural analysis. To investigate the<br />

genetic basis of biomineralization in M. bavaricum, we developed a two step<br />

whole genome amplification (WGA) process followed by pyrosequencing.<br />

Individual M. bavaricum cells were harvested via an Eppendorf<br />

micromanipulator and subsequently transferred un<strong>der</strong> microscopic control onto<br />

slides capable of thermal cycling. We obtained more than 2 µg DNA from 15-<br />

20 M. bavaricum cells per reaction. Currently the further characterization of the<br />

amplified DNA and its pyrosequencing is un<strong>der</strong> investigation.<br />

Beside genomic analysis, we investigated M. bavaricum microscopically and<br />

demonstrated the presence of multiple chains of magnetosomes with membrane<br />

enclosed crystals of magnetite, the presence of sulfur globules, unusual cell<br />

wall morphology and a furrowed surface structure. Despite of the distinct cell<br />

biology and phylogenetic position of M. bavaricum, the presence of membrane<br />

enveloped magnetite crystals suggests that the genetic basis of magnetosome<br />

formation might be homologous to other magnetotactic bacteria, which will be<br />

revealed by our current genomic analysis.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PH 01<br />

Cross-species microarray and realTime-PCR analysis of<br />

differentially regulated genes in mating-type mutants of the<br />

homothallic ascomycete Sordaria macrospora<br />

V. Klix *1 , M. Nowrousian 2 , C. Ringelberg 3 , J.J. Loros 3 , J.C. Dunlap 3 , S.<br />

Pöggeler 1<br />

1 Institut für Mikrobiologie und Genetik, Abt. Genetik eukaryotischer<br />

Mikroorganismen, Georg-August-Universität Göttingen, Göttingen, Germany<br />

2 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität, Bochum,<br />

Germany<br />

3 Departments of Genetics and Biochemistry, Dartmouth Medical School,<br />

Hanover, United States<br />

The filamentous ascomycete Sordaria macrospora is homothallic and un<strong>der</strong><br />

laboratory conditions completes its life-cycle within seven days. In contrast to<br />

heterothallic fungi, a single strain is self-fertile and S. macrospora needs no<br />

mating-partner for sexual reproduction.<br />

In the closely related, heterothallic ascomycete Neurospora crassa the mating<br />

type of the haploid mating-partners is genetically determined by the matingtype<br />

locus. In N. crassa the mat-locus is an allele pair, which can be present in<br />

two different forms (mat a and mat A).<br />

The mating type of S. macrospora consists of four genes (SmtA-1, SmtA-2,<br />

SmtA-3 and Smta-1) which share similarities to mating-type genes from N.<br />

crassa mat A- and mat a-strains. These genes code for putative transcription<br />

factors (SmtA-1, Smta-1) or proteins without characteristic DNA-binding motifs<br />

(SmtA-2, SmtA-3).<br />

Other important components in the sexual cycle of ascomycetes are<br />

pheromones. In N. crassa two pheromones (MFA-1, CCG-4) confer the ability<br />

of mating with a compatible partner. It has been shown that S. macrospora also<br />

possesses two functional pheromones (PPG-1, PPG2) that they are needed for<br />

sexual development.<br />

In this study we analysed the role of the mating-type genes SmtA-1, SmtA-2 and<br />

SmtA-3 in S. macrospora. Knockout-mutants of all three mating-type genes<br />

were generated and analysed. Additionally quantitative realTime-PCR analysis<br />

was performed to determine if the expression of pheromones and their cognate<br />

receptors is affected in the mutants. Cross-species microarray analysis with N.<br />

crassa was performed to find other differentially regulated genes within the<br />

mating-type mutants.<br />

PH 02<br />

A novel, microtubule dependent role for a formin in the<br />

filamentous fungus Ashbya gossypii<br />

M. Kemper *1 , L. Molzahn 2 , S. Buck 1 , C. Birrer 2 , M. Lickfeld 1 , H.P. Schmitz 1<br />

1 AG Genetik, Universität Osnabrück, Osnabrück, Germany<br />

2 Molekulare Mikrobiologie, Biozentrum Basel, Basel, Switzerland<br />

Assembly and organization of the actin cytoskeleton is fundamental for polar<br />

growth in many filamentous fungi. Key regulators of these processes are the<br />

Formin proteins. They contain characteristic sequence motifs termed formin<br />

homology domains that are important for subcellular localization and<br />

elongation of actin filaments.<br />

We show here that this is also true for the Formin from Ashbya gossypii.<br />

Mutation of AgBNR2 results in an instable growth axis and frequent lyses of the<br />

tip, suggesting a role in actin regulation. In agreement with this, we show that<br />

AgBnr2 is able to bind and polymerize actin.<br />

To our surprise a fusion of AgBnr2 to GFP did not only localize to the tips of<br />

hyphae but also gave a punctuate pattern throughout the whole hyphae. We<br />

were able to show that these dots are identical with the spindle pole body (SPB)<br />

of the fungal nuclei. In addition we could identify a SPB-component as binding<br />

partner of AgBnr2 and we were able to map the binding motif in AgBnr2.<br />

Furthermore microtubule-binding and microtubule-stability assays suggest a<br />

direct role for AgBnr2 in regulation of microtubule dynamics in a way that is<br />

contrary to the microtubule stabilization known from mammalian formins.<br />

In conclusion our results suggest a dual role for the formin AgBnr2, with only<br />

one of its functions <strong>bei</strong>ng related to actin and the other suggesting an<br />

involvement of AgBnr2 in the dynamics of nuclear migration via multiple<br />

microtubule interactions.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PH 03<br />

Contribution of extracellular esterases from selected wood<br />

and litter decaying ascomycetes to lignocellulose<br />

degradation<br />

N. Do Huu *1 , C. Liers 1 , F. Moritz 1 , R. Ullrich 1 , M. Hofrichter 1<br />

1 Unit of Environmental Biotechnology, International Graduate School of<br />

Zittau (IHI), Zittau, Germany<br />

The microbial degradation and recycling of persistant natural polymers such as<br />

lignin and plant cell-wall polysaccharides (cellulose, hemicellulose, pectin)<br />

plays a key role in the global carbon cycle and is the basis for the development<br />

of innovative biotechnological processes for different industries. Woody<br />

lignocellulose forms a complex but organized structure which resists microbial<br />

attack by virtue of inaccessibility and chemical composition. The most efficient<br />

degra<strong>der</strong>s of this polymer are the so-called white and brown-rotting<br />

basidiomycetes. Whereas these fungi have been studied over the last two<br />

decades and the key enzymes (peroxidases and laccases) for ligninolysis were<br />

identified and characterized, only little is known about the contribution of<br />

hydrolases to that process as well as regarding the mechanism, how<br />

ascomycetes achieve substantial destruction of wood. The structural integrity of<br />

plant cell walls is due in part to the presence of various ester linkages that<br />

originate from the coupling of hemicelluloses with the phenolic acids of the<br />

lignin molecule. Therefore, we have screened a selection of representative<br />

wood and litter decaying basidio- and ascomycetes for secretion of extracellular<br />

esterases during solid state cultivation. Mainly ascomycetes belonging to the<br />

family of Xylariaceae and Morchellaceae secreted high levels of acetylesterases<br />

(up to 1.160 U l -1 ) and phenolic acid esterases (feruloyl esterase; up to 250 l -1 ,<br />

p-coumaroyl esterase; up to 160 U l -1 ) all throughout the cultivation. In contrast<br />

no phenolic acid esterases could be detected by the tested wood-dwelling white<br />

rot fungi. The future tasks of our study are based on purification of sufficient<br />

amounts of the detected enzymes for protein characterization and in-vitro<br />

depolymerisation of milled native wood lignin.<br />

PH 04<br />

Identification of genes for a sexual cycle in the<br />

biotechnically relevant fungus Penicillium chrysogenum<br />

B. Hoff *1 , S. Pöggeler 2 , D. Janus 1 , J. Böhm 1 , U. Kück 1<br />

1 Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

2 Genetik eukaryotischer Mikroorganismen, Georg-August Universität<br />

Göttingen, Göttingen, Germany<br />

Eighty years ago, Alexan<strong>der</strong> Fleming discovered an anti-bacterial activity in the<br />

asexual mold Penicillium. Later this original “Fleming strain” was replaced by<br />

an overproducing P. chrysogenum isolate still used for industrial penicillin<br />

production today. Using a heterologous PCR approach, we identified the sex<br />

genes and demonstrated that these strains are of opposite mating types. RT-<br />

PCR analyses showed that mating-type genes are expressed and suggest that P.<br />

chrysogenum has the potential to reproduce sexually. These findings prompted<br />

us to search for homologs of pheromone and pheromone receptor genes that<br />

function in mating and pheromone signaling in sexual reproducing filamentous<br />

fungi. Indeed, a screen of a cDNA library led to the isolation of<br />

transcriptionally expressed pheromone and pheromone receptor genes in strains<br />

of both mating types. The results of our transcriptional expression data suggest<br />

the existence of a heterothallic sexual cycle in P. chrysogenum [1]. To further<br />

test the functionality of the mating-type genes in this asexual fungus, we have<br />

generated deletion strains for further analysis.<br />

[1] Hoff B, Pöggeler S, Kück U (2008) Eukaryot Cell 7: 465-470<br />

PH 05<br />

G-Protein mediated signal transduction in a filamentous<br />

fungus: Use of the yeast two-hybrid-system to identify<br />

interaction partners of the alpha subunit GSA1<br />

C. Schäfers *1 , U. Kück 1<br />

1<br />

Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

103<br />

Heterotrimeric G proteins as part of signal transduction pathways control fungal<br />

differentiation and growth. In the filamentous fungus Sordaria macrospora the<br />

three G-alpha subunits GSA1, GSA2, and GSA3 mediate processes in<br />

vegetative and sexual development. The loss of one of these subunits leads to a<br />

restricted vegetative growth. In sexual development both GSA1 and GSA2 are<br />

involved in fruiting body development and GSA3 plays a role in ascospore<br />

germination (Kamerewerd et al., (2008) Genetics 180:191). To identify

104<br />

interaction partners of the three G-alpha subunits we used the two-hybrid<br />

system from yeast to screen a cDNA library from Sordaria macrospora as well<br />

as from Neurospora crassa. To generate a bait, the gene encoding the G-alpha<br />

subunit GSA1 was fused to the gene for the binding domain of the Gal4<br />

transcription factor. In or<strong>der</strong> to test whether the GTP - or the GDP - bound form<br />

of GSA1 is interacting with any preys, we generated a constitutive active form<br />

(GSA1 Q204L ) and a constitutive inactive form of GSA1 (GSA1 G203R ). The<br />

identified interactions were further verified for example by coimmunoprecipitation<br />

studies.<br />

PH 06<br />

PRO11 and PHO1: two putative key players of the proteinprotein<br />

interaction network controlling fruiting-body<br />

development in Sordaria macrospora<br />

Y. Bernhards *1 , S. Pöggeler 1<br />

1 Institute of Microbiology and Genetics; Department Genetics of eukaryotic<br />

microorganism, Georg-August-University Göttingen, Göttingen, Germany<br />

The focus of our research work is on the interdependency of the components<br />

controlling fruiting-body development in the filamentous ascomycete Sordaria<br />

macrospora. Our actual study is based on the functional analysis of two<br />

putative key players in this complex differentiation process: pro11 and pho1.<br />

The pro11 gene encodes a multimodular WD40 repeat protein which shows<br />

significant sequence and functional homology to the mammalian protein<br />

striatin. Proteins belonging to the striatin family are thought to act as scaffolds<br />

linking signaling and eukaryotic endocytosis. It was found previously, that<br />

striatin forms a complex with the protein phocein. Little is known about<br />

phocein but in mammals it seems to be involved in vesicular trafficking<br />

processes.<br />

By yeast two-hybrid analysis we showed that the S. macrospora homologs<br />

PRO11 and PHO1 are also able to interact with each other. To get deeper<br />

insight into the cellular function of both genes, we constructed knockout strains<br />

and deleted pro11 and pho1. Both knockout strains exhibit a sterile phenotype<br />

which approves the importance of pro11 and pho1 during sexual reproduction.<br />

Here we show a detailed morphological characterization of the knockout<br />

strains. Complementation analysis of the knockout strains with truncated<br />

versions of PRO11 and PHO1 shed light on essential domains of both proteins.<br />

Furthermore, first results of Real Time PCR experiments and localization<br />

studies will be presented.<br />

PH 07<br />

Soil solid materials affect the kinetics of extracellular<br />

enzymatic reactions<br />

C. Lammirato *1 , A. Miltner 1 , M. Kästner 1<br />

1 Bioremediation/Environmental Biotechnology, Helmholtz Centre for<br />

Environmental Research-UFZ, Leipzig, Germany<br />

Soil solid materials affect the degradation of many organic compounds by<br />

decreasing the bioavailability of substrates and by interacting with degra<strong>der</strong>s.<br />

The magnitude of this effect in the environment is shown by the fact that<br />

xenobiotics which are readily metabolized in aquatic environments can have<br />

long residence times in soil. Extracellular enzymatic hydrolysis of cellobiose<br />

(enzyme: β-glucosidase from Aspergillus niger) was chosen as model<br />

degradation process. The aims of the project are: 1) quantification of solid<br />

material effect on degradation, 2) separation of the effects of minerals on<br />

enzyme (adsorption →change in activity) and substrate (adsorption →change in<br />

bioavailability). Enzymatic kinetic experiments are carried out in homogeneous<br />

liquid systems and in heterogeneous systems where solid materials (bentonite,<br />

kaolinite, goethite, activated charcoal) are suspended in a mixed liquid. The<br />

results show that, un<strong>der</strong> the experimental conditions, cellobiose is not adsorbed<br />

by the materials tested (with the exception of activated charcoal) while all the<br />

solids adsorb β-glucosidase. These results lead to the conclusion that only<br />

activated charcoal may affect the reaction rate by limiting the substrate<br />

bioavailability while all the materials tested may affect the reaction rate by<br />

limiting the enzymatic activity. The effect of kaolinite on the reaction was<br />

quantified: un<strong>der</strong> the experimental conditions the initial reaction rate in<br />

presence of the mineral was 22% less then in the control. The fraction of<br />

enzyme molecules which are adsorbed to kaolinite (60%) loses 37% of its<br />

activity. From these results we conclude that even the degradation of substrates<br />

with unrestricted bioavailability can be affected by soil solid materials.<br />

PH 08<br />

Characterization of an essential autophagy-related gene in<br />

the homothallic ascomycete Sordaria macrospora<br />

N. Nolting 1 , S. Pöggeler *1<br />

1 Abteilung Genetik eukaryotischer Mikroorganismen, Institut für Mikrobiologie<br />

und, Georg-August Universität Göttingen, Göttingen, Germany<br />

In filamentous ascomycetes, autophagy is involved in several developmental<br />

processes. Nevertheless, until now little is known about its role in fruiting-body<br />

development. We therefore isolated a gene of the homothallic ascomycete<br />

Sordaria macrospora with high sequence similarity to the Saccharomyces<br />

cerevisiae autophagy-related gene ATG7, encoding a core autophagy regulator.<br />

This is the first characterization of an ATG7 homologue in filamentous<br />

ascomycetes. A S. cerevisiae complementation assay demonstrated that the S.<br />

macrospora atg7 gene functionally replaces the yeast homologue. We were not<br />

able to generate a homokaryotic knockout mutant in S. macrospora, suggesting<br />

that atg7 is essential for viability. However, a heterokaryotic Δatg7/atg7 strain<br />

showed consi<strong>der</strong>able morphological phenotypes during fruiting-body<br />

development. Using real-time PCR, we demonstrated that in the wild type, the<br />

transcriptional expression of atg7 is markedly up-regulated un<strong>der</strong> amino acid<br />

starvation conditions and down-regulated during sexual development.<br />

PH 09<br />

Secretion of esterases by selected wood- and litterdecomposing<br />

macrofungi (basidiomycetes, ascomycetes)<br />

during solid-state fermentation of lignocelluloses<br />

D.H. Nghi *1 , C. Liers 1 , F. Moritz 1 , R. Ullrich 1 , M. Hofrichter 1<br />

1<br />

Environmental Biotechnology, International Graduate School Zittau, Zittau,<br />

Germany<br />

The microbial degradation and recycling of persistant natural polymers such as<br />

lignin and plant cell-wall polysaccharides (cellulose, hemicellulose, pectin)<br />

plays a key role in the global carbon cycle and is the basis for the development<br />

of innovative biotechnological processes for different industries. Woody<br />

lignocellulose forms a complex but organized structure that resists microbial<br />

attack by virtue of inaccessibility and chemical composition. The most efficient<br />

degra<strong>der</strong>s of this polymer are the so-called white- and brown-rot<br />

basidiomycetes. While these fungi have been studied in detail over the last<br />

three decades and the key enzymes (peroxidases and laccases) of ligninolysis<br />

were identified and characterized, little is known on the contribution of estercleaving<br />

hydrolases to this process as well as on the mechanisms, which woodrot<br />

ascomycetes use to achieve the substantial destruction of wood (including<br />

lignin). On the other hand, the structural integrity of plant cell walls is – at least<br />

in part – realized by the presence of various ester linkages originating from the<br />

coupling of hemicelluloses with the phenolic acids (e.g. ferulic acid) of the<br />

lignin molecule. Against this background, we have screened a selection of<br />

representative wood- and litter-decomposing asco- and basidiomycetes for the<br />

secretion of extracellular esterases during the growth on solid lignocelluloses<br />

(solid-state fermentation). In particular, ascomycetes belonging to the family of<br />

Xylariaceae and Morchellaceae secreted high levels of acetylesterases (up to<br />

1.160 U l -1 ) and phenolic acid esterases (feruloyl esterase; up to 250 U l -1 , pcoumaroyl<br />

esterase; up to 160 U l -1 ) throughout the whole cultivation period. In<br />

contrast, no phenolic acid esterases were detectable in case of all wooddwelling<br />

white-rot fungi tested. One particular objective of our ongoing studies<br />

is to purify sufficient amounts of the identified esterases to be used in in-vitro<br />

depolymerisation tests with different lignocelluloses.<br />

PH 10<br />

The role of superoxide dismutases in Podospora anserina<br />

lifespan control<br />

S. Zintel *1 , A. Hamann 1 , H.D. Osiewacz 1<br />

1 Institute of Molecular Biosciences, Department of Biosciences and Cluster of<br />

Excellence „Macromolecular Complexes“, J.W. Goethe University, Frankfurt<br />

am Main, Germany<br />

Molecular damage via reactive oxygen species (ROS) is a major cause of agerelated<br />

dysfunction and diseases. Fortunately, all biological systems have<br />

evolved various pathways to deal with this harmful situation. One of these<br />

pathways is the ROS scavenging system in which superoxide dismutases<br />

(SODs) play an important role. These enzymes de-toxify superoxide and<br />

convert it together with catalases and peroxidases into water. Here we report<br />

investigations analysing and modulating this system in the fungal aging model<br />

Podospora anserina. This analysis revealed: (i) the genome of P. anserina<br />

encodes three SODs localized in different cellular compartments, (ii) one<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

(PaSOD3) of two manganese containing SODs is located in mitochondria, (iii)<br />

deletion of the gene encoding the copper/zinc SOD PaSod1 results in a strongly<br />

increased sensitivity against ROS, (iv) strains lacking either the mitochondrial<br />

MnSOD or both MnSODs (PaSOD2 and PaSOD3) are viable, (v) deletion and<br />

over-expression of PaSod3 negatively affects lifespan and (vi) over-expression<br />

of either MnSOD leads to a remarked increase in activity of PaSOD1. Overall<br />

and most strikingly it appears that the enzymatic ROS scavenging system is<br />

well balanced and contains components which are co-regulated. A successful<br />

strategy for improving the healthy period in the life cycle of the fungus, the<br />

health span, has to take this situation into account.<br />

PH 11<br />

A new member of the Cdk9 kinase family interacts with a<br />

Pcl-like cyclin in Aspergillus nidulans<br />

C. Kempf *1 , S. Hettinger 1 , N. Schier 1 , R. Fischer 1 , F. Bathe 1<br />

1 Applied Microbioloy, Karlsruhe Institute of Technology, Karlsruhe, Germany<br />

Cyclin dependent kinases (CDK’s) are a large group of Serin/Threonin protein<br />

kinases that are regulated by association with a cyclin subunit. Members of the<br />

Cdk9 family (originally designated as PITALRE kinases) have been described<br />

from yeast to humans as essential components of the basal transcription<br />

elongation machinery. Their cyclin binding partners (cyclin T1, T2a, T2b and<br />

K) do not oscillate during the cell-cycle, un<strong>der</strong>lining the dependence of most<br />

cellular genes on continuous Cdk9/cyclinT activity.<br />

Based on sequence analysis, a new PITALRE kinase (PtkA) has been identified<br />

in Aspergillus nidulans. Down-regulation of ptkA expression did not affect<br />

germination, but strongly inhibited later stages of vegetative growth. A GFPtagged<br />

PtkA version showed nuclear localization and the ptkA transcription<br />

levels were constant throughout the cell-cycle and asexual development. These<br />

results are in good agreement with a basal role of PtkA in transcription<br />

elongation, as observed in other organisms. A putative cyclin T protein has<br />

been identified in the A. nidulans genome and is currently analyzed as a likely<br />

interaction partner of PtkA.<br />

Surprisingly, we identified a member of the Pcl cyclin family (PclA) as a PtkA<br />

interaction partner by various methods. PclA that is also interacting with the<br />

main cell-cycle regulator NimX Cdk1 (Schier and Fischer, FEBS Lett. 2002) was<br />

previously shown to be essential for sporulation and is regulated in a cell-cycle<br />

dependent manner (Schier et al., Mol Cell Biol. 2001). Our findings are new<br />

evidence for a possible function of Cdk9 kinases in linking transcriptional<br />

activity with cell-cycle progression and/or morphogenesis.<br />

PH 12<br />

Mitochondrial protein quality control in the ageing model<br />

Podospora anserina: Role of the Clp protease in<br />

mitochondrial function and ageing<br />

K. Luce *1 , H.D. Osiewacz 1<br />

1 Institute of Molecular Biosciences, Department of Biosciences and Cluster of<br />

Excellence “Macromolecular Complexes”, J.W. Goethe University, Frankfurt<br />

a.M., Germany<br />

Biological ageing is characterized by the accumulation of oxidatively modified<br />

proteins. Mitochondria generate the major fraction of intracellular reactive<br />

oxygen species (ROS) and thus are one of the main targets for ROS induced<br />

damage. In or<strong>der</strong> to limit damage, mitochondrial protein quality control systems<br />

are operating as an essential tool to control mitochondrial integrity. As one of<br />

the first steps in maintaining mitochondrial function, proteases recognize<br />

damaged proteins and degrade them helping to keep a population of functional<br />

mitochondria. In the mitochondrial matrix, two soluble ATP-dependent<br />

proteases, Lon and Clp, are present. While it has been demonstrated, that Lon<br />

protease preferentially degrades oxidized and misfolded proteins, almost<br />

nothing is known about the role of the Clp protease. In the filamentous<br />

ascomycete Podospora anserina, a fungus used as an experimental ageing<br />

model, homologues of ClpP and ClpX were identified. Clp protease thus is<br />

likely to be composed of a protease (ClpP) and a chaperone (ClpX) subunit.<br />

These subunits may constitute the typical hetero-oligomeric, proteasom-like<br />

structure known from other eukaryotes. In or<strong>der</strong> to elucidate the function of<br />

ClpP, we set out to modulate the abundance of the protein in P. anserina<br />

mitochondria and to characterize the resulting mutants. Both, a ClpP deletion<br />

strain and stable mutants overexpressing the ClpP subunit have been generated<br />

and confirmed by Western blot analysis and protease activity measurements.<br />

Data about the phenotype of these strains will be presented and discussed.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PH 13<br />

The calcium channels Mid1 and Cch1 in the plant pathogen<br />

Claviceps purpurea<br />

J. Bormann *1 , P. Tudzynski 1<br />

1 Institut für Botanik, Westf. Wilhelms-Universität Münster, Münster, Germany<br />

The aim of our work is to reveal the molecular pathways involved in polarized<br />

growth of the phytopathogenic ascomycete Claviceps purpurea. The fungus<br />

penetrates the cuticle of stigmatic hairs of its poacean hostplants, grows down<br />

the style and through the ovarian tissue. The fungus grows mainly<br />

intercellularly and therefore decomposes the middle lamella between the plant<br />

cells. It is known that growing fungal hyphae possess a tip high calcium<br />

gradient just as other tip growing cells like pollen tubes, axons and others do.<br />

Using GFP based calcium sensors like Cameleon we want to image calcium<br />

dynamics in vivo. We also want to address the question how the tip high<br />

calcium gradient is established. It is conceivable that the fungus follows the<br />

„calcium trail“ that is set up by the degradation of pectin during growth. For<br />

this purpose we have to postulate calcium channels located in the tip region of<br />

the hyphae. We cloned a homologue of the yeast stretch-activated, nonselective<br />

cation channel Mid1 and generated a knock-out mutant. In the Δcpmid1 mutant<br />

mycelial growth is significantly slower than in the wildtype. Interestingly the<br />

Δcpmid1 mutant is unable to penetrate and infect its host Secale cereale. The<br />

deletion mutant shows massive aggregations of cell wall material which<br />

indicates that the cell wall synthesis is affected by Mid1 function. It was shown<br />

before that in yeast Mid1 interacts with the voltage-gated calcium channel Cch1<br />

(Fischer et al. (1997), FEBS Lett 419: 259-262). We recently identified the<br />

Claviceps homologue of this gene. Its functional analysis is un<strong>der</strong> way.<br />

PH 14<br />

Analysis of conserved gene expression patterns during<br />

fruiting body development in Pyronema confluens and<br />

distantly related ascomycetes<br />

S. Gesing *1 , M. Nowrousian 1<br />

1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

Ascomycetes differentiate four major morphological types of fruiting bodies<br />

(apothecia, perithecia, pseudothecia and cleistothecia) that <strong>der</strong>ive from an<br />

ancestral fruiting body. Thus, fruiting body differentiation most likely is<br />

controlled by a set of common core genes. One way to identify such genes is to<br />

search for genes with evolutionary conserved expression patterns.<br />

Using "Suppression Subtractive Hybridization", we selected differentially<br />

expressed transcripts in Pyronema confluens (Pezizales) by comparing two<br />

cDNA libraries specific for sexual and for vegetative development,<br />

respectively. The expression patterns of a first assortment of genes were<br />

verified by real time PCR. By BLASTX analyses, corresponding orthologues in<br />

the phylogenetically more <strong>der</strong>ived ascomycete Neurospora crassa (Sordariales)<br />

were identified. Among these are NCU08605, a putative proteasome subunit,<br />

and NCU03646, which might be a beta-1,3-exoglucanase involved in cell wall<br />

synthesis. The P. confluens orthologue of the latter was already identified in a<br />

previous study in a cDNA library specific for sexual development, and a<br />

Sordaria macrospora orthologue of this gene was shown to be downregulated<br />

during vegetative development in previous microarray studies. Additionaly, we<br />

identified an orthologue of a putative N. crassa histone chaperone (NCU09436)<br />

in P. confluens by heterologous PCR. This gene was shown before to have a<br />

development-specific expression pattern in S. macrospora and Fusarium<br />

graminearum, and this is also the case in P. confluens. These data indicate<br />

conserved gene expression patterns during fruiting body development, and the<br />

corresponding genes are candidates of choice for further analysis.<br />

PH 15<br />

Characterisation of functional domains of the irondependent<br />

transcription factor HapX of Aspergillus<br />

nidulans<br />

D.H. Scharf *1 , P. Hortschansky 1 , A.A. Brakhage 1<br />

1 Leibniz Institut für Naturstoff-Forschung und Infektionsbiologie e.V.,<br />

Friedrich-Schiller-Universität Jena, Jena, Germany<br />

105<br />

Recently, a putative fourth CCAAT-binding complex (CBC) subunit with an<br />

unknown function was identified in the filamentous fungus Aspergillus<br />

nidulans and designated HapX. hapX expression is repressed by iron via the<br />

GATA-factor SreA and various iron-dependent pathways (e.g., heme<br />

biosynthesis) are repressed during iron starvation by the interaction of HapX<br />

with the CBC. These data suggest a model, in which HapX/CBC interaction is

106<br />

regulated at both transcriptional and post-translational levels. Iron starvation<br />

causes expression of hapX. Subsequent binding of HapX to the CBC results in<br />

transcriptional repression of iron-dependent pathways. During iron-replete<br />

conditions, hapX is repressed and therefore, iron-dependent pathways are<br />

<strong>der</strong>epressed. Moreover, HapX/CBC interaction is inhibited by increased iron<br />

concentrations. This post-translational mechanism allows rapid adjustment to<br />

iron availability by disruption of the HapX/CBC complex. Here, we describe<br />

the domain architecture of the HapX protein. Phylogenetic analysis revealed the<br />

conservation of certain domains within the fungi. To determine which domain<br />

is responsible for iron sensing, truncated versions of the protein were<br />

characterised further with the help of surface-plasmon-resonance. The in vivo<br />

relevance of the data was proven by complementation of a hapX deletion strain<br />

with truncated versions of the gene. Taken together, a novel iron sensing<br />

mechanism in eukaryotes was discovered.<br />

PH 16<br />

Fungal adhesion to roots - the fist step of early infection and<br />

systemic colonization of Brassica napus by Verticillium<br />

longisporum<br />

T. Tran-van *1 , S.A. Braus-Stromeyer 1 , G.H. Braus 1<br />

1 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen,<br />

Göttingen, Germany<br />

V. longisporum is a major pathogen for oilseed rape (Brassica napus ). This<br />

fungus induces early senescence and causes severe economic losses. V.<br />

longisporum infects host-plants via the roots and lives biotrophically in the<br />

xylem vessels of B. napus. Currently no fungicides are available to cure<br />

infected plants. Fungal adhesion is an important step for the early infection and<br />

colonization of many pathogenic fungi. Proteins required for adhesion of plant<br />

fungal pathogens play an important role during pre-penetration, growth, biofilm<br />

formation as well as pathogenicity.<br />

The goal of this project is to investigate the importance of adhesion of V.<br />

longisporum to the roots of host plants for successful infection and systemic<br />

colonization. Ten genes encoding putatively secreted proteins that may play a<br />

role in adhesion and interaction of the fungus with the host plant were<br />

identified from an adhesion complementation assays in Saccharomyces<br />

cerevisae and by bioinformatic analysis of a cDNA library. We could show,<br />

that 8 of 10 of these putatively secreted proteins were up-regulated when the<br />

fungus interacted with the roots of the host plant. In addition, two<br />

transcriptional regulators from V. longisporum could be identified, which<br />

induced in the complementation assay strong adhesion of S. cerevisae to the<br />

agar surface. These putative transcription factors belong to the zinc finger<br />

family and were reported to regulate many essential genes in filamentous fungi.<br />

We are currently analyzing the knock-downs of the corresponding genes in V.<br />

longisporum.<br />

PH 17<br />

Three α-subunits of heterotrimeric G proteins and an<br />

adenylyl cyclase contribute to sexual development of a selffertile<br />

fungus<br />

J. Kamerewerd *1 , M. Jansson 1 , M. Nowrousian 1 , S. Pöggeler 2 , U. Kück 1<br />

1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

2 Institut für Mikrobiologie und Genetik, Abt. Genetik Eukaryotischer<br />

Mikroorganismen, Georg-August-Universität Göttingen, Göttingen, Germany<br />

In eukaryotes, heterotrimeric GTP-binding proteins consisting of α-, β-, and γsubunits<br />

interact with heptahelical transmembrane receptors and transduce<br />

environmental signals to regulate morphogenesis and cellular response. Upon<br />

activation of a G protein-coupled receptor by an extracellular stimulus, the<br />

GDP bound by the Gα-subunit is replaced with GTP, leading to the dissociation<br />

of the α-subunit and the βγ-complex. Both Gα- and Gβγ-complex can bind and<br />

regulate effectors that then propagate signals into the cell. Filamentous fungi<br />

are ideal model systems to study the function of different Gα-subunits within<br />

multicellular eukaryotes. Many ascomycetes develop complex multicellular<br />

structures (ascocarps) to protect their meiospores. Sordaria macrospora is a<br />

self-fertile ascomycete which develops its ascocarps (perithecia) without the<br />

necessity of fertilization, thus every developmental defect is immediately<br />

apparent. Additionally, S. macrospora lacks any structures for asexual<br />

propagation, thus no overlapping developmental processes occur. Therefore S.<br />

macrospora is an ideal model organism to study aspects of sexual<br />

differentiation. In a genetic analysis we investigated the distinct roles of three<br />

gsa-genes encoding the Gα-subunits of S. macrospora in sexual development of<br />

this fungus by generating the knockout strains Δgsa1, Δgsa2 and Δgsa3 as well<br />

as all combinations of double mutants. We further addressed the question<br />

whether the Gα-subunits interact genetically with the pheromone receptors<br />

PRE1 and PRE2, the putative effector adenylyl cyclase SAC1, the<br />

developmental protein PRO41, and the putative transcription factor STE12. For<br />

this purpose, a total of 18 double mutants and one triple mutant were generated.<br />

From the sum of all our data, we propose a model for how different Gαsubunits<br />

regulate sexual development in S. macrospora.<br />

PH 18<br />

The role of the NADPH-oxidase-complex in the biotrophic<br />

interaction of Claviceps purpurea and Secale cereale<br />

D. Buttermann *1 , S. Giesbert 1 , P. Tudzynski 1<br />

1 Institut für Botanik, WWU Münster, Münster, Germany<br />

Reactive oxygen species (ROS) are important in defence reactions against<br />

pathogens in many mammalian and plant systems, where the role of the<br />

superoxide generating NADPH-oxidase-complex within this oxidative burst is<br />

well established.<br />

In fungi, the role of ROS is less well un<strong>der</strong>stood. Reactive oxygen molecules<br />

produced by phytopathogenic fungi induce oxidative stress on host organisms<br />

as they contribute to the ROS-status in planta but they also play a role in fungal<br />

differentiation processes.<br />

In Claviceps purpurea, an ecologically obligate biotroph on diverse monocots,<br />

we analyse the role of the NADPH-oxidase-complex.<br />

C. purpurea possesses two genes encoding homologues of the mammalian<br />

gp91 phox , named cpnox1 and cpnox2. Cpnox1 is a virulence factor in C.<br />

purpurea: the knockout mutant shows drastically reduced infection rates<br />

compared to the wild type. Formation of honeydew, a first macroscopic sign of<br />

infection, is strongly retarded and sclerotia, the typical fungal resting structures,<br />

have never been observed (Giesbert et al. 2008). In contrast, the knockout<br />

mutant Δcpnox2 is not affected in early colonization stages, it even shows<br />

significantly enhanced and prolonged production of honeydew, while sclerotia<br />

are not fully developed. These data indicate that both NADPH-oxidase catalytic<br />

subunits have impact on virulence: Cpnox1 plays a major role in early<br />

colonization, Cpnox2 is involved in the metabolic switch leading to<br />

development of sclerotia.<br />

Functional analysis of the C. purpurea p67 phox homologue (cpnoxR) is un<strong>der</strong><br />

way. Yeast two-hybrid experiments already showed that noxR interacts with<br />

the small GTPase Rac, suggesting that Rac is involved in regulation of the Nox<br />

complex.<br />

PH 19<br />

Regulation of tip-splitting in the filamentous fungus Ashbya<br />

gossypii<br />

M. Lickfeld *1 , H.P. Schmitz 1<br />

1 AG Genetik, Universität Osnabrück, Osnabrück, Germany<br />

During maturation, the hyphae of some filamentous fungi switch from lateral to<br />

apical branching. The molecular mechanism of this tip splitting remains in the<br />

dark. While current hypotheses of this process suggest that tip splitting is a<br />

stochastic process, our data suggests that tip splitting might be regulated. Using<br />

the filamentous fungus Ashbya gossypii as a model, we found that tip splitting<br />

can be induced by a constitutively active allele of the formin AgBNI1.<br />

However, how the formin protein is activated is still unknown. We identified<br />

several Rho-type GTPases as potential activators and we will present data from<br />

interaction and co-localization studies that sheds further light on this process.<br />

PH 20<br />

A mutation in the COX5 gene of the yeast Pichia stipitis<br />

alters both, utilization of amino acids as carbon source and<br />

ethanol formation<br />

F. Stefan *1 , V. Passoth 2 , U. Klinner 1<br />

1 Department of Biology IV (Microbiology and Genetics), RWTH Aachen<br />

University, Aachen, Germany<br />

2 Department of Microbiology, Swedish University of Agricultural Sciences<br />

(SLU), Uppsala, Sweden<br />

Public interest grows with regard to availability of alternative raw materials for<br />

fuel production in future. Mainly plant lignocellulosic materials would provide<br />

acceptable alternatives to mineral oils if efficient technologies for biochemical<br />

transformation of its monomers would be at disposal. The pentose xylose as a<br />

main component of hemicelluloses and, therefore, this „wood sugar“ could<br />

serve as raw material for the production of ethanol. The yeast P. stipitis<br />

possesses the rare natural property to can form ethanol from xylose and could<br />

obtain, therefore, in future importance for transformation of lignocellulosic<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

iomass components into ethanol. We performed random integration<br />

mutagenesis and isolated two mutants which had lost the ability to grow with<br />

glutamate as carbon source. One mutant was damaged in the COX5 gene.<br />

Mutant cells had lost the ability to grow with the amino acids glutamate, proline<br />

or aspartate and other non-fermentable carbon sources as acetic acid and<br />

ethanol. Biomass formation of the mutant cells in medium containing glucose<br />

or xylose as carbon source was lower if compared to the wild type cell.<br />

However, ethanol yields (g/g carbon source; g/g biomass) of the mutant were<br />

higher especially un<strong>der</strong> conditions of high aeration. The mutant cells showed an<br />

enhanced ADH and PDC activity and both, no cytochrome c oxidase and no<br />

cyanide sensitive respiration. However, cells expressed SHAM sensitive<br />

respiration which obviously was essential for the fermentative metabolism<br />

because SHAM completely prevented growth of the mutant cells with both,<br />

glucose or xylose as carbon source.<br />

PH 21<br />

C- and N-catabolic utilization of glutamate and related<br />

amino acids by Pichia stipitis and other yeasts<br />

S. Freese *1 , T. Vogts 1 , F. Speer 1 , B. Schäfer 1 , V. Passoth 2 , U. Klinner 1<br />

1 Department of Biology IV (Microbiology und Genetics), RWTH Aachen<br />

University, Aachen, Germany<br />

2 Department of Microbiology, Swedish University of Agricultural Sciences<br />

(SLU), Uppsala, Sweden<br />

In contrast to many other yeast species, P. stipitis and the closely related yeast<br />

Candida shehatae could utilize the L-amino acids glutamate, aspartate and<br />

proline as both, carbon and nitrogen sources. Moreover, we found this property<br />

also in several ascomycetous and basidiomycetous yeasts, including Candida<br />

albicans, C. glabrata, C. maltosa, C. utilis, C. reukaufii, C. utilis,<br />

Debaryomyces hanseniii, Kluyveromyces lactis, K. marxianus, Lod<strong>der</strong>omyces<br />

elongisporus, Pichia capsulata, P. guilliermondii, Rhodotorula rubra,<br />

Trichosporon <strong>bei</strong>gelii and Yarrowia lipolytica. The final biomass and ethanol<br />

concentrations in P. stipitis cultures grown in glucose medium containing<br />

glutamate as sole nitrogen source were consi<strong>der</strong>ably higher than in glucose<br />

medium containing ammonium as nitrogen source. Cells showed an enhanced<br />

glutamate uptake and secreted waste ammonium during growth on glutamate as<br />

sole carbon and nitrogen source. C-assimilation of glutamate was correlated to<br />

enhanced transcription and enzymatic activity of the NAD+ dependent<br />

glutamate dehydrogenase 2 (GDH2). Expression of GDH2 was also enhanced<br />

during growth on glucose/glutamate instead of glucose/ammonium. A Δgdh2<br />

disruptant was unable to utilize glutamate either as carbon or as nitrogen<br />

source. Moreover, this disruptant was also unable to utilize aspartate as carbon<br />

source. The mutation was complemented by transformation of the GDH2 ORF<br />

into the Δgdh2 strain. The results show that Gdh2p plays a dual role in P.<br />

stipitis as both, a C-catabolic and an N-catabolic enzyme, which points to the<br />

existence of an unknown regulatory interface between carbon and nitrogen<br />

metabolism of this yeast.<br />

PH 22<br />

Sexual development in zygomycetes: proteins and enzymes<br />

C. Schimek *1 , J. Wetzel 1 , Y. Rudigier 1 , K. Roth 1 , A. Burmester 1 , J.<br />

Wöstemeyer 1<br />

1 Institut für Mikrobiologie / Lehrstuhl für Allgemeine Mikrobiologie und<br />

Mikrobengenetik, Friedrich-Schiller-Universität Jena, Jena, Germany<br />

Sexual development in zygomycetes is mediated by β-carotene <strong>der</strong>ived signal<br />

compounds, the trisporoids. Trisporoid synthesis is self-regulatory, later<br />

intermediates and the terminal compound trisporic acid are only produced in<br />

the presence of compounds provided by the other mating type or in mated<br />

cultures. Trisporoids serve as pheromones involved in partner recognition and<br />

induction of developmentally committed organs, but also as internal<br />

transcription regulators. Whereas two enzymes involved in pheromone<br />

synthesis, 4-dihydromethyltrisporate dehydrogenase [1] and 4-dihydrotrisporin<br />

dehydrogenase [2], are regulated at a post-translational level after a certain<br />

stage of competence has been obtained by the mycelium, the carotene<br />

oxygenase responsible for the initial cleavage of beta-carotene is regulated at<br />

the transcriptional level [3]. Both dehydrogenase genes are functional in mating<br />

defective Phycomyces blakesleeanus mutants, indicating the possibility of<br />

mutated regulatory genes. Among the remaining unidentified trisporoid<br />

synthesis enzymes, the postulated methyl trisporate esterase, an enzyme<br />

presumably active predominantly in the (-)-mating type and catalyzing the<br />

conversion of methyl trisporate into trisporic acid, is now experimentally<br />

accessible via a multistage activity assay. Putative reaction partners involved in<br />

recognition and signal perception events are analyzed by proteome studies.<br />

Two-dimensional gel electrophoresis reveals only a small number of deviating<br />

proteins between the (+) and the (-) mating type in both cytoplasmatic and cell<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

surface-attached proteins. Several candidate proteins have been selected and are<br />

currently un<strong>der</strong> analysis.<br />

[1] Schimek et al., Fungal Genet Biol 42, 804-812, 2005<br />

[2] Wetzel et al., Eukaryotic Cell 2008, in press<br />

[3] Burmester et al., Fungal Genet Biol 44, 1096-1108, 2007<br />

PH 23<br />

Evaluation of computer predictions in the genome of the<br />

ectomycorrhizal basidiomycete Laccaria bicolor<br />

U. Kües *1<br />

1 Molecular Wood Biotechnology and Technical Mycology, Georg-August-<br />

University Göttingen, Göttingen, Germany<br />

In 2008, the sequenced genome of Laccaria bicolor was published (Martin et<br />

al. Nature 452:88). Some 19,000 genes were predicted in the genome by<br />

computer programs. Average lengths of introns and exons were calculated.<br />

Intron length was given in the paper with in average 92 bases. Having<br />

annotated as part of the Laccaria annotation consortium many genes of the<br />

fungus, this value did not correspond to the experienced observations on gene<br />

structures. Thus, 100 genes linked to the A mating type locus on a chromosome<br />

highly conserved to Coprinopsis cinerea were carefully checked for potential<br />

mistakes in their gene structure. A number of far too long introns needed<br />

corrections. Computer mistakes identified were an inability to identify a short<br />

first coding exon, an inability to identify very small exons of 3 to a few bases<br />

and wrong linkage of two different genes into one with a long predicted intron<br />

in between. More than 800 introns were analyzed: The typical intron is around<br />

50-55 bp long and only three were longer than 75 bp. Thus, the published value<br />

of 92 bp suggests that there is still a large body of false predictions in the<br />

annotated genome. Restriction of intron length between values of 35 to 75 bp in<br />

computer analysis may help out. A further observation from this study is that<br />

exon length can vary from a few bp to sizes over 1000 bp and there is no<br />

preferred exon size. Mathematical games of calculating average exon sizes are<br />

thus of little meaning.<br />

PH 24<br />

Analysis of D-lysergyl peptide synthetases of Claviceps<br />

purpurea<br />

J. Havemann *1 , I. Ortel 1 , U. Keller 1<br />

1 Institut für Chemie, Biochemie, Technische Universität Berlin, Berlin,<br />

Germany<br />

Ergot alkaloids represent a group of D-lysergic acid <strong>der</strong>ivatives produced by<br />

the ergot fungus Claviceps purpurea. These can be divided in the two groups of<br />

the ergopeptines and the simple D-lysergic acid alkylamides. In the<br />

ergopeptines, D-lysergic acid is amidated with a tripeptide chain whereas in the<br />

D-lysergic acid alkylamides the amide substituent is alaninol or ethanolamine.<br />

We have shown previously that the ergopeptine peptide chain is assembled<br />

non-ribosomally from D- lysergic acid and the three amino acids of the peptide<br />

chain by action of the peptide synthetases LPS1 (D-lysergic acid activating)<br />

and LPS2 (peptide chain assembling) [1, 2, 3]. The assembly of the simple Dlysergic<br />

acid alkylamides remained unknown. We have shown recently that the<br />

simple D-lysergic acid alkylamides are assembled by a third peptide synthetase<br />

LPS3 which in conjunction with LPS2 condenses L-alanine with D-lysergic<br />

acid and releases D-lysergylalanine as D-lysergylalaninol in an NADPHdependent<br />

manner [4]. Obviously, LPS2 can cooperate with LPS1 as well as<br />

with LPS3 in a combinatorial fashion which appears to depend on the proteinprotein<br />

interactions between different partner enzymes. From this it is to be<br />

expected that contact sites between LPS1 and LPS2 must be similar to those<br />

involved in the interaction between LPS2 and LPS3. In fact, we found that all<br />

LPS type enzymes possess highly conserved aminoterminal domains which<br />

represent condensation half-domains of NRPSs . Presently, we are engaged to<br />

expresss these domains as separate proteins and to analyse them in vitro in their<br />

behaviour as interfering probes in the protein-protein interactions between the<br />

various alkaloid peptide NRPS.<br />

[1] Rie<strong>der</strong>er et al. (1996) Chemistry & Biology 271 (44): 27524-27530<br />

[2] Walzel et al. (1997) Chemistry & Biology 4 (3): 223-230<br />

[3] Correia et al. (2003) J. Biol. Chem. 10 (12): 1281-1292<br />

[4] Ortel & Keller, in the press<br />

107

108<br />

PH 25<br />

Evaluation of GOS synthesis parameters for Fungal <strong>der</strong>ived<br />

β-Galactosidase<br />

A. Zubair *1 , S. OConnell 1 , M. Hall 1<br />

1 Shannon Applied Biotechnology Centre, Institute of Technology Tralee,<br />

Tralee, Ireland<br />

Galacto-oligosaccharides(GOS) are prebiotics that are fermented by specific<br />

colonic bacteria such as bifidobacteria and lactic acid bacteria. It is suggested<br />

that the products of fermentation of GOS in the colon, mainly short chain fatty<br />

acids, have a role in the improvement of the colonic environment. GOS are<br />

starting to command significant attention as a functional food ingredient due to<br />

encouraging clinical trial results. β-Galactosidase produce GOS but<br />

development of optimised enzymes has yet to be fully realized. β-Galactosidase<br />

<strong>der</strong>ived from Aspergillus oryzae is currently exploited for commercial purpose.<br />

Temperature, pH and stability profiles of β-Galactosidase from Aspergillus<br />

oryzae evaluating hydrolytic activity with ONPG has already been studied and<br />

reported. However there are no reports on the profiles for GOS synthesis from<br />

Aspergillus oryzae. Due to the increasing demand of application specific<br />

enzymes, this study also focussed on identification of other more suited<br />

enzymes for GOS synthesis.<br />

Evaluation of GOS synthesis profiles with varying pH, temperature and lactose<br />

concentration was carried out in this study. GOS synthesis activity was<br />

evaluated using a HPLC based assay method. The enzyme activity was<br />

processed at 10 units/mg. It was found that GOS synthesis by the commercial<br />

β-Galactosidase from Aspergillus oryzae was optimal at pH 5.5 and room<br />

temperature. There was a gradual fall of GOS produced as the temperature was<br />

increased. The starting lactose concentration facilitates increased GOS<br />

production to some extent but as the concentration was increased beyond 60%,<br />

the productivity did not increase further. This study also compared the GOS<br />

synthesis properties of β-Galactosidase from Aspergillus oryzae with that of<br />

Aspergillus carbonarius and Aspergillus niger DL002 (isolates from our lab).<br />

PH 26<br />

Fungal transposons: mobile elements as molecular tools?<br />

F. Kempken *1 , I. Braumann 1 , M. van den Berg 2<br />

1 Botanisches Institut, Christian-Albrechts-Universität zu Kiel, Kiel, Germany<br />

2 Anti-Infectives, DSM, Delft, Netherlands<br />

Transposons are mobile and mostly also repetitive sequences, which are found<br />

in all eukaryotic genomes. Basically transposons can move around their host<br />

genome in two different ways: via true transposition using a „copy and paste“<br />

or a „cut and paste“ mechanism depending on which type of transposon is<br />

consi<strong>der</strong>ed. But due to their repetitive nature transposons can also change their<br />

position via recombination resulting in genomic rearrangements of various<br />

extent.<br />

We have analyzed the transposon content in two fungal genomes, Aspergillus<br />

niger and Penicillium chryosogenum. More specific transposon mobility in the<br />

A. niger CBS513.88 genome was investigated both experimentally and in<br />

silico. A comparison of transposon distribution in different A. niger strains of<br />

the same strain lineage showed that most transposons have remained at their<br />

genomic positions during the classical strain improvement programs. The same<br />

is true for P. chrysogenum. Recombination between two copies of the A. niger<br />

retrotransposon ANiTa1 resulting in the genomic rearrangement of an<br />

approximately 45 kb region was observed.<br />

One non-autonomous element, the A. niger transposon Va<strong>der</strong>, was shown to be<br />

active during strain development. Va<strong>der</strong> mobility could also be shown in a<br />

transposon trap experiment. Due to its obvious activity and to its ability to<br />

insert into genes Va<strong>der</strong> appears to be suitable as a gene tagging tool. We<br />

present evidence for excision of Va<strong>der</strong> from a vector sequence. Finally we<br />

show data regarding the fungal transposon Restless and its use as a molecular<br />

tool.<br />

PH 27<br />

Strain-improvement und media optimization for the<br />

production of the mycotoxin alternariol from Alternaria<br />

alternata<br />

K. Brzonkalik *1 , A. Neumann 1 , C. Syldatk 1<br />

1 Institut für Bio- und Lebensmitteltechnik, Lehrstuhl für Technische Biologie,<br />

Universität Karlsruhe, Karlsruhe, Germany<br />

Black-moulds of the genus Alternaria contaminate many foodstuffs and<br />

agricultural products. In addition to the economical damage these fungi can<br />

produce harmful secondary metabolites, the Alternaria toxins. Some of these<br />

mycotoxins such as alternariol (AOH), alternariolmonomethylether (AME),<br />

altenuene (ALT) and tenuazonic acid (TA) have been described as cytotoxic,<br />

genotoxic and mutagenic in vivo and in vitro. Due to the fact that mycotoxins<br />

could be detect in many foodstuffs and that these fungi can grow even at low<br />

temperatures it is necessary to produce the mycotoxins in high amounts for the<br />

elucidation of the genotoxic, cytotoxic and mutagenic potential.<br />

Up to now the production of Alternaria toxins, especially alternariol with<br />

Alternaria alternata was only described on cultivation on solid media like e.g.<br />

rice. This kind of cultivation is strongly dependent on the kind of rice used and<br />

not transferable to bigger scales. Therefore a highly defined media and optimal<br />

culture conditions were found to make fluid submerse cultivation in shaking<br />

flask experiments possible. This is a prerequisite to make big scale cultivation<br />

in bioreactors, and production of alternariol feasible. A first study in a scale of<br />

1.5 l was already performed to investigate important parameters in a bioreactor.<br />

PH 28<br />

Effect of agitation-induced pellet morphology on product<br />

formation of Aspergillus niger<br />

P.J. Lin *1 , D.C. Hempel 1 , R. Krull 1<br />

1 Institute of Biochemical Engineering, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

In submerged cultivations, Aspergillus niger is grown either as disperse<br />

mycelia or as densely packed aggregates, so-called pellets. In pellet<br />

cultivations, there is an entangled relationship between pellet morphology,<br />

transport phenomena within the pellets and the related product formation.<br />

Various parameters such as the volumetric power input caused by agitation<br />

have significant influence on the fungal morphology and therefore the<br />

productivity.<br />

In this study, the strain A. niger AB1.13 is used to compare the effect of various<br />

volumetric power input caused by different agitation intensities on fungal pellet<br />

morphology and the formation of glucoamylase as model product. A preculture<br />

of biopellets is prepared in a 28 L stirred tank bioreactor with a working<br />

volume of 18.4 L medium containing xylose as the carbon substrate, inoculated<br />

with a suspension of conidia to give a conidial concentration of 10 6 mL -1 ,<br />

cultivated at 30°C and pH 5.5. Xylose represses the glucoamylase (glaA)<br />

promoter controlled glucoamylase synthesis. After 40 h of cultivation time, the<br />

glaA-promoter is induced by a pulse addition of maltose. Immediately, four<br />

identical 3 L stirred tank bioreactors are each filled with 2 L of the preculture<br />

broth. Pellets of the same starting point with respect to morphology and<br />

glucoamylase production are cultivated onwards in these lab-scale bioreactors<br />

at a constant aeration rate, 30°C, pH 5.5 but un<strong>der</strong> different volumetric power<br />

input caused by agitation. Samples are taken from the bioreactors at the same<br />

time in defined intervals.<br />

Quantitative morphological data such as the pellet size distribution, pellet<br />

concentration and pellet structure are obtained by digital image analysis, laser<br />

diffraction and microscopic analysis of pellet slices. Biomass growth and<br />

resulting glucoamylase activity are measured and correlated with the<br />

volumetric power input caused by agitation in dependence of the measured<br />

morphological data.<br />

PH 29<br />

Functional characterization of the mating-type locus of the<br />

asexual cephalosporin C producer Acremonium<br />

chrysogenum<br />

S. Pöggeler *1 , B. Hoff 2 , U. Kück 3<br />

1 Abteilung Genetik eukaryotischer Mikroorganismen, Georg-August<br />

Universität Göttingen, Göttingen, Germany<br />

2 Christian Doppler-Labor, Fakultät für Biologie und Biotechnologie, Ruhr-<br />

Universität Bochum, Bochum, Germany<br />

3 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,<br />

Bochum, Germany<br />

Acremonium chrysogenum, the fungal producer of the β-lactam antibiotic<br />

cephalosporin C, is classified as asexual because no direct observation of<br />

mating or meiosis has yet been reported. To assess the potential of sexual<br />

reproduction in A. chrysogenum, we screened an expressed sequence tag (EST)<br />

library of A. chrysogenum for the expression of mating-type (MAT) genes,<br />

which are the key regulators of sexual reproduction in fungi. We identified two<br />

putative mating-type genes that are homologues of the α-box domain gene<br />

MAT1-1-1 and MAT1-1-2 encoding a HPG domain protein. In addition, cDNAs<br />

encoding a putative pheromone receptor, pheromone processing enzymes as<br />

well as components of a pheromone response pathway were found. Moreover,<br />

the entire AcMAT1-1 and regions flanking the MAT region were obtained from<br />

a genomic cosmid library, and sequence analysis revealed that in addition to<br />

AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating-<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

type gene AcMAT1-1-3 encoding an HMG-domain protein. The α-box domain<br />

sequence of AcMAT1-1-1 was used to determine the phylogenetic relationship<br />

with other ascomycetes. To determine the functionality of the AcMAT1-1<br />

locus, the entire MAT locus was transferred into a MAT deletion strain of the<br />

heterothallic ascomycete Podospora anserina. After fertilization with a<br />

PaMAT1-2 (MAT+) strain, the corresponding transformants developed fruiting<br />

bodies with mature ascospores. Thus, the results of our functional analysis of<br />

the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in<br />

A. chrysogenum.<br />

PH 30<br />

Stereoselective α-hydroxylation of alkylbenzenes by<br />

Agrocybe aegerita peroxygenase<br />

M. Kluge *1 , R. Ullrich 1 , K. Scheibner 2 , M. Hofrichter 1<br />

1 Environmental Biotechnology, International Graduate School of Zittau (IHI),<br />

Zittau, Germany<br />

2 Department of Biotechnology, Lausitz University of Applied Sciences (FH),<br />

Senftenberg, Germany<br />

α-Hydroxy alkylbenzenes are of high importance as building blocks used in<br />

chemical and pharmaceutical synthesis for the production of fine chemicals and<br />

drug precursors. The enantiomeric purity of these products is essential in<br />

particular for pharmaceuticals. The agaric mushroom Agrocybe aegerita<br />

produces a heme-thiolate peroxygenase (AaP) that shares spectral and catalytic<br />

properties both with peroxidases and cytochrome P450 monooxygenases and<br />

catalyses various oxygenation reactions. Here we report on the stereoselective<br />

α-hydroxylation of alkylbenzenes (Ph-R) where -R is -C2H5; -C3H7 ; -C4H9 and<br />

-C5H11. These compounds were hydroxylated by AaP into the respective (R)-αhydroxy<br />

<strong>der</strong>ivatives to different extend and enantiomeric excesses in the<br />

presence of hydrogen peroxide. For (R)-α-phenylethanol and (R)-αphenylpropanol,<br />

the enantiomeric purity exceeded 99% e.e. and for (R)-αphenylbutanol,<br />

still 60% e.e. was achieved. With longer length of the side<br />

chain, the enantiomeric excess and total turnover decreased while the number<br />

and concentration of by-products, for instance of corresponding αphenylketones,<br />

increased. Besides α-phenylpentanol (fenipentol), five still<br />

unidentified by-products were observed in the course of pentylbenzene<br />

conversion at a total turnover of about 20%. In or<strong>der</strong> to optimize the production<br />

of (R)-α-phenylethanol, batches of AaP solutions were fed by means of a<br />

syringe pump with mixtures of the substrate leading to a final concentration of<br />

3.8 mM of the desired product. Using 18 O-labeled hydrogen peroxide, the<br />

complete incorporation of the peroxide-borne oxygen into both 1-phenylethanol<br />

and acetophenone was demonstrated. In contrast to saturated side chains, the<br />

oxygenation of vinylbenzene (styrene) proceeded via the epoxidation of the<br />

double bond to form styrene oxide (~25% e.e.) and 2-phenylethanol.<br />

PH 31<br />

Reactive oxygen species- Botrytis cinerea’s friends or foes<br />

during host infection?<br />

N. Temme *1 , P. Tudzynski 1<br />

1 Institute of Botany, Westfälische Wilhelms-Universität Münster, Münster,<br />

Germany<br />

Botrytis cinerea is a phytopathogenic ascomycete infecting a broad range of<br />

dicotyledonous plants including strawberries, grape vine, tomato and<br />

ornamental flowers. In the course of infection the necrotrophic fungus is<br />

exposed to reactive oxygen species (ROS) released by its host in the "oxidative<br />

burst", an early plant defence reaction. But B. cinerea even produces ROS itself<br />

in planta. This raises the question how the pathogen senses and responds to the<br />

host defence reaction. Do ROS released by the plant harm the pathogen at all?<br />

Investigations on the AP-1 transcription factor Bap1 revealed its role as a<br />

pivotal regulator of ROS detoxification in axenic culture un<strong>der</strong> exposure to<br />

H2O2 and Menadione, an intracellular ROS generator. Macroarray analysis<br />

revealed 99 H2O2-induced Bap1 target genes. Besides other gene products, it<br />

controls transcription of several ROS degrading enzymes as well as of the<br />

thioredoxin and the glutaredoxin system, regulators of the cellular redox status.<br />

Interestingly, Bap1 is not essential for pathogenesis and its target genes are not<br />

expressed on the host 2 days post infection indicating a minor role for H2O2<br />

degradation during infection. However, other redox-regulators like the SAPK<br />

BcSak1 or the Nox complex are essential for normal virulence. Therefore, we<br />

focus on the characterisation of factors involved in ROS signalling in or<strong>der</strong> to<br />

connect different pathways and to elucidate their regulation.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PH 32<br />

The antifungal protein AFP from Aspergillus giganteus<br />

prevents Fusarium growth of barley during the malting<br />

process<br />

H. Barakat Mohamed 1 , A. Spielvogel *1 , I. Fechter 2 , F. Rath 2 , V. Meyer 3 , U.<br />

Stahl 1<br />

1 Biotechnologie Mikrobiologie und Genetik, Technische Universität, Berlin,<br />

Germany<br />

2 Versuchs- und Lehranstalt für Brauerei in Berlin (VLB) e.V.,<br />

Forschungsinstitut für Rohstoffe, Berlin, Germany<br />

3 Leiden University, Institute of Biology Leiden, Fungal Genetics Research<br />

Group, Leiden, Netherlands<br />

Contamination and spoilage of crop and biomaterials by filamentous fungi is<br />

responsible for enormous economical losses worldwide. Globally, head blight<br />

caused by Fusarium species is mainly responsible for crop losses. However, not<br />

only crop loss is a thread but also mycotoxin formation and fungus specific<br />

metabolites cause serious food safety and quality problems. Especially,<br />

secondary growth of Fusarium species during malting is a great concern in beer<br />

production and has been made responsible for the so called gushing effect of<br />

bottled beer.<br />

The filamentous fungus Aspergillus giganteus produces a selectively acting<br />

antifungal protein, named AFP. This protein acts fungicidal at micromolar<br />

concentration against a wide range of plant-pathogenic fungi, whereby growth<br />

and viability of bacteria, yeasts, plant- or human cells remains unaffected. It has<br />

been demonstrated that especially Fusarium species are very sensitive towards<br />

AFP.<br />

We have thus tested the applicability of AFP during the malting process using<br />

barley samples naturally infested with Fusarium species. Our results indicate<br />

that AFP applied during the malting process is indeed able to inhibit the growth<br />

of Fusarium species, whereby the malt quality is not negatively affected.<br />

Current investigations are focusing on the evaluation of the impact of AFP on<br />

the end product beer; the detection of Fusarium metabolites and the analysis of<br />

the gushing potential.<br />

PH 33<br />

Degradation of dimethyl phthalate esters by a Fusarium sp.<br />

and a Trichosporon sp. isolated from mangrove sediments in<br />

Shenzhen, China<br />

Z.H. Luo *1 , K.L. Pang 2 , Y.R. Wu 1 , R.K.K. Chow 1 , J.D. Gu 3 , L.L.P. Vrijmoed 1<br />

1 Department of Biology and Chemistry, City University of Hong Kong, Hong<br />

Kong, Hong Kong<br />

2<br />

Institute of Marine Biology, National Taiwan Ocean University, Keelung,<br />

Taiwan Tajikistan<br />

3<br />

School of Biological Sciences, The University of Hong Kong, Hong Kong,<br />

Hong Kong<br />

109<br />

Dimethyl phthalate esters (DMPE) are endocrine-disrupting chemicals. Two<br />

fungal strains, Fusarium sp. DMT-5-3 and Trichosporon sp. DMI-5-1, were<br />

isolated from mangrove sediments in Futian Nature Reserve of Shenzhen,<br />

China and were able to degrade DMPE. Comparative investigations on<br />

biodegradation of three isomers of DMPE, namely dimethyl phthalate (DMP),<br />

dimethyl isophthalate (DMI), and dimethyl terephthalate (DMT), were carried<br />

out using these two fungi. DMPE could not be completely mineralized but with<br />

the transformation to respective monomethyl phthalate and phthalic acid.<br />

Biochemical degradation pathways for different DMPE isomers were different.<br />

Both fungi were able to transform DMT to monomethyl terephthlate (MMT)<br />

and further to terephthalic acid (TA) by stepwise hydrolysis of two ester bonds.<br />

They only carried out one step of ester hydrolysis to transform DMI to<br />

monomethyl isophthalate (MMI) without further degradation. Only<br />

Trichosporon sp. DMI-5-1 was able to transform DMP to monomethyl<br />

phthalate (MMP). For Fusarium sp. DMT-5-3, the optimal pH for DMI and<br />

DMT degradation was 6.0 and 4.5 respectively. While for Trichosporon sp.<br />

DMI-5-1, the optimal pH for the degradation of the three DMPE isomers was<br />

all at 6.0. Esterase activity was detected in both the cell-free supernatant and<br />

fungal mycelium. Intracellular esterases showed a much higher hydrolytic<br />

activity than extracellular esterases. This study proposes the biochemical<br />

degradation pathways of DMPE by mangrove sediment fungi and suggests that<br />

the fungal esterases responsible for hydrolysis of the two ester bonds of DMPE<br />

are highly substrate-specific.

110<br />

PH 34<br />

A vector system for production and purification of<br />

recombinant proteins in Aspergillus niger.<br />

A. Roth *1 , P. Dersch 2<br />

1 Institut of Microbiology, Technische Universität Braunschweig, Braunschweig,<br />

Germany<br />

2 Institut of Microbiology, Technische Universität Braunschweig & Helmholtz<br />

Centre for Infection Research, Braunschweig, Germany<br />

Aspergillus niger is a biotechnological important organism extensively used for<br />

the production of proteins. Nevertheless a relatively small amount of regulated<br />

expression systems for protein and enzyme production exists for this organism.<br />

In this study, a new sucrose inducible promoter Psuc1 from A. niger strain<br />

AB1.13 was characterized. To monitor gene expression, a green fluorescence<br />

protein (gfp) was cloned un<strong>der</strong> control of the recombinant Psuc1 and<br />

transformed into the protease deficient strain AB1.13. A seven fold increased<br />

fluorescence was observed after three days of growth with sucrose as sole Csource<br />

in contrast to glucose.<br />

The novel suc1 Promoter and the earlier described constitutive pkiA Promoter<br />

[1] are used for the new vector system. The basal vector ANIp8 1 is enhanced<br />

with an enlarged multiple cloning site (MCS). Affinity ligands like<br />

polyhistidine (His-tag) and streptavidin binding polypeptide (Strep-tag) are<br />

adapted to the codon usage of A. niger and added up- and downstream of the<br />

MCS for easy cloning. Stop codons are inserted in the vectors without a Cterminal<br />

tag. Therefore, it is possible to clone one fragment with the same<br />

restriction sites into all different vectors. To check the vector system the<br />

reporter protein GFP is applied. The expression and functionality of GFP<br />

including His- or Strep-tags could be proven by fluorescence measurement and<br />

immunoblot. The results show that our new vector system offers an alternative<br />

system for efficient intracellular protein production in A. niger.<br />

[1] K. McCluskey, Adv. Appl. Microbiol. 2003, 52, 245-262.<br />

PI 01<br />

A lytic cocktail from Streptomyces albus B578 for the<br />

control of lactic acid bacteria in wine<br />

K. Wirth *1 , V. Blättel 1 , H. Claus 1 , H. König 1<br />

1 Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg-<br />

Universität Mainz, Mainz, Germany<br />

Apart from yeasts, lactic acid bacteria (LAB) play an important role in<br />

vinification. Whereas Oenococcus oeni is a desirable species and is<br />

commercially used as a starter culture for the biological acid reduction in<br />

wines, others are responsible for different kinds of wine spoilage. Members of<br />

the genera Pediococcus, Leuconostoc and Lactobacillus are producers of<br />

exopolysaccharide slimes, biogenic amines, acetic acid and other off-flavours.<br />

For the control of microbial growth different procedures like thermal<br />

inactivation, addition of sulfite or lysozyme from egg white are generally<br />

applied. Because of healthy risks the application of sulfite should be reduced<br />

and lysozyme is consi<strong>der</strong>ed as an allergen and not effective against all LAB. In<br />

this study we report evidence that exoenzymes from a Streptomyces albus strain<br />

B578 lyse nearly all wine-relevant strains of LAB. The lytic enzymes are active<br />

un<strong>der</strong> wine-making conditions, like presence of sulfite and ethanol, low<br />

temperatures and low pH values. The analysis of the exoenzyme composition<br />

reveals a synergistic action of different murein hydrolases. In conclusion, the<br />

lytic cocktail of S. albus B578 is a promising tool for the control of winespoiling<br />

bacteria.<br />

PI 02<br />

The asian elephant and Teredo navalis: Excellent sources<br />

for metagenomic cellulases<br />

N. Ilmberger *1 , J. Pottkämper 1 , C. Vollstedt 1 , W. Streit 1<br />

1 Biozentrum Klein Flottbek, Universität Hamburg, Hamburg, Germany<br />

We have initiated work to investigate and to benefit from the metagenome of<br />

the faeces of the asian elephant (Elephas maximus indicus) directly and via<br />

enrichment cultures. Additionally the shipworm Teredo navalis is investigated<br />

via 16S analysis and the construction of metagenomic libraries. As faecal<br />

microbiota is very divers and both animals live on substrates rich in<br />

biopolymers, these habitats are supposed to comprise microbes with hydrolytic<br />

enzymes in high quantity and diversity.<br />

In an enrichment culture with CMC as sole carbon source (with Teredo navalis)<br />

there exists a broad diversity after four weeks, though gamma-Proteobacteria<br />

are the dominating phylum. In contradiction, in the enrichment culture with the<br />

faeces of the asian elephant, there are almost only Bacilli left. The<br />

characterisation directly of the faeces of the asian elephant reveals a much<br />

higher diversity.<br />

To analyse the overall cellulolytic activity of the two enrichment cultures, they<br />

were tested on their activity on CMC. Both cultures show significant hydrolysis<br />

of the substrate though the activity of the „Teredo navalis culture“ is more than<br />

ten-fold higher than the activity of the „elephant culture“. Metagenomic<br />

libraries are un<strong>der</strong> construction (Teredo) or screened for hydrolytic enzymes<br />

(elephant). A number of novel cellulases which are currently characterised have<br />

been identified.<br />

PI 03<br />

Immobilisation of Laccase on different carriers – a<br />

comparison<br />

A. Matura *1 , A. Mondschein 1 , S. Schachschal 2 , S. Wetzel 1 , K.H. van Pée 1<br />

1 Allgemeine Biochemie, TU Dresden, Dresden, Germany<br />

2 Makromolekulare Chemie und Textilchemie, TU Dresden, Dresden, Germany<br />

Enzymes for bioremediation and industrial bioprocesses need to show a high<br />

stability and it must be possible to run the process at low costs. For this<br />

purpose, the multiple use of the enzymes is necessary which can be achieved by<br />

employing immobilised enzymes.<br />

The immobilisation of laccase from Trametes versicolor on different carrier<br />

particles with different conjugation methods was investigated. Carrier particles<br />

of different sizes, morphologies, and with surface layers modified with different<br />

functional groups were chosen. Commercially available Sepa-Beads ® with<br />

epoxy and amino groups, temperature sensitive poly-N-isopropylacrylamide<br />

particles, and the renewable biopolymer chitosan were used. The<br />

immobilisation of laccase by entrapment polymerization and covalent linkage<br />

on the different particles was investigated. The separation of immobilised<br />

enzyme can later be realised by a magnetic core inside the particles.<br />

Efficiency of immobilisation by entrapment polymerisation depends on enzyme<br />

and cross linker concentration. For immobilisation by covalent linkage, best<br />

results were reached with chitosan as the carrier. Interestingly, a remarkably<br />

prolonged stabilisation of the enzyme was observed. The thus immobilised<br />

enzyme did not loose any activity over a period of 25 days when stored at<br />

room, whereas the free enzyme lost activity un<strong>der</strong> threes conditions. The<br />

properties of the immobilized and free laccase are compared. Free and<br />

immobilised enzyme were used for biobleaching of textile dye effluents. In<br />

cooperation with two textile factories in Saxony the bleaching of relevant<br />

textile dyes was investigated un<strong>der</strong> industrial conditions.<br />

PI 04<br />

Production of Single Cell Protein (SCP) from natural gas by<br />

Isolated<br />

F. Mirzanamadi 1 , Z. Mirzakhan *1<br />

1 Biotechnology, Razi Biotech, Tehran, Iran<br />

Methanotrophic bacteria play a major role in the global carbon cycle, degrade<br />

xenobiotic pollutions, and have great potential of biotechnological applications.<br />

Isolation and identification the microorganisms capable of using natural gas<br />

(methane) as carbon source for production SCP as protein for animal and<br />

poultry feed was the aim of our study.<br />

Samples were taken from rice fields, marshlands, oil fields, and wastewater<br />

sources from fields of Iran .they had been cultured in special medium of<br />

Methanotrophs and collection of methane oxidizing bacteria obtained. A special<br />

system was also designed for SCP production from methane. Twenty tree<br />

strains were isolated. The highest growth rate microorganism was chosen from<br />

the obtained collection. This strain was Gram negative, aerobic, nonpigmented,<br />

non-motile, and rod shape. Biochemical tests were used for<br />

recognizing the bacterium. The optimum condition for SCP production from<br />

natural gas, such as nitrogen source, pH of medium, temperature and<br />

atmosphere (air: methane) studied in flask and in fermenter.<br />

The selected bacterium cultured in fermenter and the growth rate (OD – Time)<br />

diagram drew from obtained results. Then growth acceleration calculated with<br />

mathematics software in computer and optimum condition obtained in<br />

maximum acceleration.<br />

Finally, for recognition of chosen bacterium, molecular method of partial 16S<br />

ribosomal DNA sequence analysis was used and apparent that bacterium was<br />

from unclassified β-proteobacteriase Aminomonas genus that are obligate<br />

methylotrophs. Compared with Aminomonas genus this strain can use methane<br />

as a sole carbon source and growth on nutrient agar (facultive methanotroph).<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PI 05<br />

Secretome analysis of plant growth promoting<br />

rhizobacterium Bacillus amyloliquefaciens FZB42<br />

K. Kierul *1 , X. Chen 2 , B. Voigt 3 , R. Borriss 1<br />

1 Biologie/Bakteriengenetik, Humboldt Universität <strong>zur</strong> Berlin, Berlin, Germany<br />

2 Institute for Microbiology Department of Microbial Physiology, Ernst-Moritz-<br />

Arndt-University Greifswald, Greifswald, Germany<br />

Bacillus amyloliquefaciens FZB42 is a Gram-positive bacterium which<br />

possesses the ability to competitively colonize plant roots and stimulate plant<br />

growth. Compared to other plant growth–promoting Gram-negative<br />

Pseudomonas rhizobacteria, relatively little is known about specific<br />

mechanisms of Gram-positive bacteria un<strong>der</strong>lying beneficial plant-microbe<br />

interactions.<br />

To elucidate the plant growth promoting processes of Bacillus<br />

amyloliquefaciens FZB42, a proteomic approach was used to identify secretory<br />

proteins. To simulate the growth condition in the natural environment, cells<br />

were cultured in a medium with addition of soil extract in presence or absence<br />

of root exudates in or<strong>der</strong> to evaluate the role of exudates in plant-microbe<br />

interactions. Using in silico analysis, 264 genes were found to encode proteins<br />

with putative secretion signals [1]. 91 proteins were experimentally detected in<br />

this work. Many of them possess counterparts in the secretome of B. subtilis<br />

168 [2]. Genes like ganA (predicted arabinogalactan endo-1,4-betagalactosidase),<br />

phy ( phytase), sacB (levansucrase) present in both genomes of<br />

BS168 and FZB42 could not be detected in the secretome of B. subtilis 168 in<br />

the LB-Medium [2]. Our investigation showed an over-expression of these<br />

proteins in the presence of root exudates. This findings strongly suggest that<br />

rhizobacteria like FZB42 can use plant residues as nutrients.<br />

To un<strong>der</strong>stand regulation of the secretory protein expression, we performed<br />

secretome analysis of mutants defected in synthesizing sigma factors<br />

(especially extra- cytoplasmic function (ECF) sigma factors) and global<br />

regulators such as DegU.<br />

[1] Chen et al. Comparative analysis of the complete genome sequence of the<br />

plant growth–promoting bacterium Bacillus amyloliquefaciens FZB42. Nat<br />

Biotechnol. 2007 Sep;25(9):1007-14.<br />

[2] Tjalsma et al. Proteomics of protein secretion in Bacillus subtilis: separating<br />

the‘‘secrets’’ of the secretome. Microbiol Mol Biol Rev. 2004 Jun.<br />

PJ 01<br />

Regulation of Immuno-pathological Response in Mouse<br />

Lungs by TGF-β1 After Influenza A Virus Infection<br />

V. Srivastava *1 , M. Khanna 2<br />

1 Microbiology, Auroprobe Research Institute, Delhi, India<br />

2 Respiratory Virology, V.P. Chest Institute, Delhi, India<br />

Background: TGF-β is a potent immunomodulator and regulates the<br />

inflammatory process in a complex biphasic fashion. The immune response to<br />

influenza A virus is characterized by an influx of both macrophages and<br />

lymphocytes into the lungs of the infected host. In general, the pathogenesis of<br />

influenza infection can be divided into two phases, the cellular events that<br />

precede lymphocyte invasion and those that follow it. We hypothesize that the<br />

TGF-β negatively regulates the inflammatory response by regulating<br />

lymphocyte influx to the airway and further modulating release of<br />

proinflammatory and<br />

anti-inflammatory cytokines.<br />

Methods: Eight-week-old BALB/c mice were intranasally instilled with<br />

influenza A virus (A/Udorn/317/72/H3N2), 4.1x103 PFU of virus in 50µl of<br />

allantoic fluid or mock infected 50µl of allantoic fluid. rTGF-β1 administered<br />

to mice by giving intravenous injection of rTGF-β1, 0.5µg/ Kg body weight of<br />

mouse. The mice were euthanized on days 3, 5 and 7 postinfection for the<br />

analysis of parameters.<br />

Results: We observed an increase of lymphocyte count both on 3rd and 5th day<br />

p.i however administration of rTGF-β1 with virus reduced the lymphocyte<br />

count. An increase of INF-γ level observed 3rd day of post infection however<br />

IL-10 level was maximum on 7th day and INF-γ level reduced to basal level on<br />

7th day. Simultaneous administration of rTGF-β1 with virus instillation<br />

inhibited release of INF-γ level on third day and increased level of IL-10 level<br />

seventh day.<br />

Conclusions: rTGF- β1 acts as an immunomodulatory cytokine and inhibits<br />

lymphocyte influx after virus infection and lymphocyte activation. It modulates<br />

the inflammatory process by inhibiting INF-γ a proinflammatory cytokine and<br />

increased release of IL-10, which is an anti-inflammatory cytokine. rTGF- β1<br />

affects recruitment of inflammatory cells at the site of inflammation by<br />

inhibiting lymphocyte invasion and interfering cytokines mediated<br />

inflammatory cascade by less involvement of lungs.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PJ 02<br />

Expression and physiological relevance of Agrobacterium<br />

tumefaciens phosphatidylcholine biosynthesis genes<br />

S. Klüsener *1 , M. Aktas 1 , K.M. Thormann 2 , M. Wessel 1 , F. Narberhaus 1<br />

1 Microbial Biology, Ruhr-University Bochum, Bochum, Germany<br />

2 Department of Ecophysiology, Max Planck Institute for Terrestial<br />

Microbiology, Marburg, Germany<br />

Phosphatidylcholine (PC, lecithin) is the major phospholipid in eukaryotic<br />

membranes whereas only 10 % of all bacteria are predicted to synthesize PC. In<br />

Rhizobiaceae, including the phytopathogenic bacterium Agrobacterium<br />

tumefaciens [1], PC is essential for the establishment of a successful hostmicrobe<br />

interaction.<br />

A. tumefaciens produces PC via two alternative pathways, the methylation<br />

pathway and Pcs pathway. The responsible genes pmtA (coding for a<br />

phospholipid N-methyltransferase) and pcs (coding for a PC synthase),<br />

respectively, are located on the circular chromosome of A. tumefaciens C58.<br />

Recombinant expression of pmtA and pcs in Escherichia coli revealed that the<br />

individual proteins carry out the annotated enzyme functions. Both genes and a<br />

putative ABC transporter operon downstream of PC are constitutively<br />

expressed in A. tumefaciens. The amount of PC in A. tumefaciens membranes<br />

reaches around 23 % of total membrane lipids. We show that PC is distributed<br />

in both the inner and outer membranes [2]. Loss of PC results in reduced<br />

motility and increased biofilm formation, two processes known to be involved<br />

in virulence. Our work documents the critical importance of membrane lipid<br />

homeostasis for diverse cellular processes in A. tumefaciens.<br />

[1] Wessel M., Klüsener S., Gödeke J., Fritz C., Hacker S., Narberhaus F.<br />

(2006) Mol. Microbiol. 62, 906-915<br />

[2] Klüsener S., Aktas M., Thormann K.M., Wessel M., Narberhaus F. (<strong>2009</strong>)<br />

J. Bacteriol., in press<br />

PJ 03<br />

Activation of Ingested Nitrate Reducers and Denitrifiers in<br />

the Earthworm Gut is Dependent on the Earthworm<br />

Feeding Guilds<br />

P.S. Depkat-Jakob *1 , M.A. Horn 1 , H.L. Drake 1<br />

1<br />

Department of Ecological Microbiology, University of Bayreuth, Bayreuth,<br />

Germany<br />

Denitrification occurs in the anoxic earthworm alimentary canal and is<br />

associated with the in vivo emission of N2 and the greenhouse gas N2O.<br />

Ingested soil denitrifiers appear to be selectively activated during gut passage.<br />

The effect of earthworm feeding guilds (endogeic, anecic, or epigeic) on the<br />

diversity and activity of nitrate reducers and denitrifiers in the earthworm gut<br />

was investigated. Phylogenetic analysis of the structural genes narG and nosZ<br />

indicated that gut nitrate reducers and denitrifiers originate from soil. Richness<br />

and diversity analyses of gene libraries at different evolutionary distances<br />

showed a trend towards more similar sequences in the alimentary canal and<br />

more distantly related sequences in soil, indicating a selective activation of<br />

detected soil-<strong>der</strong>ived nitrate reducers and denitrifiers in the alimentary canal. T-<br />

RFLPs of narG and nosZ were different between feeding guilds. NarG-T-RFs<br />

indicative of Actinobacteria and Proteobacteria were abundant in gut and soil<br />

samples, respectively. NosZ-T-RFs were mainly indicative of Alpha-<br />

Proteobacteria. Feeding guild-related differences in T-RFs of nosZ-transcripts<br />

were more pronounced than of nosZ genes. These collective findings suggest<br />

that earthworm feeding guilds impact on the diversity and selective activation<br />

of ingested nitrate reducers and denitrifiers.<br />

PJ 04<br />

Identifying symptom specificity determinants of S.<br />

reilianum by genome comparison<br />

H. Ghareeb *1 , E. Meyer 1 , R. Kahmann 1 , J. Schirawski 1<br />

1 Organismic interactions, Max-Planck-Institute for terrestrial Microbiology,<br />

Marburg, Germany<br />

111<br />

Smut fungi are biotrophic plant pathogens that infect economically important<br />

hosts. Typical symptoms of a smut infection are the appearance of black spores<br />

usually in the plant inflorescence. Ustilago maydis and Sporisorium reilianum<br />

are two smut fungi that parasitize the same host, but cause species-specific<br />

symptoms, e.g., U. maydis leads to tumor development, whereas S. reilianum<br />

triggers phyllody and spore formation in the inflorescence as well as an<br />

increase in the number of ears per plant.

112<br />

We recently sequenced the genomes of U. maydis, S. reilianum and Ustilago<br />

hordei, a barely pathogen. We used genome comparison to identify genes<br />

involved in symptom specificity determinants. To this end, we investigated S.<br />

reilianum-unique genes and gene clusters encoding proteins of low sequence<br />

conservation by deletion analysis. Individual deletion of 21 unique genes did<br />

not influence symptom specificity of S. reilianum. However, deletion of gene<br />

cluster 19A2 encoding four secreted proteins with low sequence conservation<br />

led to reduced virulence as well as a reduction in the number of ears per plant,<br />

indicating that this region carries symptom specificity determinants. To track<br />

the gene(s) responsible for the increase in the ear number, we have generated<br />

single gene deletions of each of the four genes of cluster 19A2. Our results<br />

suggest that symptom specificity of S. reilianum is determined by genes with<br />

low sequence conservation between closely related smut fungi.<br />

PJ 05<br />

Adhesion of an anti-inflammatory B. bifidum strain to<br />

intestinal epithelial cells is mediated by proteinaceous cell<br />

wall components<br />

M. Gleinser *1 , C.U. Riedel 1<br />

1 Institut für Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany<br />

Several beneficial effects for the host have been attributed to the presence of<br />

bifidobacteria in the intestinal tract. Adhesion of these bacteria to intestinal<br />

epithelial cells (IECs) could be an important prerequisite for their probiotic<br />

effects. Recent in vitro studies of our group showed that especially those<br />

strains of bifidobacteria that show good adhesion to IECs have a high antiinflammatory<br />

potential.<br />

We analysed the adhesive structures of B. bifidum S17 a strain that showed<br />

excellent adhesion to IECs in previous studies as well as potent inhibition of<br />

LPS-induced NF- κB activation. For this purpose, we established a method to<br />

obtain clean cell wall, membrane and cytoplasmic fractions of bifidobacteria.<br />

We were able to show, that in particular the cell wall fraction inhibits adhesion<br />

of whole cells of B. bifidum S17 to differentiated monolayers of IECs. To test<br />

whether the structures responsible for adhesion of B. bifidum S17 are cell<br />

surface proteins B. bifidum S17 was treated with pronase, lipase, and periodate<br />

and cell wall fractions were prepared after the treatment. Several bands are<br />

absent in the cell wall fraction from bacteria that were treated with pronase in<br />

comparison to the cell wall fraction from the untreated B. bifidum S17.<br />

Furthermore, treatment of B. bifidum S17 with pronase significantly decreased<br />

adhesion to IECs whereas treatment with lipase and periodate did not show any<br />

effects on adhesion. This indicates that proteinaceous cell surface components<br />

are involved in adhesion of B. bifidum to IECs.<br />

PJ 06<br />

Alkylation and accumulation of mercury in tissue of Eisenia<br />

foetida and isolated gut microorganisms<br />

B. Knopf *1 , H. König 1<br />

1 Institut for Microbiology and Wineresearch, Johannes Gutenberg University<br />

Mainz, Mainz, Germany<br />

Qualitative speciation and determination of organomercury compounds at ultra<br />

trace levels are of special interest, because toxicity, bioavailability and<br />

detoxification depend mainly on the chemical form of this element. Much is<br />

known about the mercury circle in aquatic systems and the microbial<br />

methylation but less about the bioavailability in soil and the effect on soil<br />

feeding invertebrates. Of special interest is the circle of mercury in soil because<br />

of the high methylation potential by microorganisms. Mainly sulphate reducing<br />

bacteria are responsible for the methylation of mercury. This omnipresents of<br />

microorganism in the gut of soil living and feeding invertebrates is a way to<br />

alkylated mercury and accumulate higher concentrations of organomercury<br />

compounds. The development of a food chain model which shows an increase<br />

of methyl mercury concentrations by an increasing trophic level is of special<br />

interest. This could show the way of organomercury accumulation into human<br />

tissue.<br />

For the determination the isotope specific ICP-MS coupled to gas<br />

chromatography is used because of the high sensitivity of this method for the<br />

speciation of volatile organometalic compounds. As an invertebrate model<br />

organism the annelidae Eisenia foetida was chosen. First measurements showed<br />

a methylation of mercury and an accumulation in tissue of Eisenia foetida. For<br />

detailed studies, cultures of the microbial flora of the worm gut were isolated<br />

and enriched with inorganic mercury.<br />

Water soluble organomercury compounds were transferred into peralkylated by<br />

<strong>der</strong>ivatization with sodium tetra-(n-propyl)-borate and extracted with hexane.<br />

Determination of the extracted methyl mercury was done by the isotope<br />

dilution method.<br />

PJ 07<br />

Ecological impact of biologically active metabolites<br />

produced by Laminaria saccharina associated Pseudomonas<br />

sp. strains<br />

J. Wiese *1 , A. Labes 1 , F. Goecke 1 , I. Kajahn 1 , G. Lang 1 , K. Nagel 1 , I.<br />

Schneemann 1 , V. Thiel 2 , J.F. Imhoff 1<br />

1 Kieler Wirkstoff-Zentrum am IFM-GEOMAR, IFM-GEOMAR, Kiel, Germany<br />

2 Marine Mikrobiologie, Leibniz-Institut für Meereswissenschaften, Kiel,<br />

Germany<br />

Laminaria saccharina belongs to the brown macroalgae and is distributed in<br />

temperate to polar rocky coastal ecosystems [1]. The alga was shown to<br />

harbour a quite diverse community of antimicrobially active bacteria, including<br />

Pseudomonas strains [2].<br />

In this study, biologically active secondary metabolites produced by L.<br />

saccharina associated Pseudomonas sp. strains were extracted, purified and<br />

identified. It was shown that the Pseudomonas sp. strains produce a number of<br />

secondary metabolites displaying a strong antimicrobial activity against<br />

bacteria and fungi. Among others, the polyketides rhizoxin S1 and S2, 2,4diacetyl-phloroglucinol,<br />

and pyoluteorin were identified.<br />

Due to their broad range of inhibited organisms (Gram-positive, Gram-negative<br />

bacteria as well as fungi) of the metabolites, the mode of action seems to be<br />

non-specific for different microbial species, families or classes. These<br />

secondary metabolites presumably positively effect the survival of their<br />

producers by inhibition of other competing surface-colonising microorganisms.<br />

Further, they might also protect L. saccharina against decomposing and<br />

pathogenic microorganisms. Such a positive effect of bacterial metabolites on<br />

the host algae possibly indicates a mutualistic association between algae and<br />

bacteria.<br />

[1] Bartsch, I. et al. 2007. Europ J Phycol 43:1-86.<br />

[2] Wiese, J. et al. 2008. Mar Biotech, online first.<br />

PJ 08<br />

A Sticky Guest Manipulating its Host Response: The<br />

„extracellular adherence protein (EAP)“ of S. aureus<br />

blocks fast immune responses while prolonging the overall<br />

inflammatory response.<br />

A.C.S. Sobke *1 , V.O. Frick 2 , T. Hartung 3 , K.T. Preissner 4 , M. Hannig 5 , M.<br />

Herrmann 6 , E. Straube 1 , D. Selimovic 7<br />

1 Institute of Medical Microbiology, Medical University Laboratories,<br />

Friedrich-Schiller-University, Jena, Jena, Germany<br />

2 Department of General-, Visceral-, Vascular- and Pediatric Surgery,<br />

University of Saarland Hospital, Homburg/Saar, Homburg/Saar, Germany<br />

3 Biochemical Pharmacology, University of Konstanz, Konstanz, Konstanz,<br />

Germany<br />

4 Institute of Biochemistry, Justus-Liebig-University, Giessen, Giessen,<br />

Germany<br />

5 Clinic of Operative Dentistry and Periodontology, University of Saarland<br />

Hospital, Homburg/Saar, Homburg/Saar, Germany<br />

6 Institute of Medical Microbiology and Hygiene, University of Saarland<br />

Hospital, Homburg/Saar, Homburg/Saar, Germany<br />

7 INSERM U595, Louis Pasteur University, Strasbourg, Strasbourg, France<br />

Staphylococcus aureus remains a major cause of postsurgical site infections,<br />

endocarditis and sepsis. EAP belongs to a new group of adhesins (SERAM),<br />

which are non-covalently bound and secreted into the bacterial environment.<br />

EAP was previously shown to interfere with inflammatory and angiogenic<br />

responses by inhibiting leukocyte recruitment and blocking Ras- activation.<br />

Here the effect of EAP on the inflammatory response of primary endothelial<br />

cells and monocytes, alone and in combination with other PAMPs (LPS, LTA,<br />

MDP and Pam3Cys) was examined.<br />

In HUVEC, EAP treatment resulted in a weak increase in p38- and Aktactivation<br />

and a slight, but significant increase in ICAM-1 expression (2.12<br />

over basal in qRT). This process depended on ICAM-1 activation (sensitive to<br />

blockage with inhibitory monoclonal antibody). There was a strong, synergistic<br />

effect of EAP on LPS- induced ICAM-1 expression in HUVEC (3.88 versus<br />

8.34 over basal) and PBMC (2.40 versus 6.45 over basal). The effect was<br />

specific for co-treatment with LPS and correlated with an increase in NFκB-<br />

activation (p65 nuclear translocation). Expression of the procoagulant TF,<br />

induced by TNFα in HUVEC and by LPS in PBMC, was strongly inhibited by<br />

EAP (30.55 versus 15.74 and 2.24 versus 0.78 over basal, respectively).<br />

Moreover, early (4 hrs) secretion of TNFα in LPS- or LTA- stimulated PBMC<br />

was completely blocked by EAP, whereas late secretion (14 hrs) was induced<br />

and additively enhanced.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Taken together, EAP through its negative impact on MEK/ERK-activation<br />

effectively blocks fast immune responses, while at the same time the NFκBdependent<br />

inflammatory response is actually prolonged.<br />

PJ 09<br />

Rekombinant Clostridium acetobutylicum expressing<br />

Clostridium perfringens enterotoxin (CPE) for treatment of<br />

pancreatic cancer<br />

S. König *1 , P. Dürre 1<br />

1 Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany<br />

Pancreatic cancer is one of the most malignant solid tumours. Due to the late<br />

clinical presentation, most patients only receive palliative treatment and die<br />

within 3 years. Genetically modified clostridia open a new possibility of antitumour<br />

treatment with enormous potential. Clostridial spores only germinate in<br />

the hypoxic regions of solid tumours and can deliver therapeutic proteins<br />

directly to their targets. CPE is one out of 15 toxins known from C. perfringens,<br />

is produced and released during sporulation, and was shown to interact with<br />

claudin receptors, which are 1000fold overexpressed in pancreatic carcinoma<br />

cell lines. The binding of CPE to this receptor results in the formation of pores<br />

that ultimately cause cell death. C. acetobutylicum DSM 792 was transformed<br />

with a vector carrying the cpe gene, fused with a signal peptide sequence, and<br />

controlled by the bdhA promoter. The modified strain produced and secreted<br />

500 ng/ml of the toxin into the surrounding medium. This production is<br />

independent of sporulation and starts in the early exponential growth phase.<br />

The level of production was sufficient to cause cell death in cytotoxicity tests<br />

with a pancreatic carcinoma cell line, but proved to be too low for therapy in an<br />

in vivo mouse model. Current work focuses on improved expression including<br />

the use of different signal peptides and promoter sequences. In E. coli, the<br />

signal peptide of a putative pectate lyase of C. acetobutylicum allowed highlevel<br />

expression.<br />

PJ 10<br />

Mycoplasma suis invades porcine erythrocytes<br />

K. Groebel *1 , K. Hoelzle 1 , M.M. Wittenbrink 1 , U. Ziegler 2 , L.E. Hoelzle 1<br />

1 Institute of Veterinary Bacteriology, University of Zurich, Zurich, Switzerland<br />

2 Center for Microscopy and Image Analysis, University of Zurich, Zurich,<br />

Switzerland<br />

Mycoplasma suis belongs to the hemotrophic mycoplasmas and causes an<br />

infectious anemia in pigs. According to the present state of knowledge, these<br />

organisms adhere to the surface of the red blood cells but do not invade them.<br />

We found a novel M. suis isolate that caused severe anemia in pigs with a fatal<br />

course of the disease despite of antibiotic treatment. Interestingly, only<br />

marginal numbers of the bacteria were visible on and between the erythrocytes<br />

in acridine orange-stained blood smears for acutely diseased pigs, whereas very<br />

high loads of M. suis were detected in the same blood samples by quantitative<br />

PCR. These findings indicated that M. suis is capable of erythrocyte invasion.<br />

By use of fluorescent labeling of M. suis and examination by confocal laser<br />

scanning microscopy, as well as scanning and transmission electron<br />

microscopy, we proved that M. suis was located intracellular. This organism<br />

invades erythrocytes in an endocytosis-like process and is initially surrounded<br />

by two membranes, and it was also found floating freely in the cytoplasm. In<br />

conclusion, we were able to prove for the first time that a member of the<br />

hemotrophic mycoplasmas is able to invade the erythrocytes of its host. Such<br />

colonization should protect the bacterial cells from the host’s immune response<br />

and hamper antibiotic treatment. In addition, an intracellular life cycle may<br />

explain the chronic nature of hemotrophic mycoplasma infections and should<br />

serve as the foundation for novel strategies in hemotrophic mycoplasma<br />

research (e.g., treatment or prophylaxis).<br />

PJ 11<br />

Enterobactin and Quorum Sensing (QS) in Escherichia coli<br />

( E.coli )<br />

S.I. Müller *1 , M. Valdebenito 2 , K. Hantke 1<br />

1 Microbiology/Membrane Physiology, Organismic Interactions, Eberhard<br />

Karls Universität Tübingen, Tübingen, Germany<br />

2 Microbiology/Organismic Interactions, Eberhard Karls Universität Tübingen,<br />

Tübingen, Germany<br />

Iron is an essential element for bacteria, involved in vital processes.<br />

Problematic is its bad solubility un<strong>der</strong> aerobic conditions in its oxidised form<br />

(Fe 3+ ). Many microorganisms found a solution un<strong>der</strong> iron-limiting conditions:<br />

They produce so called si<strong>der</strong>ophores, which scavenge ferric iron molecules,<br />

transport them into the cells and provide bacteria with this important element.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Enterobactin is one of these si<strong>der</strong>ophores, common in different bacterial<br />

species.<br />

In literature there is a cognate two-component system described, that is<br />

involved in QS regulatory systems. It consists of a histidine kinase, QseC, and a<br />

response regulator, QseB. They interact due to a stimulation through<br />

autoinducer-3 (QS signal molecule) or host hormones (epinephrine and<br />

norepinephrine) resultant in transcriptional changes concerning motility and<br />

virulence.<br />

In different experiments, we found a participation of QseB/C in iron uptake<br />

systems in E.coli during iron starvation.<br />

QseC downregulates the enterobactin-system; This was shown for two parts,<br />

the ferric enterobactin esterase (Fes), which releases iron out of enterobactin<br />

and the ferric enterobactin outer membrane receptor (FepA), which transports<br />

ferric enterobactin into the periplasm.<br />

In contrast, QseC stimulates the salmochelin-system, a virulence-associated<br />

si<strong>der</strong>ophore-system which provides microorganisms with the facility to escape<br />

hosts defence. Stimulation was shown for the ferric salmochelin outer<br />

membrane receptor (IroN).<br />

We think, that in addition to (nor-)epinephrine and autoinducer-3, also the<br />

catecholate si<strong>der</strong>ophores in the periplasm influence the activity of QseB/C. This<br />

results in adjustment of the iron-depending metabolism to the present situation<br />

in the host.<br />

PJ 12<br />

Who is present and who is active - sponge symbiont<br />

diversity and activity assessed by 16S rDNA and 16S rRNA<br />

analysis<br />

S. Schmitt *1 , J. Kamke 1 , M. Taylor 1<br />

1 School of Biological Sciences, University of Auckland, Auckland, New Zealand<br />

Marine sponges are well known for their association with microorganisms.<br />

These microbial communities can be highly diverse, with members of more<br />

than 20 bacterial and 2 archaeal phyla now identified in sponges. However, in<br />

contrast to our burgeoning knowledge of sponge symbiont diversity,<br />

remarkably little is known about the activity of these organisms. Using the New<br />

Zealand sponge Polymastia sp. as a model, we sought to determine which<br />

members of the bacterial community were active. 16S rDNA- and 16S rRNAbased<br />

clone libraries were constructed from DNA and RNA co-extracted from<br />

the sponge. The DNA-<strong>der</strong>ived library, which revealed the diversity that is<br />

present, was dominated by a single Alphaproteobacteria cluster while other<br />

Alphaproteobacteria, Actinobacteria and Spirochaetes were minor components<br />

of the library. The second, RNA-<strong>der</strong>ived library, identified those members of<br />

the bacterial community that are metabolically active within Polymastia sp..<br />

This active fraction included the main Alphaproteobacteria cluster, together<br />

with the Actinobacteria and Spirochaetes phylotypes. Overall, the rRNA<strong>der</strong>ived<br />

library was more diverse than the rDNA-<strong>der</strong>ived library, containing<br />

also phylotypes affiliated with the Gammaproteobacteria. These latter<br />

organisms might represent rare but highly active members of the sponge<br />

microbial community. Our data provide the first community-wide assessment<br />

of activity among marine sponge-associated microbes, and will ultimately lead<br />

to a better un<strong>der</strong>standing of the functional role of sponge symbionts.<br />

PJ 13<br />

characterisation and infection of an eye-isolated amoebal<br />

strain<br />

C. Unger *1 , M.A. Porta 2 , T. Juretzek 2 , C. Lück 1<br />

1<br />

Institut für medizinische Mikrobiologie und Hygiene, TU Dresden, Dresden,<br />

Germany<br />

2<br />

Institut für Mikrobiologie und Hygiene, Carl-Thiem-Klinikum Cottbus,<br />

Cottbus, Germany<br />

113<br />

A keratitis mostly is caused by bacteria (e.g. Staphylococcus aureus,<br />

Streptococcus pneumoniae, Listeria monocytogenes), viruses (e.g. Herpes<br />

corneae), fungi (e.g.Candida spp., Aspergillus spp.) or also amoeba, especially<br />

Acathamoeba spp.. The infection of the eye is often induced by contaminated<br />

solution used for storage of contact lenses. These tap water may contain<br />

legionellea. This might become a problem, because amoeba are the natural host<br />

of legionellae in the environment.<br />

An amoebal strain isolated from a contact-lenses associated keratitis-patient<br />

was characterized by culture and DNA-sequenzing of the 18s-rDNA-gene.<br />

After adaption to amoebal medium the isolated amoeba-strain was infected with<br />

Legionella pneumophila strains. We used a wildtyp of the Legionella<br />

pneumophila serogroup 1 strain Corby and a pad-negativ-mutant (Legionella<br />

pneumophila amoebal adhesin) CP7. In previous studies we showed, that the<br />

mutant is reduced in adherence to amoeba.

114<br />

It was found, that the infection of the isolated Acathamoeba castellanii strain is<br />

similar to the other tested amoebal strains. Furthermore the test with the CP7mutante<br />

is comparable with the results of a former test with other<br />

Acathamoeba- strains.<br />

The role of symbiotic and pathogenic bacteria multiply within Acanthamoeba<br />

spp. and their possible role in eye infection needs further investigations.<br />

PJ 14<br />

A proteogenomic comparison of bacterial endosymbionts<br />

from the deep sea tube worms Tevnia jerichonana and Riftia<br />

pachyptila<br />

A. Gardebrecht *1 , S. Markert 2 , H. Felbeck 3 , A. Thürmer 4 , H. Liesegang 4 , D.<br />

Albrecht 5 , R. Daniel 4 , S. Sievert 6 , M. Hecker 5 , T. Schwe<strong>der</strong> 1<br />

1 Institute of Pharmacy, Ernst-Moritz-Arndt-University, Greifswald, Germany<br />

2 Symbiosis Group, Institute of Marine Biotechnology, Greifswald, Germany<br />

3 Scripps Institution of Oceanography, University of California San Diego, La<br />

Jolla, United States<br />

4<br />

Göttingen Genomics Laboratory, Georg-August-University, Göttingen,<br />

Germany<br />

5<br />

Institute of Molecular Biology, Ernst-Moritz-Arndt-University, Greifswald,<br />

Germany<br />

6<br />

Microbial Ecology and Physiology Laboratory, Woods Hole Oceanographic<br />

Institution, Woods Hole, United States<br />

The vestimentiferan tube worm Tevnia jerichonana is a primary colonizer of<br />

hydrothermal vent sites in the eastern Pacific. Like the well-studied „giant“<br />

tube worm Riftia pachyptila, the smaller T. jerichonana lacks a digestive<br />

system and relies entirely on chemolithoautotrophic endosymbionts. Recent<br />

rRNA-based studies revealed that both tube worms harbor the same species of<br />

so far uncultured γ-proteobacteria. The endosymbionts are contained in a<br />

specialized organ, the trophosome, and are provided with H2S, O2 and CO2 via<br />

the worms’ blood system. The symbionts oxidize sulfide and autotrophically<br />

produce organic carbon as a nutrient source for their hosts. In this study, the<br />

genome of the T. jerichonana symbiont was sequenced and provided the basis<br />

for a comprehensive proteome analysis. More than 1000 intracellular bacterial<br />

proteins were analyzed by two-dimensional gel electrophoresis combined with<br />

mass spectrometry. This survey yielded a first impression of the uncultured<br />

Tevnia symbiont’s metabolic strategies. Furthermore, essential metabolic<br />

pathways were compared with those of the R. pachyptila symbiont, to<br />

investigate if their most abundant enzymes are involved in identical processes<br />

like sulfide oxidation and carbon fixation. This comparative genomic and<br />

proteomic approach revealed a high degree of analogy between the two<br />

symbionts. This supports the hypothesis that different tube worm genera are<br />

infected by the same symbionts from a free-living bacterial population in the<br />

hydrothermal vent ecosystem.<br />

PJ 15<br />

Neutrophil extracellular traps and Aspergillus fumigatus<br />

S. Wolke *1 , F. Lessing 1 , A. Gehrke 1 , O. Kniemeyer 1 , M. Hasenberg 2 , M.<br />

Gunzer 2 , A. Brakhage 1<br />

1 Molecular and Applied Microbiology, Leibniz Institute for Natural Product<br />

Research and Infection Biology -Hans-Knoell-Institute-, Friedrich Schiller<br />

University Jena, Jena, Germany<br />

2 Institue for Molecular and Clinical Immunology, Otto von Guericke University<br />

Magdeburg, Magdeburg, Germany<br />

Aspergillus fumigatus is the most important airborne fungal pathogen. Conidia<br />

as the infectious agent infiltrate the lungs and get in contact with the human<br />

immune system. The first line of defense is represented by alveolar<br />

macrophages and neutrophil granulocytes. From Candida albicans it is known<br />

that neutrophils are able to attack the pathogen by beneficial suicide<br />

(Brinkmann and Zychlinsky, 2007). In this ROI dependent mechanism, the<br />

neutrophils release DNA filaments covered with histones and granule proteins.<br />

These sticky filaments are known as neutrophil extracellular traps (NETs).<br />

Steinberg and Grienstein (2007) named this process NETosis. We coincubated<br />

A. fumigatus germlings with human neutrophils for up to three hours and took<br />

samples for CLSM and SEM analysis. NET like structures were clearly visible.<br />

To analyse the role of NET formation in killing of A. fumigatus during<br />

coincubation we used an XTT assay. Furthermore, we added the NADPHoxidase<br />

inhibitor DPI and the ROI scavenger glutathione to investigate the<br />

dependency of NET formation on the induction of an oxidative burst. Killing of<br />

A. fumigatus appeared within the first 30 min but clear NETs formation was not<br />

observed earlier than 120 min. Inhibition of ROI production did not influence<br />

killing, but NET formation seemed less apparent. Taken together, we showed<br />

that neutrophils form NETs after contact with A. fumigatus mycelium.<br />

Furthermore NET formation, but not killing of A. fumigatus was dependent on<br />

ROI formation. We assume that NETs have the function to agglutinate A.<br />

fumigatus hyphae, to constrain the infection and to recruite other immune cells.<br />

PJ 16<br />

A crucial role for the fibrinogen binding proteins coagulase<br />

and Efb in the Staphylococcus aureus-Candida interaction<br />

C. Fehrmann *1 , K. Schroe<strong>der</strong> 1 , C. Neumann 1 , B. Kehrel 2 , K. Jurk 2 , W. Fegeler 1 ,<br />

K. Becker 1 , C. Heilmann 1<br />

1 Institute of Medical Microbiology, University Hospital of Münster, Münster,<br />

Germany<br />

2 Experimental and Clinical Haemostaseology, University Hospital of Münster,<br />

Münster, Germany<br />

The interaction of Staphylococcus aureus with Candida species either directly<br />

or mediated by bridging molecules such as extracellular matrix and plasma<br />

proteins leading to the formation of mixed species biofilms is thought to play a<br />

critical role in S. aureus – Candida coinfections. To identify S. aureus factors<br />

involved in the interaction with Candida, we used a phage display library of S.<br />

aureus 4074, which was affinity-panned against biofilms of C. dubliniensis in<br />

the presence or absence of fibrinogen (Fg) and fibronectin. Repeatedly affinityselected<br />

and specific binding hybrid phages contained portions of the genes<br />

encoding the Fg-binding proteins coagulase or Efb. The activity of the<br />

coagulase or Efb at the surface of Candida cells in the presence of Fg might<br />

enhance phagosomal escape. The coagulase promotes the formation of fibrin<br />

from Fg that may protect the yeast against phagocytosis and Efb binds the<br />

complement component C3b that may also lead to reduced phagocytosis of<br />

Candida. To test these hypotheses, we expressed the respective genes in<br />

Escherichia coli and used the purified proteins to analyse their roles in<br />

phagocytosis of Candida cells by granulocytes in the whole blood. We found<br />

that the rate of phagocytosis was reduced by 40% and 70% in the presence of<br />

rEfb and rCoa, respectively. Additionally, the formation of fibrin at the surface<br />

of Candida mediated by the coagulase was verified by confocal laser scanning<br />

microscopy. Therefore, the presence of S. aureus in a mixed infection might be<br />

beneficial for Candida to persist.<br />

PJ 17<br />

The impact of host-adaptation on invasion and intracellular<br />

persistence of Salmonella serovars in different host species<br />

S. Maurischat *1 , K. Tedin 1 , L. Wieler 1<br />

1 FB Veterinärmedizin, Institut für Mikrobiologie und Tierseuchen, Freie<br />

Universität Berlin, Berlin, Germany<br />

Despite the availability of antibiotics and effective treatment, with over 1.3<br />

billion cases of disease annually, infections by the facultative intracellular<br />

bacterium Salmonella enterica remain of major global importance. Of the more<br />

than 2500 serovars, those belonging to subspecies 1 are mainly responsible for<br />

these infections, which can range from asymptomatic to gastroenteritis or<br />

systemic infections with fatal outcome. In addition to bacterial factors, less well<br />

investigated host factors might be of major importance concerning the outcome<br />

of infection.<br />

To characterize the nature of host-pathogen interactions, in contrast to previous<br />

studies we compared in a more systematic and extensive way the invasiveness<br />

and intracellular persistence of various Salmonella serovars with different host<br />

specificity during infection of intestinal epithelial and macrophage-like cells of<br />

human, pork and chicken origins.<br />

We found that broad host-range serovars like S. Typhimurium and S. Enteritidis<br />

showed invasion and growth rates of a similar high level in all investigated cell<br />

lines. By contrast, the host-adapted serovars (e.g. S. Dublin or S. Choleraesuis)<br />

and host-restricted serovars like S. Gallinarum behaved in a clearly different<br />

way, e.g. less invasive and/or restricted in growth in some cell lines. Further<br />

differences in infection behaviour resulted from raising the temperature during<br />

infection from 37°C to 42°C, a body temperature characteristic for chicken.<br />

Which conclusions the experiments regarding host adaptation, virulence and<br />

pathogenicity allow and how the results correlate with earlier studies especially<br />

comparable in vivo infection studies will be discussed.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PJ 18<br />

Marine Medicine: Learning from Complex Barriers and<br />

Microbiota in the Ocean<br />

R. Metzger *1 , P. Rosenstiel 2 , R.A. Schmitz-Streit 1<br />

1<br />

Institute of General Microbiology, Christian-Albrechts-Universität zu Kiel<br />

(CAU), Kiel, Germany<br />

2<br />

Institute of Clinical Molecular Biology, University Hospital Schleswig-<br />

Holstein, Kiel, Germany<br />

Marine microbial communities are highly diverse and have evolved during<br />

extended evolutionary processes of physiological adaptations un<strong>der</strong> the<br />

influence of a variety of ecological conditions and selection pressures. They<br />

also often occur in association with eukaryotes and harbour an enormous<br />

diversity of microbes with still unknown physiological characteristics.<br />

The surfaces of marine host organisms <strong>bei</strong>ng typically covered by epibiotic<br />

bacteria act as barriers where diverse interactions take place. Since this can be<br />

disadvantageous as well as beneficial for the host, as in pathogenic or<br />

symbiotic/mutualistic relationships, the eukaryote often has the ability to affect<br />

these interactions to shape its microbiota. This is especially achieved by the<br />

means of the innate immune system (e. g. antimicrobial peptides, receptors and<br />

the respective genes encoding for them) which has been kept highly conserved<br />

during evolution due to a high selective pressure. Therefore, the microbial<br />

consortia on marine multicellular host tissues are attractive model systems in<br />

or<strong>der</strong> to gain knowledge on the evolution of genes responsible for functioning<br />

of barriers and the innate immune system.<br />

Using a simple marine invertebrate – the moon jellyfish (Aurelia aurita) - as<br />

model organism, we study the ancient mechanisms of host/microbiota<br />

interactions which may allow un<strong>der</strong>standing human barrier disor<strong>der</strong>s and may<br />

provide insights into the development of so-called barrier disor<strong>der</strong>s in humans<br />

and identify new drug targets. First results studying microbial consortia on<br />

different marine multicellular host tissues including a metagenomic approach<br />

will be presented and discussed.<br />

PJ 19<br />

Assessing the genetic accessibility of Marinobacter sp.<br />

HP15w as a model organism for diatom- bacteria<br />

interactions<br />

E. Käppel *1 , A. Gärdes 1 , M. Ullrich 1<br />

1 School of Engineering and Science, Jacobs University Bremen, Bremen,<br />

Germany<br />

Aggregation of micro-algae, mainly of diatoms, is an important process in<br />

marine pelagic systems often terminating phytoplankton blooms and leading to<br />

the sinking of particulate organic matter in form of marine snow. This process<br />

has been studied extensively, but the specific role of heterotrophic bacteria,<br />

their genes, gene products, and secondary metabolite signals for this process<br />

has largely been neglected. A bilateral model system consisting of the diatom,<br />

Thalassiosira weissflogii, and the bacterial strain, Marinobacter sp. HP15w,<br />

was found suitable for an in-depth analysis by attachment assays, TEP<br />

production, and aggregation experiments. Next, the genetic accessibility of<br />

Marinobacter sp. HP15w was tested by conjugation and electroporation assays.<br />

Several of the tested broad-host range plasmids were successfully introduced to<br />

the bacterial cells and subsequently a transposon mutagenesis was conducted.<br />

Transposon mutants will be screened for randomness of insertion and for<br />

deficiency in chemotaxis and motility using appropriate assays in or<strong>der</strong> to<br />

investigate the particular role(s) of these processes in diatom-bacteria<br />

interactions. Results of this study will indicate whether or not changes in<br />

bacteria-phytoplankton interactions influence the flux and cycling of nutrients<br />

and carbon during marine snow formation and which cellular factors contribute<br />

to these interactions.<br />

PJ 20<br />

Construction of a genomic library for Marinobacter sp.<br />

HP15w<br />

S. Seebah *1 , A. Gärdes 2 , M. Ullrich 2<br />

1 International Max-Planck Research School, Max-Planck Institute for Marine<br />

Microbiologie, Bremen, Germany<br />

2 School of Engineering and Science, Jacobs University Bremen, Bremen,<br />

Germany<br />

The biological pump describes the fate of carbon in the ocean starting from CO2<br />

fixation to carbon recycling and sequestration into the ocean bed. 1-10% of<br />

sequestered CO2 is transported to the deep waters in form of marine snow,<br />

which consists of aggregates composed of inorganic particles, plankton cells,<br />

detritus and faecal material, glued together by a matrix of polymers released by<br />

bacteria and phytoplankton. Phytoplankton-bacteria interactions significantly<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

impact the production of these exopolymers and hence, marine snow formation.<br />

To further our un<strong>der</strong>standing of the molecular phytoplankton-bacteria<br />

interaction, a model system is required. Attachment assays, aggregation studies,<br />

and genetic experiments enabled us to select an in vitro model system<br />

consisting of the diatom, Thalassiosira weissflogii, and marine gammaproteobacterium,<br />

Marinobacter sp. HP15w. To improve the genetic suitability<br />

of the system, a genomic library for Marinobacter sp. HP15w was constructed<br />

using the cosmid vector, pWEB. The library contains 960 clones, each carrying<br />

inserts of approximately 36kb thus covering the genome approximately fivefold.<br />

The library represents an efficient platform to screen for genes of interest<br />

using PCR or Southern blotting and is also suitable for functional screening<br />

assays. Our current research focuses on genes required for motility such as<br />

flagella, pili, and chemotaxis genes , which could be important for the diatombacteria<br />

interaction.<br />

PJ 21<br />

SigB-dependent changes of the secretome in staphylococcus<br />

aureus and its effect on airway epithelial cells<br />

M. Gutjahr *1 , P. Hildebrandt 1 , M. Depke 1 , J. Kalinka 1 , A. Kühn 2 , J. Pané-Farré 2 ,<br />

L. Steil 1 , M. Hecker 2 , U. Völker 1 , E. Hammer 1<br />

1 Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-<br />

Arndt-University of Greifswald, Greifswald, Germany<br />

2 Institute for Microbiology, Ernst-Moritz-Arndt-University of Greifswald,<br />

Greifswald, Germany<br />

Staphylococcus aureus is associated with quite different infectious diseases (i.e.<br />

bacteremia, endocarditis, pneumonia) which is probably related to a whole<br />

collection of different various virulence factors. These proteins are expected to<br />

display niche specific expression patterns that are governed by global regulators<br />

such as agr, SigB and SarA (Sibbald et al., 2006). Some of the virulence factors<br />

seemed to be positively controlled by SigB, whereas the expression of the<br />

majority appears to be suppressed by SigB via a so far unknown mechanism.<br />

As a consequence many extracellular virulence factors are overproduced in<br />

sigB mutant strains.<br />

S. aureus RN1HG and the human bronchial epithelial cell line S9 were used as<br />

model system to study the host-pathogen-interactions. In or<strong>der</strong> to study the<br />

response of the eucaryotic cells to the pathogen without any media effects,<br />

bacterial strains were cultivated in an optimized cell culture medium and the<br />

expression and secretion of virulence factors was monitored by Northern Blot<br />

analysis and 2D-gel based and gelfree proteomics. Aliqots of bacterial cultures<br />

as well as supernatants of RN1HG wildtype and mutant cultures were used to<br />

study the response of airway epithelial cells. In addition to phenotypic changes,<br />

transcriptome and proteome data were recorded to document the gene<br />

expression changes in the eukaryotic host cells. In comparison to the wild type<br />

strain the sigB deficient strain showed significant differences in the<br />

extracellular proteome which are correlated to a higher toxicity of the culture<br />

supernatant to the S9 cells.<br />

[1] Sibbald MJ, Ziebandt AK, Engelmann S, Hecker M, de Jong A, Harmsen<br />

HJ, Raangs GC, Stokroos, I, Ahrends JP, Dubois JY, van Dijl JM. Mapping the<br />

pathways to staphylococcal pathogenesis by comparative secretomics.<br />

Microbiol Mol Biol Rev. (2006) 70: 755-88<br />

PJ 22<br />

Genetic exchange of multidrug efflux pumps among two<br />

enterobacterial species with distinctive ecological niches<br />

N. Al-Karablieh *1 , H. Weingart 1 , M.S. Ullrich 1<br />

1<br />

School of Engineering and Science, Jacobs University Bremen, Bremen,<br />

Germany<br />

115<br />

AcrAB-TolC is the major multidrug efflux system in Enterobacteriaceae and<br />

recognizes various structurally unrelated molecules including antibiotics, dyes,<br />

and detergents. Additionally, in Escherichia coli it mediates resistance to bile<br />

salts. In the plant pathogen, Erwinia amylovora, AcrAB-TolC is required for<br />

virulence and resistance towards phytoalexins. Exchange analysis of AcrAB-<br />

TolC was carried out by complementing mutants of both species defective in<br />

acrB or tolC with alleles from either species to study substrate specificities and<br />

interaction efficiencies of various AcrAB-TolC combinations. As expected, all<br />

four mutants exhibited increased susceptibility profiles for 24 different<br />

antibiotics. Interestingly, all mutants were complemented with acrAB or tolC,<br />

respectively, regardless of the taxonomic origin of the alleles.<br />

Complementation of E. amylovora mutants with respective E. coli genes fully<br />

restored virulence on apple plants. It was concluded that AcrAB and TolC of<br />

both species could interact and that these interactions did not yield in altered<br />

functions despite the divergent ecological niches, to which E. coli and E.<br />

amylovora have adopted.

116<br />

PJ 24<br />

Microarray-based detection of multidrug efflux<br />

transporters in the plant pathogen Pseudomonas syringae<br />

H. Weingart *1 , P. Wecker 2 , A. Ellrott 2 , M. Ullrich 1<br />

1 School of Engineering and Science, Jacobs University Bremen, Bremen,<br />

Germany<br />

2 Mikrobielle Genomik, MPI für Marine Mikrobiologie, Bremen, Germany<br />

Plant-pathogenic bacteria are continually exposed to deleterious chemicals<br />

naturally occurring in their environment such as antimicrobial plant metabolites<br />

and toxins produced by epiphytic microorganisms. Bacteria have developed<br />

various ways to resist the toxic effects of antimicrobial compounds. Extrusion<br />

of toxic agents from cells by multidrug efflux (MDE) is one of these<br />

mechanisms. This project aims to identify and characterize MDE pumps in the<br />

plant pathogen Pseudomonas syringae pv. tomato DC3000, the causal agent of<br />

bacterial speck on tomato and Arabidopsis.<br />

The genome sequence of P. s. pv. tomato DC3000 was used to develop a genespecific<br />

oligonucleotide microarray containing open reading frames predicted<br />

as putative multidrug pumps. The 83 oligonucleotides were selected from genes<br />

sharing sequence similarity with established MDE transporters plus efflux<br />

systems with unknown function and no similarity to characterized transporters.<br />

The microarray was used to identify transporters that are expressed during<br />

growth of P. s. pv. tomato DC3000 in Hrp-<strong>der</strong>epressing minimal medium and<br />

in AB induction medium supplemented with plant extracts and antimicrobial<br />

plant metabolites, respectively. To learn more about the natural functions of<br />

these transporters and their role during pathogenesis, we will analyze the<br />

expression of these efflux pumps during infection of the host plants.<br />

PJ 25<br />

Diseases of Caribbean sponges: "Sponge Orange Band"<br />

disease in Xestospongia muta and "Whitening" disease in<br />

Amphimedon compressa<br />

H. Angermeier *1 , G. Krohne 2 , J. Pawlik 3 , U. Hentschel 4<br />

1 Research Center for Infectious Diseases, University of Würzburg, Würzburg,<br />

Germany<br />

2 Department for Electron Microscopy, University of Würzburg, Würzburg,<br />

Germany<br />

3<br />

Center for Marine Science, University of North Carolina at Wilmington,<br />

Wilmington, Germany<br />

4<br />

Julius-von-Sachs Institute for Biological Sciences, University of Würzburg,<br />

Würzburg, Germany<br />

Microscopical and molecular studies were conducted on two sponge diseases,<br />

the "Sponge Orange Band" disease in Xestospongia muta and the "Whitening"<br />

disease in Amphimedon compressa. "Sponge Orange Band" in the barrel sponge<br />

X. muta is characterized by a gradual discolouration of the surface tissues from<br />

a healthy reddish-brown to a bleached white color which is frequently<br />

accompanied by an orange band serving as transition line. Using scanning<br />

electron microscopy severe tissue degradation was visualized in the later stages<br />

of bleaching eventually leading to sponge collapse. Molecular fingerprinting by<br />

denaturing gradient gel electrophoresis (DGGE) using cyanobacteria-specific<br />

primers revealed a distinct shift in the cyanobacterial community of the<br />

bleached tissues where the Synechococcus/Prochlorococcus clade of sponge<br />

symbionts disappeared and unspecific seawater cyanobacteria, such as<br />

Phormidium sp. and Leptolyngbya sp. appeared. So far, there is no evidence to<br />

support the involvement of a microbial pathogen and more studies are needed<br />

to determine the un<strong>der</strong>lying cause of disease.<br />

The "Whitening" disease of the rope sponge A. compressa is characterized by<br />

the occurence of distinct discoloured patches. As much as 18% of an entire A.<br />

compressa population can be affected at any given site. Electron microscopical<br />

studies showed the presence of spongin-boring bacteria solely within the white<br />

but not within the seemingly healthy regions of the same specimens. PCRamplification<br />

with specific primers revealed additionally that members of the αproteobacteria<br />

were only present within the affected tissues. This resembles the<br />

microbial participation at a disease that had previously been reported for<br />

Australian sponges (Webster et al. 2002).<br />

PK 01<br />

Are type strains from different culture collections always<br />

identical?<br />

P. Schumann *1 , T. Maier 2 , C. Spröer 1 , F. Bimet 3 , C. Bizet 3<br />

1 Molekulare Systematik, Deutsche Sammlung von Mikroorganismen und<br />

Zellkulturen GmbH, Braunschweig, Germany<br />

2 Bruker Daltonik GmbH, Leipzig, Germany<br />

3 CIP-Collection de l´Institut Pasteur, Paris, France<br />

The deposition of type strains in public culture collection is necessary in or<strong>der</strong><br />

to authenticate taxonomic descriptions and to make the respective organisms<br />

available for comparative studies and re-examination of published information.<br />

Since collections have been exchanging type strains and deposition in at least<br />

two culture collections has become obligatory for taxonomic descriptions, the<br />

type strain of a taxon is usually maintained in more than one culture collection.<br />

While recently deposited type strains are examined according to quality<br />

assurance criteria un<strong>der</strong> inclusion of mo<strong>der</strong>n identification approaches, the<br />

identity of type strains deposited in the „pre-molecular era“ remains to be<br />

confirmed by current techniques. 47 type strains acquired by CIP between 1950<br />

and 1960 which have not been exchanged with DSMZ were chosen as<br />

examples. They were compared to the corresponding type strains maintained at<br />

the DSMZ on the basis of their MALDI-TOF mass spectra. 44 pairs of strains<br />

from both collections agreed in their mass spectra with high similarity values<br />

indicating their identity. However, striking discrepancies were detected for the<br />

type strains of Pelomonas saccharophila and Campylobacter fetus subsp. fetus.<br />

The spectra of the Rhodobacter sphaeroides type strains displayed a<br />

significantly lower similarity than usually found for identical strains. Type<br />

strains with low mass spectra similarities were identified un<strong>der</strong> inclusion of<br />

independent methods like 16S rRNA gene sequence analysis and RiboPrinting<br />

and those with doubtful identity were removed from the respective collection<br />

catalogue. The study un<strong>der</strong>lined the suitability of MALDI-TOF mass<br />

spectrometry as tool for quality control in culture collections.<br />

PK 02<br />

Morphological and molecular characterization of<br />

Colletotrichum species from herbaceous plants in Iran<br />

D. Zafari *1<br />

1 plant protection, Bu Ali Sina, Hamedan, Iran<br />

Seventy-five isolates of Colletotrichum species were isolated from bean,<br />

Alfalfa, clover, potato, soybean, orange, tangerine, sweet lemon and several<br />

ornamental plants with anthracnose or black dot symptoms from different<br />

places of Iran. The aim of the persent study was thus to identify the<br />

colletotrichum species associated with anthracnose and black dot of these<br />

plants. Isolates were subjected to DNA sequence comparisons of internal<br />

transcribed spacer region (ITS1,5.8S and ITS2). Furthermore, isolates were also<br />

compared based on colony morphology, size and shape of appressoria and<br />

conidia. According molecular analysis and morphological comparision nine<br />

species and one variety of the genus Colletotrichum were identified including<br />

C. acutatum, C. boninense, C. coccodes, C. dematium, C. destructivum, C.<br />

gloeosporioides, C. gloeosporioides var. minus, C. musa, of which C.<br />

acutatum, C. boninense, C. destructivum are new for mycoflora of Iran and C.<br />

dematium from bean, C. gloeosporioides from potus C. truncatum from alfala<br />

newly are reported from these hosts in Iran. In addition Glomerella sp.<br />

teleomorph of C. boninense was formed on media and is new for mycoflora of<br />

Iran.<br />

PK 03<br />

Phylogenomics of the arthropod pathogenic bacterium<br />

Rickettsiella grylli<br />

A. Leclerque *1<br />

1 Institute for Biological Control, Julius Kühn Institute (JKI) - Fe<strong>der</strong>al Research<br />

Centre for Cultivated Plants, Darmstadt, Germany<br />

Rickettsiella grylli is an intracellular bacterial pathogen of aquatic and<br />

terrestrial arthropods. Previous determination of its 16S rRNA encoding<br />

sequence has led to the taxonomic classification of the genus Rickettsiella in<br />

the class Gammaproteobacteria, or<strong>der</strong> Legionellales, family Coxiellaceae, i.e.<br />

in close vicinity to vertebrate pathogenic bacteria of the genera Coxiella and<br />

Legionella. Using the additional information available from the recently<br />

published first whole genome sequence from this genus, this contribution<br />

critically evaluates the taxonomic classification of R. grylli beyond the 16S<br />

rRNA level. Employing phylogenetic reconstruction together with significance<br />

testing on a data basis defined by a core set of 211 previously identified<br />

families of protein-encoding genes together with a re-analysis of 16S rRNA<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

data, the present study firmly corroborates the assignment of this species to<br />

both the class Gammaproteobacteria and the or<strong>der</strong> Legionellales. However,<br />

results obtained from concatenated and single protein, single protein-encoding<br />

gene, and 16S rRNA gene data demonstrate a similar phylogenetic distance of<br />

Rickettsiella grylli to both the Coxiellaceae and the Legionellaceae and are,<br />

therefore, inconsistent with its current family-level classification.<br />

Consequently, a respective reorganization of the or<strong>der</strong> Legionellales is<br />

proposed. Detailed results are presented in FEMS Microbiology Letters 283<br />

(2008) 117-127.<br />

PL 01<br />

Evaluation of bactericidal activity of some selected food<br />

additives<br />

S. Selim *1 , S. El-Alfay 1 , M. Abdel Aziz 1 , H. Hamido 1 , M. Mashait 2 , M. Warrad 3<br />

1<br />

Botany Department, Microbiology section, Faculty of Sciences, Suez Canal<br />

University, Egypt, Ismailia, Egypt<br />

2<br />

Biology and Geology Department, Faculty of Education in Al-Arish, Suez<br />

Canal University, Egypt, Al-Arish, Egypt<br />

3<br />

Women Health Department, Faculty of Medicine, King Khaled University,<br />

Saudi Arabia, Abha, Saudi Arabia<br />

4<br />

Biology Department, Faculty of Sciences, King Khaled University, Saudi<br />

Arabia, Abha, Saudi Arabia<br />

The spread of antibiotic resistance among bacterial strains requires the<br />

development of new antibacterial agents. The present study was un<strong>der</strong>taken in<br />

or<strong>der</strong> to examine the bactericidal activity of some selected food additives<br />

against Pseudomonas aeruginosa and other pathogenic bacteria. The<br />

bactericidal activity of some selected food additives (Riboflavin; tartrazine;<br />

phloxine B; sodium nitrite; nisin; sodium benzoate; sorbic acid; ascorbic acid<br />

and monosodium glutamate) were tested for antibacterial activity in vitro<br />

against pathogenic bacteria (Bacillus cereus; Enterococcus feacalis;<br />

Escherichia coli; Salmonella enteritidis; Pseudomonas aeruginosa and<br />

vancomycin-resistant Staphylococcus aureus) using the disc diffusion method<br />

test. The most susceptible bacteria are B. cereus and other species showed<br />

variable effect. No activity was observed on studied bacteria with tartrazine;<br />

sodium nitrite and monosodium glutamate. Nisin was the most effective food<br />

additive against P. aeruginosa than other tested additives. Phloxine B was<br />

showed high anti-S. aureus activities and no activity against gram negative<br />

bacterial strains. Bactericidal studies indicated that potent tested food additives<br />

inhibit B. cereus and P. aeruginosa at concentration above 5 mg/ml and in case<br />

of S. aureus above 1 mg/ml. At twice the minimum bactericidal concentrations<br />

(MBCs), all B. cereus and P. aeruginosa were killed within 1 h with nisin while<br />

S. aureus was killed in 40 min with phloxine B. Our result showed that the food<br />

additive nisin has bactericidal activity against P. aeruginosa as well as other<br />

Gram-positive bacteria. Phloxine B selectively kills gram-positive bacteria,<br />

making it a candidate for inclusion in the use of selective media for the<br />

cultivation of gram-negative bacteria for use in medical and food microbiology.<br />

PL 03<br />

Double trouble: Cryo-electron tomography and vitreous<br />

sections uncover the native structure of mycobacterial cell<br />

walls<br />

C. Hoffmann *1 , A. Leis 1 , M. Nie<strong>der</strong>weis 2 , J. Plitzko 1 , H. Engelhardt 1<br />

1<br />

Molekulare Strukturbiologie, Max-Planck-Institut für Biochemie, Martinsried,<br />

Germany<br />

2<br />

Department of Microbiology, Univerity of Alabama at Birmingham,<br />

Birmingham, AL, United States<br />

The cell wall of mycobacteria forms an exceptional permeability barrier and it<br />

is essential for virulence. It contains extractable lipids and long-chain mycolic<br />

acids that are covalently linked to peptidoglycan via an arabinogalactan<br />

network. The lipids were thought to form an asymmetrical bilayer of<br />

consi<strong>der</strong>able thickness, but this could never be proven directly by microscopy<br />

or other means. By cryo-electron tomography of unperturbed cells of M. bovis<br />

BCG and M. smegmatis embedded in vitreous ice we revealed the native<br />

organization of the cell envelope with several distinct layers and identified the<br />

‘outer layer’ as a morphologically symmetrical lipid bilayer of limited<br />

thickness. For the first time membrane bilayer structures were ren<strong>der</strong>ed visible<br />

in tomograms of intact cells what denotes that the tomographic resolution must<br />

be clearly better than 3 nm. The 3-D data was confirmed by the investigation of<br />

detergent-treated cells and ultrathin frozen-hydrated cryosections of M. bovis<br />

BCG, M. smegmatis and the related actinomycete Corynebacterium<br />

glutamicum. To get a better un<strong>der</strong>standing of the contribution of distinct lipids<br />

to this membrane, several mutants of M.smegmatis were analyzed in the same<br />

way.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

The combination of cryo-electron tomography, which preserves the native state<br />

of the cell, and electron microscopy of vitreous sections, which allows to<br />

dissect structures at a higher resolution, necessitates a consi<strong>der</strong>able revision of<br />

the current view of the mycobacterial cell wall-architecture. Conceivable<br />

models are proposed and discussed. These results are crucial for the<br />

investigation and un<strong>der</strong>standing of transport processes across the mycobacterial<br />

cell wall, and they are of particular medical relevance for pathogenic<br />

mycobacteria.<br />

PL 04<br />

LOV-based fluorescence reporters in biotechnology<br />

B. Kopka 1 , M. Kaschner 1 , K. Klein *1 , M. Pohl 1 , K.E. Jaeger 1 , U. Krauss 1<br />

1 Institut für Molekulare Enzymtechnologie, Heinrich-Heine Universität<br />

Düsseldorf im Forschungszentrum Jülich, Jülich, Germany<br />

Genetically encoded fluorescence reporters of the green fluorescence protein<br />

(GFP) family are widely used in science. Applications, among others, include<br />

the monitoring of gene expression and protein localization as well as the study<br />

of protein-protein interactions, both in vivo and in vitro. In biotechnology,<br />

chemical fluorescent labels prove to be valuable tools to study e.g. enzyme<br />

distribution as well as physico-chemical parameters in immobilized enzyme<br />

carriers. However, fluorescent reporter proteins have been rarely used for this<br />

latter purpose.<br />

One major drawback of GFP and its <strong>der</strong>ivatives is their strict dependency on<br />

molecular oxygen for fluorophore maturation. This deficit recently lead to the<br />

construction of a set of small (~15 kDa), oxygen-independent, flavin<br />

mononucleotide (FMN)–binding fluorescent proteins (FbFPs), based on<br />

bacterial or plant light, oxygen, voltage (LOV) domains.<br />

In biotechnology, FbFPs might provide a non-invasive method to study the fate<br />

of biocatalysts in situ in the employed reaction system. Thereupon, we decided<br />

to explore this possibility by fusing the LOV-domain of the Bacillus subtilis<br />

YtvA-protein (BsFbFP) to the Lipase A (BSLA) of B. subtilis. Both N- and C-<br />

terminal fusions of BSLA with BsFbFP were constructed. To establish whether<br />

the LOV-BSLA fusions were functional, the proteins were heterologously<br />

overexpressed in Escherichia coli. Fluorescence measurements on whole cells<br />

and in solution demonstrated functionality of the fluorescent reporter (BsFbFP).<br />

Activity tests, based on the hydrolysis of the triacylglyceride tributyrin in agar<br />

plates as well as the p-Nitrophenylpalmitate (pNPP) assay system, revealed<br />

functionality of the lipase in the fusion.<br />

PM 01<br />

Evaluation of Common and New Transport Media for<br />

Helicobacter pylori in Gastric Biopsy Specimens<br />

A. talebi bezmin abadi *1 , A. mohabati mobarez 1<br />

1 Department of bacteriology, Tarbiat Modares University, tehran, Iran<br />

117<br />

Background:Stability of biopsy specimens of H. pylori during transport to<br />

laboratory for susceptibility testing remain a limiting factor.Few studies<br />

regarding the transport medium and its optimal transport conditions have been<br />

done yet (3,4).We have tested 3 new modified medium for H.pylori in our<br />

study.<br />

Material and Methods:Our transport mediums were<br />

included:T.M1:Saline,T.M2: Phosphate-Buffered Saline,T.M3:Thioglycolate<br />

Broth,T.M4: Brucella Broth supplemented with 2% fetal calf serum(FCS) and<br />

2% Glucose,T.M5:Thioglycolate Broth supplemented with 2% FCS and 2%<br />

Glucose.We tested the survival and viability of 36 biopsy specimens for each<br />

medium after endoscopy of 180patients.After incubation at 37°C un<strong>der</strong><br />

microaerophilic conditions for 7 days in anaerobe jars plates were checked<br />

optically for the presence of H. pylori colonies.Urease,oxidase, and catalase<br />

tests were carried out for further identification of H. pylori.<br />

Results:The best recovery rate of H. pylori from biopsy specimens after one<br />

hour incubation at 4°C was belonged to T.M4 with94.4%.T.M1 in all storage<br />

during had lowest recovery rates with55.5%, 55.5%, 38.8% ,5.5% ,5.5%).<br />

Indeed the recovery rate of H. pylori from biopsy specimens after one hour<br />

storage at 4°C in T.M1 and T.M4 were lowest(5.5%) and highest(94.4).<br />

Conclusion:Application of fetal calf serum and Glucose,even in low<br />

concentration were effective for increasing the recovery rate after hours in<br />

T.M4 and recommended for future studies.

118<br />

PM 02<br />

Nucleoid Occlusion and Septum Placement in<br />

Corynebacterium glutamicum<br />

A. Schwaiger *1 , M. Bramkamp 1<br />

1 Institut für Biochemie, Universität zu Köln, Köln, Germany<br />

Cell division is an important and tightly regulated process. During cell division<br />

in bacteria negative acting factors protect the cell poles and the nucleoid from<br />

aberrant division. Most rod-shaped bacteria have at least a dual system to<br />

ensure correct division. Cell poles are usually protected by the Min system and<br />

nucleoids are protected by DNA binding proteins that act as FtsZ<br />

polymerization inhibitors. Strikingly, some rod-shaped bacteria (e.g.<br />

Corynebacteria, Mycobacteria) have no Min system and hence division site<br />

selection completely rely on an elaborated nucleoid occlusion system. Here, we<br />

describe the identification of a chromosome partitioning system in<br />

Corynebacterium glutamicum that is involved in regulation of nucleoid<br />

occlusion.<br />

The chromosome partitioning system is composed of a ParAB system. Unlike<br />

most other organisms C. glutamicum possesses apart from the canonical parAB<br />

operon an orphan parA2 gene. Up-regulation of both ParA ATPases lead to<br />

significant cell elongation. Investigation of sub-cellular localization of ParA1,<br />

ParA2, and ParB revealed that they co-localize with the polar localized origins,<br />

thereby forming a centromere-like structure. ParA1 polymers spread from there<br />

over the nucleoids, while ParA2 mostly remains at the origin region.<br />

Surprisingly, biochemical studies showed that both ParA ATPases have an<br />

inhibitory effect on FtsZ polymerization and seem to fulfill a role in nucleoid<br />

occlusion and therefore act as negative regulators of septum placement. Based<br />

on our in vivo and in vitro results we propose a model that links division site<br />

selection and chromosome segregation in Corynebacteria.<br />

PM 03<br />

In vivo study of the two-component signalling network in E.<br />

coli<br />

E. Sommer *1 , V. Sourjik 1<br />

1 Zentrum für Molekulare Biologie Heidelberg, Universität Heidelberg,<br />

Heidelberg, Germany<br />

Two-component systems are the most widespread sensing systems in<br />

prokaryotes and lower eukaryotes, with multiple members of this class <strong>bei</strong>ng<br />

present in one organism. We are interested in investigating the interconnection<br />

among different two-component signalling pathways in Escherichia coli. To<br />

map interactions between the pathways in vivo and to study relative cellular<br />

distribution of their proteins, we assay real-time dynamics of protein<br />

interactions and their dependencies on stimulation using fluorescence imaging<br />

and fluorescence resonance energy transfer (FRET)- and fluorescence recovery<br />

after photobleaching (FRAP)-microscopy. Additionally, intracellular<br />

processing of sensed stimuli with regard to amplification, integration and<br />

possible cross-talk between the systems will be investigated. Such analysis will<br />

help to establish an integral picture of cell signalling performed by prokaryotic<br />

organisms.<br />

PM 04<br />

Specific and global stress response in bacterial populations<br />

M. Braendle *1 , S. Kirchen 1 , T. Schwartz 1 , U. Obst 1<br />

1 Institute for Technical Chemistry / Microbiology of natural and technical<br />

surfaces department, Forschungszentrum Karlsruhe, Eggenstein-<br />

Leopoldshafen, Germany<br />

Bacterial stress responses in the natural environment are barely un<strong>der</strong>stood, so<br />

the aim of our work is the investigation of specific and global response to<br />

starvation, osmotic shock and the stationary growth phase of hygienic relevant<br />

bacteria and biofilms.<br />

The physiology of bacteria showed an increase of the total RNA and decrease<br />

in colony forming units during the exposure to stress in Gram-positive bacteria<br />

and there was certain evidence for a VBNC state in E. faecalis.<br />

We investigated the sigma factor rpoS of P. aeruginosa and rcsA gene, which is<br />

involved in biofilm formation of E. coli. Furthermore we studied pbp5 which<br />

participates in the peptidoglycan synthesis of E. faecium and E. faecalis and<br />

might be a marker for VBNC state. As a common putative stress marker we<br />

studied also the universal stress protein usp.<br />

During gene expression analysis by Real-Time PCR and Northern-Blot we<br />

found an induction rpoS and rcsA un<strong>der</strong> stress conditions, while there was<br />

mostly a repression of pbp5. The gene expression of usp partly differs from the<br />

pattern of species specific genes. Analysis of biofilms indicated a similar<br />

expression pattern compared with planktonic bacteria.<br />

For examination of global stress response RAPD-PCR were used. Gram-<br />

negative bacteria showed clear distinctions during starvation. E. faecalis had a<br />

stronger stress response compared to E. faecium indicating a stronger stress<br />

adaption of E. faecium. These results suggest that RAPD-PCR is a promising<br />

method for finding stress patterns without investigation of specific genes.<br />

PM 05<br />

The cell division protein FtsN is functionally conserved<br />

among proteobacteria<br />

A. Moell *1 , M. Thanbichler 1<br />

1 Independent Junior Research Group Prokaryotic Cell Biology, Max Planck<br />

Institute for Terrestrial Microbiology, Marburg, Germany<br />

The division of the bacterial cell is a complex process which is still poorly<br />

un<strong>der</strong>stood. Many components of the cell division complex, or divisome, have<br />

been identified over the last years, but much is still unknown about their precise<br />

functions, their interactions and the mechanism of their assembly. Additionally,<br />

while some of the divisome components are widely conserved, others are only<br />

found in bacterial subgroups. FtsN is the last essential cell division protein to<br />

localize to the septum in Escherichia coli [1]. It has been suggested that it is<br />

involved in septal peptidoglycan synthesis [2]. FtsN is poorly conserved outside<br />

the enteric bacteria. Here, we describe the Caulobacter crescentus homologue<br />

of FtsN. In Caulobacter, FtsN localizes to the swarmer cell pole and is relocalized<br />

to midcell at the initiation of cell division. Its recruitment to the<br />

divisome occurs late in the cell cycle in comparison with other cell division<br />

proteins and is dependent on FtsZ. Depletion of FtsN results in filamentation.<br />

We determined the structural elements necessary for function and localization<br />

of the protein. We further provide experimental evidence that FtsN is indeed<br />

conserved among proteobacteria and that several so far unannotated<br />

homologues exist.<br />

[1] Addinall, S.G., Cao, C. and Lutkenhaus, J. (1997). FtsN, a late recruit to the<br />

septum in Escherichia coli. Mol Microbiol 25, 303-9.<br />

[2] Müller, P. et al. (2007). The essential cell division protein FtsN interacts<br />

with the murein (peptidoglycan) synthase PBP1B in Escherichia coli. J Biol<br />

Chem 282, 36394-402.<br />

PM 06<br />

Control of lipopolysaccharide biosynthesis by FtsHdependent<br />

proteolysis<br />

S. Langklotz *1 , M. Schäkermann 1 , F. Narberhaus 1<br />

1 Institute for Microbial Biology, Ruhr-University Bochum, Bochum, Germany<br />

A proper equilibrium between lipopolysaccharides (LPS) and phospholipids in<br />

the outer membrane is crucial for viability of Gram-negative bacteria and<br />

provides defense against external influences [1]. To avoid toxic accumulation<br />

of LPS in Escherichia coli, the membrane-bound, ATP-dependent and essential<br />

AAA + -protease FtsH degrades two enzymes in the LPS biosynthesis pathway:<br />

LpxC and KdtA [2, 3]. LpxC catalyzes the first committed step in biosynthesis<br />

of lipid A, which forms the hydrophobic anchor of LPS. KdtA attaches the<br />

KDO sugar core moieties to lipid A. Regulated proteolysis of LpxC by the<br />

FtsH protease requires a C-terminal degradation signal (LAxxxxxAVLA)<br />

consisting of six non-polar amino acids within the last eleven residues [4, 5].<br />

Since any deviation from normal amount of LpxC are toxic for E. coli, LpxC is<br />

discussed as a potential drug target in Gram-negative bacteria Therefore a<br />

detailed un<strong>der</strong>standing of posttranscriptional LpxC regulation by proteolysis<br />

might help to develop new antibiotics influencing cellular LpxC amounts.<br />

Here we demonstrate that (i) proteolysis of LpxC by the FtsH protease is a<br />

common but not entirely conserved mechanism in Gram-negative bacteria and<br />

(ii) LpxC stability is regulated in response to the growth-rate ensuring the<br />

adequate biosynthesis of membrane components when cells grow rapidly.<br />

[1] Raetz (1990) Annu. Rev. Biochem., 59: 129-170<br />

[2] Ogura et al (1999), Mol. Microbiol., 31: 833-44<br />

[3] Katz and Ron (2008), J. Bacteriol., 190: 7117-22<br />

[4] Führer et al (2006), Mol. Microbiol., 59: 1025-36<br />

[5] Führer et al (2007), J. Mol. Biol., 372: 485-96<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PM 07<br />

Proteolysis of the cell division protein FtsL in Bacillus<br />

subtilis<br />

I. Wadenpohl *1 , M. Bramkamp 1<br />

1 Institut für Biochemie, Universität zu Köln, Köln, Germany<br />

Cell division in Bacillus subtilis is initiated by assembly of a cytokinetic ring<br />

followed by recruitment of different transmembrane proteins. The late division<br />

protein FtsL is highly unstable and was shown to be rate limiting for cell<br />

division. FtsL may stabilize the division complex by protein-protein<br />

interactions and might be involved in transmembrane signaling events. FtsL is<br />

presumably cleaved by the site-2-protease RasP within its transmembrane<br />

domain. At which time point during cell cycle FtsL cleavage occurs is yet<br />

unclear. Using fluorescent microscopy the localization and co-localization of<br />

FtsL and the protease RasP during cell cycle is investigated.<br />

Co-expression of FtsL and RasP in E. coli led to rapid degradation of FtsL. Coexpression<br />

of FtsL and an inactive mutant of RasP, however, had no effect on<br />

FtsL stability. The membrane spanning division proteins DivIB and DivIC are<br />

discussed as possible interaction partners of FtsL. Null mutations of DivIB are<br />

viable, but cells are not able to divide at higher temperatures. This effect can be<br />

overcome by overexpression of FtsL, suggesting heat protection of FtsL by<br />

DivIB. DivIC stability is directly dependent on FtsL. To analyze whether DivIC<br />

and DivIB have a stabilizing effect on FtsL co-expression experiments of FtsL,<br />

DivIC, DivIB, and RasP in E. coli are carried out.<br />

PM 08<br />

Ploidy in Proteobacteria and Methanogenic Archaea<br />

C. Hildenbrand *1 , V. Pecoraro 1 , D. Morbitzer 1 , J. Soppa 1<br />

1 Institute for Molecular Biosciences, Goethe-University, Frankfurt, Germany<br />

Polyploidy, the existence of multiple copies of the normal set of chromosomes,<br />

is common in higher eukaryotes, especially in plants, fishes and amphibians. By<br />

contrast, it is assumed that most prokaryotes are monoploid. However, several<br />

prokaryotic species were found to contain more than one copy of the<br />

chromosome, e.g. recently it was reported that the halophilic archaea<br />

Halobacterium salinarum and Haloferax volcanii are polyploid [1].<br />

To determine the genome copy number in further groups of prokaryotes<br />

quantitative Real-Time PCR analyses were performed. Surprisingly, two<br />

methanogenic archaea, Methanosarcina acetivorans and Methanococcus<br />

maripaludis, were found to be highly polyploid and harbor more than 200<br />

genome copies. These results constitute the first examples for such a high<br />

ploidy in prokaryotes. It remains to be shown whether genome copy numbers<br />

above 200 are common among methanogenic archaea and whether they are<br />

specific for certain growth conditions.<br />

To verify that the Real Time PCR method can also be used to characterize the<br />

ploidy level of bacterial species, it was validated with Escherichia coli. Growth<br />

rate-dependence of the chromosomal copy number and merooligoploidy at high<br />

growth rates could be verified. Additional species were selected to represent -<br />

together with the results of previous studies - all groups of proteobacteria.<br />

Caulobacter crescentus and Wolinella succinogenes were shown to be<br />

monoploid. In contrast, Pseudomonas putida contains on average 20 copies of<br />

the chromosome. Taken together, proteobacteria contain monoploid, oligoploid,<br />

merooligoploid and polyploid species, indicating that monoploidy is the<br />

exception rather than the rule in prokaryotes.<br />

[1] Breuert S., Allers T., Spohn G., Soppa J. (2006). Regulated Polyploidy in<br />

Halophilic Archaea. PLos ONE 1 (1): e92<br />

PM 09<br />

Expression analysis and localization of the MreB-like<br />

proteins in S.coelicolor A3(2)<br />

A. Heichlinger *1 , A. Latus 1 , W. Wohlleben 1 , G. Muth 1<br />

1 Microbiology/Biotechnology, Eberhard Karls Universität Tübingen, Tübingen,<br />

Germany<br />

The mre gene cluster encompassing the mreB, mreC and mreD genes is<br />

required for elongation growth and cell-shape determination in rod-shaped<br />

bacteria [1]. Three mreB homologous genes (mreB, mbl, SCO6166) have been<br />

detected in the genome of the mycel forming Streptomyces coelicolor. MreB<br />

and Mbl showed high similarity (42%) while SCO6166 has only 26% amino<br />

acid sequence similarity. Analysis of other actinomycetes suggested that an<br />

mre-cluster is only present in those actinomycetes that sporulate by septating<br />

aerial mycelium but is missing in non-sporulating ones.<br />

In contrast to all other bacteria deletion of mreB or mbl is tolerated in<br />

S.coelicolor. Vegetative growth was not affected but parts of the aerial hyphae<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

lysed, irregular septation took place and spores were swollen. On SM-Agar<br />

with 10% sucrose the phenotype was more serious. Deletion of SCO6166 led to<br />

a similar but weaker phenotype.<br />

An eGFP-fusion localized MreB specifically to the sporulation septa in young<br />

aerial hyphae. Afterwards MreB-eGFP assembled at the poles of prespores and<br />

finally formed a shell like structures at the inner surface of the spores [2]. A Cterminal<br />

Mbl-eGFP fusion localized at division septa in young aerial mycelium<br />

similar to MreB.<br />

The mreBCD gene cluster is transcribed as an operon which is un<strong>der</strong> control of<br />

three promoters, two constitutive ones and one strongly induced during<br />

sporulation [3]. RNA isolation at different time points of the life cycle of<br />

S.coelicolor and RT-PCR analysis showed that expression of mreB and mbl<br />

increased during sporulation, while expression of SCO6166 decreased.<br />

[1] Errington, J. 2003. Nat.Cell Biol. 5:175-178<br />

[2] Mazza, P. et al. 2006. Mol Microbiol. 60:838-852<br />

[3] Burger et al. 2000. Mol.Gen.Genet. 263:1053-1060<br />

PM 10<br />

Conjugal Plasmid-transfer in Streptomyces-<br />

The FtsK-like motor protein TraB and SpdB2 enable DNAtranslocation<br />

J. Vogelmann *1 , T. Roth 1 , W. Wohlleben 1 , G. Muth 1<br />

1 Dpt. of Microbiology/Biotechnology, University, Tuebingen, Germany<br />

The plasmid-encoded DNA-translocator TraB is the only plasmid encoded<br />

protein essential for conjugation in Streptomyces. Proteins that belong to the<br />

DNA translocator family are membrane associated ATPases involved in<br />

segregation of ds chromosomal DNA during cell division (FtsK) and<br />

sporulation (SpoIIIE).<br />

Conjugal DNA transfer in streptomycetes most probably proceeds via a TraB<br />

complex at the hyphal tip from donor to recipient mycelium. First evidence for<br />

a hexameric pore like structure was obtained by chemical crosslinking and EM<br />

analysis of a soluble TraBpSVH1.<br />

TraB binds specifically to a non coding 50bp plasmid sequence (clt)(1,2). This<br />

sequence contains three 8 bp imperfect direct repeats and a 14 bp direct repeat.<br />

A similar mechanism is used for FtsK binding to 8bp KOPS-motif that enables<br />

FtsK to read polarity of the chromosome. TraBs encoded by different plasmids<br />

only bind to their corresponding clt locus but do not bind to clt loci of other<br />

plasmids. By analyzing truncated and chimeric TraB proteins (TraBpSVH1,<br />

TraBpSG5, TraBpIJ101)a region responsible for specific DNA-binding could be<br />

identified at the very C-terminus. This region contains a wHTH motif typically<br />

for DNA-binding proteins.<br />

Since Streptomyces show mycelial growth, primary plasmid transfer is followed<br />

by a secondary transfer in the recipient from one compartment to the next one.<br />

This results in spreading of the plasmid in the recipient mycelium.<br />

SpdB2, a plasmid encoded integral membrane protein is involved in this<br />

process. In a lipid bilayer assay it was shown to form pores for plasmid<br />

translocation.<br />

PM 11<br />

Protein translocation to the periplasm without a signal<br />

peptide. Evidence for a third pathway?<br />

M. Krehenbrink *1 , A. Edwards 2 , J.A. Downie 2<br />

1 Unité de Génétique Moléculaire, Institut Pasteur, Paris, France<br />

2 Molecular Microbiology, John Innes Centre, Norwich, United Kingdom<br />

119<br />

Proteins are translocated to the periplasm by either the general export pathway<br />

(GEP) or the twin-arginine translocation pathway (TAT). The targeting of<br />

soluble proteins to either pathway is dependent on the presence of N-terminal<br />

and largely hydrophobic signal peptides that are usually cleaved by a signal<br />

peptidase. A systematic analysis of the Rhizobium leguminosarum bv viciae<br />

3841 secretome showed that the Fe/Mn superoxide dismutase SodA is a soluble<br />

periplasmic protein, despite its complete lack of such a signal peptide. Instead,<br />

the N-terminus of SodA is highly hydrophilic and remains uncleaved. The<br />

translocation of SodA to the periplasm was highly sequence-dependent, and the<br />

first 59 amino acids were sufficient to target a reporter protein to the periplasm.<br />

SodA/B orthologues of certain Gram-negative bacteria are also periplasmic, but<br />

in these strains the protein carries either a TAT or GEP signal peptide. SodA is<br />

also known to be exported in a few Gram-positive bacteria in a SecA2dependent<br />

manner, but no SecA2 orthologue is encoded in the R. l. bv viciae<br />

3841 genome, and SodA translocation in R. l. bv viciae 3841 is unaltered in a<br />

tatC as well as a secDF null mutant. Since neither of the currently known<br />

translocation pathways can account for the presence of SodA in the periplasm,<br />

the possibility of a third route to the periplasm has to be consi<strong>der</strong>ed. The ability

120<br />

to translocate SodA to the periplasm was found to be not limited to the genus<br />

Rhizobium, but was also present in other proteobacteria. The involvement of<br />

SodA in the virulence of many pathogens makes the SodA translocation<br />

pathway not only interesting from a fundamental perspective, but also makes it<br />

a potential target for novel antimicrobials. Here we present the identification of<br />

SodA as a periplasmic protein and our first results towards the identification of<br />

the translocation machinery.<br />

PM 12<br />

Cellular microbiology: mechanics, engineering and<br />

architecture at the molecular level.<br />

B.J. Tindall *1<br />

1 DSMZ, DSMZ, Braunschweig, Germany<br />

Our view of cellular systems is changing at a dramatic rate. Cells are highly<br />

organised systems based on features more akin to engineering and architecture<br />

than our classical view of prokaryotes based on genetics and<br />

biochemistry/physiology. While the components of the cell, nucleic acids,<br />

proteins, polysaccharides and lipids (or combinations therefore) are generally<br />

viewed from a biochemical point of view there is an increasing need to examine<br />

them in a structural and functional context. Rather than studying the individual<br />

components it is also important to view them as parts of complexes, many of<br />

which are in dynamic interaction with each other. These complexes may, in<br />

turn also interact with one another. Un<strong>der</strong>standing the structural and functional<br />

role of the components of the cell also allows us to examine the evolutionary<br />

aspects that un<strong>der</strong>lie the complexity of mo<strong>der</strong>n cells. An important aspect of<br />

these developments has been the ability to observe the location of various<br />

components at the sub-cellular level. This must also be accompanied by an<br />

appreciation of the fact that examining cellular and sub cellular structure and<br />

function must be studied at the molecular level, where the term molecular is<br />

used in a wi<strong>der</strong>, chemical sense rather in the sense of genetics/genomics. While<br />

there is a danger of introducing further reductionist philosophy it is important to<br />

realise the fundamental basis of biology and cellular systems.<br />

PM 13<br />

A bacterial flotillin homologue involved in the early stages<br />

of sporulation in Bacillus subtilis<br />

C. Donovan *1 , M. Bramkamp 1<br />

1 Institute of Biochemistry, University of Cologne, Cologne, Germany<br />

Here we describe a novel protein, YuaG, a eukaryotic reggie/flotillin<br />

homologue that is involved in the early stages of sporulation of the Grampositive<br />

model organism Bacillus subtilis. YuaG is encoded in the yuaFGI<br />

operon and localizes in discrete foci in the membrane and these foci are highly<br />

dynamic. Purification of detergent resistant membranes (DRM) revealed that<br />

YuaG is firmly associated with negatively charged phospholipids. Transcription<br />

of the yuaFGI operon begins during the early stationary phase and persists until<br />

the late stationary phase. Endospore formation in Bacillus subtilis requires the<br />

generation of two distinct cell types, a forespore and larger mother cell. The<br />

development of these cell types is controlled and regulated by cell-type specific<br />

gene expression, activated by a sigma factor cascade, which is coupled with the<br />

completion of polar septation. A YuaG “knock-in” strain, demonstrated that<br />

expression of YuaG begins one hour after cells are induced to sporulate. Thus,<br />

the transcription of YuaG begins even before the asymmetric septum has<br />

formed. According to the data presented, YuaG plays a role during the early<br />

stages of sporulation. A yuaG mutant exhibits decreased levels of Spo0A~P, the<br />

master regulator and therefore a delay in the onset of sporulation. However, this<br />

strain is only slightly impaired in the production of viable spores. Our results<br />

indicate that YuaG is involved in the early stage of spore development, most<br />

likely playing a role in the signalling cascade at the onset of sporulation.<br />

PM 14<br />

Analysis of MraY and MurG from Staphylococcus aureus<br />

and Chlamydia pneumoniae<br />

C. Pöllinger *1 , J. Esche 1 , D. Alborn 1 , B. Henrichfreise 1 , T. Schnei<strong>der</strong> 1 , I.<br />

Wiedemann 1 , K. Mölleken 2 , H.G. Sahl 1<br />

1 IMMIP/Pharmaceutical Microbiology Unit, University of Bonn, Bonn,<br />

Germany<br />

2 Institute for Functional Genomics of Microorganisms, University of<br />

Duesseldorf, Duesseldorf, Germany<br />

Cell wall biosynthesis is a vital process for almost all bacteria and has no<br />

counterpart in eukaryotic cells with respect to biosynthesis of the polymer.<br />

To analyse in detail the peptidoglycan biosynthesis machinery we use Grampositive<br />

and -negative model systems based on S. aureus and C. pneumoniae,<br />

respectively.<br />

We started to investigate the fundamental membrane associated steps of<br />

peptidoglycan biosynthesis which lead to the formation of the lipid-bound<br />

precursors lipid I and lipid II.<br />

Therefore, the integral membrane protein MraY and the membrane associated<br />

protein MurG from S. aureus and<br />

C. pneumoniae were overproduced in Escherichia coli and purified. We<br />

demonstrate in vitro activity of MraY and MurG from both species and present<br />

further data on substrate specificities and coupled assays.<br />

A deeper insight into the peptidoglycan biosynthesis machinery on molecular<br />

level will provide valuable information for the design of new antibiotics.<br />

PM 15<br />

The actin-like protein MamK is involved in magnetosome<br />

biomineralisation and chain assembly in Magnetospirillum<br />

gryphiswaldense<br />

E. Katzmann *1 , J.M. Plitzko 2 , D. Schüler 1<br />

1 Microbiology, Ludwig Maximilians-University, Biocenter, Munich, Germany<br />

2 Molecular Structural Biology, Max Planck Institute of Biochemistry,<br />

Martinsried, Germany<br />

Magnetosomes of magnetotactic bacteria are magnetic organelles , which<br />

assembly into complex chain-like structures. Proper chain assembly in<br />

magnetospirilla was recently shown to depend on the acidic repeat protein<br />

MamJ that attaches magnetosomes to the cytoskeletal “magnetosome filament”<br />

that is thought to be formed by the actin-like MamK protein. To reconcile the<br />

seemingly inconsistent phenotypes of deletion mutants of mamJ and mamK in<br />

M. gryphiswaldense (clustered magnetososomes) and M. magneticum<br />

(sactterered magnetosomes), respectively, a ΔmamK deletion mutant was<br />

created in M. gryphiswaldense. Cryo-electron tomography revealed the<br />

presence of cytoskeletal structures resembling the magnetosome filaments in<br />

the wildtype, which were absent in the mutant. Surprisingly, the phenotype of<br />

the mamK deletion in M. gryphiswaldense did neither resemble the mamK<br />

mutant of nor the mamJ phenotype in M. grpyhiswaldense. Instead, we<br />

observed multiple short chains that were spaced by large gaps and delocalized<br />

from their usual midcell position. Another unexpected finding was that mamK<br />

mutants accumulated less iron, and on average synthesized only 54 % of the<br />

magnetosome crystal numbers found in the wildtype, which could be restored<br />

by homologous and heterologous transcomplementation.<br />

Our preliminary data are not fully consistent with the previous hypothesis that<br />

MamK is only forming a rigid backbone to which magnetosome particles are<br />

attached for chain stabilization. Instead, MamK might play a more complex<br />

role which could involve the dynamic organization of magnetosome chains, and<br />

which also affects the biomineralization of magnetite.<br />

PM 16<br />

A dual stator system drives a single polar flagellum in<br />

Shewanella oneidensis MR-1<br />

A. Paulick *1 , A. Koerdt 1 , K.M. Thormann 1<br />

1 Department Ecophysiology, Max Planck Institute for Terrestrial Microbiology,<br />

Marburg, Germany<br />

Shewanella oneidensis MR-1 is motile by means of a single polar flagellum,<br />

but harbors two different functional stator complexes. One of these stator<br />

complexes is sodium- (PomAB) and one is proton-driven (MotAB). Homology<br />

studies indicate that the MotAB stator complex has been acquired by lateral<br />

gene transfer driven by motility adaptation to a low-sodium environment.<br />

Studies on corresponding deletion mutants demonstrated that both stator<br />

complexes are solely sufficient to drive the flagellar rotation. However, PomAB<br />

appears to be the dominant stator complex.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

By using promoter-Gfp fusions we showed that both PomAB and MotAB are<br />

expressed simultaneously in each cell. Localization studies with mCherry<br />

fusions revealed that PomAB localizes to the flagellated pole independent of<br />

the sodium concentration, whereas functional localization of MotAB decreases<br />

in response to high sodium conditions. In addition, further studies demonstrated<br />

that the stator subunits in S. oneidensis do not form functional hybrid-stators<br />

(e.g. a PomA-MotB stator complex) but putatively a functional hybrid motor.<br />

This motor probably consists of PomAB and MotAB in the stator-ring system,<br />

simultaneously using protons and sodium ions. We observed that two additional<br />

proteins, MotX and MotY are important for flagellar rotation. However, in<br />

contrast to the orthologous proteins in Vibrio alginolyticus, both proteins are<br />

not important for the recruitment of the stator complexes.<br />

In conclusion, we have demonstrated that S. oneidensis MR-1 harbors a dual<br />

stator system, which is regulated at the protein level in response to salt<br />

conditions.<br />

PM 17<br />

Regulation of cell division by MipZ: How to establish a<br />

protein gradient within a prokaryotic cell<br />

D. Kiekebusch *1 , K. Michie 2 , L.O. Essen 3 , S. Wick 1 , J. Löwe 2 , M. Thanbichler 1<br />

1<br />

Independent Junior Research Group Prokaryotic Cell Biology, Max Planck<br />

Institute for Terrestrial Microbiology, Marburg, Germany<br />

2<br />

Laboratory of Molecular Biology, Medical Research Council, Cambridge,<br />

United Kingdom<br />

3<br />

Department of Chemistry, Philipps-University, Marburg, Germany<br />

Successful cell division requires close coordination of chromosome dynamics<br />

with the positioning and assembly of the cell division apparatus. In many<br />

bacteria this objective is achieved by the combined action of nucleoid occlusion<br />

and oscillatory waves of MinCDE, which inhibit FtsZ ring formation at the<br />

poles and thus restrict establishment of the division apparatus to midcell.<br />

Caulobacter crescentus, which lacks homologues of these proteins, has recently<br />

been shown to control formation of the FtsZ ring near midcell by means of a<br />

newly identified essential ATPase, called MipZ. In pre-divisional cells MipZ is<br />

positioned at both poles by interaction with the origin-bound ParB•parS<br />

complex. As a consequence, a gradient of MipZ is established, with its<br />

concentration <strong>bei</strong>ng highest at the poles and lowest at midcell. Due to an<br />

inhibitory effect of MipZ on FtsZ polymerization, FtsZ is directed towards<br />

midcell, where it initiates cell division by polymerizing into a stable FtsZ ring.<br />

Using several mutant MipZ <strong>der</strong>ivatives, we show that a dynamic equilibrium<br />

between the ATP-bound, dimeric state and the ADP-bound or nucleotide-free,<br />

monomeric state is crucial for the function of MipZ. We demonstrate that MipZ<br />

does not only interact with FtsZ and ParB but is also able to bind unspecifically<br />

to the nucleoid. Based on our in vivo and in vitro data, we present a model that<br />

explains how ATP binding and hydrolysis modulate the interaction of MipZ<br />

with its different binding partners, thus facilitating coordination of chromosome<br />

segregation with cell division both in a temporal and spatial manner.<br />

PM 18<br />

In vivo localization of GFP labelled magnetosome proteins<br />

in Magnetospirillum gryphiswaldense reveals new insights<br />

into targeting mechanisms of magnetosome membrane<br />

proteins and magnetosome chain assembly<br />

C. Lang *1 , K. Junge 1 , D. Schüler 1<br />

1 Department Biologie I; Abteilung Mikrobiologie, Ludwig-Maximillians<br />

Universität München, München, Germany<br />

The assembly of magnetosomes in magnetotactic bacteria is a unique example<br />

for the development of a bacterial organelle. Magnetosome formation is a<br />

highly complex, cell biological process comprising (i) the<br />

compartmentalization of magnetosome vesicles, (ii) the assembly of the<br />

magnetosome vesicles in chains and (iii) the biomineralization of magnetite<br />

nanocrystals. Proteomic studies identified a set of approximately 20<br />

magnetosome membrane associated proteins (MMP), whose function is mostly<br />

unknown. To investigate targeting mechanisms of MMP and the assembly of<br />

the magnetosome chain during cell cycle we studied GFP fusions of MamC,<br />

MamF and MamM. While MamC- and MamF-GFP localized exclusively in the<br />

magnetosome membrane, MamM-GFP was additionally detected in the<br />

cytoplasmic membrane in the wild type. The same localization pattern was<br />

observed un<strong>der</strong> biomineralization inducing and un<strong>der</strong> non-inducing conditions,<br />

which suggests that the chain-like arrangement of magnetosomes does not<br />

depend on the presence of magnetite crystals. In ΔmamGFDC and ΔmamC<br />

mutants, which still form magnetosomes, the localization of MamM- and<br />

MamC-GFP was reminiscent of the localization in the wild type, whereas the<br />

protein localization was limited to the cytoplasmic membrane (MamM-GFP) or<br />

to inclusion bodies (MamC-GFP) in the non-magnetic mutants MSR-1B, which<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

lacks almost all magnetosome genes and ΔmamB. These results indicate that<br />

protein localization and potentially vesicle formation are inhibited in the<br />

ΔmamB mutant.<br />

In addition, we will present preliminary results on life cell imaging of<br />

magnetotactic bacteria un<strong>der</strong> microoxic and oxic growth conditions to study the<br />

dynamics of protein localization and magnetosome chain assembly during cell<br />

cycle.<br />

PM 19<br />

Investigation of bacterial cytoskeletal elements in a<br />

heterologous system<br />

F. Dempwolff *1 , P.L. Graumann 1<br />

1 Faculty of Biology/ Microbiology, University of Freiburg, Freiburg, Germany<br />

All types of ctytoskeletal elements known in eukaryotes are present in bacteria.<br />

They play an essential role in cell shape determination as well as in the cell<br />

cycle, and frequently show defined subcellular localization patterns. For<br />

example, MreB and intermediate filament-type proteins form filamentous<br />

structures un<strong>der</strong>neath the cell membrane.<br />

We are interested in the intrinsic features of cytoskeletal elements, e. g.<br />

filament formation or localization dynamics. To this end, we express these<br />

proteins in eukaryotic Schnei<strong>der</strong> S2 cells that serve as a heterologous model<br />

system. We find, that MreB localizes to the cell membrane where it is forming<br />

straight polymers. For IF-like proteins we show that they polymerize into<br />

helical fibres in the cytoplasm.<br />

In addition we expressed bacterial integral membrane proteins that form patch<br />

like structures in B. subtilis and these proteins also accumulate in large<br />

assemblies in S2 cells. These data indicate that many bacterial proteins have<br />

intrinsic localization and assembly properties.<br />

PM 20<br />

Dynamics of DNA-binding proteins in live Bacillus subtilis<br />

cells<br />

L. Simon *1 , S. Welling 1 , P.L. Graumann 1<br />

1 Faculty of Biology/Microbiology, University of Freiburg, Freiburg, Germany<br />

Chromosomes must be compacted about 1000 fold to fit into the bacterial cell<br />

and the topology of the DNA must be regulated to allow efficient transcription<br />

and replication. Compaction of the DNA is achieved by a number of proteins<br />

including histone-like proteins, the SMC complex and topoisomerases. To<br />

investigate the dynamics of different DNA-binding proteins, Fluorescence<br />

Recovery after Photobleaching (FRAP) experiments were carried out. The<br />

SMC complex, consisting of SMC, ScpA and ScpB, is essential for<br />

chromosome segregation. It localizes in a bipolar manner to discrete centers on<br />

the nucleoid. FRAP experiments of SMC-GFP revealed full recovery of the<br />

bleached foci within a few minutes, showing that there is an ongoing exchange<br />

of the complexes between the foci, indicating a transient nature of the complex.<br />

Contrarily, the non-specifically DNA-binding histone-like HBsu did not<br />

recover after bleaching, but has long lived binding kinetics throughout the<br />

nucleoid.<br />

Topoisomerase II introduces supercoils into DNA and localizes throughout the<br />

nucleoid, with a transient formation of accumulations at the replication forks.<br />

FRAP studies showed that it is also highly dynamic. Interestingly, we found<br />

that one subunit, GyrA, interacts with the DNA repair protein RecN, a member<br />

of the SMC protein family and together with RecN assembles in so called DNA<br />

repair centers after induction of DNA breaks. These experiments suggest that<br />

gyrase may be recruited to repair centers by RecN following DNA damage.<br />

As a further example for a protein that forms specific assemblies on the<br />

chromosome, we studied Spo0J, which forms nucleoprotein complexes at<br />

several parS sides near the origin of replication. Spo0J is involved in the<br />

regulation of replication initiation and of chromosome segregation. Performing<br />

FRAP experiments we were able to visualize the highly dynamic nature of<br />

Spo0J, showing that many specific assemblies on the bacterial chromosome are<br />

highly dynamic in vivo.<br />

PM 22<br />

Effect of Iron on Fusaricidin Production by Paenibacillus<br />

polymyxa<br />

W. Raza *1 , H. Wu 1 , Q. Shen 1<br />

1 College of Resource and Environmental Sciences, Nanjing Agriculture<br />

University, Nanjing, China<br />

121<br />

Paenibacillus polymyxa strains produce fusaricidin type compounds that are<br />

active against a wide variety of gram-positive bacteria and fungi. Growth and<br />

production of fusaricidin type antifungal compounds by P. polymyxa SQR-21<br />

were compared in tryptone broth supplemented with four concentrations (25,<br />

50, 100 and 200 µM) of iron. The data revealed that the growth of P. polymyxa

122<br />

was increased by 3-8% with the increase in concentration of ferric ion (Fe+3)<br />

and fusaricidin type compounds production was increased by 7-23% only up to<br />

50 µM Fe+3 while at the highest level of Fe+3 (200 µM), 7% decrease in<br />

antifungal activity was measured. Increase in the concentration of Fe+3 in the<br />

liquid culture increased the intracellular protein (2%) and carbohydrate contents<br />

(14%) and extracellular protein (7%) and polysaccharide contents (14%) while<br />

the intracellular lipid contents were increased (11%) only up to 50 µM Fe+3. In<br />

addition, the regulatory effects of Fe+3 were also reflected by the increase in<br />

total RNA contents and relative expression of the six module-containing<br />

nonribosomal peptide synthetase (FusA) up to 50 µM Fe+3 after that a<br />

continuous decrease was observed. It is first report that depicted the direct or<br />

indirect correlation of Fe+3 with the production of fusaricidin type antifungal<br />

compounds and other cellular processes of P. polymyxa.<br />

PM 23<br />

Interplay between LscR and the H-NS-like proteins, MvaT<br />

and MvaU, determines temperature-dependent expression<br />

of levansucrase in Pseudomonas syringae<br />

D. Zhurina *1 , A. Arndt 2 , M. Brocker 3 , H. Weingart 1 , M. Bott 3 , B. Eikmanns 2 ,<br />

M. Ullrich 1<br />

1<br />

School of Engineering and Science, Jacobs University, Bremen, Germany<br />

2<br />

Institut für Mikrobiologie und Biotechnologie, University of Ulm, Ulm,<br />

Germany<br />

3<br />

Institut für Biotechnologie 1, Forschungszentrum Jülich, Jülich, Germany<br />

In pathogenic bacteria, exopolysaccharides (EPS) play an important role in<br />

successful colonization of the host. In the plant pathogen, Pseudomonas<br />

syringae pv. glycinea PG4180, synthesis of the EPS, levan, is mediated by two<br />

levansucrases LscB and LscC, which are 97% identical and exhibit a<br />

temperature-dependent mode of expression. Maximum mRNA synthesis for<br />

these genes occurs at the virulence-promoting temperature of 18°C and is<br />

repressed at the optimal growth temperature of 28°C. DNA affinity<br />

chromatography revealed that two H-NS-like repressor proteins termed MvaT<br />

and MvaU bound upstream of lscB at 28°C whereas at 18°C no such binding<br />

occured. Functional screening of a genomic library of P. syringae in P. putida<br />

carrying lscB allowed the identification of a third transcriptional regulator<br />

named LscR, which gave rise to transcription of lscB in the same temperaturedependent<br />

manner as in its native host, P. syringae. The observed phenomenon<br />

was found to be due to removal of MvaT from the lscB upstream sequence by<br />

LscR. Surprisingly LscR was not binding to the lscB promoter region at any<br />

tested temperature. This result suggested that LscR might not interact with the<br />

DNA target but rather with MvaT directly. It was concluded that lack of lscB<br />

expression at 28°C is based on reversible binding of MvaT and MvaU to its<br />

upstream sequence and that this binding might in turn be regulated by the<br />

activator protein, LscR, at 18°C. The provided results give novel insights into<br />

mechanisms, by which certain H-NS like proteins might regulate their target<br />

genes.<br />

PM 24<br />

Quantitative proteome analysis of the effects of induction of<br />

alginate synthesis in genetically engineered Pseudomonas<br />

fluorescens strains<br />

F. Schmidt *1 , M. Gesell Salazar 1 , L. Steil 1 , R. Dam 1 , U. Lissner 1 , S. Valla 2 , U.<br />

Völker 1<br />

1 Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-<br />

Arndt-University of Greifswald, Greifswald, Germany<br />

2 Department for Biotechnology, Norwegian University of Science and<br />

Technology, Trondheim, Norway<br />

In this study we combined two complementary proteomic approaches to<br />

explore the consequence of permanent activation of the alternative sigma factor<br />

Alg4 and alginate production onto the expression pattern of P. fluorescens. In<br />

or<strong>der</strong> to separate pleiotropic effects of induction of the Alg4 regulon from those<br />

of metabolic alginate production and synthesis of alginate biosynthesis<br />

enzymes the analysis included a wild type strain and four isogenic mutants with<br />

defects on mucA, algC, mucA algC and mucA PalgD. The study was<br />

performed in or<strong>der</strong> to provide data for the modeling of the effects of<br />

perturbations of the metabolic networks resulting from controlled induction of<br />

alginate synthesis in P. fluorescens. Comparative 2D-DIGE/MALDI-MS as<br />

well as gel-free nanoLC/FT-ICR-MS analysis were applied. The combination<br />

of both approaches resulted in the identification of approx. 25% of the<br />

predicted proteome of approx. 6,144 coding regions and allowed us to (i)<br />

differentiate the wild type and mutants, (ii) describe the mucA regulon, (iii)<br />

study the effect of burden aginate production and enzyme production, and (iiii)<br />

separate pleiotropic effects.<br />

PM 25<br />

Horizontal gene transfer of eukaryotic cytoskeletal elements<br />

within a complex bacterial community<br />

A. Guljamow *1 , N. Tandeau de Marsac 2 , E. Dittmann 1<br />

1<br />

Molecular Ecology, Institute of Biology, Humboldt University, Berlin,<br />

Germany<br />

2<br />

Departement de Microbiologie Fondamentale et Medicale, Institut Pasteur,<br />

Paris, France<br />

In or<strong>der</strong> to respond to environmental conditions and to invade ecological<br />

niches, prokaryotic communities are able to dynamically rearrange their genetic<br />

equipment by means of horizontal gene transfer. The genome of the<br />

cyanobacterium Microcystis aeruginosa contains a potentially mobile genomic<br />

island encoding two components of the eukaryotic actin cytoskeleton. One is<br />

actin itself, the other is profilin, an actin binding protein only known in<br />

eukaryotes. Phylogenetic data show that both proteins have not arisen from<br />

ancestral cyanobacterial sequences but rather were introduced into the<br />

Microcystis lineage later in evolution, exemplifying an otherwise rarely<br />

documented and poorly un<strong>der</strong>stood case of eukaryote-to-prokaryote horizontal<br />

gene transfer.<br />

The mobile genetic element is only detectable in one single strain in culture and<br />

in DNA preparations generated from field samples of this strain’s original<br />

habitat in a Dutch coastal region. Since Immunofluorescence micrographs<br />

suggest that actin is localized towards the cell’s periphery possibly playing a<br />

role in cell stabilisation, we conclude that this genomic island confers a<br />

selective advantage specific to this environment. Intending to obtain a more<br />

detailed insight into the mechanisms un<strong>der</strong>lying the genetic cross-talk within<br />

complex bacterial communities, we have focussed on a metagenomic approach<br />

to determine the species distribution and possible degree of variation of this<br />

genomic island in field samples taken from the original habitat and from a<br />

number of neighbouring waters.<br />

PM 26<br />

Structure-functional analysis of the Dictyoglomus cell<br />

envelope<br />

W. Theilmann 1 , M. Hoppert *1 , M. Valdez 1 , P. Dutow 1 , S. Zeides 1 , K. Döring 1 ,<br />

H. Pförtner 1 , W. Liebl 2<br />

1 Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen,<br />

Germany<br />

2 Lehrstuhl für Mikrobiologie, Technische Universität München, München,<br />

Germany<br />

Several closely related strains of the thermophilic ancient eubacterium<br />

Dictyoglomus have been isolated from various hot springs on the Philippine<br />

archipelago. Cell envelopes of all Dictyoglomus strains show several peculiar<br />

features that are apparently unique for the genus Dictyoglomus, as well as<br />

features that are common for other "ancient" eubacteria. We documented the<br />

formation of these multicellular structures, reaching dimensions in the 100-µm<br />

range, by electron microscopy, and, in an anaerobic incubation chamber at 60<br />

°C, by light microscopy. The filamentous cells develop pili at the cell poles,<br />

guided by large columnar protein assemblies that traverse the periplasm.<br />

Filamentous protein complexes span the periplasmic space at the longitudinal<br />

sides of the cell, similar to the Thermotoga Omp α protein. By the end of the<br />

exponential growth phase, Dictyoglomus strains form multicellular aggregates<br />

inside a compartment surrounded by a single, continuous outer envelope,<br />

similar to members of the Thermotogales-group ("rotund bodies"). The<br />

transition between unicellular and multicellular lifestyle can be explained by<br />

the partial detachment of a protoplast from the outer envelope during cell<br />

division. When the outer envelope is partially detached from the protoplast,<br />

mechanical forces, apparently generated by protoplast elongation, drive cell<br />

rearrangement of daughter cells inside the compartment. During the following<br />

rounds of cell division, the overall shape of the compartment changes from<br />

spindle-like to globular geometry.<br />

PN 01<br />

Detection of certain food-borne pathogen in some Egyptian<br />

meats<br />

E.M. El-Safey *1 , A.A. Aldahash 2<br />

1<br />

Botany and microbiology, Al-Azhar university, Assuit, Egypt<br />

2<br />

Commuity college of Al Majma ah, King saud university, Al Majma ah, Saudi<br />

Arabia<br />

In this study 48 samples of processed meat e.g. luncheon, Basterma and ground<br />

beef were collected from retail supermarkets in Assiut city, middle Egypt. In<br />

three consecutive periods, 16 sample per period. The first goal was designed on<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

the prevalence of total aerobic and anaerobic mesophiles, and aerobic<br />

psychrophiles in addition to aerobic and anaerobic spore former bacteria. The<br />

second goal was designed for looking for the presence of certain food borne<br />

bacterial pathogens e.g. Bacillus cereus, Listeria monocytogenes, E. coli<br />

O157:H7 and Staphylococcus aureus. The population of aerobic mesophiles,<br />

aerobic psychrophiles, and anaerobic mesophiles were counted at the average<br />

of 6.177, 2.618 and 5.773 Log 10(CFU/g) respectively. However, the<br />

population of aerobic spore formers, and anaerobic spore formers were counted<br />

at the average of 97.103 and 108.236 spore per gram respectively. E .coli<br />

O157:H7 was isolated from 2 of 6 (33.33%)beef burger, but was not found in<br />

18 beef luncheon, 12 chicken luncheon, 6 ground beef and 6 Basterma ,<br />

Bacillus cereus was isolated from 3 of 18 (16.66%) beef luncheon, but could<br />

not be detected in 12 chicken luncheon, 6 ground beef 6 beef burger, and 6<br />

Basterma, Listeria monocytogenes was recovered from 1of 6 (16.66%) beef<br />

burger, 1 of 6(16.66%) Basterma, but not found in 18 beef luncheon, 12<br />

chicken luncheon, and 6 ground beef, and Staphylococcus aureus was isolated<br />

from 2 of 18 (11.11%) beef luncheon, 2 of 12 (16.66%) chicken luncheon, 1 of<br />

6 (16.66%) ground beef , but could not be detected in 6 beef burger, and 6<br />

Basterma.<br />

The data highlighted the need for a more systematic approach to ensuring safe<br />

food through implementing quality control methods to prevent the entry and<br />

proliferation of pathogen in meat and meat products especially during<br />

processing, handling and storing.<br />

PN 02<br />

Metabolic activities of selected lactobacilli in amaranth<br />

sourdough fermentations<br />

A. Vogel *1 , Y. Sterr 1 , A. Weiss 1 , H. Schmidt 1<br />

1 Fachgebiet Lebensmittelmikrobiologie, Institut für Lebensmittelwissenschaft<br />

und Biotechnologie, Universität Hohenheim, Stuttgart, Germany<br />

Amaranth is characterized by a high nutritional value of essential amino acids<br />

and minerals. This special composition leads to different aroma components<br />

with influence on bread taste and odor. The aim of our study was to<br />

characterize two strains of the genus Lactobacillus with positive effects on the<br />

pseudocereal sourdoughs.<br />

L. plantarum AL30 and L. paralimentarius AL28, isolated from previous<br />

fermentations with amaranth, were used as single strains and in combination.<br />

As control one spontaneous fermented dough was treated simultaneously. The<br />

fermentations were performed over a period of 48 h at 30°C with a dough yield<br />

of 200. At intervals of 2 to 3 h the pH-value, the total titratable acidity values,<br />

metabolic profiles, proteolysis and the cultural dominance were measured. To<br />

evaluate the dough microflora different growth media were used.<br />

Single strains as well as the combined strains dominated the microflora after 12<br />

h in all three inoculated doughs. Colony counts ranged between 10 9 and 10 10<br />

cfu/g sourdough. Stable pH- and total titratable acidity values were observed<br />

after 15 h and 24 h, respectively. Increased proteolytic activities could not be<br />

detected in comparison to the data of the spontaneous fermented dough.<br />

Both strains alone as well as the combination show a good potential for an<br />

application as sourdough starters on account of their dominance, rapid<br />

acidification and competitiveness.<br />

PN 03<br />

Quantification of key genes steering the microbial nitrogen<br />

cycle in the rhizosphere of sorghum cultivars in tropical<br />

agro-ecosystems<br />

B. Hai *1 , F. Haesler 2 , K. Schauss 3 , J.C. Munch 4 , M. Schloter 1<br />

1 Institute of Soil Ecology/ Terrestrial Ecogenetics, Helmholtz Zentrum<br />

München - German Research Center for Environmental Health, Neuherberg,<br />

Germany<br />

2 Chair of Soil Ecology, Technical University Munich, Neuherberg, Germany<br />

3 Institute of Soil Ecology, Helmholtz Zentrum München - German Research<br />

Center for Environmental Health, Neuherberg, Germany<br />

The effect of agricultural management practices on geochemical cycles in<br />

mo<strong>der</strong>ate ecosystems is by far better un<strong>der</strong>stood than in semi-arid regions,<br />

where fertilizer availability and climatic conditions are less favorable. We<br />

studied the impact of different fertilizer regimes in an agricultural long-term<br />

observatory in Burkina Faso at three different plant development stages (early<br />

leaf development, flowering and senescence) of sorghum cultivars. Using realtime<br />

PCR functional microbial communities involved in key processes of the<br />

nitrogen cycle (nitrogen fixation, ammonia oxidation and denitrification) in the<br />

rhizosphere were investigated. The results indicate that fertilizer treatments and<br />

plant development stages combined with environmental factors affected the<br />

abundance of the targeted functional genes in the rhizosphere. While nitrogen-<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

fixing populations were dominating the investigated communities when<br />

applying organic fertilizers (manure and straw), their number was<br />

comparatively reduced in urea-treated plots. In contrast, ammonia-oxidizing<br />

bacteria (AOB) were not only increased in absolute numbers but also in relation<br />

to the other bacterial groups investigated in the urea-amended plots. Ammoniaoxidizing<br />

archaea (AOA) exhibited higher numbers compared to AOB<br />

independent from fertilizer application. Similarly, also denitrifiers were more<br />

abundant in the urea-treated plots. Our data imply as well that, more than in<br />

mo<strong>der</strong>ate regions, water availability shapes microbial communities in the<br />

rhizosphere, as low gene abundance data for all genes measured at the<br />

flowering stage were obtained, when water availability was very limited.<br />

PN 04<br />

ATP-generation within the archaeal coculture of Ignicoccus<br />

hospitalis and Nanoarchaeum equitans<br />

U. Kueper *1 , F. Mayer 1 , K. Pisa 2 , R. Rachel 3 , V. Mueller 2 , H. Huber 1<br />

1 Institute for Microbiology, University of Regensburg, Regensburg, Germany<br />

2 Institute for Molecular Bio Sciences, University of Frankfurt / Main, Frankfurt<br />

/ Main, Germany<br />

3 Centre of Electron Microscopy, University of Regensburg, Regensburg,<br />

Germany<br />

123<br />

Ignicoccus hospitalis is a hyperthermophilic crenarchaeal coccus that serves as<br />

a host organism for Nanoarchaeum equitans thereby forming the first and only<br />

known intimate association of two Archaea.<br />

Since the annotation of the N. equitans genome revealed only a (if at all)<br />

rudimentary ATPase/synthase consisting of just five different subunits, we<br />

started a project on the investigation of this enzyme in N. equitans and in its<br />

host. The annotation of the genome of I. hospitalis revealed 8 to 9 different<br />

subunits (A, B, C, D, E, F, I, K and ?) of a membrane bound A1AO<br />

ATPase/synthase with a postulated molecular mass of 660 kDa. For a first<br />

characterization of the I. hospitalis enzyme we purified the A1-subcomplex<br />

(440 kDa). The enrichment of this complex was 22 fold with a final specific<br />

activity of 44 U / mg. All purification steps were controlled by western blot<br />

analysis, native PAGE (hrCNE), and in-gel-assay for ATP hydrolysis. In<br />

addition, purification of the A1-subcomplex was verified by electron<br />

microscopic analysis.<br />

The dependency of ATP synthesis in I. hospitalis on the transmembrane proton<br />

gradient was shown by "pH-jump" experiments with whole cells. An artificial<br />

proton gradient (ΔpH) was able to drive ATP synthesis, while protonophores<br />

like Tetrachlorosalicylanilide (TCS) were able to uncouple the proton gradient.<br />

The raising of antibodies against the A1-subcomplex is in progress to obtain<br />

information not only on the localization of the enzyme in I. hospitalis but also<br />

in N. equitans.<br />

PN 05<br />

Customizing PCS biofilm reactor for enrichment and<br />

isolation of thermophilic bacterium producing lipase<br />

M.A. Khiyami *1 , E.J. Alyamani 2<br />

1 General Directorate of Research Grants, King Abdulaziz City for Science and<br />

Technology, Riyadh, Saudi Arabia<br />

2 Natural Resources & Environment Research Institute, King Abdulaziz City for<br />

Science and Technology, Riyadh, Saudi Arabia<br />

Biofilms represent a natural form of cell immobilization, in which microbial<br />

cells are attached to a solid surface. A Plastic Composite Supports (PCS)<br />

stimulates microbial attachment and serves as a vehicle for slow release of<br />

nutrients during long term fermentation.<br />

In this study, samples were collected from hot springs and oil reservoirs. The<br />

samples were chemically analyzed for minerals and salts. PCS bioreactor was<br />

designed to enhance thermophilic biofilm formation. The bioreactor<br />

temperature was set at >70°C controlled by using sand baths. Repeated batch<br />

fermentation method employing both rich and minimal media was used to<br />

isolate bacteria grew from 45 to 70°C. Spent media was checked for pH and<br />

Gram staining every 24hr and changed every 7 days. Also, biological activity<br />

was monitored via cell count using plating on 2% gelrite. The isolates images<br />

were captured by scanning electron microscopy. The optimal growth conditions<br />

of the strains were determined. The isolates were examined for hydrolytic<br />

enzyme. The isolates were identified by 16S rDNA sequence analysis.<br />

The PCS containing 50% polypropylene, 40% ground soyhulls, 5% yeast<br />

extract, and 5% salts were used for enrichment of lipase-producing<br />

thermophilic bacterium. The isolates were aerobic with lipase activity. The<br />

500bp sequencing of bacterial 16S rDNA gene indicated the isolates were<br />

Aneurinibacillus migulanus and Bacillus thermoamylovorans.

124<br />

PN 06<br />

Effect of rice cultivars on root-associated methanotrophic<br />

communities<br />

C. Lüke *1 , P. Frenzel 1<br />

1 Department of Biogeochemistry, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

Rice agriculture represents a major source of the greenhouse gas methane.<br />

However, a large amount of methane is oxidized by methanotrophic bacteria<br />

before <strong>bei</strong>ng released to the atmosphere. Methanotrophs are characterized by<br />

their unique ability to use methane as sole source for carbon and energy. They<br />

are located at oxic-anoxic interfaces where methane and oxygen are present,<br />

such as the rhizosphere. Although they have been studied extensively in the<br />

past, only little is known about natural or anthropogenic factors influencing<br />

their large diversity.<br />

In this study, we investigated the effect of 20 different rice cultivars on<br />

methanotrophic communities associated with the roots of rice plants. The<br />

pmoA gene encoding a subunit of the particulate methane monooxygenase<br />

(catalyzing the first step of methane oxidation) was used as a functional and<br />

phylogenetic marker and analyzed using two different fingerprinting methods.<br />

The well established terminal restriction fragment length polymorphism (T-<br />

RFLP) analysis was compared to results obtained using a diagnostic pmoA<br />

microarray.<br />

Both methods indicated that type Ib (Methylococcus/Methylocaldum) and type<br />

II (Methylosinus/Methylocystis) were the predominat methanotrophs located on<br />

rice roots. Interestingly, analysis of pmoA transcripts suggested<br />

Methylobacter/Methylomonas (type Ia) to present the actively methane<br />

oxidizing population in this environment.<br />

PN 07<br />

Effects of repeated applications of sulfadiazinecontaminated<br />

manure on nitrogen turnover processes in<br />

two arable soils<br />

K. Schauss *1 , A. Kotzerke 2 , S. Thiele-Bruhn 3 , B.M. Wilke 2 , M. Schloter 1<br />

1<br />

Department of Terrestrial Ecogenetics, Helmholtz Zentrum München,<br />

Neuherberg, Germany<br />

2<br />

Institute of Ecology, Berlin University of Technology, Berlin, Germany<br />

3<br />

Department of Soil Science, University of Trier, Trier, Germany<br />

Sulfadiazine (SDZ) is a frequently used antibiotic in agricultural husbandry.<br />

Via manuring of excrements of medicated animals the drug reaches the soil and<br />

might impair important biochemical transformation processes performed by<br />

microbes, e.g. the nitrogen turnover. We studied in a laboratory experiment the<br />

effect of repeated applications of pig manure and pig manure spiked with two<br />

different concentrations of SDZ on microbes <strong>bei</strong>ng involved in nitrogen cycling<br />

in two arable soils. The manures were applied three times within 130 days and<br />

samplings were performed 3, 18, 32, and 60 days after each amendment.<br />

Besides observing nitrous oxide and carbon dioxide fluxes, we investigated the<br />

concentrations of SDZ, ammonium, nitrate, and dissolved organic nitrogen and<br />

carbon in the soils, determined the potential nitrification and denitrification<br />

activities, and quantified functional genes of nitrification and denitrification by<br />

real-time PCR. The antibiotic effect of SDZ was clearly reflected e.g. in<br />

delayed or reduced ammonium and nitrate turnover, repressed nitrification<br />

activity, and altered nitrous oxide flux pattern in both soils. Moreover, we<br />

assessed also differences in the response of microbes in the silty loam and the<br />

loamy sand soil to the addition of manure and the SDZ-amended manure.<br />

PN 08<br />

Microbial Interactions in Marine Systems<br />

C. Klockow *1 , B. Öztürk 1 , H. Teeling 1 , J. Waldmann 1 , F.O. Glöckner 1<br />

1<br />

Microbial Genomics, Max Planck Institut for Marine Microbiology, Bremen,<br />

Germany<br />

The aim of the project “Microbial Interactions in Marine Systems”, short<br />

MIMAS, is to explore the seasonal changes of the microbial communities at<br />

long term ecological research sites in the North Sea (Helgoland Roads) and the<br />

Baltic Sea (Gotland Deep). An integrated, state of the art, approach will be<br />

applied using the full spectrum of currently available molecular tools:<br />

Ribosomal RNA sequencing and single cell in situ hybridization to unravel the<br />

diversity and abundance of organisms, metagenomics to address the genetic<br />

potential, as well as metatranscriptomics and metaproteomics to explore the<br />

active set of genes.<br />

A central step is the analysis of the metatranscriptome via pyrosequencing,<br />

which will give clues regarding the active part of the communities and their<br />

seasonal changes. This approach requires challenging method development<br />

regarding sampling techniques, downstream processing and bioinformatics.<br />

Marine bacterial communities represent 96% of the world´s prokaryotes and<br />

play an important role in element cycling and contain a large number of<br />

unexplored enzymes. This holistic approach will overcome the fragmentary<br />

un<strong>der</strong>standing of bacterial communities and shed light on their functions and<br />

abilities as well as their response to environmental changes such as climate<br />

change.<br />

PN 09<br />

Microbial communities in the gut of Daphnia magna<br />

H.M. Freese *1 , B. Schink 1<br />

1 Microbial Ecology, Department of Biology, University of Konstanz, Konstanz,<br />

Germany<br />

In freshwater lakes, free and attached bacteria play a key role in carbon<br />

mineralisation, but it is unknown, if they have a similar role in the guts of<br />

zooplankton. In other eukaryotes like humans, cetoniid beetles, and termites,<br />

microorganisms break down more complex organic matter to easily absorbable<br />

organic substances which sustain the host while they live symbiotically in<br />

anoxic guts.<br />

Our research focuses on the gut microbiology of Daphnia magna. Daphnia are<br />

even smaller than termites, which are known to have an anoxic gut<br />

environment, and their gut is surrounded by oxic water. This leads to the<br />

question whether anoxic or oxic bacteria colonise the gut and whether they<br />

participate in digestion or compete with Daphnia for organic food constituents.<br />

Therefore, among others, aerobic and anaerobic bacteria were isolated from<br />

Daphnia guts as well as from the surrounding water. The obtained isolates were<br />

identified and compared to the gut microbial community via terminal restriction<br />

fragment length polymorphism. Preliminary results indicate that the community<br />

was dominated by few types of bacteria whereas isolated strains so far did not<br />

or only to a small extent contribute to microbial community.<br />

Since Daphnia can graze on bacteria, we are especially interested if the<br />

microbial community in Daphnia guts is stable when exposed to varying<br />

conditions or if it is continuously restructured. Experiments are un<strong>der</strong> way to<br />

detect if the community composition changes when clones of Daphnia were<br />

starved or fed with different known (isolated) bacteria in comparison to normal<br />

treatment during a defined time course.<br />

PN 10<br />

Living and non-living bacteria in groundwater and<br />

drinking water<br />

G. Preuß *1 , E. Ziemann 1 , N. Zullei-Seibert 1<br />

1 Institut für Wasserforschung GmbH, Zum Kellerbach 46, Schwerte, Germany<br />

Several fluorescence staining methods were proofed to determine bacteria in<br />

water samples collected from a groundwater catchment area used for drinking<br />

water production. The investigation aimed at comparing the ability of<br />

microscopic methods for the assessment of hygienic drinking water quality.<br />

The samples were treated with DNA-binding dyes (DAPI, SYBR Green II,<br />

SYTO 62) to estimate total bacteria counts and with PI, that penetrates the<br />

membranes of non-living cells only. Additionally cFDA was used for viability<br />

analysis after incubation at 30°C. Colony forming units (cfu/ml) were estimated<br />

using cultivating methods according to the German drinking water regulation.<br />

In surface water dyes with DAPI, SYBR Green II and SYTO 62 showed<br />

comparable results between 1,6*10 6 and 2,8*10 6 cells/ml. In groundwater and<br />

drinking water cell counts were between 3,9*10 4 and 8,4*10 4 cells/ml. In these<br />

samples the highest results were found with SYBR Green II, detecting 43%<br />

more cells than the assay with DAPI.<br />

Using SYTO 62 or SYBR Green II gave the same results as the sum of cFDAactive<br />

cells and dead cells detected with PI. The rate of active cells was<br />

between 7,8% in surface water and 1,4% in drinking water. Only 0,02% of<br />

these cells could be detected on nutrient plates.<br />

This results confirm the actual knowledge about viable but not cultivable<br />

bacteria (VBNC) in nutrient-poor water. In addition of the standard culture<br />

methods fluorescence microscopic techniques can be used as sensitive<br />

parameters to assess the efficiency of water treatment processes.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 11<br />

From Deep-sea Hydrothermal Vent Habitats: Primary<br />

Productivity Based on Hydrogen Oxidation and Diversity of<br />

Uptake Hydrogenases<br />

M. Perner *1 , R. Seifert 2 , J. Petersen 3 , W. Streit 1<br />

1<br />

Microbiology and Biotechnology, University of Hamburg, Hamburg, Germany<br />

2<br />

Institute of Biogeochemistry and Marine Chemistry, University of Hamburg,<br />

Hamburg, Germany<br />

3<br />

Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen,<br />

Germany<br />

The aim of this study was to link chemistry, phylogenetic and metabolic<br />

diversity with productivity of fluids from two low-temperature hydrothermal<br />

systems. We chose two hydrothermal vent sites that differ significantly in their<br />

fluid chemistry. The first site, Quest, has high hydrogen and low hydrogen<br />

sulfide concentrations. The second site, Clueless, has low hydrogen and high<br />

hydrogen sulfide concentrations. Our approach included molecular studies of<br />

16S rRNA and functional gene diversity, and physiological studies of substrate<br />

uptake and inorganic carbon fixation in the vent fluids. Incubation experiments<br />

were performed ex situ by supplementing the natural hydrothermal fluids with<br />

hydrogen or hydrogen sulfide. We incubated the hydrogen sulfide-amended<br />

fluids un<strong>der</strong> anoxic conditions, and compared both oxic and anoxic incubations<br />

of hydrogen-amended fluids. We found a substantially higher diversity of PCRamplified<br />

uptake hydrogenase genes from the hydrogen-rich site Quest.<br />

Unexpectedly, the highest CO2 fixation rates in hydrogen-amended fluids were<br />

from the Clueless site, where hydrogen concentrations in the natural fluids are<br />

relatively low. We therefore found a negative correlation between diversity of<br />

hydrogenases and in situ hydrogen concentration, and primary productivity<br />

based on hydrogen oxidation. Although the Epsilonproteobacteria are<br />

consi<strong>der</strong>ed to be the primary colonizers at vent habitats because of their<br />

metabolic flexibility, they were out-competed by other Proteobacteria in all<br />

incubation experiments. CARD-FISH analyses of the fluid incubations<br />

identified the Gammaproteobacteria as the most successful un<strong>der</strong> many of<br />

conditions tested, indicating their ability to exploit the resources provided in ex<br />

situ incubation experiments.<br />

PN 12<br />

Shift of the bacterial community by bioaugmentation<br />

during the start up phase of a biogas fermenter fed with<br />

renewable biomass.<br />

L. Neumann *1 , M. Unbehauen 1 , P. Scherer 1<br />

1 Research and Transfer Center for Renewable Energy and Process Efficiency,<br />

Hamburg University of Applied Sciences, Hamburg, Germany<br />

The start up phase of a biogas plant shows generally a great effect on the whole<br />

process. The influence of the bacterial composition in the inoculum on the<br />

former process is often discussed and it is known that an optimal inoculum can<br />

accelerate the start up phase of a biogas plant. In this study the start up phase of<br />

the biogas fermentation was simulated in 2 liter batch reactors. The aim of this<br />

work was to study the effect of hot rot compost on anaerobic digestion as<br />

observed previously in the biogas lab of the Hamburg University of Applied<br />

Sciences. This influence was investigated in combination with sewage sludge<br />

as main inoculum, un<strong>der</strong> mesophilic (40°C) as well as thermopilic (60°C)<br />

conditions. The experiment was performed by two feeding steps with sugar<br />

beet mash as substrate for 28 days. Following process parameters were<br />

measured online: gas production, methane content and pH value. Volatile fatty<br />

acids were analyzed at the start and the end of the experiment. With the<br />

distance of 1 week the samples for molecular analysis were taken. For the<br />

characterization and quantification of the bacterial community shift the<br />

fluorescence in situ hybridization (FISH) was applied, in combination with a<br />

special pretreatment for environmental samples. A large probe set with over 30<br />

different organisms was applied to investigate the dynamics in the archaea and<br />

bacteria population. Dramatic positive effects of compost as inoculum on the<br />

gas production could be observed. Also the methane content of the gas<br />

increased, especially for the thermopilic fermentation. It will be discussed, if<br />

the positive effect by bioaugmentation depended more on the hydrolytic or<br />

methanogenic bacteria from the hot rot compost.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PN 13<br />

Analysis of nitrifying bacteria in bioreactors treating<br />

landfill leachate reveals the presence of an unusual<br />

Nitrospira sp.<br />

S. Bathe *1 , J. Winter 1 , F.H. Frimmel 2 , E. ter Haseborg 2<br />

1 Institute of Biology for Engineers and Wastewater Biotechnology, University<br />

of Karlsruhe, Karlsruhe, Germany<br />

2 Engler-Bunte-Institute, Chair of Water Chemistry, University of Karlsruhe,<br />

Karlsruhe, Germany<br />

125<br />

Landfill leachates are demanding wastewaters containing high amounts of<br />

ammonia, recalcitrant humic polymers, and a BOD/N ratio < 1. Treatment of<br />

such wastewaters in two reactor systems investigated here is <strong>bei</strong>ng conducted in<br />

oxic-anoxic reactor combinations (one with pre-, the other one with postnitrification)<br />

at temperatures of 35-40°C, high solid concentrations of 2.5%,<br />

and substantial additional carbon source feeding to support denitrification. Due<br />

to these unusual conditions, the populations of nitrifying bacteria were<br />

investigated by PCR-DGGE and FISH.<br />

General DGGE analyses revealed bacterial communities containing relatively<br />

few dominant bands. Group-specific investigations of ammonia-oxidizing<br />

bacteria showed simple AOB communities dominated by bands with sequences<br />

related to Nitrosomonas nitrosa. Whereas Nitrobacter spp. could not be<br />

detected in the samples, Nitrospira-specific primers yielded strong products.<br />

Subsequent DGGE analysis showed only one dominant band in samples from<br />

the two systems. The phylogenetic position of the corresponding sequence was<br />

outside the established lineages I to IV and diverged more than 5% from the<br />

most closely related database sequence, indicating the possible presence of a<br />

novel Nitrospira species.<br />

FISH results showed that the sludges were dominated by β-proteobacteria, with<br />

Nitrosomonas and Nitrospira representing significant proportions of the total<br />

microbial communities. A probe to specifically target the new Nitrospira sp.<br />

was designed to confirm the dominance of this organism within the nitriteoxidizer<br />

populations of the systems.<br />

Further work concerning the evaluation of the designed FISH probe as well as<br />

enrichment of this nitrite-oxidizer followed by investigation of its physiological<br />

characteristics is un<strong>der</strong> way.<br />

PN 14<br />

Matrix composition and community structure analysis of a<br />

novel bacterial pyrite leaching community<br />

S. Ziegler *1 , S. Ackermann 2 , J. Göttlicher 3 , J. Majzlan 2 , J. Gescher 1<br />

1<br />

Institut für Biologie II/ Mikrobiologie, Albert-Ludwigs-Universität, Freiburg,<br />

Germany<br />

2<br />

Mineralogisch-Geochemisches Institut, Albert-Ludwigs-Universität, Freiburg,<br />

Germany<br />

3<br />

Institut für Synchrotron Strahlung ISS, ANKA, Karlsruhe, Germany<br />

Pyrite is the most abundant sulfide mineral in Earth’s crust. The oxidation of<br />

pyrite leads to the release of ferric iron and - via several steps - of sulfuric acid.<br />

This causes a dramatic decrease of the pH values which in turn lead to serious<br />

environmental problems. This process called acid mine drainage (AMD) is<br />

greatly accelerated by bacteria that catalyze the rate limiting step of pyrite<br />

oxidation, namely the reaction from ferrous to ferric iron. We found a novel<br />

bacterial community that is embedded in a matrix of organic substances and<br />

bio/geochemical products of pyrite oxidation. This community grows in<br />

stalactite-like structures on the ceiling of an abandoned pyrite mine at pH<br />

values of 2.2-2.6. We measured sulfate concentrations of 200 mM and total iron<br />

concentrations of 60 mM in the soluble fraction of the matrix. Micro-X-ray<br />

diffraction analysis showed that jarosite is the major mineral embedded in the<br />

biofilm matrix. X-ray absorption near-edge structure experiments at the ANKA<br />

(Karlsruhe) SUL-X beamline revealed three different sulfur species, whereby<br />

the major signal was caused by sulfate. The other two peaks might correspond<br />

to organic sulfur compounds. Via restriction fragment length polymorphism<br />

analysis, we elucidated the community structure. It consists mainly of iron and<br />

probably sulfur oxidizing microorganisms (Leptospirillum ferrooxidans and<br />

Ferrovum myxofaciens like strains) but also of bacteria that might be involved<br />

in the reverse reactions (dissimilatory sulfate and dissimilatory iron reducers).<br />

Therefore, we hypothesize that a complete iron and sulfur cycle might be<br />

accomplished within this biofilm community.

126<br />

PN 15<br />

Identification of local microbial communities in the North<br />

Atlantic Ocean by T-RFLP<br />

R. Hahnke *1 , C. Probian 1 , J. Har<strong>der</strong> 1<br />

1<br />

Dept. of Microbiology, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

The dispersal of microbes is mainly resulting from the movement of air or<br />

water masses. We analyzed the microbial populations in the phototrophic zone<br />

of the North Atlantic Ocean far from coastal shelfs by T-RFLP. Totally 467<br />

individual peaks were found, by definition the OTU diversity. We applied a full<br />

statistical approach to identify local OTU populations and correlated these<br />

results with environmental parameters. The terminal restriction fragment<br />

pattern revealed individual populations north and south of the Gulf stream as<br />

well as individual populations along a depth gradient, likely corresponding to<br />

the light intensity. Currently we explore in silico methods to link terminal<br />

restriction fragments with phylogenetic information via the statistics of the<br />

restriction fragment intensity. The mathematical approach and first results will<br />

be presented.<br />

PN 16<br />

Unexpected diversity within the Rimicaris exoculata<br />

epibiotic community - Insights into sulfur and carbon<br />

metabolism<br />

M. Hügler *1 , J.M. Struck 2 , N. Dubilier 2 , J.F. Imhoff 3 , S.M. Sievert 4<br />

1 Mikrobiologie, DVGW-Technologiezentrum Wasser, Karlsruhe, Germany<br />

2 Molekulare Ökologie, MPI für marine Mikrobiologie, Bremen, Germany<br />

3 Marine Mikrobiologie, IFM-GEOMAR, Kiel, Germany<br />

4 Biology Department, Woods Hole Oceanographic Institution, Woods Hole,<br />

United States<br />

The shrimp Rimicaris exoculata harbors a specialized epibiotic community in<br />

its gill chamber. We analyzed specimens from the SnakePit hydrothermal vent<br />

field on the Mid-Atlantic-Ridge by complementing a 16S rRNA based diversity<br />

assessment with a survey of metabolic genes involved in carbon, sulfur and<br />

hydrogen metabolism. In addition to epsilonproteobacteria, the epibiotic<br />

community unexpectedly also harbors deltaproteobacteria of a single<br />

phylotype, closely related to the genus Desulfocapsa. These results are in<br />

congruence with functional gene analyses, which indicate the presence of<br />

autotrophic sulfur-oxidizing epsilonproteobacteria and sulfur-metabolizing<br />

deltaproteobacteria. In addition, hydrogen metabolism might also play an<br />

important role as hydrogenases from both groups could be identified. Our data<br />

further suggest that autotrophic carbon fixation is primarily carried out by<br />

epsilonproteobacteria using the reductive tricarboxylic acid cycle. Interestingly,<br />

the community composition of the gills seems to differ from that of the inner<br />

lining of the gill chamber. Overall, our results show that the epibiotic<br />

community is more diverse than previously assumed. The co-occurrence of<br />

sulfur-oxidizing and sulfur-reducing epibionts indicates that these are involved<br />

in the syntrophic exchange of sulfur, possibly increasing the overall efficiency<br />

of the symbiosis.<br />

PN 17<br />

Microbial communities releasing methane in abandoned<br />

coal mines<br />

S. Beckmann *1 , M. Krüger 2 , H. Cypionka 1 , B. Engelen 1<br />

1 ICBM, University of Oldenburg, Oldenburg, Germany<br />

2 BGR, Department of Geomicrobiology, Hannover, Germany<br />

Worldwide, about seven percent of the annual methane emissions originate<br />

from coal mining. In many coal deposits, stable isotopic analyses have shown a<br />

mixed thermogenic and biogenic origin of methane [1]. Previously, we have<br />

identified microbially produced methane as a recent source of mine gas [2].<br />

Hard coal and mine timber predominantly harbored acetoclastic<br />

Methanosarcinales and so far uncultivated members of the Crenarchaeota.<br />

Coal and mine timber degradation via acetate as a central intermediate is<br />

probably catalyzed by a broad spectrum of fermenting and nitrate- or sulfatereducing<br />

bacteria belonging to the Firmicutes, Tenericutes, Beta-, Gamma-, and<br />

Deltaproteobacteria. Fungi known for mediating the degradation of wood were<br />

isolated from mine timber samples. Quantitative PCR assays were performed to<br />

determine the abundance of Fungi, Bacteria, Archaea and key genes indicative<br />

for methanogenesis and sulfate reduction (mcrA, dsrA). In all coal and timber<br />

samples, Bacteria dominated over Archaea by a ratio of 1100:1 and 700:1,<br />

respectively. While 45% of all Archaea harbored the mcrA-gene, approximately<br />

34% accounted for the Methanosarcinales. Fungi reached values of 10 7 - 10 8<br />

and 10 5 -10 6 copy numbers g -1 timber and coal, respectively. In conclusion, coal<br />

mines harbor diverse communities consisting of all domains of life mediating<br />

the degradation of coal and timber to methane.<br />

[1] Thielemann et al. (2004) Coalbed methane in the Ruhr Basin, Germany: a<br />

renewable energy resource? Organic Geochemistry 35, 1537-1549.<br />

[2] Krüger et al. (2008) Microbial methane formation from coal and mine<br />

timber in abandoned coal mines. Geomicrobiology Journal 25, 315-321.<br />

PN 18<br />

Microbial ecology on the microcosm level: Activity and<br />

population dynamics of methanotrophic bacteria during<br />

early succession in a flooded rice field soil<br />

S. Krause *1 , P. Frenzel 1<br />

1<br />

Biogeochemistry, Max Planck Institute for Terrestrial Microbiology, Marburg,<br />

Germany<br />

Methane oxidizing bacteria (methanotrophs) play an important role in natural<br />

wetlands and rice fields preventing large amounts of methane from escaping<br />

into the atmosphere. The occurrence of both type I and type II methanotrophs in<br />

the soil surface layer has been demonstrated in many studies. However, there is<br />

no profound un<strong>der</strong>standing which of them are responsible for the oxidizing<br />

activity and how they differ ecologically. Hence, a gradient microcosm system<br />

was applied simulating oxic-anoxic interfaces of water saturated soils to<br />

unravel population dynamics in early succession of methanotrophs in a flooded<br />

rice paddy. Additionally, environmental parameters were analyzed to link<br />

environment, populations, and their specific activity. We measured pmoA-based<br />

(particulate methane monooxygenase) terminal restriction fragment length<br />

polymorphism (T-RFLP) profiles both on DNA and mRNA. DNA T-RFLP<br />

patterns showed no major differences in the methanotrophic community<br />

structure remaining relatively constant over time. In contrast the active<br />

methanotrophic community structure as detected by pmoA mRNA T-RFLP<br />

analysis clearly demonstrated a distinct pattern from DNA T-RFLP profiles.<br />

While type II represented the most prominent group on the population level it<br />

seems to play a minor role on the activity level. Furthermore there were no<br />

clear implications towards a link between soil parameters (e.g. NH4 +<br />

concentration) and methanotrophic community structure.<br />

PN 19<br />

Cultivation and molecular analyses of methanotrophic<br />

enrichments from Siberian permafrost-affected soils<br />

J. Bischoff *1 , S. Liebner 2 , D. Wagner 1<br />

1 Geomicrobiology in Periglacial Regions, Alfred Wegener Institute for Polar<br />

and Marine Research, Potsdam, Germany<br />

2 Institute for Biogeochemistry and Pollutant Dynamics (IBP), Fe<strong>der</strong>al Institute<br />

of Technology (ETH), Zurich, Switzerland<br />

The Arctic plays a key role in Earth’s climate system as global warming is<br />

predicted to be most pronounced at high latitudes. Thawing of permafrost could<br />

release large quantities of greenhouse gases into the atmosphere, thus further<br />

increasing global warming and transforming the Arctic tundra ecosystems from<br />

a carbon sink to a carbon source. Therefore, an un<strong>der</strong>standing of the aerobic<br />

methane oxidizing community, as the major sink for methane in permafrost<br />

environments, is of particular interest. Active layer samples from different<br />

horizons were taken on Samoylov Island (Lena Delta, Siberia) for cultivation<br />

and further molecular characterization of the methane oxidizing bacteria<br />

(MOB). Conditions of cultivation in mineral-salt-media with methane as sole<br />

carbon source were different in terms of temperature, concentration of methane<br />

and presence of copper. Amplification of bacterial 16S rRNA genes was carried<br />

out using the universal primers 27F and GC_907R and 1492R, respectively,<br />

followed by DGGE or clone library analyses. DGGE fingerprints showed an<br />

effect of different cultivation conditions on the composition of the enriched<br />

communities. In addition to MOB, heterotrophic microorganisms were detected<br />

that belong to the phyla Bacteroidetes and Proteobacteria. Clone library<br />

analyses showed that cultures incubated at 28°C contained regularly<br />

Methylocella tundrae. In contrast, cultures grown at 10°C were of higher<br />

methanotrophic variety with members related to Methylobacter psychrophilus<br />

and Methylobacter tundripaludum, whose presence in permafrost-affected soils<br />

was confirmed by former clone library analyses of environmental DNA.<br />

Further studies will be conducted on an isolation of relevant methane oxidizing<br />

bacteria using optimized cultivation conditions.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 21<br />

Natural biofilms in drinking water systems use dark repair<br />

mechanisms to restore DNA injuries after UV disinfection<br />

C. Jungfer *1 , T. Schwartz 1 , U. Obst 1<br />

1 Institute of Technical Chemistry - Water Technology and Geotechnology<br />

Division, Forschungszentrum Karlsruhe, Karlsruhe, Germany<br />

Repair mechanisms in bacteria could be responsible for the regeneration or regrowth<br />

of bacteria in drinking water facilities after disinfection treatment.<br />

Within this work different disinfection and process techniques (chlorine<br />

disinfection, UV disinfection without/ with increased turbidity, UV oxidation)<br />

were integrated in a pilot scale simulating a drinking water distribution system.<br />

Natural biofilms were studied with a specific concern on recA-dependent dark<br />

repair activities. The recA gene is highly conserved among bacteria and<br />

regulates the SOS systems. DNA strands alterations are responsible for an<br />

induction of recA expression in bacteria.<br />

The expression of recA gene in these natural communities was analysed via<br />

RNA extraction, reverse transcription and PCR quantification. Additionally, the<br />

biofilms were analysed for total cell counts, metabolic activities, and bacterial<br />

composition on different pipe materials with molecular finger print techniques.<br />

The results demonstrated an induced recA-specific dark repair when UV<br />

irradiation was used for drinking water treatment indicating an increased<br />

regeneration of bacteria. In accordance to these results, biofilms from untreated<br />

systems and after UV treatment did not significantly change their composition.<br />

In case of UV application with increased turbidity the above mentioned<br />

observations were enforced. In contrast, no clear induced regeneration potential<br />

was detected during chemical disinfection. As a possible consequence,<br />

significant alterations in bacterial composition of the downstream grown<br />

biofilms were detected together with decreased metabolic activities. All these<br />

results generated with natural grown biofilms confirmed the results from<br />

previous studies about dark repair mechanisms in reference bacteria and labscaled<br />

UV reactors (Jungfer et al., 2007).<br />

PN 22<br />

Activity and diversity of phylogenetically novel acidtolerant<br />

nitrate reducer and denitrifier communities in an<br />

N2O-emitting fen<br />

K. Palmer *1 , H. Drake 1 , M. Horn 1<br />

1<br />

Lehrstuhl Ökologische Mikrobiologie, Universität Bayreuth, Bayreuth,<br />

Germany<br />

Acidic wetlands emit N2O. The nitrate reducer and denitrifier communities<br />

potentially linked to the emission of N2O by a regional acidic fen (pH 4.2-5.5)<br />

was evaluated. MPN-counts of denitrifiers from 0-10 cm and 30-40 cm fen<br />

depths approximated 10 8 cells*gDW -1 , indicating that denitrifiers were abundant.<br />

Phylogenetic analyses of the functional genes narG and nosZ revealed 8 and 6<br />

distinct clusters containing phylogenetically novel genes for these depths,<br />

respectively. tRFLP analyses of narG and nosZ indicate that the structure of the<br />

nitrate reducer and denitrifier communities was independent of soil depth. Fen<br />

soil produced and consumed N2O in unsupplemented anoxic microcosms.<br />

Supplemental nitrate stimulated denitrification. Denitrification rates decreased<br />

with depth. Despite similar MPN values obtained for each soil depth, vmax<br />

values for different depths ranged from 1-24 nmol N2O*h -1 *gDW -1 .<br />

Denitrification occurred at 2-60°C and at pH 2.0-6.5 with highest rates at in situ<br />

pHs (i.e., at pH 4-5). N2O approximated 40% of total produced N gases at in<br />

situ pH. Formate, acetate, and ethanol enhanced denitrification. Nitrate reducers<br />

capable of N2O-production were isolated from the fen. N2O was consumed by<br />

soils in anoxic microcosms supplemented with N2O ranging from subatmospheric<br />

concentrations to 0.2% (v/v). The collective data indicate that (i) a<br />

novel highly diverse denitrifier community capable of complete denitrification<br />

and consumption of atmospheric N2O at in situ pH occurs in acidic fens and (ii)<br />

novel, acid-tolerant nitrate reducers might contribute to N2O emission.<br />

PN 23<br />

Combination of microcalorimetry and stable-isotope<br />

probing to identify fermentative microorganisms in anoxic<br />

tidal flat sediments<br />

J. Graue *1 , S. Kleindienst 2 , B. Engelen 1 , H. Cypionka 1<br />

1<br />

ICBM, Universität Oldenburg, Oldenburg, Germany<br />

2<br />

Max-Planck-Institut für marine Mikrobiologie, Bremen, Germany<br />

Organic matter in anoxic sediments is initially decomposed by the hydrolysis of<br />

polymers followed by different fermentation steps. The terminal degra<strong>der</strong>s are<br />

sulfate-reducing bacteria and methanogenic archaea. Until now, investigations<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

predominantly focused on these physiological groups as they are easily<br />

detectable by molecular methods. Their abundance was determined by<br />

analyzing key genes indicative for methanogenesis and sulfate reduction [1].<br />

Generally, fermentative organisms are more abundant than the terminal<br />

degra<strong>der</strong>s. However, no specific molecular targets for this heterogeneous group<br />

are known. Here we present a novel strategy, the combination of<br />

microcalorimetry and stable-isotope probing, which was used to identify<br />

fermentative microorganisms in anoxic tidal flat sediments.<br />

Microcalorimetry allows to follow successive degradation steps via a highly<br />

sensitive detection of heat production during stimulation experiments. Thus this<br />

technique enables to detect different degradation phases and provides the<br />

possibility for precisely timed sampling. Microorganisms that are mediating<br />

different degradation steps were identified by RNA-based stable-isotope<br />

probing followed by PCR-DGGE. To unravel the fermentation processes, short<br />

chain fatty acids, alcohols and gases were analyzed by HPLC and gas<br />

chromatography.<br />

In a first experiment a close relative to Stenotrophomonas maltophilia was<br />

identified as the dominant fermenting organism, when 13 C-labeled glucose was<br />

added as sole carbon source. In the following experiment 13 C-labeled Spirulina<br />

was used as a complex substrate to simulate more natural conditions.<br />

[1] Wilms et al. FEMS Microbiol. Ecol. (2007)<br />

PN 24<br />

Monitoring the alkane monooxygenase gene alkB in<br />

different soil interfaces during plant litter degradation of<br />

C3 and C4 plants<br />

S. Schulz *1 , J.C. Munch 2 , M. Schloter 1<br />

1 Department Terrestrial Ecogenetics, Helmholtz Zentrum München, German<br />

Research Center for Environmental Health, Munich, Germany<br />

2 Institute of Soil Ecology, Helmholtz Zentrum München, German Research<br />

Center for Environmental Health, Munich, Germany<br />

127<br />

Hydrocarbons like n-alkanes are ubiquitous in the environment as a result of<br />

anthropogenic contamination (e.g. oil spills) as well as a part of an ecosystem’s<br />

biomass. For example n-alkanes become released during plant litter<br />

degradation; consequently they become a high abundant carbon source for<br />

microorganism. One possibility for the prokaryotic hydrocarbon metabolisation<br />

is an aerobic degradation pathway where the initial step is catalysed by the<br />

membrane bound alkane monooxygenase AlkB.<br />

We analysed the influence of alkanes on the abundance of the alkB gene in<br />

different interfaces of the litter-soil system during the degradation of maize and<br />

pea litter. Therefore soil samples of a sandy and a loamy soil have been<br />

incubated with straw of maize and pea plants up to 30 weeks with constant soil<br />

moisture and temperature. Using quantitative real-time PCR we were able to<br />

monitor the changes of the abundance and the expression rates of alkB. In our<br />

experiments we focused on the straw layer, the litter/soil interface and the soil 1<br />

cm below this interface (bulk soil). Our results clearly demonstrate time and<br />

space dependent abundance patterns of alkB genes and transcripts in the<br />

different layers studied, which are additionally shaped by the soil type used.<br />

PN 25<br />

Antibiotic pulse experiments in a 3-species mixed culture<br />

C. Riedele *1 , L. Geisler 2 , U. Reichl 3<br />

1 Bioprocess Engineering, Otto-von-Guericke-University, Magdeburg, Germany<br />

2 Bioprocess Engineering, Max Planck Institute for Dynamics of Complex<br />

Technical Systems, Magdeburg, Germany<br />

3 Bioprocess Engineering, Otto-von-Guericke University and Max Planck<br />

Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany<br />

Pseudomonas aeruginosa, Burkhol<strong>der</strong>ia cepacia and Staphylococcus aureus<br />

are opportunistic infectants, which occur as mixed cultures in the lungs of<br />

cystic fibrosis (CF) patients. Knowledge on possible interactions and growth<br />

characteristics of this microbial community in the lung obviously cannot be<br />

obtained in situ. It would be very desirable to be able to predict the mixed<br />

culture’s reaction, e.g. on antibiotic treatment, for optimal therapy of patients.<br />

We established mixed culture cultivations in a chemostat bioreactor, which are<br />

used as a model system related to the microbial community in CF lungs [1].<br />

Our experimental setup ensures defined and controllable conditions for such<br />

cultures. With comprehensive quantitative analytical methods we study<br />

bacterial growth characteristics and metabolic activity. For quantification of<br />

species specific cell number a T-RFLP method was developed and optimized<br />

[1]. In combination with mathematical modelling we focus on identifying<br />

microbial interactions and analyze the dynamics of the system, e.g. by<br />

disturbing the quasi steady state in chemostat experiments.

128<br />

Here, we show results of antibiotic pulse experiments in batch culture. We use<br />

mathematical models to describe the results of the single culture experiments<br />

and compare it with results from mixed culture experiments.<br />

[1] Schmidt, J.K. et al.: (2007) BiotechBioeng 96(4):738-756<br />

PN 26<br />

Main biogeochemical transformations in marine pelagic<br />

redoxclines are related to only a few archaeal or bacterial<br />

key species<br />

M. Labrenz *1 , F. Toetzke 1 , A. Zumsteg 1 , J. Grote 1 , G. Jost 1 , K. Jürgens 1<br />

1<br />

IOW - Leibniz Institute for Baltic Sea Research, University of Rostock,<br />

Rostock, Germany<br />

Marine pelagic redoxclines are areas of pronounced biogeochemical cycling<br />

and inhabited by distinct functional groups of prokaryotes. Interestingly, for<br />

Baltic Sea redoxclines it has been demonstrated that only one Sulfurimonasrelated<br />

key species, representing up to 22% of the total cell counts, could<br />

explain the occurring denitrification coupled to oxidation of reduced sulphur<br />

compounds. We were interested if an analogous phenomenon could be<br />

observed for the suboxic, aerobic ammonium oxidation zone, potentially based<br />

on crenarchaeal activity. Thus, we investigated the diversity, distribution and<br />

abundance of Archaea using 16S rRNA cloning, 16S rRNA fingerprinting and<br />

CARD-FISH. Based on fingerprints, several crenarchaeal and euryarchaeal<br />

sequences were detected. However, clone libraries were dominated by only one<br />

distinct crenarchaeal group with 97% sequence similarity to Candidatus<br />

Nitrosopumilus maritimus. Application of specific CARD-FISH probes<br />

revealed that this group dominated crenarchaeota by practically 100 % and<br />

reached up to 25% of total prokaryotic cell numbers slightly above the<br />

chemocline. This presents further evidence that the main biogeochemical<br />

transformations in marine pelagic redoxclines are driven by only a few key<br />

species.<br />

PN 27<br />

Temperature-dependent Shift of the Bacterial Community<br />

in a Thermophilic Biogas Reactor<br />

K. Satke *1 , N. Krakat 1 , P. Scherer 1<br />

1 Lifetec Process Engineering, Hochschule für angewandte Wissenschaften<br />

Hamburg, Hamburg, Germany<br />

Commercial operators of biogas plants are increasingly interested in an<br />

optimization of the biogas process. The key to this lies in the un<strong>der</strong>standing of<br />

the biology of this yet unresolved "black box".<br />

A thermophilic biogas reactor has been driven for more than five years. Beet<br />

silage has been used as sole substrate without any supplementation of manure.<br />

The population of the bacterial community was investigated for five<br />

representative reactor states, covering a period of 640 days. This was achieved<br />

by means of the Amplified Ribosomal DNA Restriction Analysis (ARDRA).<br />

Of each clone library, 120 clones were selected and phylogenetically<br />

investigated.<br />

Previous investigations (Westphal 2007) of the same reactor showed a mostly<br />

uniform population within the domain Archaea. Hydrogen-utilizing<br />

methanogenic Archaea of the or<strong>der</strong> Methanobacteriales dominated on each<br />

investigated reactor day. When present, acetate-utilizing Methanosarcinales<br />

contributed only to a minor extent. However, our results show that species<br />

richness and diversity among the Bacteria were significantly higher. In<br />

addition, the bacterial population was consi<strong>der</strong>ably more dynamic. Bacterial<br />

Phyla like Firmicutes and Bacteroidetes were always present in high, but<br />

varying amounts. Several other phyla were found quite often, but Deinococcus-<br />

Thermus, Gemmatimonadetes, Spirochaetes and Thermotogae were only<br />

observed once, all in the same period.<br />

Slight changes of the process parameters seemed to affect the composition of<br />

the Bacteria population significantly; especially a short substrate shift to<br />

unensiled fod<strong>der</strong> beet juice yielded a sustainable increase in bacterial diversity.<br />

Furthermore, a temperature shift from 55°C to 60°C established a more<br />

dissimilar and more diverse bacterial community.<br />

PN 28<br />

A glimpse un<strong>der</strong> the rim – the composition of microbial<br />

biofilm communities in domestic toilets<br />

M. Egert *1 , I. Schmidt 1 , K. Bussey 1 , R. Breves 1<br />

1 Microbiology Department, Henkel AG & Co. KGaA, Düsseldorf, Germany<br />

The microbial community of biofilms collected from domestic toilets was<br />

characterized with a molecular approach. Genomic DNA was extracted from<br />

six biofilm samples originating from households around Duesseldorf, Germany.<br />

While no archaeal 16S rRNA genes or fungal ITS genes were detected by PCR,<br />

T RFLP fingerprinting of bacterial 16S rRNA genes revealed a diverse<br />

community in all the samples. These communities also differed consi<strong>der</strong>ably<br />

between the six biofilms. 275 cloned partial 16S rRNA sequences were<br />

assigned to eleven bacterial phyla and 104 bacterial genera. Only 15 genera<br />

(representing 121 sequences affiliated with Acidobacteria, Actinobacteria,<br />

Bacteroidetes, Planctomycetes, and Proteobacteria) occurred in at least half of<br />

the samples or contributed at least 10% of the sequences in a single biofilm.<br />

Theses sequences were defined as "typical" for toilet biofilms, and they were<br />

examined in detail. On a 97% sequence similarity level these sequences<br />

represented 56 species. Twelve of these were closely related to well described,<br />

cultured bacterial species and only two of them, Stenotrophomonas maltophilia<br />

and Brevundimonas diminuata, were categorized as belonging to risk group 2.<br />

No 16S rRNA genes of typical faecal bacteria were detected in any of the<br />

samples. Virtually all typical clones were found to be closely related to bacteria<br />

or to sequences obtained from environmental sources, implicating that the<br />

flushing water is the main source of recruitment.<br />

In view of the great diversity of mostly as yet-uncultured bacteria, and the<br />

consi<strong>der</strong>able differences between individual toilets, unspecific strategies appear<br />

to be most suited for the removal and prevention of toilet biofilms.<br />

PN 29<br />

Temperature influences population structure of nitrite<br />

oxidizing bacteria in activated sludge<br />

M. Alawi 1 , S. Off 1 , M. Kaya 1 , M. Klimova 2 , E. Spieck *1<br />

1<br />

Biozentrum Klein Flottbek, Abteilung Mikrobiologie, Universität Hamburg,<br />

Hamburg, Germany<br />

2<br />

Biochemistry and Physiology of Cell, Voronezh State University, Voronezh,<br />

Russia<br />

Population structure and activity of nitrifying microorganisms are highly<br />

influenced by temperature. Whereas nitrification in thermal environments like<br />

hot springs is a current topic with Archaea identified as dominant ammonia<br />

oxidizers, detailed investigations about nitrifiers in cold habitats are scarce.<br />

Studies of the N-cycle in permafrost-affected soils in Siberia showed that a<br />

novel betaproteobacterium is one of the primary organisms responsible for<br />

nitrite oxidation here [1]. In this study, we started to enrich nitrite oxidizing<br />

bacteria (NOB) from activated sludge of a municipal wastewater treatment<br />

plant at temperatures between 10°C and 28°C. Electron microscopy revealed<br />

the existence of a novel nitrite oxidizer in enrichments at 10°C and 17°C<br />

together with Nitrospira. At higher temperatures of 28°C only members of<br />

Nitrospira were found. The temperature-dependent shift in the population<br />

structure was confirmed by DGGE and TGGE. The cold-adapted nitrite<br />

oxidizer was identified to be closely related to ‘Candidatus Nitrotoga arctica’<br />

by 16S rDNA sequencing. Using specific primer sets, Nitrotoga was detected in<br />

laboratory cultures grown at 10°C, Nitrospira at 10°C, 17°C and 28°C and<br />

Nitrobacter was enriched at 17°C as well as 28°C. In activated sludge without<br />

further cultivation a coexistence of Nitrotoga and Nitrospira was revealed.<br />

These findings confirmed the dominance of Nitrospira in activated sludge but<br />

also showed that the population structure of NOB is more complex than<br />

previously known and strongly responded to long-term changes in temperature.<br />

[1] Alawi et al. (2007). Cultivation of a novel cold-adapted nitrite oxidizing<br />

betaproteobacterium from the Siberian Arctic. The ISME Journal 1: 256-264<br />

PN 30<br />

Acidobacteria and Rubrobacteridae as potential key players<br />

in semiarid savanna soils<br />

B.U. Fösel *1 , E. Romann 1 , M. Mayer 1 , J. Overmann 1<br />

1 Bereich Mikrobiologie, Depertment Biologie I, Ludwig-Maximillians-<br />

Universität München, Planegg-Martinsried, Germany<br />

Besides natural factors like soil type or climate, anthropogenic alterations such<br />

as farming or logging affect soil bacterial community composition and activity.<br />

To date interdependence among land use, soil properties, and bacterial<br />

populations is poorly un<strong>der</strong>stood. This especially holds true for subtropical<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

soils in semiarid climates. In forest savanna soils from the Kavango region in<br />

northeastern Namibia, members of the poorly characterized Acidobacteria and<br />

Rubrobacteridae were found to be highly abundant. In a 16S rRNA gene<br />

library from Kavango soil, sequences assigned to these two groups accounted<br />

for 15 and 26%, respectively, of all 570 clones sequenced; their high abundance<br />

was confirmed by quantitative PCR. Culture-independent assessment of<br />

substrate utilization by Acidobacteria and Rubrobacteridae using stable isotope<br />

probing indicated that Acidobacteria were capable of assimilating glucose, but<br />

not acetate, bicarbonate, or benzoate. Phylotypes detected in the 13 C-enriched<br />

fractions belonged to subgroup 3, 4, and 6. In contrast, members of the<br />

subphylum Rubrobacteridae were found to incorporate only bicarbonate. By a<br />

high throughput cultivation approach and two different growth media it was<br />

possible to recover several novel strains of Acidobacteria and Rubrobacteridae.<br />

Acidobacteria pure cultures obtained so far belong to subgroup 1 and 4;<br />

Rubrobacteridae isolates are distantly related to Solirubrobacter soli and<br />

Conexibacter woesei.<br />

PN 31<br />

High-pressure adaptation of sulfate-reducing bacteria from<br />

fluid-influenced sediments of the deep subseafloor<br />

biosphere<br />

K. Ziegelmüller 1 , J. Logemenn 1 , J. Fichtel 1 , J. Rullkötter 1 , H. Cypionka 1 , B.<br />

Engelen *1<br />

1 Institute for Chemistry and Biology of the Marine Environment (ICBM),<br />

University of Oldenburg, Germany, Oldenburg, Germany<br />

Marine sediments were recovered during IODP Exp. 301 at a hydrologically<br />

active flank of the Juan de Fuca Ridge (northeast Pacific) to examine the<br />

importance of crustal fluids in driving the deep biosphere. At this site, sulfatecontaining<br />

fluids diffuse from the ocean crust into the overlying sediments<br />

resulting in enhanced cell counts and metabolic activities [1].<br />

Several sulfate reducers belonging to the Firmicutes and the<br />

Deltaproteobacteria were enriched from upper, seawater-influenced sediments<br />

(1-30 meter below seafloor, mbsf). In contrast, from deep fluid-influenced<br />

sediment layers (240-262 mbsf) only members of the non-spore forming<br />

Deltaproteobacteria were cultivated. One strain affiliated to Desulfovibrio<br />

aespoeensis which was firstly isolated from a deep granitic rock aquifer,<br />

confirming similarities between the marine and the terrestrial deep biosphere.<br />

High pressure experiments showed that our isolates are adapted to near in situ<br />

conditions of 30 MPa (300 bar) and 60°C. At 45°C cells were piezophilic,<br />

forming highest biomass at high pressure. At 20°C they rather behaved<br />

piezotolerant, as the growth yield was pressure-independent. To maintain<br />

membrane fluidity, cells adapted the chain length of fatty acids, the<br />

contribution of branched and unsaturated chains, the content of ornithine lipids<br />

as well as the quantitative ratio of the major phospholipid head groups<br />

(phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol). These<br />

results and the finding, that the sulfate-reducing bacteria still possess the ability<br />

to grow un<strong>der</strong> high pressure after years of <strong>bei</strong>ng decompressed, indicate their<br />

deep-biosphere origin.<br />

[1] Engelen and Ziegelmüller et al. (2008) Geomicrobiol. J. 25 :56-66<br />

PN 32<br />

Methane oxidation un<strong>der</strong> oxygen limitation in Lake<br />

Constance sediments<br />

J. Deutzmann *1 , B. Schink 1<br />

1 Microbial Ecology, Universität Konstanz, Konstanz, Germany<br />

Freshwater lakes contribute with 10-16% to the global non anthropogenic<br />

methane emissions. In Lake Constance, a well-studied model for oligotrophic<br />

lakes, aerobic methane oxidation was investigated in detail. Recently also<br />

anaerobic oxidation of methane has been reported in different freshwater<br />

habitats. Sulfate-dependent methane oxidation has been described in Lake<br />

Plußsee, a small eutrophic lake, and methane oxidation coupled to nitrate<br />

reduction was evident in enrichment cultures obtained from a canal in the<br />

Netherlands loaded with agricultural runoff. Until now it is unclear to what<br />

extent anaerobic oxidation of methane plays a role to mitigate methane<br />

emissions from freshwater habitats.<br />

Previously measured high-resolution oxygen and methane profiles from Lake<br />

Constance sediments indicated that methane was consumed in depth layers<br />

where no oxygen was present anymore. Furthermore, nitrate profiles measured<br />

in profundal sediment revealed that nitrate penetrates deeper into the sediment<br />

than oxygen, thus it might serve as electron acceptor for methane oxidation.<br />

Tracer assays for measurement of anaerobic methane oxidation with 14 C<br />

labelled methane were initiated. Comparison of calculated rates of methane<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

oxidation in the presence of various electron acceptors allows to assess the<br />

importance of anaerobic methane oxidation in Lake Constance and comparable<br />

oligotrophic freshwater habitats.<br />

PN 33<br />

Isolation of novel iron-oxidizing bacteria from a recently<br />

characterized community in an acid mine water treatment<br />

plant<br />

S. Hedrich *1 , J. Seifert 1 , M. Schlömann 1<br />

1 Institute of Bioscience, Environmental Microbiology, TU Bergakademie<br />

Freiberg, Freiberg, Germany<br />

In the Lusatia area (Germany) a pilot plant for the treatment of iron and<br />

sulphate rich acidic mine waters by microbial iron oxidation and a concomitant<br />

schwertmannite precipitation is operated. Molecular based studies of the acidic<br />

water and mineral samples revealed the presence of 16S rRNA gene sequences<br />

from various undescribed iron-oxidizing bacteria (IOB), especially two groups<br />

of β-Proteobacteria related to Gallionella ferruginea [1]. The community was<br />

very stable during the operation.<br />

Physiological characterisation of these IOB will help to optimize the pilot plant<br />

processes and improve waste water remediation. Therefore, different samples<br />

from the pilot plant were plated onto various solid media, whereas several<br />

strains could be isolated. Phylogenetic analysis showed that one strain was<br />

related to the so far undescribed Betaproteobacterium "Ferrovum myxofaciens",<br />

which dominates the pilot plant community. Furthermore, a species affiliating<br />

to the genus Thiomonas and many strains related to Acidithiobacillus<br />

ferrooxidans, were isolated. Successful enrichment of Gallionella related<br />

species was performed with the gradient tube technique and monitored via T-<br />

RFLP and 16S rDNA sequencing.<br />

Mixed cultures of acidophilic Proteobacteria were used to optimize iron<br />

oxidation rates in lab experiments and monitored by FISH using specific<br />

probes. To enhance the biomass in the pilot plant by immobilizing the bacteria,<br />

their attachment to different carrier materials was tested. All results <strong>der</strong>ived<br />

from the various investigations provide a deeper insight into the pilot plant<br />

processes.<br />

[1] Heinzel, E., et al., Bacterial diversity in a plant for mine water treatment.<br />

Appl. Environ. Microbiol., 1. December 2008. doi:10.1128/AEM.01045-08.<br />

129<br />

PN 34<br />

Spatial and temporal dynamics of microbial activities in a<br />

tar oil-contaminated aquifer<br />

A. Bayer *1 , B. Anneser 1 , C. Griebler 1<br />

1<br />

institute of groundwater ecology, Helmholtz Zentrum Muenchen, Neuherberg,<br />

Germany<br />

For effective bioremediation of contaminated aquifers profound knowledge on<br />

the hydrogeology of the site, its un<strong>der</strong>lying redox processes, the degra<strong>der</strong><br />

community and the factors limiting contaminant conversion is crucial. Previous<br />

work in our group has shown that in anoxic petroleum-hydrocarbon plumes<br />

most of the microbial degradation activities are found in the plume’s fringe.<br />

This is due to the distribution of the electron donor (the organic contaminant)<br />

and the electron acceptors (oxygen, nitrate, sulfate), which may only meet in<br />

this area. The identification of small-scale gradients and the precise localization<br />

of biodegradation processes have been elucidated by a high-resolution multilevel<br />

well at a tar oil-contaminated sandy aquifer in Duesseldorf-Flingern,<br />

Germany. The vertical distribution of redox-typical species across the vertical<br />

transect at various sampling dates suggests the co-occurrence of different redox<br />

processes, e.g. Fe(III)- and sulfate reduction, rather than a clear succession of<br />

thermodynamically favorable reactions. Comparing the results of several<br />

sampling campaigns showed pronounced vertical shifts of physical-chemical<br />

and microbial gradients including the spreading of the contaminants with time<br />

although hardly any fluctuation of the groundwater table took place. The<br />

timescale of the temporal and spatial changes with the distribution of redox<br />

species and microbial activities is focus of the current research. One aspect is to<br />

study how attached microorganisms can cope with these dynamics and how fast<br />

they can adapt to the changing redox conditions.

130<br />

PN 35<br />

A molecular comparison of the nitrifying bacterial<br />

communities in WSB ® process biofilms and in activated<br />

sludges of municipal wastewater treatment plants<br />

C. Steinbrenner *1 , M. Eschenhagen 1 , R. Böttger 1 , N. Fichtner 2 , W. Triller 2 , I.<br />

Röske 1<br />

1<br />

Institut für Mikrobiologie, TU Dresden, Dresden, Germany<br />

2<br />

Martin Bergmann Umwelttechnik, Bergmann clean Abwassertechnik GmbH,<br />

Penig, Germany<br />

The WSB ® technology (fluid bed biofilm process) is a biofilm process without<br />

a recirculation of activated sludge into the biological stage and with a stable<br />

nitrification at temperatures far below 12°C as opposed to other processes for<br />

wastewater treatment.<br />

In this study we compared the nitrifying bacterial communities present in the<br />

biofilms of the WSB ® process with evolving biofilms on two different floating<br />

carriers and in activated sludges of municipal wastewater treatment plants.<br />

The microbial biofilms on the two types of carriers were characterized using<br />

different biochemical and physical methods. Thereby we investigated the<br />

ability of active bacteria to hydrolyse fluorescein diacetat (FDA) by<br />

intracellular esterases, the total cell count, the content of dry weight and of<br />

protein content on the carriers.<br />

The diversity of ammonia-oxidizers in the microbial communities was<br />

investigated using molecular approaches like FISH, amoA-specific PCR<br />

combined with cloning/sequencing and DGGE. FISH was performed with<br />

group-specific oligonucleotide probes targeting Proteobacteria and particularly<br />

with specific probes for ammonia oxidizing bacteria. The first results of amoAspecific<br />

PCR showed a dominance of the genus Nitrosospira in the biofilms.<br />

This could be confirmed by FISH. Using this method we detected 10 % of the<br />

total cell count with the probe Nsv443, which is specific for the genus<br />

Nitrosospira. In addition to these assays we compared the ammonia oxidation<br />

rates in the biofilms with the rates in activated sludges.<br />

PN 36<br />

Earthworms enhance the microbial degradation of the<br />

herbicide MCPA in soil<br />

Y. Liu *1 , A. Zaprasis 1 , H. Drake 1 , M. Horn 1<br />

1<br />

Department of Ecological Microbiology, University of Bayreuth, Bayreuth,<br />

Germany<br />

The herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) is a potential<br />

groundwater and soil contaminant. The drilosphere, i.e., the soil influenced by<br />

earthworms including burrow walls, gut contents, and casts of earthworms,<br />

represents a hotspot of microbial activity in soil. The objective of the present<br />

study was to test the hypothesis that the burrowing activities of earthworms<br />

enhance aerobic MCPA degra<strong>der</strong>s in agricultural soil. Quantitative (reverse<br />

transcriptase) [q(RT)] PCR of 16S rRNA genes and 16S rRNA indicated that<br />

the activity of soil bacteria was higher in the presence of earthworms. q(RT)<br />

PCR of tfdA (which encodes a alpha-ketoglutarate dependent dioxygenase that<br />

catalyzes the first step of MCPA degradation) and tfdA transcripts indicated that<br />

earthworms enhanced the abundance and expression of tfdA. Analysis of tfdA<br />

from bulk soil revealed 5 distinct clusters containing phylogenetically diverse<br />

sequences. MCPA degradation was stimulated by the presence of earthworms,<br />

especially in upper soil layers. Although most probable numbers of MCPA<br />

degra<strong>der</strong>s in bulk soil and burrow walls were similar (ca. 5 x 10 7 gdw -1 ), MCPA<br />

degradation was faster in burrow walls than in bulk soil. Earthworm gut<br />

contents did not facilitate the degradation of MCPA un<strong>der</strong> oxic conditions.<br />

These collective findings indicated that the burrowing activities of earthworms<br />

enhanced aerobic MCPA degra<strong>der</strong>s in agricultural soil.<br />

PN 37<br />

Peptidoglycan catabolism in aquatic bacteria:<br />

Identification of cell wall sugar transporters of Gramella<br />

forsetii und Rhodopirellula baltica<br />

I. Schütze *1 , C. Mayer 1<br />

1 Department of Biology, University of Konstanz, Konstanz, Germany<br />

Cell wall recycling, the uptake of peptidoglycan fragments <strong>der</strong>ived from the<br />

endogenous cell wall and their reutilization, is a known phenomenon in E. coli<br />

but unknown in aquatic bacteria. Because of the sparse offering of nutrients in<br />

aquatic environments bacteria in these habitats are very likely forced to<br />

improve cell wall recycling and/or to apply efficient strategies to scavenge cell<br />

wall material from the environment. We chose two organisms of bacterial<br />

families that are abundant in aquatic environments and for which the genome<br />

sequences are available as model systems: Gramella forsetii, a Gram-negative<br />

bacterium of the Bacteriodetes and Rhodopirellula baltica, which belongs to<br />

the Planctomycetes, a unique group of bacteria that lack peptidoglycan. In both<br />

organisms, we identified gene clusters that contain homologues of cell wall<br />

recycling genes of E. coli, including the MurNAc etherase. In close proximity<br />

we identified genes that encode so far uncharacterised secondary transporters,<br />

which were cloned and expressed in E. coli. Selection systems for MurNAc and<br />

anhydroMurNAc were created to investigate the specificity of these<br />

transporters. Our preliminary results indicate that the R. baltica transporter<br />

might scavenge MurNAc as carbon and energy source. In contrast, G. forsetii<br />

likely transports anhydroMurNAc from the endogenous cell wall during cell<br />

wall recycling.<br />

PN 38<br />

Nitrous oxide (N2O) emission and spatial separation of<br />

denitrification and DNRA in the highly compartmentalized<br />

gut of soil-feeding termites<br />

D. Ngugi K. *1 , A. Brune 1<br />

1 Max Planck Institute for Terrestrial Microbiology, Department of<br />

Biogeochemistry, Marburg, Germany<br />

Soil-feeding termites play important roles in the dynamics of carbon and<br />

nitrogen in tropical soils. They effectively mineralize nitrogenous soil organic<br />

matter, which results in the production of enormous amounts of ammonia.<br />

Ammonia is subsequently oxidized to nitrate, which accumulates in the<br />

intestinal tract. 15 N tracer experiments revealed high potential rates for nitrate<br />

reduction in the different gut compartments. The highest rates of dissimilatory<br />

nitrate reduction to ammonia were observed in the anterior gut, whereas<br />

denitrification to N2O or N2 prevailed in the posterior hindgut. Virtually no<br />

nitrate-reducing activities were detected in the alkaline gut regions. Also living<br />

termites emitted N2O, and rates increased up to 17-fold in the presence of<br />

acetylene, un<strong>der</strong>scoring the importance of denitrification in vivo. In soil<br />

microcosms, N2O production was enhanced in the presence of termites, with<br />

rates surpassing that of methane emission by up to two or<strong>der</strong>s of magnitude.<br />

Collectively, our results indicate that both dissimilatory nitrate reduction to<br />

ammonia and denitrification are important processes in the intestinal tracts of<br />

soil-feeding termites, and that besides earthworms, the guts of soil-feeding<br />

termites are also important biological sources of the greenhouse gas N2O.<br />

PN 39<br />

Determination of active microorganisms in the gas reservoir<br />

Altmark and their role in CO2 turnover<br />

C. Gniese *1 , M. Krüger 2 , J. Frerichs 2 , A. Kassahun 3 , N. Hoth 4 , J. Seifert 1<br />

1<br />

Institute of Bioscience, Environmental Microbiology, TU Bergakademie<br />

Freiberg, Freiberg, Germany<br />

2<br />

Department Geomicrobiology, Fe<strong>der</strong>al Institute for Geoscience and Natural<br />

Resources (BGR), Hannover, Germany<br />

3<br />

DGFZ, Dresdner Ground Water Research Center e.V., Dresden, Germany<br />

4<br />

Institute for Drilling and Fluid Mining, TU Bergakademie Freiberg, Freiberg,<br />

Germany<br />

The almost depleted Altmark gas field operated by GDF SUEZ E&P Germany<br />

GmbH is located at the southern edge of the Northeast German Basin. The<br />

reservoir horizons belong to the Perminan Rotliegend formation (Saxon) and<br />

have an average depth of about 3300 m. The Altmark site is known to have<br />

favourable geological properties for the storage of natural gas and thus, is<br />

currently investigated by GDF SUEZ and the BMBF-Geotechnologien<br />

RECOBIO-2 project.<br />

The gas field fluids are characterized by slightly acidic pH-values (5 to 6.5),<br />

low redox potentials (-300 to –100mV) and high salinities. Iron is mainly<br />

present as ferrous iron (150 to 300 mg/L) and the sulphate concentration is in<br />

the range of the detection limit. The fluids were sampled at the wellheads and<br />

were chosen with respect to conveyance and chemical properties, i.e. a part of<br />

the sampled wells are continuously treated with chemical foams to enhance the<br />

gas lift.<br />

The microbial 16S rRNA diversity of all sampled gas field fluids was analysed<br />

by T-RFLP. The results showed a mo<strong>der</strong>ate bacterial diversity and a rather low<br />

archaeal occurrence. Microscopic analysis and CARD-FISH showed a variety<br />

of cell morphologies in the microbial communities.<br />

The potential CH4 and CO2 formation as well as the sulphate reduction rate was<br />

studied in cultivation experiments. Cultures with H2/CO2, acetate and methanol,<br />

respectively, and controls were made to determine the activities of the<br />

autochthonous microbial community in the gas field fluid.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 40<br />

Variability of the algae-associated bacterial communities<br />

among coexisting Fucus vesiculosus (Phaeophyceae) and<br />

Delesseria sanguinea (Rhodophyta) in the Baltic Sea<br />

F. Goecke *1 , J. Wiese 1 , A. Labes 1 , J.F. Imhoff 1<br />

1 Kieler Wirkstoff-Zentrum am IFM-GEOMAR, IFM-GEOMAR, Kiel, Germany<br />

Biotic surfaces frequently harbor species-specific microbial communities that<br />

can be highly variable and distinct from those found in the surrounding<br />

environment [1]. We investigate the bacterial communities associated with two<br />

coexisting seaweeds, the brown algae Fucus vesiculosus and the red algae<br />

Delesseria sanguinea in the Kieler Fjord (Baltic Sea). For this purpose we used<br />

restriction fragment length polymorphism (RFLP) analysis, cloning and<br />

analysis of 16S rDNA sequences. It is suggested that a specific bacterial<br />

community can be found associated with different groups of marine algae<br />

growing in the same habitat un<strong>der</strong> the same environmental conditions. The<br />

possible reasons of such differences and the ecological significance of naturally<br />

occurring epiphytic bacterial communities are discussed.<br />

[1] Staufenberger et al. 2008. FEMS Microbial Ecology 64: 65-77.<br />

PhD. Scholarships by the German Academical Exchange Service (DAAD) and<br />

Comite Nacional de Ciencia y Tecnologia (CONICYT), Chile.<br />

PN 41<br />

Comparative analysis of initial attachment during biofilm<br />

formation in Shewanella oneidensis MR-1<br />

J. Gödeke *1 , K.M. Thormann 1<br />

1 Department Ecophysiology, Max Planck Institute for Terrestrial Microbiology,<br />

Marburg, Germany<br />

Bacteria form architecturally complex communities, referred to as biofilms,<br />

where cells are connected by an extracellular matrix. Initial attachment to a<br />

surface marks the onset of this bacterial life style switch. Here we investigate<br />

the differences in biofilm development in Shewanella oneidensis MR-1, a metal<br />

ion-reducing microorganism, un<strong>der</strong> static and hydrodynamic conditions,<br />

respectively.<br />

By cultivating S. oneidensis MR-1 in static environment the formation of a<br />

flexible, yet sturdy network of cells without three-dimensional structures could<br />

be observed. Remarkably, living cell counts demonstrated that a subpopulation<br />

of cells died immediately subsequent to initial attachment.<br />

Cell death and lysis possibly leads to the release of biofilm-mediating<br />

compounds or electron transfer shuttles like flavins in S. oneidensis MR-1.<br />

DDAO-staining and confocal microscopy confirmed the presence of<br />

extracellular genomic DNA (eDNA) in ol<strong>der</strong> biofilms as a stabilizing factor of<br />

three-dimensional structures un<strong>der</strong> hydrodynamic conditions. Experiments<br />

involving DNase I treatment of S. oneidensis MR-1 cultivated in microtitre<br />

trays showed a strongly reduced biofilm formation, implying the essential role<br />

of eDNA in biofilm stability also un<strong>der</strong> static growth conditions. Evidence that<br />

eDNA is released via cell lysis was obtained through measurements of the<br />

release of extracellular β-galactosidase.<br />

In conclusion, our data indicate that biofilm appearance highly varies in static<br />

and hydrodynamic environments. We hypothesize that cell death during initial<br />

attachment is mediating biofilm formation at least in static environment.<br />

Further analyses are focused on identifying factors potentially controlling or<br />

triggering DNA release.<br />

PN 42<br />

Adaptation of terrestrial microbial communities to elevated<br />

CO2 concentrations<br />

J. Frerichs 1 , I. Möller 1 , F. May 1 , M. Krüger *1<br />

1 Fe<strong>der</strong>al Institute for Geosciences and Natural Resources, Section<br />

Geomicrobiology, Hannover, Germany<br />

From the IPCC report on global warming, it is clear that large-scale solutions<br />

are needed immediately to reduce emissions of greenhouse gases. CO2 capture<br />

and storage offers one option for reducing greenhouse gas emissions. Our study<br />

aims at investigating the environmental impact of CO2 leakage from deep<br />

reservoirs into near-surface terrestrial environments. Therefore, an ecosystem<br />

study has been conducted on a natural CO2 leak at the Laacher See, Germany.<br />

CO2 is produced below this extinct volcanic cal<strong>der</strong>a. The CO2 releasing vent<br />

located on an agricultural field at the western shore of the lake is clearly visible<br />

due to a 5m wide core of exposed soil.<br />

The determination of environmentally important microbial activities, eg. CO2<br />

and methane production, sulfate reduction and methane oxidation, showed<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

differences between the CO2-rich (>90 % of soil gas) medium (20%) and the<br />

control site with background CO2 concentrations. Especially in deeper soil<br />

layers, rates of methane production and sulfate reduction increased with<br />

increasing CO2 in the soil gas. Methane oxidation activity was highest at the<br />

control site. In accordance with the activities, also the microbial communities<br />

analysed by DGGE with general bacterial, archaeal and primers for functional<br />

genes of the C- and N-metabolism showed a shift in species composition<br />

between CO2-rich and control sites. However, all results indicate that effects of<br />

the gas vent are spatially limited. The ecosystem appears to have adapted to the<br />

different conditions through species substitution or adaptation, showing a shift<br />

towards anaerobic and acidophilic species un<strong>der</strong> elevated CO2 concentrations.<br />

PN 43<br />

Quantitative distribution and significant chemoautotrophic<br />

activity of Epsilonproteobacteria in sulfidic waters of the<br />

Baltic Sea and Black Sea redoxclines<br />

J. Grote *1 , M. Labrenz 1 , G. Jost 1 , G.J. Herndl 2 , K. Jürgens 1<br />

1 Biological Oceanography, Leibniz Institute for Baltic Sea Research,<br />

Warnemünde, Germany<br />

2 Biological Oceanography, Royal Netherlands Institute for Sea Research, Den<br />

Burg, Netherlands<br />

131<br />

In marine pelagic redoxclines of the Black Sea and Baltic Sea autotrophic<br />

production measured as dark CO2 fixation rates contributes significantly to<br />

overall primary production. Recent studies indicated that chemoautotrophic<br />

Epsilonproteobacteria might play an important role, especially as anaerobic or<br />

microaerophilic dark CO2 fixing organisms. However, knowledge of their<br />

distribution and abundance as actively CO2 fixing microorganisms in<br />

redoxclines is still rare. For central Baltic Sea redoxclines, a specific<br />

Sulfurimonas subgroup, group GD17, was proposed to play a key role in<br />

chemoautotrophic denitrification. We determined the contribution of group<br />

GD17 and of Epsilonproteobacteria as a whole to dark CO2 fixation in the<br />

sulfidic area of a Black Sea and a central Baltic Sea redoxcline by combining<br />

CARD-FISH with microautoradiography using [ 14 C]bicarbonate. 29% of the<br />

Baltic prokaryotic cells fixed 14 CO2 and 12% of the Black Sea prokaryotes,<br />

respectively. 14 CO2 incorporating cells belonged to the domain Bacteria.<br />

Among these, approximately 70% in the central Baltic and up to 100% in the<br />

Black Sea were members of the Epsilonproteobacteria. For the Baltic Sea, the<br />

Sulfurimonas subgroup GD17 was the most abundant group fixing CO2. The<br />

genome analysis as well as autecological studies with an isolate of group GD17<br />

should provide more information about its metabolic potential.<br />

PN 44<br />

Incorporation of carbon atoms into proteins for<br />

simultaneous identification of bacterial species and<br />

determination of metabolic activity in a microbial<br />

community by protein-based stable isotope probing<br />

(Protein-SIP)<br />

N. Jehmlich *1 , F. Schmidt 2 , M. Taubert 1 , M. von Bergen 1 , H.H. Richnow 3 , C.<br />

Vogt 3<br />

1<br />

Department of Proteomics, Helmholtz Centre for Environmental Research -<br />

UFZ, Leipzig, Germany<br />

2<br />

Interfaculty Institute for Genetics and Functional Genomics, University of<br />

Greifswald, Greifswald, Germany<br />

3<br />

Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental<br />

Research - UFZ, Leipzig, Germany<br />

A multitude of microbial species in environmental systems can not be isolated<br />

by standard methods which make it hard to identify the metabolically active<br />

key players. However, identification of metabolically active species can be<br />

analysed by feeding the microbial culture with stable isotope labeled substrates<br />

and subsequently tracing their incorporation into the biomass. DNA- and RNAstable<br />

isotope probing techniques lack sensitivity for low incorporation levels.<br />

Therefore, the protein-based stable isotope probing (Protein-SIP) approach was<br />

developed to detect the incorporation from the substrate into the proteins. For<br />

proof of principle, Protein-SIP was successfully applied to analyse proteins<br />

from a pure culture of Pseudomonas putida strain ML2 that had grown un<strong>der</strong><br />

aerobic conditions with the substrates 12 C-benzene, 13 C-benzene or 15 Nammonium<br />

and 12 C-benzene [1].<br />

In a second step, a more complex artificial culture consisting of the anaerobic<br />

growing Aromatoleum aromaticum strain EbN1 and an enrichment culture<br />

known as UFZ-1 were used to detect the active species by determining the<br />

incorporation of stable isotopes into proteins [2]. After harvesting the proteins<br />

were analysed by 2-D gel electrophoresis (2-DE) and mass spectrometry (MS)

132<br />

to characterise their identity, as well as their 13 C content as an indicator for<br />

function and activity of the host organism.<br />

In total, 60 unique proteins were identified by MALDI MS/MS. From 38<br />

proteins the levels of 13 C incorporation were determined, demonstrating the<br />

suitability of used method to identify metabolic active species with specific<br />

properties within a mixed culture with sensitivity down to 2%. In summary,<br />

Protein-SIP can be used to sensitively assess structure and function of microbial<br />

communities.<br />

[1] Jehmlich et al., (2008) RCM 22(18): 2889-97.<br />

[2] Jehmlich et al., (2008) Isme J 2(11): 1122-33.<br />

PN 45<br />

Novel Microbial Build-Ups on the Seafloor in the Northern<br />

Black Sea<br />

M. Siegert *1 , R. Seifert 2 , K. Weitbrecht 3 , B. Oppermann 2 , M. Friedrich 3 , M.<br />

Blumberg 2 , W. Michaelis 2 , M. Krüger 1 ,<br />

1<br />

Geomikrobiologie, Bundesanstalt für Geowissenschaften und Rohstoffe,<br />

Hannover, Germany<br />

2<br />

Institut für Biogeochemie und Meereschemie, Universität Hamburg, Hamburg,<br />

Germany<br />

3<br />

Molekulare Geomikrobiologie, Max Planck Institut für terrestrische<br />

Mikrobiologie, Hannover, Germany<br />

The Black Sea is the world’s largest anoxic meromictic seawater basin. It is<br />

therefore an ideal area for the investigation of anoxic microbial processes such<br />

as the anaerobic oxidation of methane (AOM). On R/V Meteor research cruise<br />

M72-1 2007, novel microbial mats have been discovered in a water depth of<br />

780 m in the northern Black Sea. The so-called "Troll Field" is situated directly<br />

on an active gas seep. A major component of the released gases is methane.<br />

Hence, culturing experiments with homogenates from the tube like structures<br />

showed methane dependent sulfate reduction. Here we compare sulfate<br />

reduction rates of microbial mats previously described as AOM-Chimneys<br />

(Michaelis et al. 2002) and the surrounding sediments, with and without<br />

pressure. The microbial community of the Trolls and the surrounding sediment<br />

was further analysed using molecular techniques such as 16S rRNA clone<br />

libraries for Bacteria and Archaea and T-RFLP. A comparison of the inner and<br />

the outer part of these microbial structures revealed a high bacterial, but not<br />

archaeal diversity. Most members of the archaeal domain were affiliated to<br />

ANME2 archaea, known for their ability to oxidise methane anaerobically. The<br />

bacterial community showed a great diversity with a large number of species in<br />

addition to AOM-related SRB of the Desulfosacina group.<br />

[1] Michaelis et al. "Microbial Reefs in the Black Sea Fueled by Anaerobic<br />

Oxidation of Methane", Science 2002, 297, 1013-1015<br />

PN 46<br />

Some Properties of Acidophilic Iron-oxidizing Bacteria<br />

Enriched from Acid Mine Drainage Generating Mine waste<br />

H. Korehi *1 , T. Wright 1 , A. Breuker 1 , A. Schippers 1<br />

1<br />

Geomicrobiology, Fe<strong>der</strong>al Institute for Geosciences and Natural Resources<br />

(BGR), Hannover, Germany<br />

The use of acidophilic, chemolithotrophic iron(II) - and sulfur-oxidizing<br />

microorganisms in processes to recover metals from certain types of copper-<br />

and gold-bearing minerals or mineral concentrates is now well established. Ten<br />

unclassified acidophilic iron(II) oxidizing microorganisms were isolated from<br />

different sites of acid mine drainage (AMD) and subjected to 16S rRNA gene<br />

sequence analysis. The results showed the presence of gram-positive Bacteria<br />

not related to described iron(II)-oxidizing Bacteria. Others belonged to the<br />

genera Acidithiobacillus, Ferrimicrobium and Leptospirilium. The<br />

physiological properties of the ten strains such as substrate spectrum, pH- and<br />

temperature optimum and range, and anaerobic growth have been investigated<br />

as well. Results revealed that all of the strains grew lithoautotrophically with<br />

iron(II) and also heterotrophically with glucose, acetate or yeast extract as a<br />

substrate. Six strains grew autotrophically with elemental sulfur as a substrate.<br />

Mixotrophic growth with iron(II) and an organic substrate was also observed<br />

for several strains. The leaching of the metal sulfides by six strains was also<br />

tested. All six strains were able to grow with pyrite, three with sphalerite, and<br />

only one with chalcopyrite. The characterization and description of the novel<br />

acidophilic iron(II) - oxidizing Bacteria is the goal of this study.<br />

PN 47<br />

Long term analysis of microbial communities in saline<br />

industrial wastewater<br />

K. Sahm 1 , P. John *1 , I. Zorll 1 , G. Antranikian 1<br />

1<br />

Institute of Technical Microbiology, Technical University Hamburg-Harburg,<br />

Hamburg, Germany<br />

Microbiall composition of three saline industrial wastewater reactors was<br />

monitored and analysed over a period of 20 months in regular intervals of app.<br />

4 months. Denaturing gradient gel electrophoresis showed stable community<br />

structure over the whole period of time. More than twenty isolates were<br />

obtained on medium based on waste water. All isolates belonged to the γ-<br />

Proteobacteria. Based on 16S rDNA sequences from these isolates, we<br />

designed specific probes for fluorescence in situ hybridisation (FISH) for<br />

subgroups of Alcanivorax and Halomonas . Furthermore, we applied probes<br />

targeting γ-Proteobacteria, Methylophaga, and Bacteria.<br />

For all three reactors 70 – 79% of all DAPI-stained cells belonged to the<br />

bacterial domain. Of these more than 30% could be attributed to γ-<br />

Proteobacteria for two reactors. In the third reactor the fraction of γ-<br />

Proteobacteria was even higher, between 79 and 87%. The γ-Proteobacteria<br />

were dominated by Halomonas. More than 80% of the γ-Proteobacteria in all<br />

three reactors belonged to the subgroup of Halomonas targeted by our probe.<br />

These results suggest, that the Halomonas isolates we obtained from the<br />

reactors are suitable model organisms for optimisation and monitoring of<br />

reactor performance.<br />

PN 48<br />

Diversity, physiological and biogeochemical characteristics<br />

of Acidobacteria in soil across a land use gradient<br />

V. Nägele *1 , A. Nähter 2 , M.W. Friedrich 2 , J. Overmann 1<br />

1 Bereich Mikrobiologie, Department Biologie I, Ludwig-Maximilians-<br />

Universität München, München, Germany<br />

2 Fachbereich 2 Biologie & Chemie, Universität Bremen, Bremen, Germany<br />

Acidobacteria account for up to 80% of all soil bacteria. Yet their diversity,<br />

physiological characteristics and biogeochemical relevance have remained<br />

obscure to date. In or<strong>der</strong> to investigate the functional implications of<br />

Acidobacteria in soil, the factors controlling their diversity were initially<br />

assessed. To this end, 16S rRNA gene sequences of different Acidobacteria<br />

communities were analyzed across a land use gradient as the major variable,<br />

employing soil samples from three DFG Biodiversity Exploratories which<br />

comprised different land use types. Genomic DNA was extracted from the<br />

samples and analyzed by PCR-DGGE with primers Acido31f and Uni341r-GC,<br />

which are specific for the 16S rRNA gene of Acidobacteria. The resulting<br />

DGGE profiles were statistically correlated to biotic and abiotic factors to<br />

determine the impact of land use on the composition of Acidobacteria. To<br />

quantify the abundance of Acidobacteria in the different soils, we used a 16S<br />

rDNA based qPCR assay on the same genomic DNA as employed for DGGE.<br />

Subsequently, Acidobacteria isolates, which dominate in the different natural<br />

environments, were selected by comparison of cultured phylotypes with<br />

environmental 16S rRNA gene sequences. The respective isolates were then<br />

subjected to a detailed physiological analysis in or<strong>der</strong> to identify their<br />

physiological key traits and ecological niches.<br />

PN 49<br />

Cultivation of sulfate-reducing bacteria from marine<br />

sediments of the coastal upwelling area off Namibia<br />

B. Kraft 1 , B. Engelen 1 , H. Cypionka 1 , M. Könneke *1<br />

1 ICBM, Universität Oldenburg, Oldenburg, Germany<br />

Sediments of coastal upwelling areas are generally characterised by a high<br />

content of organic carbon that is mainly degraded via anaerobic microbial<br />

processes including sulfate reduction as major terminal oxidation step. A recent<br />

molecular survey on sediment off coast Namibia revealed many unknown 16S<br />

rRNA gene sequences (Schäfer et al. Aquat. Microb. Ecol. 2007) Furthermore,<br />

sulfate reduction rates in this area have been found to be higher than in<br />

neighbouring coastal areas and correlate with water and sediment depth<br />

(Ferdelman et al. Limnol. Oceanogr. 1999) Here, we applied a cultivationbased<br />

approach using selective enrichment conditions to study the diversity and<br />

distribution of SRB in sediments of the Benguela upwelling system. Sediment<br />

cores were recovered during the Meteor-cruise M76/1 along a transect<br />

perpendicular to the continental slope off coast Namibia at water depths<br />

between 130 m and 3800 m. In or<strong>der</strong> to promote growth of the most abundant<br />

SRB from the surface down to 5 m depth, dilution series were set up with<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

sediment from five different depth intervals and were amended with hydrogen,<br />

acetate or a mixture of monomers representing typical substrates for SRB.<br />

Sulfide production and thus growth of SRB could be detected with all electron<br />

donors provided and from sediment layers down to 4 m depth. Potentially in<br />

situ abundant SRB from highest dilutions showing sulfide formation were<br />

analysed using 16S rRNA genes based DGGE. Extracted bands are currently<br />

<strong>bei</strong>ng sequenced and phylogenetically characterized. In addition, deep agar<br />

dilution series are used to obtain sulfate-reducing pure cultures. This study shall<br />

provide phylogenetic and physiological information of the yet unknown SRB<br />

from a highly productive, marine upwelling system.<br />

PN 50<br />

Microbial degradation of a biocide (Cu-HDO) in soil<br />

contact<br />

D. Jakobs *1 , M. Helena 1 , W. Pritzkow 2 , W.R. Abraham 3 , M. Noll 1<br />

1<br />

BAM, Fe<strong>der</strong>al Institute for Materials Research and Testing, Division IV.12,<br />

Berlin, Germany<br />

2<br />

BAM, Fe<strong>der</strong>al Institute for Materials Research and Testing, Division I.53,<br />

Berlin, Germany<br />

3<br />

Hemholtz Centre for Infection Research, Research Group Chemical<br />

Microbiology, Braunschweig, Germany<br />

The wood protection industry has refined their products from chrome, copper<br />

and arsenate based wood preservatives towards solely copper based<br />

preservatives, which are combined with organic biocides. One of these is Cu-<br />

HDO that contain the chelate of the fungicidal components copper and Ncyclohexyl-diazeniumdioxy<br />

(HDO) as co-biocide. Although this wood<br />

preservative is highly effective in wood preservation, its degradation once<br />

released from the wood remains unclear. In this work, we have investigated the<br />

fate of carbon stable isotope-labeled ( 13 C) and non-labeled ( 12 C) Cu-HDO in<br />

sterilized and non-sterilized soil at two concentrations (5 µg and 20 µg Cu-<br />

HDO / g soil dry weight). We monitored the fate of HDO by HPLC, and the δ<br />

13 C in respired CO2 and in total carbon of the soil by EA-IRMS over a period of<br />

three months. No significant decrease in Cu-HDO concentration occurred in<br />

sterilized soil, while already after two days of incubation Cu-HDO decreased<br />

significantly in both concentrations in non-sterilized soil. In addition, the δ 13 C<br />

in respired CO2 increased steadily to more than 500 ‰ after 16 days of<br />

incubation and slightly decreased afterwards. These results indicate a strong<br />

microbial degradation process of Cu-HDO. Currently, we are analyzing the<br />

microbial phospholipid fatty acids including their carbon isotope ratios to<br />

identify the key-players of degradation.<br />

PN 51<br />

Molecular biology control of aimed denitrifcation processes<br />

for raw water improvement<br />

A. Karolewiez *1 , T. Schwartz 1 , U. Obst 1<br />

1 Institute for Technical Chemistry, Water- and Geotechnology Division,<br />

Microbiology of natural and technical surfaces Department,<br />

Forschungszentrum Karlsruhe, Karlsruhe, Germany<br />

Within the framework of this project microbial denitrification acitivities are<br />

used for nitrate degradation in contaminated raw water sources. Adapted<br />

bacterial communities are forming biofilms on a non-toxic synthetic material<br />

like Polycaprolactone, which is used as carbon source and substrate for biofilm<br />

growth, respectively.<br />

To control the process gene expression analyses are important to evaluate the<br />

critical steps during nitrate degradation, and rDNA based populations analyses<br />

are required for the characterization of the biofilm compositions or dynamics.<br />

Therefore different gene expression analyses were performed with specifically<br />

designed primers targeting key genes involved in the denitrification process.<br />

Pseudomonas aeruginosa and Acidovorax caeni previously identified as<br />

denitrifiers were used as reference bacteria. The molecular biology tools were<br />

applied to natural biofilms grown on Polycaprolactone in an up-scaled<br />

denitrification reactor.<br />

The expressions of two different nitrate reductase genes (nar and nap) and two<br />

different nitrite reductase genes (nirS and nirK) were quantified in specific<br />

reference bacteria and in natural biofilm communities.<br />

The results demonstrated no common expression pattern for the reference<br />

strains Pseudomonas aeruginosa and Acidovorax caeni targeting the nitrate<br />

reductase. It became obvious that the natural biofilms of the up scaled<br />

dentrification reactor exhibited a constitutive like expression of the nitrate<br />

reductase gene and a significant up-regulation of nitrite reductase when nitrite<br />

accumulated in the system.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PN 52<br />

Impact of Sulfurimonas – related bacteria on the microbial<br />

food web in pelagic redoxclines of the Baltic and Black Sea<br />

as determined by 13 C analyses<br />

S. Glaubitz *1 , K. Jürgens 1 , G. Jost 1 , T. Lü<strong>der</strong>s 2 , M. Labrenz 1<br />

1<br />

Biological Oceanography / Molecular Microbial Ecology, Leibniz Institute for<br />

Baltic Sea Research, Rostock, Germany<br />

2<br />

Institute of Groundwater Ecology, Helmholtz Center Munich, Neuherberg,<br />

Germany<br />

Baltic Sea and Black Sea pelagic redoxclines are usually characterized by high<br />

dark CO2 fixation rates below the chemocline, i.e. the first appearance of<br />

sulphide. For the Baltic Sea dark CO2 fixation rates can account up to 30 % of<br />

surface primary production. Ribosomal RNA Stable Isotope Probing (rRNA-<br />

SIP) analyses based on 13 C bicarbonate incorporation demonstrated for this<br />

habitat already that chemolithoautotrophic activity was driven by Gamma- and<br />

Epsilonproteobacteria, with the latter ones phylogenetically closely related to<br />

Sulfurimonas spp. (Glaubitz et al., 2008). In pelagic redoxclines of the Black<br />

Sea similar Epsilonproteobacteria were detected by T-RFLP analyses<br />

combined with 16S rRNA cloning and sequencing (Vetriani et al., 2003). The<br />

aim of our 13 C rRNA-SIP based study was to verify the importance of members<br />

of the Sulfurimonas cluster for dark CO2 fixation in redoxclines of the Black<br />

Sea. Our results revealed that also in this habitat especially members of the<br />

Sulfurimonas cluster actively fixed CO2. However, we also detected some<br />

chemolithoautotrophic Gammaproteobacteria, phylogenetically related to<br />

known sulphide-oxidizing endosymbionts. Moreover, and comparable to the<br />

Baltic Sea, the 13 C-isotope signal in the Black Sea samples was transferred to<br />

the second trophic level after 72 h of incubation, probably by grazing as well as<br />

endosymbionts. Thus, rRNA-SIP provided direct evidence for the contribution<br />

of chemolithoautotrophic production to the microbial food web in both systems,<br />

emphasising the importance of dark CO2-fixing members of the Sulfurimonas<br />

cluster in marine and sulphidic pelagic redoxclines.<br />

[1] Glaubitz et al. (2008) EM DOI: 10.1111/j.1462-2920.2008.01770.x.<br />

[2] Vetriani et al. (2003) AEM 69:6481-6488<br />

PN 53<br />

Community composition and abundance of archaeal and<br />

bacterial ammonia oxidizers in an acidic fen<br />

M. Herrmann *1 , A. Hädrich 1 , K. Küsel 1<br />

1 Institute for Ecology - Limnology/Aquatic Geomicrobiology -, Friedrich<br />

Schiller University Jena, Jena, Germany<br />

133<br />

Nitrification in fens and bogs is often hampered by low pH, high content of<br />

humic acids, and lack of oxygen in the water-logged peat soils. These<br />

environments have been predicted to un<strong>der</strong>go more frequent drying and<br />

rewetting un<strong>der</strong> the conditions of a changing global climate, resulting in water<br />

table draw-down, improved aeration in the surface soil layers, and increased<br />

mineralization and mobilization of nitrogen compounds. So far, only little is<br />

known about microbial communities involved in nitrification in peat soils. The<br />

goals of this study are (i) to assess the effect of experimental lowering of the<br />

water table on nitrification in an acidic fen and (ii) to investigate the microbial<br />

groups involved in ammonia oxidation, the first step of nitrification, in the peat<br />

soil. In the acidic fen Schlöppnerbrunnen (Fichtelgebirge/Bavaria) a water table<br />

manipulation experiment was carried out in summer 2008 resulting in a<br />

substantial accumulation of nitrate in the upper peat horizons. Microbial<br />

communities of ammonia oxidizing archaea (AOA) and ammonia oxidizing<br />

bacteria (AOB) are analyzed based on the amoA gene, encoding ammonia<br />

monooxygenase, the key enzyme of ammonia oxidation. Preliminary results<br />

indicate a numerical predominance of ammonia oxidizing archaea in the peat.<br />

Community profiles obtained by Denaturant Gradient Gel Electrophoresis<br />

suggest depth-dependent changes in AOA community composition along the<br />

peat profile.

134<br />

PN 54<br />

Novel Microbial Build-Ups on the Seafloor in the Northern<br />

Black Sea<br />

M. Siegert *1 , K. Weitbrecht 3 , R. Seifert 2 , M. Friedrich 3 , M. Krüger 1 , B.<br />

Oppermann 2 , M. Blumberg 2 , W. Michaelis 2<br />

1<br />

Geomikrobiologie, Bundesanstalt für Geowissenschaften und Rohstoffe,<br />

Hannover, Germany<br />

2<br />

Molekulare Geomikrobiologie, Max Planck Institut für terrestrische<br />

Mikrobiologie, Hannover, Germany<br />

3<br />

Institut für Biogeochemie und Meereschemie, Universität Hamburg, Hamburg,<br />

Germany<br />

The Black Sea is the world’s largest anoxic meromictic seawater basin. It is<br />

therefore an ideal area for the investigation of anoxic microbial processes such<br />

as the anaerobic oxidation of methane (AOM). On R/V Meteor research cruise<br />

M72-1 2007, novel microbial mats have been discovered in a water depth of<br />

780 m in the northern Black Sea. The so-called „Troll Field“ is situated<br />

directly on an active gas seep. A major component of the released gases is<br />

methane. Hence, culturing experiments with homogenates from the tube like<br />

structures showed methane dependent sulfate reduction. Here we compare<br />

sulfate reduction rates of microbial mats previously described as AOM-<br />

Chimneys (Michaelis et al. 2002) and the surrounding sediments, with and<br />

without pressure. The microbial community of the Trolls and the surrounding<br />

sediment was further analysed using molecular techniques such as 16S rRNA<br />

clone libraries for Bacteria and Archaea and T-RFLP. A comparison of the<br />

inner and the outer part of these microbial structures revealed a high bacterial,<br />

but not archaeal diversity. Most members of the archaeal domain were affiliated<br />

to ANME2 archaea, known for their ability to oxidise methane anaerobically.<br />

The bacterial community showed a great diversity with a large number of<br />

species in addition to AOM-related SRB of the Desulfosacina group.<br />

[1] Michaelis et al. „Microbial Reefs in the Black Sea Fueled by Anaerobic<br />

Oxidation of Methane“, Science 2002, 297, 1013-1015<br />

PN 55<br />

Biofilm-associated versus water column-associated<br />

nitrification and ammonia oxidizer community composition<br />

in creek ecosystems<br />

A. Scheibe 1 , M. Herrmann *1 , S. Avrahami 1 , K. Küsel 1<br />

1 Institute for Ecology - Limnology/Aquatic Geomicrobiology -, Friedrich<br />

Schiller University Jena, Jena, Germany<br />

Creek ecosystems are of great importance for inorganic nitrogen<br />

transformation, preventing the accumulation of agriculturally-<strong>der</strong>ived ammonia<br />

and nitrate and eutrophication of natural waters. Here, we focus on nitrification<br />

as a key process. The first step of nitrification, the oxidation of ammonia, is<br />

carried out by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing<br />

archaea (AOA). The relative importance of biofilm-associated versus water<br />

column-associated nitrification in creek ecosystems at different ammonia<br />

concentrations was studied using a set up of experimental flow channel systems<br />

with clay tiles simulating a stream bed substrate. Water was obtained from<br />

three creek ecosystems differing in their in-situ ammonia concentrations.<br />

Changes in potential nitrification activity and ammonia oxidizer community<br />

composition were followed during biofilm development. Community<br />

composition of AOB and AOA was analyzed using a combined<br />

cloning/sequencing approach and Denaturant Gradient Gel Electrophoresis<br />

targeting the amoA gene, which encodes ammonia monooxygenase, the key<br />

enzyme of ammonia oxidation. In contrast to other studies, potential<br />

nitrification activity in the flow channels was generally higher in the water<br />

column compared to the biofilm. However, the variation in ammonia oxidizer<br />

community composition between biofilm and water column was not sufficient<br />

to explain the variation observed in potential nitrification activity between these<br />

two compartments. Ammonia availability had a clear effect on the community<br />

composition of both AOA and AOB with an increasing importance of<br />

Nitrosomonas-related AOB at high ammonia concentrations.<br />

PN 56<br />

Wastewater treatment plants as hotspots for horizontal<br />

gene transfer: Community analysis combined with plasmid<br />

replicon based fingerprinting<br />

S. Caucci *1 , B. Kiesel 1 , A. Chatzinotas 1 , T. Berendonk 2 , P. Krebs 3 , H. Harms 1<br />

1<br />

Department of Environmental Microbiology, Helmholtz Centre for<br />

Environmental Research UFZ, Leipzig, Germany<br />

2<br />

University of Leipzig, Department of Biology II, University of Leipzig, Leipzig,<br />

Germany<br />

3<br />

Dresden University of Technology, Institute for Urban Water Management,<br />

Dresden University of Technology, Dresden, Germany<br />

The widespread increase of bacterial resistance to antibiotics, caused by the<br />

extensive use of antibiotics in clinical therapy and animal husbandry creates<br />

severe public health problems. Bacteria can evolve rapidly and often acquire<br />

genes from each other, including resistance genes, which leads to better<br />

survival in the presence of antibiotics. Wastewater treatment plants (WWTP)<br />

can be described as substrate rich ecosystems harbouring abundant<br />

autochthonous environmental bacteria as well as sewage water bacteria exposed<br />

to antibiotics, thus they represent ideal hotspots for horizontal gene transfer<br />

(HGT) by conjugation processes.<br />

As the hygienisation of WWTP effluents is not fully effective, new antibiotic<br />

resistant bacteria could reach via the outflow the aquatic ecosystems. The<br />

project "HGT in wastewater treatment plants" aims towards a better<br />

un<strong>der</strong>standing of the role of WWTP as a reservoir for antibiotic resistance<br />

genes and as a source for their release into the environment. Focus of the work<br />

is to elucidate the risk for resistance transfer to autochthonous environmental<br />

bacteria. To un<strong>der</strong>stand the interdependencies between plasmids carrying<br />

resistance genes and WWTP microorganisms, we analyzed samples from<br />

sequential treatment steps in the municipal WWTP in Dresden. In addition to<br />

endogenous plasmid replicon screening we monitored the overall microbial<br />

community composition and diversity by T-RFLP of phylogenetic marker<br />

genes (16S and 18S rRNA genes). First results will be linked to physicochemical<br />

parameters and discussed in the frame of the current microbiological<br />

wastewater technologies debate.<br />

PN 57<br />

Novel xylanase enzyme <strong>der</strong>ived from a thermophilic<br />

microbial community<br />

S. Wittenberg *1 , A. Angelov 2 , M. Mientus 2 , J. Schuldes 1 , R. Daniel 1 , W. Liebl 2<br />

1<br />

Institute of Microbiology and Genetics, Georg-August-Universitaet<br />

Goettingen, Goettingen, Germany<br />

2<br />

Department of Microbiology, Technische Universitaet Muenchen, Muenchen,<br />

Germany<br />

The degradation of the main plant cell wall polysaccharides cellulose and<br />

hemicellulose has been studied mainly on isolated microorganisms or their<br />

isolated enzyme systems. However, in natural environments this process is<br />

carried out by complex microbial communities whose enzyme complements<br />

and interactions are only poorly un<strong>der</strong>stood.<br />

A functional screen for cellulolytic and xylanolytic activities was performed<br />

with 5.200 E. coli fosmid clones, carrying approximately 208 Mbp of<br />

environmental DNA <strong>der</strong>ived from a naturally heated and slightly alkaline<br />

sample from the crater of Avachinsky in the Kamchatka region. Analysis of<br />

fosmids conferring xylanase activity by low coverage sequencing and primer<br />

walking led to the identification of an ORF coding for a potential xylanase,<br />

xyl1015. The encoded polypeptide possessed a signal peptide sequence and<br />

displayed high level of amino acid sequence similarity (40% identity) to a<br />

„glycoside hydrolase, family 10 protein“ from Caldicellulosiruptor<br />

saccharolyticus. The xyl1015 gene was cloned in the expression vector pET101<br />

and the encoded enzyme overproduced in E. coli BL21. A two - step<br />

purification, heat treatment followed by affinity chromatography, led to an<br />

essentially pure enzyme preparation. Recombinant Xyl1015 was active against<br />

oat spelts xylan (13.4 U/mg), with an apparent temperature optimum of 96°C at<br />

pH 8.0. The enzyme retained 33% of its activity even at 105°C (15 min assay)<br />

and 50% of the initial activity could be measured after a 6h incubation at 96°C.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 58<br />

Correlation between materials, water qualities, and biofilm<br />

diversity in drinking water biofilms<br />

R. Roe<strong>der</strong> *1 , P. Tarne 1 , K. Heeg 1 , U. Szewzyk 1<br />

1<br />

Department of Environmental Microbiology, University of Technology Berlin,<br />

Berlin, Germany<br />

Biofilm communities grown on different drinking water pipe materials (Copper,<br />

different PE and rubber qualities) at different sites of Germany (reduced<br />

groundwater, recharged groundwater, river bank filtration) are analysed. The<br />

objective was to correlate factors from the water, the different materials and the<br />

composition of the resulting biofilms.<br />

The biofilms of the pilot plant (DVGW Research Center TUHH) were also<br />

contaminated with Pseudomonas aeruginosa and Legionella pneumophila and<br />

were treated mechanically in combination with chemical disinfection (chlorine<br />

dioxide). Additional evaluations on the effect of other disinfectants on biofilm<br />

communities, were carried out with biofilms grown on silicone.<br />

The biofilm communities on different materials were compared with a<br />

fingerprinting method (DGGE) and cloning (16S rDNA). The different biofilm<br />

communities had high diversity on rubber and comparative low diversity on<br />

PE-X. DGGE patterns and cloning results of the same materials exposed at<br />

different sites showed a relative low similarity. Consequently, the composition<br />

of the biofilm population is influenced both by the material and the origin of the<br />

drinking water. A contamination with water relevant pathogens resulted in a<br />

changed population structure. Chemical treatment induced a selection pressure<br />

and resulted in a new biofilm population.<br />

Acknowledgements:<br />

Part of this investigation was financially supported by the German Fe<strong>der</strong>al<br />

Ministry of Education and Research (BMBF).<br />

We would like to thank our research partners (www.biofilmhausinstallation.de)<br />

for providing biofilm samples.<br />

PN 59<br />

Metal Mobilization by Fe(III)-reducing Microbial<br />

Communities in Contaminated Creek Soils<br />

E.M. Burkhardt *1 , D. Akob 1 , S. Bischoff 1 , J. Kostka 2 , K. Küsel 1<br />

1<br />

Institute of Ecology, Friedrich Schiller University Jena, Jena, Germany<br />

2<br />

Department of Oceanography, Florida State University, Tallahassee, United<br />

States<br />

Uranium mining in the area of Ronneburg, Germany, led to contamination of<br />

soil and groundwater with metals and radionuclides. Geochemical conditions in<br />

a metal-enriched horizon of contaminated creek soil indicated that microbial<br />

Fe(III)-reduction could influence heavy metal mobility. Metal measurements in<br />

anoxic soil microcosms, biostimulated with ethanol or lactate, revealed<br />

mobilization of Mn, Co, Ni, Zn, As, and U during Fe(III)-reduction. Direct<br />

reduction of Mn and As and reductive dissolution of Fe(III)-oxides likely<br />

caused the release of sorbed metals, with unexpected mobilization of U<br />

observed. During Fe(III)-reduction the active microbial population was<br />

characterized by DNA stable isotope probing, terminal restriction fragment<br />

length polymorphism (TRFLP) analysis, and cloning/sequencing. The Fe(III)reducing<br />

population was dominated by δ-Proteobacteria related to Geobacter<br />

in the 13 C-ethanol microcosms. In the 13 C-lactate microcosms the microbial<br />

community was more diverse and taxa related to Acidobacteria (Geothrix),<br />

Firmicutes (Pelosinus), δ-Proteobacteria (Pelobacter), and β-Proteobacteria<br />

(Dechloromonas) were detected using TRFLP analysis. Sequencing confirmed<br />

the dominance of Acidobacteria and Firmicutes taxa, whereas, the other<br />

phylogenetic groups were less abundant. Populations of Fe(III)-reducing<br />

bacteria in enrichment cultures were found to have a low tolerance to metal<br />

stress caused by the presence of Ni, Zn, Cu, and Cd. Our results suggested that<br />

Fe(III)-reducing communities, stimulated with ethanol or lactate, facilitated<br />

metal mobility and could potentially cause metal-enriched soil horizons to be a<br />

source of metal contaminants to groundwater. However, the low tolerance of<br />

Fe(III)-reducers to metal stress indicated that metal mobilization may be slowed<br />

due to inhibition of Fe(III)-reduction in situ.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PN 60<br />

Bacterial and eukaryotic community variations in different<br />

filter compartments during slow sand filtration of<br />

wastewater<br />

K. Euringer *1 , K. Pfannes 1 , B. Michaela 1 , R. Meckenstock 1 , K. Langenbach 2 ,<br />

M. Kästner 2<br />

1<br />

Institute of Groundwater Ecology, Helmholtz Zentrum München, Neuherberg,<br />

Germany<br />

2<br />

Department of Bioremediation, UFZ– Helmholtz Centre for Environmental<br />

Research, Leipzig, Germany<br />

Especially in arid regions, wastewater reuse is becoming more and more<br />

important because of overexploitation, pollution of natural water resources, and<br />

water scarcity. A simple technology for the removal of pathogens and particles<br />

from wastewater is slow sand filtration (SSF), a low-tech method used in<br />

drinking-water purification for centuries. The Schmutzdecke of sand filters has<br />

been shown to be the biologically most active compartment, where more than<br />

90% of pathogen indicator organisms are <strong>bei</strong>ng removed.<br />

We extracted DNA of different compartments (inflow, supernatant,<br />

Schmutzdecke, sand and water samples of different depths, effluent) of SSF<br />

filters fed with secondary effluents of a wastewater treatment plant and<br />

investigated bacterial and eukaryotic community variations in these<br />

compartments via T-RFLP fingerprinting of 16S and 18S rRNA genes. By<br />

qPCR, we also assessed respective gene copy numbers in these samples.<br />

We showed that specific sediment-associated bacterial and eukaryotic<br />

communities had developed in the filter matrix, which differed clearly from<br />

those detected throughout the water samples. This indicates an accumulation<br />

but also proliferation of sessile organisms at surfaces, which was also proven<br />

by the results of the qPCR. Additionally, distinct community members were<br />

eliminated, mostly in the upper part, while others were passing right through<br />

the sand filters.<br />

Our findings substantiate the importance of the Schmutzdecke and the upper<br />

zones of filters, where processes seem to be located on surfaces. Future<br />

identification of community members (e.g. predators) in different<br />

compartments will shed more light on biological processes responsible for the<br />

hygienisation efficiency of slow sand filters.<br />

PN 61<br />

Changes of the microbial community during self-heating<br />

and artificial heating in a composting process<br />

K. Worm *1 , M. Schmidt 1 , U. Lechner 1<br />

1 Institute for Biology / Microbiology, Martin-Luther-University<br />

Halle/Wittenberg, Halle, Germany<br />

135<br />

Biowaste including animal material, paperboard and sawdust were composted<br />

in 400- to 750-l bioreactors. During composting the temperature increased up to<br />

65°C by self-heating. For the elimination of pathogenic microorganisms, a<br />

constant temperature of 70°C for one hour is required. This temperature<br />

increase was obtained with a casing-integrated heater. The aims of this study<br />

were the elucidation of changes of the microbial community structure during<br />

composting in regard to self-heating and hygiensiation, and the identification of<br />

thermophilic key players for their potential use as starter cultures. Thermophilic<br />

bacteria were isolated, which were also identified in 16S rRNA gene clone<br />

libraries established from DNA extracts of hot compost. Their occurrence and<br />

relative frequency during the composting process were followed by terminal<br />

restriction fragment length polymorphism (t-RFLP) analyses. The identified<br />

thermophilic bacteria belonged to the Firmicutes (e.g. Ureibacillus<br />

thermosphaericus, Geobacillus sp.) and to the Proteobacteria (e.g.<br />

Pseudoxanthomonas taiwanensis, Pseudomonas sp.) and were abundant in the<br />

self-heating phase. The community structure changed slowly during the<br />

subsequent cooling phase. In contrast, the hygienisation caused a more drastic<br />

change of the microbial community structure, particularly after a delayed start<br />

of hygienisation in the cooling phase at temperatures below 50°C. Attempts<br />

were made to differentiate live and dead cells in the compost matrix by a<br />

staining procedure and a quantitative PCR approach using propidium<br />

monoazide.

136<br />

PN 62<br />

Diversity and localization of bacterial symbionts associated<br />

with Trichonympha flagellates in lower termites<br />

T.H.G. Wienemann 1 , W. Ikeda-Ohtsubo 1 , N. Faivre 1 , S. Frankenberg 1 , T.<br />

Köhler *1 , A. Brune 1<br />

1<br />

Dept. of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology,<br />

Marburg, Germany<br />

Cellulolytic flagellates are the key players in the digestion of lignocellulose in<br />

the hindgut of lower termites. The flagellates are typically colonized by hostspecific<br />

lineages of ectosymbiotic and endosymbiotic bacteria. Previous studies<br />

have shown that Trichonympha flagellates, a parabasalid genus that dominates<br />

the gut microbiota of many termites, harbor more than one type of symbiont.<br />

Using an rRNA-based approach, we comprehensively investigated the<br />

phylogeny and subcellular locations of the bacterial populations associated with<br />

Trichonympha flagellates. Endomicrobia, the most abundant endosymbionts<br />

from the new Elusimicrobia phylum, were present only in Trichonympha<br />

Cluster I, comprising gut flagellates of the termite families Termopsidae and<br />

Rhinotermitidae. In contrast, the bacterial assemblage associated with<br />

Trichonympha Cluster II was dominated by a novel, hitherto undetected lineage<br />

of Actinobacteria. With a few exceptions, each flagellate species contained also<br />

a second population of bacterial symbionts, whose phylogeny was much more<br />

varied. Depending on the flagellate lineage, they represented hitherto<br />

uncultivated phylotypes, e.g., Deltaproteobacteria or Verrucomicrobia, closely<br />

related to bacteria previously detected in termite hindguts, whose association<br />

with flagellates was so far undocumented. In one flagellate species, we detected<br />

a novel lineage of the genus Prevotella (Bacteroidales), whose presence in<br />

insect guts had not been observed before. Using fluorescent in situ<br />

hybridization, we localized the different phylotypes in specific subcellular<br />

compartments of their Trichonympha hosts. The identical location of<br />

phylogenetically distinct lineages of bacterial symbionts in several termite<br />

species suggests a specific interaction with their host flagellates.<br />

PN 63<br />

Attempts for an integrative concept for the ecological<br />

assessment of groundwater ecosystems – microbes as<br />

bioindicators<br />

C. Kellermann *1 , S. Richter 2 , C. Griebler 1<br />

1 Institute of Groundwater Ecology, Helmholtz Center Munich - German<br />

Research Center of Environmental Health, Neuherberg, Germany<br />

2 General Aspects of Water Quality and Management, Groundwater Protection,<br />

UBA – Fe<strong>der</strong>al Environment Agency, Dessau, Germany<br />

Healthy aquifers deliver important ecosystem services e.g. the purification of<br />

infiltrating water and the storage of high quality water over decades in<br />

significant quantities. Also the functioning of terrestrial and surface aquatic<br />

ecosystems directly depends on groundwater and vice versa. Nowadays,<br />

legislation in many parts of the world has started to consi<strong>der</strong> groundwater not<br />

only as a resource but as a living ecosystem. However, to our opinion, the<br />

assessment of ecosystems requires consi<strong>der</strong>ation of ecological criteria. So far,<br />

such criteria are not available for groundwater systems. In the framework of a<br />

project supported by the German Fe<strong>der</strong>al Environment Agency (UBA), a first<br />

concept for the ecological assessment of groundwater ecosystems is developed,<br />

with a strong focus on microbes as potential bioindicators. Steps to be taken are<br />

introduced, including the typology of groundwater ecosystems, (ii) the<br />

<strong>der</strong>ivation of natural background values, (iii) the identification of potential<br />

bioindicators, and (iv) the development of an assessment model. Successes and<br />

difficulties associated with these challenges, e.g. the lack of simple correlations<br />

between abiotic and biotic variables, are discussed on the basis of two data sets<br />

from local and regional aquifers of two different groundwater landscapes in<br />

Germany. The need for collaboration between ecologists, hydrogeologists and<br />

geochemists, as well as the application of multivariate statistics, is emphasized.<br />

PN 64<br />

Microbial community dynamics of key contaminant<br />

degra<strong>der</strong>s and sulphate reducers in a tar-oil contaminated<br />

aquifer as driven by hydrogeochemical change<br />

G. Pilloni *1 , T. Riedel 1 , A. Bayer 1 , C. Griebler 1 , T. Lue<strong>der</strong>s 1<br />

1<br />

Institute of Groundwater Ecology, Helmholtz Zentrum Muenchen, Neuherberg,<br />

Germany<br />

The aim of this project is to develop a better un<strong>der</strong>standing of the in situ<br />

ecology of microbial communities involved in natural attenuation at sites<br />

polluted with aromatic hydrocarbons. In contaminated aquifers, contaminant<br />

plumes usually spread with groundwater flow, forming characteristic redox<br />

zones along as well as transversal to the main flow direction. Un<strong>der</strong> stationary<br />

conditions, specialized microbial assemblages establish within these redox<br />

zones, which oxidize hydrocarbons un<strong>der</strong> respiration of locally available<br />

electron acceptors. The implications of hydrogeochemical dynamics (e.g.<br />

increased recharge, shifts in the groundwater table) for degra<strong>der</strong>s and<br />

degradation activities, however, are poorly un<strong>der</strong>stood at present. Such<br />

dynamics can be hypothesized to either increase (by increasing the mixing) or<br />

decrease (by imposing unfavorable conditions on established sessile degra<strong>der</strong>s)<br />

net contaminant removal. The capacity of locally established degra<strong>der</strong>s to adapt<br />

to hydrogeochemical change is the key for the un<strong>der</strong>standing of these<br />

couplings.<br />

In this study, for the first time, these questions are addressed by repeated<br />

sampling of sediments and groundwater in a tar-oil contaminated aquifer. Via<br />

T-RFLP fingerprinting and qPCR of ribosomal and catabolic genes we show<br />

that the distribution of key contaminant degra<strong>der</strong>s and sulphate reducers (SRB)<br />

involved in hydrocarbon degradation is dynamic in space and time within the<br />

aquifer, and surprisingly tightly coupled to the dynamics of local redox<br />

regimes. These findings indicate interdependence between habitat dynamics<br />

and locally established degra<strong>der</strong> populations, and thus open a new window to a<br />

better un<strong>der</strong>standing of the controls of contaminant degradation in groundwater<br />

environments.<br />

PN 65<br />

Molecular characterisation of the microbial community<br />

structure of phosphate accumulating bacteria in municipal<br />

wastewater treatment plants<br />

L. Mehlig *1 , M. Eschenhagen 1 , M. Petzold 1 , S. Müller 2 , S. Günther 2 , I. Röske 1<br />

1<br />

Department of Microbiology, Dresden University of Technology, Dresden,<br />

Germany<br />

2<br />

Department of Environmental Microbiology, Helmholtz Centre for<br />

Environmental Research, Leipzig, Germany<br />

The enhanced biological phosphorous removal (EBPR) is a common<br />

technology in wastewater treatment. Despite this fact it is still largely unknown<br />

which microbial species are responsible for phosphorus accumulation and<br />

release. This acknowledgement is necessary for the improvement and to<br />

provide an efficient and environmentally friendly wastewater treatment process.<br />

The emphasis of this study was to analyze the polyphosphate accumulating<br />

organisms (PAO) community structure in wastewater treatment plants and<br />

furthermore the identification of microorganisms responsible for the EBPR<br />

process.<br />

Therefore polyphosphate granules containing bacteria were separated out of<br />

activated sludge from the aeration basin using flow cytometric cell sorting.<br />

In addition to semi quantitative fluorescence in situ hybridization (FISH)<br />

analysis molecular techniques where used to yield additional information and to<br />

verify the results from FISH analysis.<br />

Despite the different modes of operation only minor differences in the bacterial<br />

composition of the investigated plants were detected by FISH analysis.<br />

On the other hand T-RFLP analysis showed major differences of the bacterial<br />

communities. Characteristic community fingerprints for each of the<br />

investigated plants and comparative 16S rDNA analysis indicated highly<br />

diverse microbial communities in all plants suggesting substantial differences<br />

in their microbial structure.<br />

In our case Tetrasphaera spp., Microlunatus spp., members of the Rhodocyclus<br />

group and other PAOs had been also detected by cloning and sequencing.<br />

These organisms were found in all plants, including those without EBPR,<br />

obviously they are constituents of the microbial community structure of<br />

activated sludge systems.<br />

PN 67<br />

Microbial community changes and plasmid ecology in nearnatural<br />

remediation systems for BTEX- and MTBEcontaminated<br />

groundwater<br />

M. Kaulfersch 1 , A. Chatzinotas 1 , H. Harms 1 , B. Kiesel *1<br />

1 Dept. Environmental Microbiology, Helmholtz Centre for Environmental<br />

Microbiology-UFZ, Leipzig, Germany<br />

Aromatic contaminants are persistent un<strong>der</strong> anoxic conditions and, therefore,<br />

the transfer of contaminated groundwater from anaerobic into aerobic<br />

environments induces significant changes in degradation but also in community<br />

composition and the potential for horizontal gene transfer. A pilot-scale plant<br />

was set up at a former refinery site near Leuna, Germany and receives<br />

groundwater mainly contaminated with BTEX (15 mg/l) and MTBE (4 mg/l).<br />

The primary task of this Compartment Transfer (CoTra) project is to evaluate<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

five near-natural remediation systems for efficient low-cost contaminant<br />

biodegradation.<br />

In this study we aimed at un<strong>der</strong>standing (i) the changes within the microbial<br />

communities as well as the (i) involvement of plasmids and their potential for<br />

transferring catabolic genes of interest in the constructed wetlands (AP2) and<br />

the aerobic trenches (AP5) of the pilot plant.<br />

16S rRNA gene based T-RFLP community patterns indicated that compartment<br />

transfer of the groundwater community resulted in different community<br />

compositions, whereby larger changes were recorded in AP5 than in AP2. All<br />

three communities were found to be well equipped with upper and lower<br />

pathway genes for degradation of aromatic compounds. Interestingly, plasmid<br />

analysis revealed that all sites contained plasmids of the IncP1 group, but no<br />

IncP7 and IncP9 group plasmids. IncP1 plasmids in groundwater did not carry<br />

any catabolic genes, while the plasmid pools of AP2 and AP5 were found to be<br />

equipped with upper pathway genes coding for subunits of the monooxygenases<br />

TMBD and TMOA. For AP5 also a toluene/biphenyl – dioxygenase<br />

(BEDe/BEDm) was found. The role of horizontal gene transfer for spreading<br />

catabolic plasmids will be discussed.<br />

PN 68<br />

Response of chemotactic bacteria to air-born<br />

chemoattractants<br />

J. Hanzel *1 , M. Thullner 1 , H. Harms 1 , L. Wick 1<br />

1<br />

Environmental Microbiology, Helmholtz Centre For Environmental Research -<br />

UFZ, Leipzig, Germany<br />

Bacterial chemotaxis, i.e. directed bacterial swimming along chemical<br />

gradients, has been proposed as a relevant mobilizing mechanism during the<br />

biodegradation of hydrophobic organic contaminants (HOC) in soil.<br />

Chemotaxis decreases the distance between the microorganisms and the<br />

pollutant source and, hence, increases both HOC mass transfer to the cells and<br />

HOC-biodegradation. Whereas chemotaxis in aqueous systems is fairly welldocumented,<br />

not much is known on the influence of air-born chemoattractant<br />

gradients on chemotactic response of water-bound bacteria. In this study we<br />

compared the influence of air-born vs. agar-born (water-born) gradients of the<br />

semi-volatile chemoeffector naphthalene on the dispersal of chemotactic,<br />

naphthalene-degrading Pseudomonas putida PpG7 (NAH7). Strain PpG7 was<br />

spot-inoculated onto swimming agar plates at variable distances to the solid<br />

chemoeffector, <strong>bei</strong>ng either submerged in the agar or placed above the agar in<br />

the plates’ headspace, and the spatiotemporal dispersal of strain PpG7<br />

quantified at growth and non-growth conditions. Whereas positive chemotaxis<br />

to water-born naphthalene gradients was detected, negative chemotaxis to airborne<br />

naphthalene was found when microbial growth was excluded. This<br />

observation is explained by the strain’s avoidance of potentially toxic air-born<br />

naphthalene and hence points at an increased bioavailability when naphthalene<br />

was exposed by the gas phase. Our data suggest that gaseous chemical<br />

gradients significantly influence the spatio-temporal distribution and growth of<br />

catabolically active bacteria and, finally, the biodegradation of semivolatiles in<br />

the vadose zones.<br />

PN 69<br />

Metaproteomic Investigation of Biofouling in Membrane<br />

Bioreactors<br />

D. Benndorf *1 , R. Kuhn 2 , E. Rapp 3 , U. Reichl 3 , B. Devreese 4 , A. Pollice 5 , G.<br />

Laera 2 , L.L. Palese 5<br />

1<br />

Bioprocess Engineering, Otto von Guericke University, Magdeburg, Germany<br />

2<br />

IRSA CNR, National Water Research Institute, Bari, Italy<br />

3<br />

Bioprocess Engineering, Max Planck Institute for Dynamic of technical<br />

complex systems, Magdeburg, Germany<br />

4<br />

Dept. of Protein Biochemistry, University of Ghent, Ghent, Belgium<br />

5<br />

Dept. of Medical Biochemistry, Biology and Physics, University of Bari, Bari,<br />

Italy<br />

MBRs (membrane bioreactors) applied for municipal wastewater treatment, are<br />

equipped with a set of membrane modules that allow the separation of<br />

processed sewage from activated sludge. One of the main advantages in the use<br />

of MBRs is the option to handle both non-flocculating and flocculating<br />

bacteria, which results in operations at high loading rates with an excellent<br />

effluent quality. However, MBRs still tend to membrane biofouling. Biofouling<br />

is mainly caused by extracellular polymeric substances (EPS) and soluble<br />

microbial products (SMP) accumulating on and inside membranes causing<br />

permeate flux deterioration. Besides, polysaccharides, EPS and SMP also<br />

contain proteins that play a key role in the biofouling process. Therefore,<br />

proteins were extracted from MBR sludge and separated by conventional 2D-<br />

PAGE. The protein pattern gave a first insight to the steady state of MBR<br />

biomass. Furthermore, microbial dynamics and recovery were studied with a<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

137<br />

NaCl shock load experiment. Subsequently, ten different MBR operation plants<br />

from bench-scale up to full-scale were compared. As expected, all sludge<br />

samples showed a unique protein pattern, but also several common proteins.<br />

These proteins were analysed by (nano)-LC-ESI-MS/MS and MALDI-<br />

TOF/TOF. Proteins were identified by database search. Some proteins were<br />

related to environmental bacteria. Interestingly human elastase IIIA and high<br />

amounts of keratin were detected as well. In a reference, MBR biomass only<br />

fed with synthetic wastewater, proteins of human source were not detectable.<br />

The function of those proteins to the MBR bioconsortium is still unknown.<br />

PN 70<br />

Activity and Identification of Sulfate Reducing Prokaryotes<br />

in Heavy Metal and Radionuclide Contaminated Creek Soil<br />

J. Sitte 1 , D. Akob *1 , C. Kaufmann 1 , K. Pollok 2 , K. Finster 3 , J. Kostka 4 , F.<br />

Langenhorst 2 , K. Küsel 1<br />

1<br />

Institute of Ecology, Friedrich Schiller University Jena, Jena, Germany<br />

2<br />

Bavarian Research Institute of Experimental Geochemistry and Geophysics,<br />

University of Bayreuth, Bayreuth, Germany<br />

3<br />

Department of Microbial Ecology, Institute of Biological Science, Århus<br />

University, Århus, Denmark<br />

4<br />

Department of Oceanography, Florida State University, Tallahassee, United<br />

States<br />

Uranium mining in the area of Ronneburg, Germany led to the contamination<br />

of surrounding aquifers with sulfate, heavy metals and radionuclides. Sulfate<br />

reducing prokaryotes (SRP) can affect the mobility of metals directly via<br />

reduction/oxidation reactions or indirectly via immobilization as metal sulfides.<br />

Consequently, the activity of SRP may act to decrease contaminant inputs to<br />

adjacent ecosystems. The objectives of this study were (1) to assess the<br />

influence of SRP on metal mobility and (2) to characterize active and heavy<br />

metal tolerant SRP in heavy metal contaminated creek soil. Sulfate reduction<br />

rates, determined by the 35S tracer techniques, in soil samples were up to 142<br />

nmol cm -3 day -1 at in situ sulfate concentrations. Sulfate reduction was<br />

stimulated in lactate amended, anoxic soil microcosms and lactate was oxidized<br />

to propionate and acetate. Nickel and cobalt were immobilized, whereas<br />

uranium was mobilized during the microcosm incubation. A combination of<br />

several molecular methods including DNA stable isotope probing, terminal<br />

restriction fragment length polymorphism (TRFLP) analysis, and<br />

cloning/sequencing techniques, revealed significant differences in microbial<br />

communities composition between the early and the late sulfate reducing<br />

activity. Resident SRP were resistant to several heavy metals, such as Ni, Zn,<br />

and Co, and a stable enrichment culture growing on 22 mM Ni could be<br />

obtained. The formation of amorphous and nanocrystalline nickel sulfides was<br />

confirmed by TEM and EDX analysis. Our results indicate that an active and<br />

diverse community of SRP is present in contaminated creek soils, which may<br />

influence metal mobility through sulfide formation.<br />

PN 71<br />

Characterization of microbial communities by FT-IR<br />

spectroscopy<br />

R. Schäwe *1 , A. Tönniges 1 , I. Fetzer 1 , W. Geyer 2 , H. Harms 1 , A. Chatzinotas 1<br />

1<br />

Department of Environmental Microbiology, Helmholtz-Centre for<br />

Environmental Research - UFZ, Leipzig, Germany<br />

2<br />

Department Analytical Chemistry, Helmholtz-Centre for Environmental<br />

Research - UFZ, Leipzig, Germany<br />

Infrared spectroscopy potentially offers a fast and effective characterization of<br />

microrganisms due to its high specific information content. Development of<br />

Fourier-Transformation infrared spectroscopy (FT-IR) resulted in a significant<br />

improvement suitable for identification and classification of microorganisms.<br />

So far this method has successfully been applied for the characterization of<br />

monocultures but up to now little investigation has been done to analyze<br />

complex microbial communities by infrared techniques.<br />

In this study we used FT-IR spectroscopy for the characterization of mixed<br />

bacterial model cultures of Pseudomonas putida and Rhodococcus ruber. We<br />

aimed to evaluate the application of infrared measurements to i) quantify the<br />

relative abundance of species within mixed communities and ii) differentiate<br />

within population growth characteristic (exponential growing vs. stationary<br />

phase). Analyses were carried out with transmission Micro-FT-IR spectrometer<br />

followed by chemometrics including multivariate analyses.<br />

We could show that FT-IR spectroscopy is a powerful method to<br />

simultaneously determine the structure of simple mixed microbial communities<br />

estimating relative abundance and overall physiological status of populations<br />

with high accuracy.

138<br />

PN 72<br />

Effect of elevated CO2 on the composition of methanotrophs<br />

in meadow soil<br />

P.M. Shrestha *1 , C. Kammann 2 , W. Liesack 1<br />

1 Biogeochemistry department, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

2 Institute for Plant Ecology, Justus-Liebig-University, Giessen, Germany<br />

A detailed un<strong>der</strong>standing of how elevated CO2 affects methane oxidation in<br />

grassland soils is critical to predict their contributions to the future atmospheric<br />

methane budget. We assessed the composition of methanotrophic bacteria in a<br />

meadow soil that has been treated with elevated CO2 (450 ppmv) since 1998, in<br />

comparison to control plots with ambient CO2. The community composition of<br />

methanotrophs was compared in the different treatment and control plots by<br />

molecular fingerprinting (T-RFLP) and comparative sequence analysis of pmoA<br />

gene fragments. Both elevated-CO2 and control plots were dominated by two<br />

subpopulations (JR1, RA14) of the „Upland Soil Cluster alpha“ (USCα). This<br />

as-yet-uncultivated methanotroph group is assumed to be specialized on<br />

atmospheric methane oxidation, which agrees well with the CH4 and O2 profiles<br />

measured in the test plots. In T-RFLP analysis, the two USCα subpopulations<br />

were characterized by 34-bp (RA14) and 80-bp (JR1) T-RFs. The relative<br />

abundance of the 34-bp and 80-bp T-RFs varied with the CO2 concentration.<br />

The 80-bp T-RF was increased relatively to the 34-bp T-RF in the T-RFLP<br />

patterns obtained from the elevated-CO2 plots, suggesting that RA14 and JR1<br />

respond differently to changes in the CO2 concentration. Depth-related changes<br />

in the methanotrophic community were observed neither in the plots enriched<br />

with CO2 nor in those exposed to ambient CO2.<br />

PN 73<br />

Linking phylogeny with function in microbial symbionts of<br />

marine sponges using whole genome amplifications<br />

A. Siegl *1 , U. Hentschel 2<br />

1<br />

Zentrum für Infektionsforschung, Universität Würzburg, Würzburg, Germany<br />

2<br />

Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg,<br />

Würzburg, Germany<br />

Whole genome amplification (WGA) based approaches hold great promise for<br />

the field of microbial ecology. These molecular techniques provide genomic<br />

information on single bacterial cells in a cultivation-independent manner. Here<br />

we present results from the application of WGA to microbial symbionts of<br />

marine sponges. For that purpose, the microbial consortia of the Mediterranean<br />

sponge Aplysina aerophoba were sorted by fluorescence activated cell sorting<br />

(FACS) and then subjected to genome amplification by use of the phi29<br />

polymerase. A cosmid library consisting of ~8,000 clones was constructed<br />

using the amplified genomic DNA <strong>der</strong>ived from two bacterial cells, one<br />

member of the Chloroflexi and one member of the sponge-specific candidate<br />

phylum Poribacteria. Library screening led to the genomic characterization of<br />

three cosmid clones, encoding a Chloroflexi 16S rRNA gene, a non-ribosomal<br />

peptide synthetase (NRPS) and a polyketide synthase (PKS), respectively.<br />

PCR-screening of amplified genomes from additional, FACS-sorted microbial<br />

symbiont cells resulted in the assignment of the PKS to the Poribacteria and<br />

the putatively novel NRPS to the Chloroflexi. Furthermore, nif and nas genes<br />

were identified in WGA products of members of the Chloroflexi, indicating that<br />

sponge-associated Chloroflexi have the genomic potential of nitrogen fixation<br />

and assimilatory nitrate reduction. This promising single cell-based approach<br />

will enable linking phylogeny and function of sponge-associated microbes and<br />

lead to a better un<strong>der</strong>standing of their symbiosis.<br />

PN 74<br />

Effect of elevated CO2 on the composition of methanotrophs<br />

in meadow soil<br />

P.M. Shrestha *1 , C. Kammann 2 , W. Liesack 1<br />

1 Biogeochemistry department, Max Planck Institute for Terrestrial<br />

Microbiology, Marburg, Germany<br />

2 Institute for Plant Ecology, Justus-Liebig-University, Giessen, Germany<br />

A detailed un<strong>der</strong>standing of how elevated CO2 affects methane oxidation in<br />

grassland soils is critical to predict their contributions to the future atmospheric<br />

methane budget. We assessed the composition of methanotrophic bacteria in a<br />

meadow soil that has been treated with elevated CO2 (450 ppmv) since 1998, in<br />

comparison to control plots with ambient CO2. The community composition of<br />

methanotrophs was compared in the different treatment and control plots by<br />

molecular fingerprinting (T-RFLP) and comparative sequence analysis of pmoA<br />

gene fragments. Both elevated-CO2 and control plots were dominated by two<br />

subpopulations (JR1, RA14) of the “Upland Soil Cluster alpha” (USCα). This<br />

as-yet-uncultivated methanotroph group is assumed to be specialized on<br />

atmospheric methane oxidation, which agrees well with the CH4 and O2 profiles<br />

measured in the test plots. In T-RFLP analysis, the two USCα subpopulations<br />

were characterized by 34-bp (RA14) and 80-bp (JR1) T-RFs. The relative<br />

abundance of the 34-bp and 80-bp T-RFs varied with the CO2 concentration.<br />

The 80-bp T-RF was increased relatively to the 34-bp T-RF in the T-RFLP<br />

patterns obtained from the elevated-CO2 plots, suggesting that RA14 and JR1<br />

respond differently to changes in the CO2 concentration. Depth-related changes<br />

in the methanotrophic community were observed neither in the plots enriched<br />

with CO2 nor in those exposed to ambient CO2.<br />

PN 75<br />

Singlet oxygen affects aquatic bacterioplankton activity and<br />

composition<br />

J. Glaeser *1 , H.P. Grossart 2 , S. Glaeser 1<br />

1<br />

Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität<br />

Giessen, Giessen, Germany<br />

2<br />

Abteilung Limnologie Geschichteter Seen, Leibniz-Institut für<br />

Gewässerökologie und Binnenfischerei, Neuglobsow, Germany<br />

Photolysis of dissolved organic matter (DOM) leads to contrasting effects in<br />

aquatic bacterial communities. In this process production of low molecular<br />

weight substrates stimulates bacterial activity and simultaneously generated<br />

reactive oxygen species (ROS) cause inhibition of bacteria. The bacterial<br />

response to DOM photolysis is poorly un<strong>der</strong>stood and in particular the role of<br />

short-lived ROS as singlet oxygen ( 1 O2) has scarcely been investigated. In<br />

aquatic habitats humic acids serve as photosensitizer and mediate the light<br />

dependent generation of 1 O2 and other ROS. We observed that bacterial carbon<br />

production as determined by 14 C-leucine uptake experiments is inhibited in a<br />

humic acid rich lake during hours of high solar irradiance. In or<strong>der</strong> to<br />

investigate the effects of 1 O2 on bacterioplankton activity we gradually<br />

increased 1 O2 steady state concentrations in situ and thereby determined<br />

threshold levels for total bacterioplankton inhibition. Analysis of metabolically<br />

active bacterioplankton species using RT-PCR DGGE fingerprints with<br />

Bacteria and group specific primers was used to determine thresholds for<br />

specific predominant bacterioplankton species. Our experiments show that<br />

changes in 1 O2 steady state concentrations affect bacterioplankton activity and<br />

species composition in a humic acid rich lake and that predominant bacterial<br />

species are affected by different 1 O2 steady state concentrations. The analysis of<br />

culturable bacteria by MPN dilution series, staining of damaged cells using the<br />

LIFE/DEAD staining kit, and pure culture studies support our findings. Overall<br />

our data indicate that generation of ROS is an important natural stress factor for<br />

bacteria and affects bacterioplankton species composition in aquatic habitats.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 76<br />

Determination of the influence of plant litter-<strong>der</strong>ived<br />

hexadecane on soil microbial communities<br />

J. Giebler *1 , A. Chatzinotas 1 , L.Y. Wick 1 , H. Harms 1<br />

1 Department Environmental Microbiology, Helmholtz Centre for<br />

Environmental Research, Leipzig, Germany<br />

Aim of the study is to investigate the influence of plant litter-<strong>der</strong>ived<br />

hydrocarbons on the diversity of n-alkane degrading microbial communities<br />

and alkane-biodegradation at soil-interfaces. The spatio-temporal influence of<br />

hexadecane (C16) emanating from decaying maize litter on the microbial<br />

diversity in microcosms containing agricultural soil was assessed using<br />

conventional enrichment. Alternatively, Teflon membrane-based transfer<br />

procedures were applied to select for hydrophobic C16-degrading bacteria. To<br />

determine diversity changes during the enrichment, C16-degrading isolates<br />

were characterised by MPN and amplified rDNA restriction analysis (ARDRA)<br />

of 16S rRNA gene amplicons and subsequent sequence analysis. ARDRA<br />

resulted in 102 different restriction patterns (operational taxonomic units, OTU)<br />

after 8 weeks incubation of the soil microcosms with maize litter. Comparisons<br />

of the number of OTU before and after 8 weeks of incubation showed a strong<br />

increase in OTU in all sampling depths over time. A higher increase in OTU<br />

was observed during enrichment with hydrophobic Teflon-membranes<br />

compared to common liquid enrichment. Sequence analysis of at least one<br />

representative sequence per OTU allowed the phylogenetic affiliation of<br />

bacteria capable to grow with C16 as sole carbon source. Results indicate that<br />

cultivable C16-degrading bacteria show distinct dynamics over time and depths<br />

and differ as a result of the enrichment strategy. More than 20 different genera<br />

were identified, including genera typically found at oil-contaminated sites like<br />

Burkhol<strong>der</strong>ia or Rhodococcus, while MPN enumeration showed a clear<br />

influence of litter on the quantity of cultivable C16-degrading organisms. The<br />

enriching effect of plant litter on the n-alkane degrading community in soil<br />

could constitute a new bioremediation strategy for oil contaminated sites.<br />

Possible physiological adaptations of the bacteria to hydrophobic substrates<br />

could further enhance the degradation activity and will be assessed in future<br />

studies.<br />

PN 77<br />

Adaptive response to singlet oxygen in Alpha - and<br />

Betaproteobacteria isolated from the humic acid rich Lake<br />

Grosse Fuchskuhle<br />

S. Glaeser *1 , M. Grosz 1 , H.P. Grossart 2 , J. Glaeser 1<br />

1 Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität<br />

Gießen, Gießen, Germany<br />

2 Limnologie geschichteter Seen, Leibniz-Institut für Gewässerökologie und<br />

Binnenfischerei, Neukobsow, Germany<br />

The photolysis of dissolved organic matter (DOM) is an important process in<br />

freshwater habitats enhancing the bioavailability of recalcitrant DOM.<br />

Although bacterioplankton species benefit from carbon substrates generated by<br />

this process they have to cope with reactive oxygen species (ROS) generated<br />

simultaneously. We investigated in situ short-term effects of artificially<br />

increased singlet oxygen ( 1 O2) formation on the bacterioplankton in the SW<br />

basin of the humic acid rich Lake Grosse Fuchskuhle. Changes in the structure<br />

of metabolically active, predominant bacteria upon 1 O2 exposure were<br />

unraveled by RT-PCR using Bacteria specific 16S rRNA gene targeting<br />

primers followed by denaturing gradient gel electrophoresis (DGGE). DNA<br />

bands affiliated to Novosphingobium acidiphilum (Aphaproteobacteria) and<br />

Herbaspirillum and Curvibacter spp. (Betaproteobacteria) were most intense<br />

in the incubation with increased 1 O2 concentrations whereas a DNA band<br />

affiliated to Methylocapsa spp. (Alphaproteobacteria) was only observed in the<br />

control incubation experiment. We were able to isolate bacterial species<br />

representing those DNA bands and performed pure culture studies using spot<br />

assay experiments to investigate the response to 1 O2 treatments in greater detail.<br />

Those isolates representing major DNA bands in the 1 O2 treatment were more<br />

resistant to 1 O2 than the isolate representing a DNA band not observed after this<br />

treatment. Pre-incubation with non-inhibitory amounts of 1 O2 induced an<br />

adaptive response to increased 1 O2 concentrations in the more resistant isolates.<br />

We propose that an adaptive response of heterotrophic bacteria to 1 O2 is<br />

potentially important for species selection in humic acid rich lakes.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PN 78<br />

Microbial biofilms on stone monuments<br />

C. Hallmann *1 , M. Hoppert 2 , J. Ruedrich 3 , M. Enseleit 2 , T. Friedl 1<br />

1<br />

Experimentelle Phykologie und Sammlung von Algenkulturen, Universität<br />

Göttingen, Göttingen, Germany<br />

2<br />

Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen,<br />

Germany<br />

3<br />

Geowissenschaftliches Zentrum Göttingen, Abteilung<br />

Strukturgeologie/Geodynamik, Universität Göttingen, Göttingen, Germany<br />

Microbial biofilms, consisting of green algae, filamentous fungi and bacterial<br />

organisms cover solid surfaces, such as plastic material, glass and natural stone.<br />

In the present study, samples from a marble stone monument were taken and<br />

subjected to analysis of the microbial communities. The biofilm consists mainly<br />

of algal and fungal organisms, as revealed by 18S-rDNA analysis of the<br />

community DNA. Some of the algal organisms were also isolated and subjected<br />

to further structure-functional analysis. A strain of the green alga Dilabifilum,<br />

also known as a phycobiont of freshwater lichens, could be isolated from the<br />

biofilm. In unialgal culture, the organisms form branched, interwoven<br />

filaments. A thin layer of extracellular polymers on the algal cell wall provides<br />

the matrix for biofilm formation and attachment to the material surface.<br />

PN 79<br />

Study of a specific phage-host-system with a biocontrol host<br />

strain in the rhizosphere of oilseed rape<br />

K. Ulbricht 1 , A. Wolf *1<br />

1<br />

Institute of Biological Sciences/ Microbiology, University of Rostock, Rostock,<br />

Germany<br />

Verticillium dahliae is a very important phytopathogen and with chemicals<br />

hardly to destroy because of resistant microsclerotia in the soil. Therefore it is<br />

necessary to find more effective non-toxic alternatives to get rid of this<br />

problem. One possibility is the application of a Biological Control Agent<br />

(BCA), like Serratia plymuthica HRO-C48, an already certified biocontrolproduct<br />

(RhizoStar ® ). This rhizobacterium inhibits the fungus effective to grow<br />

and improved plant health. Bacteriophages are ecologically important by<br />

controlling bacterial numbers and activity. In this study the influence of a lytic<br />

phage on the establishment of the biocontrol strain HRO-C48 in the rhizosphere<br />

of oilseed rape was investigated in the greenhouse. The phage belonged to the<br />

Myoviridus morphotype with a latent period of about 65 min and a burst size of<br />

20 phage particles per cell in minimal medium. In liquid culture it shows a lytic<br />

character and lysed the host culture in dependence of the initial multiplicity of<br />

infection (moi).<br />

In greenhouse phage and host strains were applied to the rhizosphere of oilseed<br />

rape at different moi. The fungus was added with about 10 4 cells g -1 soil. First<br />

results showed a rapid increase in phage multiplication after the addition of the<br />

host strain with a minimal host concentration of about 10 4 CFU g -1 . Here the<br />

host strain population was influenced significantly by phage lysis. As the host<br />

number droped un<strong>der</strong> this limit no phage multiplication occurred. Phageinsensitive<br />

cells occurred. The biocontrol activity of the strain against the<br />

phytopathogen was stable in vitro. The plant health has to be estimated.<br />

PN 80<br />

Population dynamics of host bacteria and phages in the<br />

rhizosphere of oilseed rape un<strong>der</strong> greenhouse and field<br />

conditions<br />

A. Wolf *1 , F. Schulz 1<br />

1<br />

Institute of Biological Sciences / Microbiology, University of Rostock, Rostock,<br />

Germany<br />

139<br />

Infection by virulent bacteriophages can control the numbers and biocontrol<br />

activity of beneficial bacteria introduced in the plant rhizosphere to suppress<br />

plant diseases. Here the influence of a specific phage on the population size of<br />

the Biological Control Agent (BCA) Serratia plymuthica HRO-C48 was<br />

investigated in the rhizopshere of oilseed rape un<strong>der</strong> greenhouse and field<br />

conditions. The BCA showed antagonistic activity against the phytopathogenic<br />

fungus Verticillium dahliae KLEB. The phage belongs to the Myoviridae<br />

morphotype with a head size of 65 nm and a tail length of 165 nm. It shows a<br />

high specifity for its host and formed clear plaques of about 3 mm.<br />

In the greenhouse phage and host strains were applied to the rhizosphere of<br />

oilseed rape at different initial multiplicities of infection (moi). The fungus was<br />

added with about 10 4 cells g -1 soil. First results showed a rapid increase in<br />

phage numbers after the addition of the host strain with a minimal<br />

concentration of 10 4 CFU g -1 . In this case the number of the host strain<br />

population was influenced significantly by phage lysis. Phage-insensitive cells

140<br />

occurred and were characterized. The biocontrol activity of the strains against<br />

the phytopathogen was stable in vitro.<br />

In field trail the BCA was added to the seeds by seed priming resulting in a<br />

concentration of about 10 6 CFU per grain of seed. Also in the field the number<br />

of the host strain was reduced significantly by phage attack until four weeks.<br />

The number of naturally occurring phages of HRO-C48 increased rapidly<br />

within seven days. The phages were characterized. The plant health has to be<br />

estimated.<br />

PN 81<br />

Susceptibility of adherent Streptococcus sanguinis to<br />

antibiotic agents applied against peri-implantitis<br />

M. Astashov-Frauenhoffer *1 , I. Hauser-Gerspach 1 , N. Zitzmann 2 , T. Waltimo 1<br />

1 Institute of Preventive Dentistry and Oral Microbiology, University of Basel,<br />

Basel, Switzerland<br />

2 Clinic for Periodontology, Endodontology and Cariology, Department of Oral<br />

Surgery, University of Basel, Basel, Switzerland<br />

Aims: To assess the in vitro susceptibility of an important primary colonizer of<br />

dental implants to common antibiotic agents.<br />

Material and methods: An anaerobic flow chamber model with bacteria<br />

circulating in simulated body fluid (SBF) was developed. Polished titanium<br />

plates (Straumann) were used as substrates for adherence. Streptococcus<br />

sanguinis ATCC20068 grown overnight in Schaedler broth (Oxoid) at 37°C<br />

was harvested, resuspended in SBF (10 8 CFU/mL) and allowed to adhere on the<br />

saliva-serum-coated specimens for 2h. They were aseptically removed, rinsed<br />

and placed in various concentrations (1-100xMIC, CLSI-protocol) of<br />

amoxicillin, metronidazole and minocycline solutions anaerobically at 37°C for<br />

24h. SBF without antibiotics served as a control. Adherent bacteria were<br />

stained (Live/Dead BacLight Bacterial Viability Kit, MoBiTec) and counted<br />

un<strong>der</strong> fluorescence microscope (5x0.02mm 2 /specimen, 5 parallels; Provis<br />

AX70, Olympus). Mean log10 of adherent bacteria ±SD per mm 2 was<br />

calculated. Percentages of vital bacteria were compared and statistically<br />

analyzed using one-way ANOVA.<br />

Results: The mean adherence in the logarithmic scale was 5.0±4.2<br />

bacteria/mm 2 . The control vitality percentage was 96.98 ± 0.85. MICs were 0.5,<br />

128.0 and 1.0 µg/ml for amoxicillin, metronidazole and minocycline,<br />

respectively. All three agents revealed statistically significant reduction of<br />

vitality in concentrations of ≥10xMIC. Metronidazole was significantly more<br />

efficient (mean reduction of vitality up to 99%) than the amoxicillin (5%) and<br />

minocycline (15%).<br />

Conclusion: Within the limitations of this in vitro study, metronidazole seems<br />

to be more efficient against adherent S. sanguinis than amoxicillin and<br />

minocycline.<br />

PN 82<br />

Microbial activity in terrestrial mud volcanoes from the<br />

Northern Apennines<br />

S. Kokoschka 1 , A. Dreier *1 , C. Heller 2 , V. Krukenberg 1 , C. Wrede 1 , M.<br />

Taviani 3 , J. Reitner 2 , M. Hoppert 1<br />

1<br />

Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen,<br />

Germany<br />

2<br />

Geowissenschaftliches Zentrum, Abteilung Geobiologie, Universität<br />

Göttingen, Göttingen, Germany<br />

3<br />

Instituto di Scienze Marine (ISMAR), Consiglio Nationale delle Richerche<br />

(CNR), Bologna, Germany<br />

Mud volcanoes can be observed in terrestrial and marine areas worldwide and<br />

are often localized in tectonic zones of compression. It is known that several<br />

types of archaeal and bacterial microorganisms are able to metabolize<br />

hydrocarbons from the gas and fluid phases. Methane is oxidized un<strong>der</strong><br />

anaerobic conditions (AOM) by methane oxidizing microbial consortia. Here,<br />

we describe the geochemical background (main and trace elements, pH, δ 13 C)<br />

of the phases seeping out of the mud volcanoes from the Northern Apennines,<br />

and we present first results of biomarker analysis with respect to AOM.<br />

Several organisms could be identified and isolated and may serve as model<br />

organisms for biomineral formation in terrestrial and marine mud volcano<br />

environments. The formation of biominerals un<strong>der</strong> reducing conditions were<br />

also studied in model systems. It is obvious that, at least in model systems,<br />

biopolymers, secreted by the organisms, are involved in precipitation of iron<br />

sulfides.<br />

PN 83<br />

Potential of nitrate addition to control the activity of<br />

sulfate-reducing prokaryotes in high-temperature oil<br />

production systems - a comparative study on a nitratetreated<br />

and an untreated system<br />

A. Gittel *1 , K. Sørensen 2 , T. Skovhus 2 , K. Ingvorsen 1 , A. Schramm 1<br />

1<br />

Microbiology Group, Department of Biological Sciences, University of<br />

Aarhus, Aarhus, Denmark<br />

2<br />

Center for Chemistry and Water Technology, Danish Technological Institute,<br />

Aarhus, Denmark<br />

Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial<br />

corrosion and reservoir souring in seawater-injected oil production systems.<br />

Adding nitrate to the injection water is applied to control SRP activity by<br />

favoring the growth of heterotrophic, nitrate-reducing bacteria (hNRB) and<br />

nitrate-reducing, sulfide-oxidizing bacteria (NR-SOB). Microbial diversity,<br />

abundance of Bacteria, Archaea and sulfate-reducing prokaryotes (SRP) and<br />

the potential activity of SRP were studied in production water samples from a<br />

nitrate-treated and an untreated system with similar physicochemical<br />

characteristics. At both sites, Archaea and Archaeoglobus-related SRP<br />

dominated the total prokaryotic and the sulfate-reducing community,<br />

respectively. It was however indicated from clone libraries and the<br />

quantification of 16S rRNA and dsrAB gene copies that Archaeoglobus-related<br />

SRP were less prominent at the nitrate-treated site than at the untreated site. In<br />

return, thermophilic bacterial SRP appeared to be more abundant (2 and 8 % of<br />

all SRP, respectively). They were related to members of the genera<br />

Desulfacinum and Desulfoglaeba (system without nitrate) and<br />

Desulfotomaculum (system with nitrate). In samples from the untreated site, the<br />

presence of active SRP was supported by demonstrating their activity<br />

(incubations with 35 S-sulfate) and growth in batch cultures at pipeline<br />

temperature. No SRP activity was detected at reservoir temperature and in<br />

samples from the nitrate-treated site. In addition, potential competitive nitrate<br />

reducers of the genus Sulfurospirillum (NR-SOB) and the or<strong>der</strong><br />

Deferribacterales (hNRB) were exclusively detected at the nitrate-treated site.<br />

It is therefore indicated that nitrate addition resulted in decreased SRP activity,<br />

an increase in diversity of Bacteria and bacterial SRP and a stimulation of<br />

nitrate-reducing competitors.<br />

PN 84<br />

Tracing Population Dynamics and Species Interactions of a<br />

Heterogeneous Microbial Culture by Proteome Analysis<br />

S. Kluge 1 , M. Hoffmann 1 , D. Benndorf *2 , E. Rapp 1 , U. Reichl 1<br />

1 Bioprocess Engineering, Max Planck Institute for Dynamics of Complex<br />

Technical Systems, Magdeburg, Germany<br />

2 Bioprocess Engineering, Otto von Guericke University, Magdeburg, Germany<br />

Proteome analysis is mostly applied to pure cultures. In or<strong>der</strong> to demonstrate its<br />

application to a microbial community we use a defined mixed culture of<br />

Burkhol<strong>der</strong>ia cepacia, Pseudomonas aeruginosa and Staphylococcus aureus.<br />

All species cause lung infections related to the genetic disease cystic fibrosis.<br />

From about 1100 proteins detected in 2D-gel of the mixed culture, 292 proteins,<br />

206 proteins and 183 have been assigned by comparison with 2D-gels of pure<br />

cultures to Burkhol<strong>der</strong>ia cepacia, Pseudomonas aeruginosa and<br />

Staphylococcus aureus, respectively. The correct assignment was confirmed by<br />

mass spectrometry. Subsets of well separated and high abundant proteins of<br />

each species were taken to trace the changes in the community during batch<br />

experiments with the optional addition of the antibiotic ceftazidime. Although<br />

Pseudomonas aeruginosa is thought to be sensitive against ceftazidime, the<br />

population was dominated by Pseudomonas aeruginosa in both cases.<br />

Differently expressed proteins between the antibiotic pulse and the control<br />

indicate for adaptation to ceftazidime and are probably in antibiotic resistance.<br />

Furthermore, several proteins that exclusively occur in the mixture of the three<br />

species and may be involved in the interactions were identified.<br />

Finally, the integration of these results into mathematical models is an ongoing<br />

project to improve our un<strong>der</strong>standing of interactions in microbial communities<br />

and hopefully contribute to the optimisation of the therapy of lung infections<br />

related to cystic fibrosis.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PN 85<br />

Investigation of Lactobacilli variability by MALDI-TOF<br />

MS profiling<br />

A. Borovskaya 1 , E. Ilina 1 , V. Govorun 1 , A. Letarov 2 , A. Isaeva 2 , T. Maier 3 , M.<br />

Kostrzewa *3<br />

1 Research Institute for Physical-Chemical Medicine, Moscow, Moscow, Russia<br />

2 Institute for Microbiology RAS, Moscow, Moscow, Russia<br />

3 Abteilung Bioanalytik, Bruker Daltonik GmbH, Bremen, Germany<br />

Lactobacilli are the dominant bacteria of healthy women’s vagina. The growing<br />

interest in using them as probiotics against urogenital tract infections requires<br />

the description of their species variability for healthy women and for women<br />

with bacterial vaginosis. Speciation of Lactobacilli sometimes is difficult, and<br />

simple and rapid methods are lacking. The goal of this study is to demonstrate<br />

the applicability of MALDI-TOF profiling for species identification of<br />

Lactobacilli, both for epidemiological and applied microbiology purposes.<br />

24 clinical isolates of Lactobacilli from 14 patients were studied by MALDI-<br />

TOF MS. Bacterial colonies were picked from MRS selective agar and<br />

transferred into 300 µl of water. After precipitation with ethanol (900 µl) and<br />

centrifugation the pellet was suspended in 20 µL of 50% acetonitrile, 35%<br />

formic acid, and analyzed in a microflexTM (Bruker Daltonics, Germany).<br />

Species identification was done using the MALDI Biotyper 2.0 software<br />

(Bruker Daltonics, Germany). In addition, 16S rRNA genes sequencing was<br />

performed for all samples.<br />

Of 24 isolates investigated, 18 were reliably identified as different species from<br />

genus Lactobacillus (log(score) ≥ 2.0) – L.fermentum (n=3), L.gasseri (n=2),<br />

L.crispatus (n=10), L.delbrueckii (n=3). The remaining five isolates were<br />

identified as L.fermentum (n=3) and L.jensenii (n=2) with a non-reliable<br />

log(score) < 2.0. One isolate was identified as Bacillus coagulans. All results<br />

were confirmed by 16S rRNA gene sequencing. Moreover, according to<br />

BLAST search the average identity value was higher for the first 18 isolates<br />

(98.1%) than for the other five (95.8%), reflecting the natural variability of<br />

Lactobacilli also detected by MALDI-TOF profiling.<br />

PN 86<br />

Community comparison of clogging related bacteria in<br />

Berlin water wells<br />

O. Thronicker *1 , M. Popiol 1 , U. Szewzyk 1<br />

1 Department of Environmental Microbiology, TU Berlin, Berlin, Germany<br />

Well biofouling is a complex and yet not sufficiently un<strong>der</strong>stood process. Water<br />

wells represent a unique habitat, since they create a link between the anaerobic<br />

ground water, containing Fe(II) and the aerobic surface. This groundwater is<br />

rich in soluble Fe(II) and the presence of trace amounts of free oxygen in the<br />

well screens sets ideal conditions for the growth of iron bacteria. Their<br />

ochreous deposits not only block the filter area, but also the adjacent gravel<br />

pack or even parts of the aquifer and result in a steady decrease of well<br />

performance. In this project the bacterial communities of several Berlin wells<br />

have been compared using standard microscopic techniques and molecular<br />

techniques like DGGE.<br />

By phylogenetic comparisons of different wells in Berlin and also of different<br />

parts of a well itself, we found some similarities in the composition of the<br />

bacteria community structures. We also found some bands that are present in<br />

nearly every well. Further work is <strong>bei</strong>ng un<strong>der</strong>taken to determine those bacteria<br />

which are most relevant for the clogging process.<br />

Acknowledgements:<br />

The project WellMa is coordinated by the KWB and financed by the Berliner<br />

Wasserbetriebe (BWB) and Veolia<br />

PN 87<br />

Hydrostatic pressure effects on growth of marine bacteria<br />

H.P. Grossart *1 , G. Gust 2<br />

1<br />

Limnology of Stratified Lakes, Leibniz-Institute of Freshwater Ecology and<br />

Inland Fisheries, Stechlin, Germany<br />

2<br />

Institut für Meerestechnik, TU Hamburg-Harburg, Hamburg-Harburg,<br />

Germany<br />

In a purely differential experiment the response of 5 strains of shallow-water<br />

microbes is explored to changing hydrostatic pressure simulating a sinking at<br />

1000m/d from surface waters to 4000 m depth in an isothermal ocean.<br />

Technology and methods to execute this task are a combination of a new<br />

pressure laboratory with computer control to simulate, among others, oceanic<br />

settings along typical depth curves of T-S-diagrams, together with classical and<br />

new genetic tools to evaluate the species responses of size, number,<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

consumption, activities and species shift in a time series pattern and regular,<br />

non-saturating feeding.<br />

Al<strong>bei</strong>t our results the initial step in a quest for quantifying particularly the role<br />

of hydrostatic pressure in oceanic microbial distributions observed, the results<br />

indicate that the species selected respond differently to pressure. Common is a<br />

strong physiological response leading to reduced bacterial numbers of all<br />

selected strains at 4000 m depth. Pressure changes (during sinking) with<br />

maxima and minima in number and activity of the selected strains are not seen<br />

in the ambient pressure control and a reshuffling of species in the subsequent<br />

steady-state mode at 4000 m depth take place over a three-day span. Likewise,<br />

between control and pressure, the activities and sizes of all tested strains differ<br />

resulting in great changes in species distribution. The effects indicate that<br />

sampling and laboratory protocols have to be carefully explored where pressure<br />

effects have to be incorporated, using autoclaved equipment, and where not.<br />

Furthermore, time series experiments with non-intrusive sampling features<br />

rather than end-member experiments become mandatory to reveal physiological<br />

responses.<br />

PN 88<br />

Bacterial communities of drinking water system (collecting<br />

and distributing network) of Budapest (Hungary)<br />

E. M Tóth *1 , Z.G. Homonnay 1 , G. Török 1 , K. Márialigeti 1<br />

1<br />

Department of Microbiology, Eötvös Loránd University of Science, Budapest,<br />

Hungary<br />

Drinking water of Budapest originates from riverbank filtration wells. Wells<br />

supply so clean water that thereafter only chlorination is needed before<br />

distributing it to the consumers. In the present work bacterial communities of<br />

the drinking water network were investigated. Water samples were taken from<br />

3 different bank filtration wells, 3 points of the collection network, 3 pump<br />

stations of the distribution network (containing chlorinated water) and the<br />

central pump station. Water samples were taken 6 times at all sampling sites<br />

from March 2007 to October 2007. Bacterial cell count and CFU values of each<br />

sample were determined by fluorescent microscopy, by classical standard<br />

methods and even by using oligotrophic media. DNA was isolated from 10 – 14<br />

litres of water in case of each sample. After partial amplification of 16S rRNA<br />

gene T-RFLP analysis was performed, and samples were grouped by statistical<br />

analyses based on the number and relative quantity of TRFs. 16S rDNA clone<br />

libraries were constructed, TRF lengths and sequences of the clones were<br />

determined to identify the peaks in community TRFLP-profile. Chlorination<br />

usually decreased the direct cell counts and CFU numbers of the samples by<br />

102 thereshold. Unchlorinated water in the wells and in the collecting system<br />

was charaterised by diverse communities, dominated by oligo-heterotroph<br />

(Sphingomonas spp., Bradyrhizobium spp.) and chemolitotroph bacteria<br />

(Gallionella sp. Nitrospira sp.). Diversity of chlorinated water samples was<br />

lower, and dominance of non-tuberculous Mycobacterium species and<br />

Methylocella sp. could be detected. This divergence between chlorinated and<br />

non-chlorinated samples could be observed during all sampling period. Some<br />

seasonal differences also appeared: in the middle of summer community of<br />

chlorinated water samples has changed, diversity increased, which may be<br />

explained by hot weather and high water temperature.<br />

PO 01<br />

Genetic diversity of Stenotrophomonas maltophilia<br />

M. Adamek *1 , J. Winter 1 , S. Bathe 1<br />

1 Institute of Biology for Engineers and Wastewater Biotechnology, University<br />

of Karlsruhe, Karlsruhe, Germany<br />

141<br />

Stenotrophomonas maltophilia is a γ-β proteobacterium that is ubiquitously<br />

distributed in the environment. The species contains strains that act as<br />

nosocomial pathogens causing infections among immunocompromised patients.<br />

S. maltophilia strains display a high genetic diversity, which so far has<br />

primarily been evaluated for clinical strains. The possible appearance of<br />

sublineages and ways to differentiate them should be investigated further.<br />

In the present study, 180 S. maltophilia strains of clinical and environmental<br />

origin have been investigated by rep-PCR using the primers BoxA1R and<br />

(GTG)5. Furthermore, isolates falling into a distinct 16S rRNA clade containing<br />

purely environmental strains and sequences (group E2, Minkwitz and Berg<br />

2001 [1]) were identified by a specific PCR assay.<br />

The fingerprints revealed a high diversity, but showed also some defined<br />

groups, one containing clinical respiratory isolates as well as environmental<br />

isolates, and another containing the more diverse clade of group E2 strains.<br />

A subsample of the strain collection was further analyzed by partial gyrB gene<br />

sequencing (approx. 500 bp). Most S. maltophilia strains fell within a cluster<br />

containing several subgroups, but distant from the other Stenotrophomonas<br />

species. Remarkably, closest neighbours of group E2 strains were strains of S.

142<br />

rhizophila. This finding is in discordance with the 16S rRNA gene<br />

relationships, where E2 strains cluster together with the other S. maltophilia<br />

strains, well separate from S. rhizophila.<br />

Additional work is un<strong>der</strong>way to clarify the taxonomic status of group E2 strains<br />

and to compare genetic relationships with phenotypic characteristics.<br />

[1] Minkwitz A, Berg G. 2001. Comparison of antifungal activities and 16S<br />

ribosomal DNA sequences of clinical and environmental isolates of<br />

Stenotrophomonas maltophilia. J Clin Microbiol 39:139-45.<br />

PO 02<br />

In-situ measurements and transcriptome analysis of<br />

atmospheric methane oxidizers in desert soils<br />

R. Angel *1 , R. Conrad 1<br />

1 Max-Planck-Institute for Terrestrial Microbiology, Philipps-Universität<br />

Marburg, Marburg, Germany<br />

Upland soils are a biological sink for atmospheric methane; however, the<br />

activity of desert soils and the presence of methanotrophs in these soils have<br />

hardly been studied. We studied on-site atmospheric methane consumption<br />

rates as well as the diversity and expression of the pmoA gene, coding for a<br />

subunit of the particulate methane monooxygenaze, in arid and hyper-arid soils<br />

in the Negev Desert, Israel. Methane uptake was only detected in undisturbed<br />

soils in the arid zone (~80 mm yr-1) at rates ranging from 1.2 (un<strong>der</strong> shrub) to<br />

1.9 mg CH4 m-2 day-1 (open field). Vertical methane profiles in soil showed<br />

the active layer to be 0-30 cm. No methane uptake was detected in the hyperarid<br />

soils (~20 mm yr-1) as well as in disturbed soils in the arid zone (i.e<br />

agricultural field and a mini-catchment). Molecular analysis of the<br />

methanotrophic community using T-RFLP and cloning of the pmoA gene<br />

detected methanotrophs in the active soils, whereas the inactive ones were<br />

dominated by sequences of the homologous gene amoA, coding for a subunit of<br />

the ammonia monooxygenaze. Even in the active soils, methanotrophs (as well<br />

as in-situ activity) could not be detected in the soil crust – the biologically most<br />

important layer in desert soils. All pmoA sequences belonged to yet uncultured<br />

strains, two of which closely related to type II methanotrophs and one to a yet<br />

unclassified cluster. Transcriptome analysis showed dominance of sequences<br />

clustering within the JR3 but also members of USCγ. Our results show that<br />

although active methanotrophs are prevalent in arid soils they seem to be absent<br />

or inactive in hyper-arid and disturbed arid soils.<br />

PO 03<br />

Diversity of the strains of Streptomyces causing potato scab<br />

disease from Iran<br />

G. Khodakaramian *1 , H. Kinashi 2 , K. Arakawa 2<br />

1<br />

Department of Plant Protection, College of Agriculture, Bu-Ali Sina<br />

University, Hamedan, Germany<br />

2<br />

Department of Molecular Biotechnology, Hiroshima University, Higashi-<br />

Hiroshima, Japan<br />

Number of Streptomyces species pathogenic on potato are increasing. Main<br />

pathogenicity factor of this gram positive soil habitat bacteria is thaxtomin but<br />

concanamycin and FD-891 also reported as pathogenicity compounds.<br />

Pathogenic streptomyces strains from main potato growing area in Iran are<br />

diverse and were belong to different groups based on their phenotypic,<br />

genotypic and virulence factors. At lease four groups of the strains were<br />

recognized based on their types of symptoms on potato tubers and phenotypic<br />

charactristics. They induced raised, deep pitted and netted lesion on potato<br />

tubers. Some strains produced thaxtomin but some did not produced<br />

thaxthomin and also did not hybridized to thaxtomin biosynthetic gene probe<br />

txtA. Based on the primer designed for nec1 gene they showed same pattern but<br />

they showed different pattern for other pathogenicity genes. A non-thaxtomin<br />

producing representative strain which induced deep-pitted lesion on potato<br />

tuber was used for isolation of new pathogenicity compound. we found a new<br />

fraction and compound as a pathogenicity factor. This factor showed growth<br />

inhibitory activity against all examined plants.<br />

PO 04<br />

Quantification of Bacterial Communities in Environmental<br />

Samples with High Content of Fibrous Plant Material by<br />

Molecular FISH – Technique.<br />

L. Neumann *1 , P. Scherer 1<br />

1 Research and Transfer Center for Renewable Energy and Process Efficiency,<br />

Hamburg University of Applied Sciences, Hamburg, Germany<br />

The heterogeneity of environmental samples as well as its chemical<br />

components often affect an optical analytical method, e.g., bacteria stick in the<br />

matrix. Organic matter from biogas plants fed with material from renewable<br />

energy plants, show extreme conditions. Total cell counts around 10 11 cells/ml,<br />

high amount of fibrous plant material, bacterial aggregates and a high content<br />

of humic substances can often be found. Therefore, at present no procedure is<br />

described to quantify bacteria by fluorescence in situ hybridization (FISH) in<br />

such samples. In this work a quantitative analytical method with a special<br />

dispersing system and an enlarged wash step as pretreatment for FISH was<br />

developed. To exemplify this adapted and improved method, material from<br />

three different biogas reactors were tested, one mesophilic and thermopilic<br />

reactor fed with a mixture of sugar beet mash and a mesophilic reactor fed with<br />

fod<strong>der</strong> beed silage. All reactors were processed automatically un<strong>der</strong> with a high<br />

loading rate. For the quantitative FISH-technique a probe set for the major<br />

methanogenic genera (Methanobacteriales, Methanomicrobiales,<br />

Methanosarcina, Methanisaeta and Methanococcales) was used. The efficiency<br />

of the hybridization was determined with probes for the domain archaea<br />

(ARCH915), bacteria (EUB338[I-III]) and universal (UNIV1390) in<br />

comparison to the total cell count with SYBR-Green I. The main aim of this<br />

study was the maximization of the probe efficiency in the reactor material while<br />

minimizing the total cell loss by the homogenization step and the reduction of<br />

matrix substances influences. With this new protocol it was possible to create a<br />

fast, semi-automatic and quantitative population screening for biogas plants<br />

working with renewable biomass.<br />

PO 05<br />

Diversity of PHB-producing bacteria associated with crop<br />

plants<br />

I. Gasser *1 , H. Müller 1 , G. Berg 1<br />

1 Institute for Environmental Biotechnology, TU Graz, Austria, Graz, Austria<br />

Among terrestrial ecosystems, plant-associated habitats represent<br />

microenvironments with high microbial activity. Especially in the rhizosphere,<br />

according to root exudation, plants are densely colonised by specific bacterial<br />

and fungal communities. Along with the growth of the roots, the biotic and<br />

abiotic conditions dynamically change. Indigenous microorganisms must be<br />

adapted to the changing conditions of their environment and to fluctuations in<br />

the concentration of nutrients exuded by plant roots. Bacteria are known to<br />

produce Polyhydroxybutyrate (PHB) as a storage substance to resist detrimental<br />

periods.<br />

In the present study, rhizosphere-associated bacteria from oilseed rape, wheat<br />

and sugar beet were tested on their ability to produce PHBs by applying a<br />

multiphasic approach. By the cultivation-dependent approach, bacterial isolates<br />

originated from different plants were screened on their ability to form PHBs in<br />

vitro as well as on the presence of PHB synthase genes using polymerase chain<br />

reaction (PCR). To get an overview about the diversity of PHB-producing<br />

bacteria, total DNA extracts from rhizosphere samples were analysed for the<br />

occurrence of the PHB synthesis gene phaC by Single Strand Conformation<br />

Polymorphism analysis (SSCP). SSCP profiles of phaC revealed a high<br />

specificity for each plant species. Comparing the results obtained by single<br />

colony PCR, sugar beet was found to be associated with a higher number (90%)<br />

of PHB-producing bacteria than oilseed rape (32%) and wheat (31%).<br />

PO 06<br />

Microbial diversity as resource and tool for biological<br />

disease control in grapevine<br />

F. Schmid *1 , G. Berg 1<br />

1<br />

Institute of Environmental Biotechnology, Technical University Graz, Graz,<br />

Austria<br />

As the causative agent of grey mould disease in grapevine the ascomycetous<br />

fungus Botrytis cinerea PERS. leads to high losses in yield. In or<strong>der</strong> to control<br />

Botrytis traditionally, high amounts of botryticides or copper compounds are<br />

used in conventionally or organically treated vineyards respectively in or<strong>der</strong> to<br />

control Botrytis. Due to the emergence of resistances and to ecological reasons<br />

the interest in efficient and sustainable alternatives to traditional pesticides<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

ises. The intention of biological control is to use microorganisms with effects<br />

beneficial to plants that reduce the abundance and the damaging effects of<br />

pathogens on plants by several mechanisms.<br />

The aim of this study was to investigate the native microbial flora of<br />

organically and conventionally treated grapevine in or<strong>der</strong> to find differences<br />

between the two ways of treatment. Furthermore the abundance and the<br />

composition of antagonists towards B. cinerea on different parts of grapevine<br />

plants was examined, with the objective to find and characterise new<br />

antagonistic microorganisms. Microbiological and molecular methods, such as<br />

single strand conformation polymorphism analysis (SSCP) of the rRNA<br />

encoding genes were used to determine the microbial communities and to<br />

isolate and characterise the antagonistic organisms. Statistically significant<br />

differences between the two treatments were found in microorganism<br />

population size, population composition and the distribution of antagonists<br />

among different parts of the plant. Furthermore a high number of promising<br />

new antagonistic strains against B. cinerea were isolated.<br />

PO 07<br />

Acidobacteria diversity changes and their functional<br />

interrelation to land use<br />

A. Näther *1 , V. Nägele 2 , J. Overmann 2 , M.W. Friedrich 1<br />

1<br />

Fachbereich 2 Biologie & Chemie, Universität Bremen, Bremen, Germany<br />

2<br />

Biozentrum, Department Biologie I, Mikrobiologie, Ludwig-Maximilian-<br />

Universität München, Planegg-Martinsried, Germany<br />

Studies of the functional significance of prokaryotic diversity in soil have<br />

mainly been limited to a few groups of Proteobacteria implicated in N-cycling<br />

and to rhizosphere bacteria. Yet, Acidobacteria represent a second abundant<br />

bacterial phylum in soil. They can account for 50 to 80% of all soil bacteria and<br />

are physiologically active in situ. However, the functional implication of the<br />

diversity of this major bacterial group and its coupling to land use is unknown.<br />

Using a land-use gradient as the major variable, we investigate the<br />

composition, physiological key traits and the functional implications of<br />

Acidobacteria diversity in soil. RNA was extracted from soil of 9 grassland and<br />

9 forest plots of each of the three DFG Biodiversity Exploratories (Biosphere<br />

Reserve Schorfheide-Chorin in Brandenburg, National Park Hainich with<br />

surrounding area in Thuringia and designated Biosphere Reserve Schwäbische<br />

Alb in Baden-Württemberg, Germany), respectively. In or<strong>der</strong> to compare all<br />

experimental plots sampled, 16S rRNA fingerprints of the whole Bacteria<br />

population and particularly Acidobacteria were generated by PCR using the<br />

specific primers Bact27f/Acido31f and 907r. Diversity changes and<br />

physiologically active types of Bacteria and Acidobacteria were monitored<br />

using T-RFLP analysis of 16S rRNA. To identify the dominant populations,<br />

16S rRNA clone libraries will be constructed and clones sequenced.<br />

PO 08<br />

Microbial role in the decomposition of outdoor pine wood<br />

chip piles<br />

M. Noll *1 , M. Malow 2 , F. Ferrero 2<br />

1<br />

BAM, Fe<strong>der</strong>al Institute for Materials Research and Testing, Division IV.12,<br />

12205, Germany<br />

2<br />

BAM, Fe<strong>der</strong>al Institute for Materials Reserach and Testing, Division II.22,<br />

Berlin, Germany<br />

Fast growing softwood species, i.e. pine, are preferred for biomass-based heat<br />

and/or electricity production and are stored in large-scale. To model exothermic<br />

processes within such piles, we monitored a newly established pine-wood<br />

debris pile (approx. 400 tons fresh weight, size 20 x 15 x 6 m) and analyzed the<br />

same material in a small-scale laboratory experiment. CO2-concentration<br />

increased rapidly within the first days of both storages. Simultaneously, we<br />

measured inside the outdoor pile a temperature increase up to 65°C and O2concentration<br />

decrease, which increased back to ambient value after one week.<br />

CO2-concentrations decreased in both experiments after one week and<br />

additional carbon dioxide peaks were observed after approx. 35 and 120 days.<br />

We assessed the structure and composition of the bacterial and fungal<br />

community by T-RFLP analyses of the SSU rRNA gene and of the fungal<br />

intragenic transcribed spacer, respectively. While the fungal community<br />

structure remained almost unchanged after one week of incubation, the<br />

bacterial community structure was characterized in continuous shifts over time.<br />

The early bacterial community was dominated by members of the genera<br />

Sphingomonas, Pseudomonas and Luteibacter, which are all known to be<br />

metabolic versatile in terms of their organic carbon degradation. The late stage<br />

was more diverse and comprised by members of the genera Acidocella,<br />

Nocardioides and the class of Acidobacteria and enterobacterial genera such as<br />

Enterobacter or Yersinia. Taken together, these observations suggest that the<br />

early bacterial diversity is a key player in exothermic processes; and this<br />

diversity was replaced by highly adapted microorganisms.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PO 09<br />

In situ abundance and seasonality of Verrucomicrobia in<br />

oxic and anoxic waters of the humic lake Große Fuchskuhle<br />

J. Arnds *1 , K. Knittel 1 , U. Buck 1 , R. Amann 1<br />

1 Molekulare Ökologie, Max-Planck-Institut für marine Mikrobiologie, Bremen,<br />

Germany<br />

Molecular tools have provided ecologists with many insights into the diversity<br />

of microbial communities in nature. Dominant microbial players have been<br />

identified in both terrestrial and aquatic habitats. However, some microbes were<br />

missed using these approaches due to limited coverage of conventional primers<br />

and probes. For example, the general bacterial probe EUB338 commonly used<br />

for fluorescence in situ hybridization (FISH) does not target the bacterial phyla<br />

Planctomycetes and Verrucomicrobia. As a matter of fact, quantitative data on<br />

the abundance of Verrucomicrobia in nature are still rare. Isolates are<br />

phylogenetically, morphologically, and physiologically diverse. They gain<br />

energy from different carbon compounds, e.g., various sugars or methane.<br />

Here, we report data on the abundance and distribution of verrucomicrobial<br />

populations in different water basins (northwest versus southeast) and layers<br />

(oxic versus anoxic) of the humic lake Grosse Fuchskuhle (Germany). Seasonal<br />

data were obtained by FISH with a set of newly developed 16S rRNA-targeted<br />

oligonucleotide probes. We could show that between 0.2% and 9% of all cells<br />

were Verrucomicrobia. In-depth analysis with subgroup-specific probes<br />

revealed the presence of (i) members of the Verrucomicrobia-Prosthecobacter<br />

group, (ii) the Opitutus group, and, most likely, (iii) Xiphinematobacter like<br />

species. The seasonal variability of the three subgroups will be discussed with<br />

respect to the well studied lake hydrogeology, biogeochemistry and microbial<br />

community.<br />

PO 10<br />

Genome analysis of Elusimicrobium minutum, the first<br />

cultivated representative of the Elusimicrobia phylum<br />

(formerly Termite Group 1)<br />

D.P.R. Herlemann *1 , O. Geissinger 1 , W. Ikeda-Ohtsubo 1 , V. Kunin 2 , U.G.<br />

Maier 3 , P. Hugenholtz 2 , A. Brune 1<br />

1 Biogeochemistry, Max Planck Institute for Terrestrial Microbiology, Marburg,<br />

Germany<br />

2 Microbial Ecology Program, DOE Joint Genome Institute, Walnut Creek,<br />

United States<br />

3 Biology, University of Marburg, Marburg, Germany<br />

143<br />

Insect intestinal tracts harbor several novel, deep-rooting lineages of hitherto<br />

uncultivated bacteria, whose biology is typically completely obscure. One<br />

lineage, the candidate phylum Termite group 1 (TG-1), is regularly encountered<br />

in termite guts but is present also in many other habitats. We recently isolated<br />

the first representative of this phylum from the gut of a humivorous scarab<br />

beetle larva. Elusimicrobium minutum is an obligately anaerobic<br />

ultramicrobacterium that grows heterotrophically on glucose and produces<br />

acetate, hydrogen, and ethanol as major products. Based on the complete<br />

genome sequence (1.6 Mbp), we reconstructed the organism’s metabolism. E.<br />

minutum has all genes required for uptake and fermentation of sugars via the<br />

Embden-Meyerhoff pathway, including several hydrogenases, and an unusual<br />

peptide degradation pathway comprising transamination reactions and leading<br />

to the formation of alanine, which is excreted in substantial amounts. The<br />

presence of genes encoding lipopolysaccharide biosynthesis is consistent with<br />

ultrastructural evidence of a Gram-negative cell envelope and the presence of a<br />

pathway for peptidoglycan formation. Even though electron micrographs<br />

showed no cell appendages, the genome encodes many genes putatively<br />

involved in pilus assembly. We assigned some to a type-II secretion system, but<br />

the function of almost 60 pilE-like genes remains to be investigated. Numerous<br />

genes with only hypothetical functions, e.g., polyketide synthesis, transport of<br />

antibiotics, and protection from oxygen stress, indicate the presence of hitherto<br />

undiscovered physiological traits. Comparative analysis of 22 concatenated<br />

single-copy marker genes un<strong>der</strong>scored the phylum-level division of the Termite<br />

Group 1, which was accordingly renamed to Elusimicrobia phyl. nov.

144<br />

PO 11<br />

Colonizing New Worlds: The (Dis)ability of Methanotrophs<br />

to Recover from Disturbances.<br />

A. Ho *1 , P. Frenzel 1<br />

1<br />

Max Planck Institute for Terrestrial Microbiology / Biogeochemistry, Marburg<br />

University, Marburg, Germany<br />

Biodiversity is claimed to be essential for ecosystem functioning. However,<br />

most experiments on biodiversity and ecosystem functioning (BEF) were made<br />

on higher plants, while only few studies have dealt with microbial<br />

communities. Overall microbial diversity may be very high, and general<br />

functions like aerobic carbon mineralization are assumed to be carried out by<br />

highly redundant communities. Therefore, studies should be directed towards<br />

more specialised functions. We focused on aerobic methane oxidation in a rice<br />

paddy, where >90% of potentially emitted methane may be oxidized in the oxic<br />

surface layer. This community is presumed to consist of 10-20 taxa more or less<br />

equivalent to species. We focused on the ability of methanotrophs to recover<br />

from a disturbance causing a significant die-off of all microbial populations.<br />

This was simulated by mixing native with sterile soil in two ratios (1:4 and<br />

1:40). Microcosms were incubated and the temporal shift of the methanotrophic<br />

communities was followed by pmoA-based Terminal Restriction Length<br />

Polymorphism (T-RFLP), qPCR, and a pmoA-based diagnostic microarray. We<br />

consistently observed distinctive temporal shifts between Type I<br />

(Methylococcacea) and Type II (Methylocystaceaea), a rapid population growth<br />

leading to the same or even higher cell numbers as in microcosms made from<br />

native soil alone, but no effect on the amount of methane oxidized. Similarly,<br />

the ratio of different methanotrophs changed with treatment, while the number<br />

of taxa stayed nearly the same. Overall, methanotrophs showed a remarkable<br />

resilience compensating for die-offs.<br />

PO 12<br />

Bacterial communities from mineral soils located on<br />

Livingston Island, South Shetland Islands, Antarctica<br />

L. Ganzert *1 , A. Lipski 2 , C. Pimpirev 3 , H.W. Hubberten 1 , D. Wagner 1<br />

1<br />

Periglacial Research, Alfred Wegener Institute for Polar and Marine<br />

Research, Potsdam, Germany<br />

2<br />

Department of Biology and Chemistry, University of Osnabrueck, Osnabrueck,<br />

Germany<br />

3<br />

Bulgarian Antarctic Institute, Sofia, Bulgaria<br />

Microorganisms can be found in very different cold soil environments playing a<br />

major role in nutrient cycling in these habitats. We studied the dominant<br />

bacterial composition from nine soil profiles located on Livingston Island,<br />

Antarctica. Two vegetated sites (moss-covered) and seven mineral soil sites<br />

were analysed. Total carbon (TC) and total nitrogen (TN) values were up to<br />

26.50 % and 0.84%, respectively, for vegetated soils decreasing with depth<br />

whereas values for mineral soils were

(desulfonating) bacteria in the Damma glacier forefield on the basis of the key<br />

gene asfA by terminal restriction fragment length polymorphism (T-RFLP)<br />

analysis and clone libraries. Diverse populations of desulfonating bacteria were<br />

identified in 8 and 70 year old ice-free forefield soils that differed significantly.<br />

Protein sequences of AsfA affiliated to Polaromonas and Acidovorax were<br />

predominantly found in the more recent ice-free soils and a group of<br />

unidentified sequences was found to be dominating in the matured soils.<br />

However, the desulfonating bacterial diversity was not affected by varying<br />

levels of sulfate concentrations in matured soil samples. In addition, we studied<br />

desulfonating bacterial communities in rhizospheres of pioneering plants<br />

colonizing both types of ice-free soils. Soil age had an effect on the<br />

desulfonating rhizophere community of Agrostris rupestris, but not of<br />

Leucanthemopsis alpina. Therefore, we conclude that the desulfonating<br />

community in bulk soil samples was predominantly affected by soil age, while<br />

the rhizosphere was governed by both soil age and plant. The level of asfA<br />

diversity in recently deglaciated soils suggests that desulfonating bacteria are a<br />

critical factor in sulfur cycling with defined groups dominating at different<br />

stages of soil formation.<br />

PO 16<br />

Taxonomy of myxobacteria – gliding from morphology to<br />

phylogeny<br />

E. Lang *1<br />

1 Mikrobiologie, DSMZ - Deutsche Sammlung von Mikroorganismen und<br />

Zellkulturen GmbH, Braunschweig, Germany<br />

Myxobacteria are an efficient source for novel bioactive compounds.<br />

Taxonomy of the myxobacteria had been developed almost exclusively on the<br />

basis of the morphology of colonies or swarms, vegetative cells, fruiting bodies<br />

and myxospores. A first survey of the 16S rRNA gene sequences revealed a<br />

striking correlation of morphological characteristics and phylogenetic grouping.<br />

Meanwhile, salt dependent marine isolates and anaerobic relatives were isolated<br />

forming new phylogenic lineages. H. Reichenbach described new taxa in<br />

Bergey’s Manual. However, not all of those have been accepted as Names with<br />

Standing in Nomenclature, and the 16S rRNA gene sequences were not<br />

determined for all of the strains proposed as type strains. For several species,<br />

herbarium specimen or strains which are no more available to the scientific<br />

community are currently set as type material. On the other hand, sequencing of<br />

the genomes of eight different myxobacteria is ongoing or has been completed<br />

already. A schedular survey of the current state of the taxonomy of the<br />

Myxococcales and future tasks will be presented.<br />

PO 17<br />

Choosing appropriate substrate and salt concentrations in<br />

biochemical testing systems for water isolates<br />

E. Lang *1 , I. Brandes 1<br />

1 Mikrobiologie, DSMZ - Deutsche Sammlung von Mikroorganismen und<br />

Zellkulturen GmbH, Braunschweig, Germany<br />

Physiological taxonomic test systems have been developed mainly for strains<br />

isolated from clinical samples. It was assumed that the cells are adapted to the<br />

patient habitats relatively rich in organic substances. According to the salt<br />

content of the body fluids, suspensions of cells in 0.8% NaCl solution is<br />

routinely used for inoculation of the testing systems. This is applied<br />

successfully to most environmental isolates, too. When studying a strain<br />

isolated from pure water we did not obtain any positive answer in routine<br />

systems testing for substrate utilization. This result was in accord with the<br />

passiveness described for the nearest phylogenetic neighbour of the strain, also<br />

isolated from drinking water. However, after decreasing the salt content of the<br />

suspension fluid and of the mineral basis medium of the testing systems, good<br />

growth was observed on several substrates. The influence of the substrate and<br />

salt concentration on the test result will be presented.<br />

It will be helpful to consi<strong>der</strong> a possible „halophobicity“ for isolates from sites<br />

with very low salt content but this finding will not assure an answer to<br />

problems in all cases. For ultramicrobacterial strains isolated from limnic sites<br />

and belonging to the genus Polynucleobacter, utilization of defined single<br />

substrates could only be demonstrated in a medium containing, in addition, low<br />

amounts of nutrient broth, yeast extract and peptone (Hahn et al. Emended<br />

description of the genus Polynucleobacter .....Int J System Evolut Microbiol,in<br />

press).<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PO 18<br />

Molecular abundance and diversity of bacterial alkane<br />

monooygenase genes (alkB) in a forest area historically<br />

contaminated with mineral oil hydrocarbons<br />

A. Pérez-de-Mora *1 , S. Schulz 1 , M. Schloter 1<br />

1 Institute of Soil Ecology/Dept. of Terrestrial Ecogenetics, HelmholtzZentrum<br />

münchen, Neuherberg, Germany<br />

Alyphatic n-alkanes are major contaminants of soil ecosystems as a result of<br />

uncontrolled oil spills and negligent disposal of wastes. Precise ecological risk<br />

assessment and remediation of affected sites is often very limited due to little<br />

un<strong>der</strong>standing of abiotic-biotic interactions occurring in situ between<br />

contaminants and biological targets. To gain a better insight into the ecology of<br />

hydrocarbon degradation at contaminated sites we investigated the abundance<br />

and diversity of the bacterial alkB gene coding for rubredoxin dependent alkane<br />

monooxygenase in a forest area contaminated with hydrocarbons for more than<br />

60 years. This enzyme is responsible for the first step (activation) of alkane<br />

degradation un<strong>der</strong> aerobic conditions. We developed a real-time PCR approach<br />

based on a degenerative primer set to quantify the abundance of the alkB gene<br />

in soil samples without obvious discrimination of any known bacterial group<br />

carrying this gene. In addition, a fingerprinting method based on tRFLPs<br />

(terminal restriction length polymorphisms) was developed to assess the<br />

diversity of alkB in environmental samples. Analysis of tRFLPs fingerprints<br />

using multivariate statistics allowed further selection of specific samples to<br />

construct clone libraries. This approach enables a culture-independent<br />

screening of the alkB gene in the environment, which can be used to assess the<br />

intrinsic natural attenuation potential of a site or to follow up on-going progress<br />

of bioremediation assays.<br />

PO 19<br />

Characterization of nitrite oxidizing bacteria of the genus<br />

Nitrospira in complex ecosystems by means of fatty acid<br />

profiles in combination with 16S- rRNA cloning<br />

M. Kruse *1 , E. Spieck 2 , A. Lipski 3 , E. Bakker 1<br />

1 Mikrobiologie, Universität Osnabrück, Osnabrück, Germany<br />

2 Mikrobiologie, Universität Hamburg, Hamburg, Germany<br />

3 Mikrobiologie, MicroPro GmbH, Gommern, Germany<br />

Nitrification is a two step process and includes the biological oxidation of<br />

ammonia to nitrite followed by the oxidation of nitrite to nitrate. Most of the<br />

nitrifying bacteria are obligate chemolithoautotrophic organisms. For the<br />

biosynthesis of organic carbon and for growth they <strong>der</strong>ive energy from the<br />

oxidation of ammonium or nitrite. The genus Nitrospira is only distantly related<br />

to other bacteria, including all known nitrite oxidizing bacteria, hence it is<br />

classified as a new phylum Nitrospira [1].<br />

Chemotaxonomic and molecular methods have shown that Nitrospira is present<br />

and active in different habitats. Nitrospira contains up to three for the phylum<br />

characteristic major fatty acids, the cis 7 and cis 11 isomers of hexadecenoic<br />

acid and 11-methyl-hexadecanoic acid [2]. Different Nitrospira species carry<br />

different ratios of these three fatty acids. Labeling experiments with 13 C-labeled<br />

carbonate as a substrate indicated the presence of a metabolically-active<br />

autotrophic bacteria community in activated sludge from waste water treatment<br />

plant and biofilter material from aqua culturing plants. The specific major fatty<br />

acids cis 7 and cis 11 isomers of hexadecenoic acid were present and labeled in<br />

these habitats, indicating the presence of active Nitrospira. The ratio of these<br />

acids suggested the presence of hitherto unknown Nitrospira species. For the<br />

molecular identification of these species the 16S rRNA occurring in these<br />

habitats was cloned with specific primers.<br />

[1] Spieck et al. (2006), Environ. Microbiol. 8, 405-415.<br />

[2] Lipski et al. (2001), System. Appl. Microbiol. 24, 377-384.<br />

PO 20<br />

Biogeography and genetic diversity of phyllosphere pinkpigmented<br />

facultative methylotrophs<br />

S.A. Wellner *1 , N. Lod<strong>der</strong>s 1 , P. Kämpfer 1<br />

1<br />

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Gießen,<br />

Gießen, Germany<br />

145<br />

Phyllosphere methylotrophic bacteria are Gram-negative organisms, which<br />

inhabit plant surfaces, mainly the leaves. They use compounds such as methane<br />

or methanol as their sole carbon and energy source and play an important role<br />

in the methanol cycle by utilizing the methanol emitted by plants. On the other

146<br />

hand, they can produce plant growth promoting substances like auxins,<br />

cytokinins, and vitamin B12.<br />

The objective of this project is to analyse the genetic diversity, biogeography,<br />

and functional role of phyllosphere methylotrophs in dependence of inhabited<br />

plant species, land-use intensity and geographic location of the sampling sites.<br />

Cultivation of pink-pigmented facultative methylotrophs (PPFM) was<br />

performed after isolation from leaf samples of two different plants, Trifolium<br />

repens and Cerastium holosteoides. For each plant, samples were taken from<br />

sites with five different land-use intensities. From 16 representative isolates the<br />

16S rRNA gene sequences were analysed and phylogenetic analyses were<br />

performed using the neighbour-joining method.<br />

The concentration of PPFM per g leaf was significantly higher for Trifolium<br />

repens (2.7x10 7 CFU / g leave) than for Cerastium holosteoides (1.5x10 6 CFU /<br />

g leave). Land-use intensity had no significant influence on the concentration of<br />

PPFM on leaf surfaces. Analysis of the 16S rRNA gene sequences confirmed<br />

that all isolates belong to the genus Methylobacterium with similarities between<br />

97.3 and 100% to described species.<br />

The intraspecies diversity will be studied in the near future by sequencing two<br />

housekeeping genes (mxaF and fhcD) of selected strains. Additionally, the<br />

diversity of methylotrophs will be further analysed by cultivation-independent<br />

methods (e.g. SSCP).<br />

PO 21<br />

Bacterial diversity in arsenic contaminated overburden<br />

soils and the corresponding genes of arsenite oxidases<br />

M. Sultana *1 , S. Vogler 1 , J. Seifert 1 , M. Schlömann 1<br />

1 Institute of Bioscience, TU Bergakademie Freiberg, Freiberg, Germany<br />

Cultivation and molecular methods were applied to investigate the microbial<br />

community in highly arsenic contaminated abandoned mine area at Rauchblöße<br />

in Freiberg, Germany. Soil samples, collected at four different points of arsenic<br />

load, were subjected to enrichment using heterotrophic and autotrophic mineral<br />

media containing arsenite.<br />

In parallel, total community DNA was extracted and bacterial 16S rDNA genes<br />

and genes for the arsenite oxidase (AoxB) were amplified by PCR. A 16S<br />

rDNA and an AoxB clone library wer created from a sample with an original<br />

arsenic concentration of about 2700 mg/kg un<strong>der</strong> autotrophic enriched<br />

condition. The AoxB sequences obtained by clone studies showed a novel<br />

branch of an AoxB cluster beyond the already published genes. The various<br />

16S rDNA clones containing bacterial sequences were used to designate<br />

terminal restriction fragments (T-RFs). Correspondence analysis of T-RFLP<br />

patterns in all soil samples showed diverse bacterial communities in arsenic<br />

contaminated overburden soils and also a discrete change in bacterial<br />

community was visible in original and enriched samples at different time<br />

points.<br />

From enrichments, various heterotrophic bacteria with the capability of arsenite<br />

resistance and transformation were isolated and representative bacteria were<br />

analysed phylogenetically. The PCR based 16S rDNA analysis revealed that the<br />

isolates belong to alpha and beta proteobacterial group closely related to Bosea,<br />

Rhizobium, Burkhol<strong>der</strong>ia and Alcaligenes. The AoxB phylogeny generates a<br />

novel taxonomic group of arsenite oxidase for isolates closely related to<br />

Burkhol<strong>der</strong>ia. Hence, detailed analysis of arsenite oxidase enzyme of the<br />

isolates would add a new diversity within the arsenite oxidase family.<br />

PO 22<br />

Morphological Studies on Thiomargarita spp.<br />

V. Salman *1 , H. Schulz-Vogt 1<br />

1 Mikrobiologie, Max Planck Institut für Marine Mikrobiolgie, Bremen,<br />

Germany<br />

Thiomargarita namibiensis, until today the largest bacterium measuring up to<br />

750 µm in diameter, is a spherical, chain-forming facultative aerobic sulfideoxidizer.<br />

Storage of sulfur globules and the production of a robust mucus<br />

sheath are typical. Up to 98 % of the biovolume is composed of a central<br />

vacuole filled with nitrate for anaerobic respiration.<br />

Several undescribed morphotypes were discovered in sediment samples taken<br />

in 2004 and 2008 from the coast off Namibia. Cell compositions and inner<br />

structures of the different morphotypes were investigated by fluorescent<br />

staining using confocal laser scanning electron microscopy (CLSM),<br />

fluorescence in situ hybridization (FISH) and scanning electron microscopy<br />

(SEM), revealing a similar cell structure as known for T. namibiensis cells.<br />

Besides binary fission of the main morphotype we discovered smaller single<br />

cells dividing in three planes forming clusters of up to eight cells. They strongly<br />

resemble features commonly known from eukaryotic cell division. Also, even<br />

smaller cells densely packed with sulfur globules and held together by a<br />

common envelope were discovered. They have similarities with some of the<br />

fossils from the Doushantuo Formation interpreted as the first eukaryotic<br />

embryos.<br />

After obtaining the complete sequence of the 16S rRNA gene of Thiomargarita<br />

namibiensis we now want to get a hold of the according sequences of single<br />

chains belonging to the different morphotypes. This phylogenetic information<br />

will show in more detail the relationship of the cells showing abberant cell<br />

shape, size and the ability to produce different kinds of mucus.<br />

PO 23<br />

Nitrospira – the main nitrite oxidizing bacteria in marine<br />

recirculation aquaculture systems<br />

S. Keuter *1 , M. Alawi 2 , E. Spieck 1<br />

1 Mikrobiologie, Uni Hamburg, Hamburg, Germany<br />

2 Abteilung Bio-Geoengineering, Helmholtz-Zentrum Potsdam - Deutsches<br />

Geoforschungszentrum, Potsdam, Germany<br />

Beginning in 1998, several authors identified representatives of the genus<br />

Nitrospira to play the major role in the important step of nitrite oxidation in<br />

biofilters of marine recirculation aquaculture systems (RAS). We investigated<br />

nitrifying bacteria from biofilter material (recycled high density polyethylene)<br />

of two RAS located at the North Sea and the Baltic Sea. In the marine RAS<br />

turbot and sea bass were cultured at 17°C, and shrimps were grown at 28°C in<br />

the brackish RAS. Using electron microscopy (EM) we could observe dense<br />

colonies of diverse ammonia oxidizing bacteria and nitrite oxidizing bacteria<br />

(NOB) of the genus Nitrospira in close vicinity within the thick biofilm on the<br />

filter material. In the samples obtained from the marine RAS we could<br />

discriminate two phenotypes of Nitrospira. 16S rRNA gene analyses of NOB<br />

originating from these samples revealed two new strains with different<br />

relationships to Nitrospira marina, the only described marine member of the<br />

genus Nitrospira. Physiological tests with a highly enriched culture of one of<br />

these new strains revealed elevated tolerances against nitrite, nitrate and<br />

ammonia concentrations as well as higher activities than N. marina. The culture<br />

exposed strong capability to develop biofilms with a high affinity to glass.<br />

Thus, the enriched Nitrospira-like strain seems to be well adapted to the<br />

attached lifestyle on biofilter material as well as to the high nitrogenous load<br />

prevailing in the effluents of the fish tanks of RAS.<br />

PO 24<br />

Quantitative determination of the non-cultured bacterial<br />

flora of ground water with fluorescent probes<br />

A. Ultee 1 , H. König *1<br />

1 Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg<br />

Universität Mainz, Mainz, Germany<br />

Ground water is despite its low nutrient content a habitat for different bacteria.<br />

Although bacterial counts are low compared to surface water, a transformation<br />

and degradation of different compounds in ground water is expected, leading to<br />

sensoric problems of the final drinking water. The present study describes the<br />

isolation and identification of different bacteria (culturable and non-culturable)<br />

in ground water of waterwork Hof Schönau in the neighbourhood of Mainz<br />

(Germany). The cultured species (1-5% of the total counts) belonged to the<br />

Actinobacteridae, Fermicutes, Bacteroidetes, α-, β- and γ-Proteobacteria. The<br />

other 95-99%, which could not be cultivated on the media used, belonged to the<br />

same group as the cultured species, however, δ- and ε-Proteobacteria were also<br />

identified. Specific 16S rRNA probes were designed for 19 non-cultured<br />

species and, depending on their binding site, also some non-specific “helperoligonucleotides”<br />

were constructed. With the aid of fluorescence in situ<br />

hybridisation, the titre of these 19 species was determined in spring and autumn<br />

as percentage of the total count. 90-92 % of the bacteria were quantified with<br />

the specific probes. The highest counts were observed for Methylophilus<br />

methylotrophus (7-20%), Ferribacterium limneticum (10-12%), Oxalobacter<br />

sp. (7-8%), Desulfovibrio sp. (5-8%) and strain K-S10-Sep02 (6-7%) at both<br />

sampling times.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PO 25<br />

Sulphate-reducing bacteria in hydrocarbon-rich sediments<br />

from the Gulf of Mexico<br />

S. Kleindienst *1 , B. Orcutt 2 , T. Treude 3 , A. Boetius 4 , R. Amann 1 , K. Knittel 1<br />

1<br />

Molecular Ecology, Max Planck Institute for Marine Microbiology, Bremen,<br />

Germany<br />

2<br />

Department of Marine Sciences, University of Georgia, Georgia, Germany<br />

3<br />

Department of Marine Biogeochemistry/Marine Geosystems, Leibniz Institute<br />

of Marine Sciences, Kiel, Germany<br />

4<br />

Department Geosystem, Alfred Wegener Institute for Polar and Marine<br />

Research, Bremerhaven, Germany<br />

Sulphate-reduction (SR) is the dominant process for organic matter<br />

mineralization in ocean margin sediments, accounting for up to 50% of CO2<br />

production. Hot spots of SR occur in cold seep sediments, where methane,<br />

short-chain alkanes or oily components dominate. In methane seeps SR is<br />

tightly coupled to the anaerobic oxidation of methane (AOM) whereas in<br />

sediments with seepage of higher hydrocarbons in addition to methane, SR gets<br />

decoupled from AOM. At these sites, methane-dependent SR drops to less than<br />

10% of total SR rates. The key sulphate-reducing bacteria (SRB) have neither<br />

been identified nor quantified yet. Recently, first SRB using propane and<br />

butane as sole growth substrate could be isolated (Kniemeyer, 2007. Nature<br />

449: 898-901).<br />

We have investigated different hydrocarbon-rich seep sediments from the Gulf<br />

of Mexico using geochemical and molecular techniques to determine which<br />

microbes dominated each system. Integrated SR rates ranged between 0.04 and<br />

10.8 mmol m -2 d -1 . Diversity of deltaproteobacterial SRB was high, especially<br />

at oily sites. Therefore, these bacteria were studied by CARD-FISH using<br />

probes for Deltaproteobacteria and additional newly developed probes for seepspecific<br />

clusters (SRB-SEEP1-4). SRB were highly abundant with up to 15% of<br />

total cells. Only a minor fraction could be assigned to the otherwise abundant<br />

Desulfosarcina/Desulfococcus indicating the relevance of yet unknown SRB.<br />

The correlation of biogeochemical and molecular data from sediments with<br />

tight coupling of AOM and SR to those with loose coupling will allow us to<br />

elucidate which SRB were involved in non-methane hydrocarbon degradation,<br />

methane oxidation or organic matter mineralization.<br />

PO 26<br />

Acetobacteraceae: Comparative Sequence Analyses of 23S<br />

rRNA-Genes<br />

A.C. Geisel *1 , K. Rappl 1 , W. Ludwig 1 , K.H. Schleifer 1<br />

1 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising,<br />

Germany<br />

Acetic acid bacteria (AAB) are used in industrial vinegar production because of<br />

their capability to produce acetic acid by oxidation of ethanol and their high<br />

resistance to acetic acid. However, AAB species occurring in vinegar are not<br />

well known and there are only few ecological studies investigating the<br />

microbial composition of different vinegars. It is known that vinegar oxidation<br />

is mainly due to strains belonging to the genera Acetobacter,<br />

Gluconacetobacter and Gluconobacter, but it is quite difficult to isolate and<br />

preserve AAB out of industrial vinegar.<br />

The 16S rRNA-gene is highly conserved within the species of one genus and<br />

the pairwise comparison of the 16S rRNA-gene sequences of AAB shows high<br />

similarity up to 99.9%. The 23S rRNA-gene offers the same advantages as the<br />

16S rRNA-gene, yet it includes additional diagnostic sequence stretches and<br />

possibly provides better phylogenetic resolution because of greater sequence<br />

variation.<br />

In this study, 23S rRNA-genes of several AAB type strains, roughly reflecting<br />

the diversity of the Acetobacteraceae family, were sequenced and compared<br />

applying the ARB software package. Frateuria aurantia sharing phenotypic<br />

similarities with some AAB was included as out-group. Agarose embedded<br />

genomic DNA of some AAB representatives was digested using the restriction<br />

endonuclease I-CeuI, a homing endonuclease which specifically cuts 23S<br />

rRNA-genes. The individual restriction patterns were visualized and compared<br />

via pulsed field gel electrophoresis and can serve to distinguish most of the<br />

respective species and to determine the number of rRNA-operons in their<br />

genomes.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PO 27<br />

Acidobacteria PhyloChip Analyses<br />

A culture-independent survey studying the diversity of<br />

Acidobacteria in Namibian soils<br />

K. Rappl *1 , W. Ludwig 1 , K.H. Schleifer 1<br />

1 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising,<br />

Germany<br />

In soil, Acidobacteria are present in high numbers. Their metabolic activity<br />

implies that these bacteria might be involved in biogeochemical cycles.<br />

Therefore, Acidobacteria are consi<strong>der</strong>ed <strong>bei</strong>ng of great ecological importance.<br />

Acidobacteria were observed in a wide variety of environments. Despite their<br />

ubiquity and abundance, only a few species were cultivated and described up to<br />

now. However, molecular methods provide the opportunity to identify<br />

Acidobacteria in microbial communities based on 16S rRNA gene sequences.<br />

Almost all currently known acidobacterial sequences have been recovered from<br />

uncultivated microorganisms in soil samples.<br />

Moreover, Acidobacteria show a phylogenetic diversity as high as compared<br />

with the phylum Proteobacteria. This pyhlogenetic complexity of the<br />

Acidobacteria phylum indicates a wide physiological spectrum of its members.<br />

We analyzed the diversity of representatives of the phylum Acidobacteria in<br />

Namibian soil samples collected at sites that are subjected to different human<br />

land use managements as well as non-impacted, pristine control sites.<br />

A prototype of an Acidobacteria PhyloChip allowed the rapid screening of soils<br />

via microarray hybridizations using a probe set for monitoring Acidobacteria<br />

on the subdivision level. The subdivision-specific chip contains 51<br />

acidobacterial probes, roughly covering the phylogenetic depth of the<br />

Acidobacteria phylum. The probes were designed based on the multiple probe<br />

concept detecting different regions of the 16S rRNA gene. Hence, a definite<br />

identification of the different Acidobacteria subdivisions is possible via<br />

characteristic hybridization patterns.<br />

PO 28<br />

Oxidation of atmospheric methane in upland forest soils<br />

J. Pratscher *1 , R. Conrad 1<br />

1<br />

Max Planck Institute for Terrestrial Microbiology, Biogeochemistry, Marburg,<br />

Germany<br />

Methane (CH4) is an important greenhouse gas with a global warming potential<br />

20 times more effective than CO2. Its current atmospheric concentration is 1.7<br />

ppmv. One major sink for atmospheric methane are upland soils, where<br />

oxidation of CH4 is performed by methanotrophic bacteria. The key step of this<br />

process, the initial oxidation of CH4 to methanol, is catalysed via the methane<br />

monooxygenase enzyme which occurs as a particulate, membrane bound form<br />

(pMMO), and as a soluble, cytosolic form (sMMO). Phylogenetic analyses of<br />

soils revealed that these „high affinity“ methanotrophic bacteria represent<br />

novel sequence lineages of PmoA and were therefore named Upland Soil<br />

Cluster (USC) α and γ. Unfortunately, only little is known about the molecular<br />

phylogeny and function of these microorganisms. So far no successful isolation<br />

approach has been reported.<br />

In this study we tested a different approach to unravel the 16S rRNA phylogeny<br />

of those organisms able to oxidize atmospheric methane. We investigated the<br />

ability of an upland forest soil near Marburg to oxidize atmospheric methane.<br />

Based on the pmoA gene sequences of the USCα obtained from this soil,<br />

polynucleotide probes where generated and applied for both recognition of<br />

individual genes (RING)-FISH and catalyzed reporter deposition (CARD)-<br />

FISH to laboratory cultures, clones, and environmental samples. This approach<br />

in combination with cell separation based on magnetic beads or fluorescence<br />

could lead to the identification of these Upland Soil Clusters and to a better<br />

un<strong>der</strong>standing of their function in the environment.<br />

PO 29<br />

Diversity of eukaryotic microorganisms in anoxic microbial<br />

mats from the Black Sea<br />

K. Weitbrecht *1 , M. Krüger 2 , R. Seifert 3 , M.W. Friedrich 1<br />

1<br />

Fachbereich Biologie/Chemie, Universität Bremen, Bremen, Germany<br />

2<br />

Referat Geomikrobiologie, Bundesanstalt für Geowissenschaften und<br />

Rohstoffe, Hannover, Germany<br />

3<br />

Institute of Biogeochemistry and Marine Chemistry, University of Hamburg,<br />

Hamburg, Germany<br />

147<br />

The Black Sea is the largest meromictic body of water resulting in an anoxic<br />

zone that takes up 90% of its volume. In the anoxic waters of the Black Sea<br />

northwestern shelf microbial mats exist on massive carbonate structures, which<br />

are fueled from cold methane seeps [1]. These mats apparently thrive

148<br />

exclusively by anaerobic oxidation of methane coupled to sulfate reduction.<br />

Anaerobically methane-oxidizing Archaea (ANME-I and ANME-II) and<br />

sulfate-reducing bacteria appear to be the predominant microorganisms in this<br />

unique, completely anoxic ecosystem.<br />

Prokaryotic biomass typically is controlled by protistan grazing and viral lysis<br />

in well-aerated pelagic marine environments. Microplanktonic eukaryotes have<br />

been found at oxic-anoxic interface of the Black Sea water column [2] and in<br />

anoxic sediments [3]. However, not much is known about eukaryotic<br />

microorganisms in anoxic waters and sediments and their potential role in<br />

grazing.<br />

Therefore, we have started analyzing the presence and diversity of eukaryotic<br />

microorganisms in samples from anaerobic methane-oxidizing mats.<br />

First PCR surveys suggest the presence of eukaryotic 18S rRNA. To<br />

characterize the community structure of the eukaryotes present genetic<br />

fingerprints from mat samples will be presented in conjunction with sequence<br />

data from clone libraries.<br />

[1] Michaelis, R. Seifert, et al.Microbial reefs in the Black Sea fueled by<br />

anaerobic oxidation of methane. Science 297 (5583):1013-1015, 2002.<br />

[2] M.V. Zubkov, A.F. Sazhin, M.V. Flint, The microplankton organisms at the<br />

oxic-anoxic interface in the pelagial of the Black Sea, FEMS Microbiology<br />

Letters 101(4): 245-250, 1992<br />

[3]Stoeck T, Epstein S, Novel eukaryotic lineages inferred from small-subunit<br />

rRNA analyses of oxygen-depleted marine environments, Applied and<br />

Environmental Microbiology 69(5), 2003<br />

PO 30<br />

Influence of lake trophic status on pelagic microbial<br />

community composition and microbial and viral abundance<br />

C. Rathmann *1 , M. Herrmann 1 , P. Stolle 1 , M. Ackermann 1 , J. Pust 2 , G. Auling 1<br />

1 Institut für Mikrobiologie, Leibniz Universität Hannover, Hannover, Germany<br />

2 Außenstelle Heiliges Meer, Westfälisches Landesmuseum, Hannover, Germany<br />

Seasonal changes in physicochemical parameters have a strong impact on<br />

spatio-temporal patterns of pelagic microbial populations in freshwater lakes.<br />

We have monitored diverging vertical profiles of inorganic nitrogen<br />

concentrations in summer and early autumn by comparison of an oligotrophic<br />

and a neighboring eutrophic lake. Therefore, we expect a strong effect of lake<br />

trophic status on microbial communities involved in the transformation of<br />

nitrogen, a key parameter of lake trophy. Vertical patterns of microbial<br />

community composition will be analyzed by Denaturing Gradient Gel<br />

Electrophoresis and Fluorescence in Situ Hybridization.<br />

Total cell counts using SYBR Green II staining revealed higher microbial<br />

abundances in the eutrophic compared to the oligotrophic lake. HPLC-based<br />

techniques [1] and SYBR Green II staining indicated depth-dependent changes<br />

of viral abundances as well. Noteworthy, an important impact of viruses on<br />

freshwater bacterial population dynamics was suggested recently [2].<br />

[1] Rathmann, C., Stolle, P., Auling, G. (2008): Microbial Ecology Revised due<br />

to New Methods for Quantification of Bacteriophages, RAISEBIO-HIGRADE-<br />

Summerschool Leipzig, Germany 22 - 25/09/2008<br />

[2] Filippini, M., Buesing, N. & Gessner, M. O. (2008): Temporal dynamics of<br />

freshwater bacterio- and virioplankton along a littoral-pelagic gradient.<br />

Freshwater Biology 53: 1114-1125.<br />

PO 31<br />

Mechanisms of species differentiation in bacteria<br />

M. Heppe *1 , H. Siemens 1 , H. Chen 1 , J. Overmann 1<br />

1 Microbiology, LMU, Munich, Germany<br />

The objective of the present study is to elucidate the role of recombination,<br />

adaptation and selection in shaping bacterial diversity, using aquatic bacteria<br />

from the family Sphingomonadaceae (subphylum Alphaproteobacteria) as a<br />

model system. To investigate species differentiation, closely related lineages<br />

with up to 100 % 16s rRNA gene sequence identity (ecotypes) originating from<br />

lakes were analysed. The ecotypes were cultivated from winter and summer<br />

samples by a high throughput multidrop technique and screened by a<br />

Spingomonadaceae specific PCR. 30% of the cultures grouped with the<br />

Sphingomonadaceae. The 117 analysed 16S rRNA gene sequences cluster in<br />

six subgroups with more than 97 % 16s rRNA gene sequence identity. The<br />

dominant fraction comprised 86 sequences with identical 16s rRNA gene<br />

sequences. Phylogenetic analysis of the 16s rRNA gene and the ITS1 region<br />

from cultures and a clone-library based on seasonal samples, was used to<br />

elucidate factors governing bacterial evolution and diversity within the model<br />

group. To analyse the significance of recombination, a multilocus sequence<br />

analysis of housekeeping genes in our culture collection was employed. The<br />

genetic analysis combined with a physiological characterisation allowed<br />

conclusions regarding the recombination, adaptation and selection processes<br />

during the differentiation of closely related bacterial lineages.<br />

PO 32<br />

Activity and Composition of Microbial Communities in<br />

Methane Seep Influenced Sediments off Sumatra<br />

M. Siegert *1 , B. Teichert 2 , A. Schippers 1 , M. Krüger 1<br />

1<br />

Geomikrobiologie, Bundesanstalt für Geowissenschaften und Rohstoffe,<br />

Hannover, Germany<br />

2<br />

Meeresgeologie, Bundesanstalt für Geowissenschaften und Rohstoffe,<br />

Hannover, Germany<br />

As a result of high seismic activity, sediment basins are formed in the fault<br />

between the Indo-Australian and the Eurasian plate off the island Sumatra.<br />

These basins contain several hundreds of meters thick sediments, providing<br />

conditions with high pressure and low substrate fluxes and are characterised by<br />

a high content of organic matter. During the R/V Sonne cruise SO189-2 in<br />

September 2006, several stations have been investigated for dissolved gases,<br />

the associated microbial communities and biogeochemical processes in the<br />

sediment. Samples from a methane seep in the Simeulue basin were taken in<br />

high resolution from the sediment surface by a multicorer and from deeper<br />

sediments using a gravity corer at a water depth of 1135 m. Living cultures of<br />

psychrophilic anaerobic degra<strong>der</strong>s of methane could be recovered. Anaerobic<br />

oxidation of methane (AOM) was measured by sulfate reduction in cultures<br />

from superficial sediments in rates of up to 17.6 µmol cm -3 d -1 (±2.9 µmol cm -3<br />

d -1 ). The microbial composition and functional genes have been analysed using<br />

real-time PCR, DGGE and total cell counting. Additionally, a zone of ceased<br />

AOM activity could be discovered in approximately 2.5 meters below the<br />

seafloor at the seep margin.<br />

PO 33<br />

Phylogenetic Relationship of Purple Sulfur Bacteria<br />

According to pufL and pufM Genes<br />

M. Tank *1 , V. Thiel 1 , J.F. Imhoff 1<br />

1<br />

Marine Mikrobiologie, Leibniz-Institut für Meereswissenschaften, Kiel,<br />

Germany<br />

Purple Sulfur Bacteria (PSB) are photoautotrophic bacteria phylogenetically<br />

grouped to the or<strong>der</strong> Chromatiales within the Gammaproteobacteria. They<br />

perform anoxygenic photosynthesis un<strong>der</strong> anaerobic conditions generally using<br />

reduced sulfur compounds (e.g. H2S, S 2- , thiosulfate) as e - -donator. PSB are<br />

ubiquitously distributed but mainly restricted to aquatic environments<br />

containing adequate light conditions, low/no oxygen tension and mo<strong>der</strong>ate<br />

sulfide concentrations. pufL and pufM are essential in photosynthesis of PSB<br />

and encode for polypeptides of the photosynthetic reaction centres which are<br />

located in intracytoplasmic membranes (ICM).<br />

In this study we analysed the pufL and pufM genes as well as the 16S rRNA<br />

gene of 66 PSB strains concerning their phylogenetic relationship. The dataset<br />

covers 16 of the 25 known PSB genera with 29 validly proved type strains,<br />

isolates from various habitats and our culture collection and database <strong>der</strong>ived<br />

sequences. The puf inferred phylogenetic tree topologies (nucleotides and<br />

amino acids) are in good agreement with the 16S rRNA gene phylogeny of the<br />

analysed PSB. All groups found in the 16S rRNA gene phylogenetic tree are<br />

found in the puf phylogenetic trees, as well. Members of the<br />

Ectothiorhodospiraceae and Chromatiaceae cluster well separated in all<br />

phylogenetic trees. Chromatiaceae harbouring bacteriochlorophyll b form a<br />

separate clade within the Chromatiaceae. Furthermore puf nucleotide<br />

similarities of 86% mark the limit for the genus level. Horizontal gene transfer<br />

of puf genes as suggested for PNSB and AAPB could not be observed within<br />

the PSB. We demonstrated that puf genes support 16S rRNA gene phylogeny<br />

and are suitable as phylogenetic marker for PSB.<br />

PO 34<br />

Prokaryotic communities in the Eastern Mediterranean<br />

deep sea<br />

M. Blümel *1 , J. Süling 1 , J.F. Imhoff 1<br />

1<br />

Marine Mikrobiologie, Leibniz-Institut für Meereswissenschaften, Kiel,<br />

Germany<br />

The Eastern Mediterranean Sea is one of the world´s most oligotrophic oceanic<br />

regions featuring comparably high deep-water temperatures of >13°C and<br />

extremely low nutrient concentrations. In the present study, the prokaryotic<br />

community composition in deep water samples (4000 m) from the Ierapetra<br />

Trough (34°31.514 N, 26°13.085 E) originating from 1998, 1999 and 2007 was<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

investigated by 16S rRNA gene based analysis. Alpha- and<br />

Gammaproteobacteria dominated the bacterial community, followed by<br />

uncultured representatives of the Thermomicrobia, Planctomycetales,<br />

Deltaproteobacteria, Verrucomicrobia, Actinobacteria, Bacteroidetes and<br />

Chaldithrix lineages. Only two archaeal phylotypes were found, one was<br />

affiliated to the marine group II (Euryarchaeota) and one to the marine group I<br />

(Crenarcheota). The results indicate a temporally stable prokaryotic<br />

community. Interestingly, a great proportion of the bacterial sequences was<br />

highly related to uncultured representatives found close to various<br />

hydrothermal systems. Thus, the bacterial community in the deep waters of the<br />

Eastern Mediterranean Sea may be dominated by rather mesophilic than<br />

psychrophilic bacteria. Bacterial adaptation to the prevailing nutrient situation<br />

in the deep Ierapetra basin was investigated by enrichment with different<br />

nutrients and analyzed by 16S rRNA gene based RFLP. The community was<br />

shown to be well adapted to oligotrophic conditions indicated by a quick<br />

response to nutrient addition even at low concentrations and short incubation<br />

times.<br />

PO 36<br />

Characterization of two groups of polyphosphate<br />

accumulating bacteria in eight municipal wastewater<br />

treatment plants<br />

M. Eschenhagen *1 , L. Mehlig 1 , L. Wächter 1 , K. Röske 1 , I. Röske 1<br />

1 Institut für Mikrobiologie, TU Dresden, Dresden, Germany<br />

Although activated sludge systems with Enhanced Biological Phosphorus<br />

Removal (EBPR) represent state-of-the-art technology for phosphate removal<br />

from domestic wastewater it is largely unknown which bacteria are responsible<br />

for the EBPR process.<br />

The aim of this study was to characterize two groups of possible polyphosphate<br />

accumulating organisms (PAO) in eight municipal wastewater treatment plants<br />

(WWTP) with three different kinds of phosphate elimination systems, EBPR<br />

without chemical precipitation, chemical precipitation and without advanced P<br />

removal. Three different molecular methods (FISH, cloning/sequencing and<br />

DGGE) were used for the investigation of the Rhodocyclus and Tetrasphaera<br />

related PAOs.<br />

With exception of the WWTP without advanced P removal, for all WWTPs a<br />

significant P release and uptake could be verified in batch trials using acetate or<br />

casein hydrolysate as carbon source. The highest proportions were detected for<br />

sludge from WWTP with EPBR (up to 10 mg PO4-P/g DW), but otherwise,<br />

there were partly no differences between the results from WWTP with chemical<br />

precipitation and WWTP with EBPR provided by additional chemical<br />

precipitation to reach very low residual content.<br />

Based on these results it was not surprising that members of both investigated<br />

PAO groups could be detected by FISH in all WWTP with an abundance of 2-<br />

12%, also in the WWTP without advanced P removal. This result indicates that<br />

the presence of possible PAOs is no evidence for an established EBPR. The<br />

species composition of the PAOs detected by molecular methods<br />

(cloning/sequencing and DGGE) showed no correlation to the mode of P<br />

elimination and probably depends on the wastewater composition.<br />

PO 37<br />

Diversity of a collection of iron oxidizing bacteria from a<br />

various sulfidic mine waste dumps<br />

A. Breuker *1 , B. Anna 1 , K. Bosecker 1 , A. Schippers 1<br />

1 Abteilung für Geomikrobiologie, Bundesanstalt für Geowissenschaften und<br />

Rohstoffe, Hannover, Germany<br />

More than 80 strains of acidophilic Fe(II)- and sulfur-oxidizing microorganisms<br />

from mine waste dumps in 10 different countries all over the world have been<br />

maintained in liquid culture in the BGR-strain collection for many years.<br />

Characterization by 16S rDNA analysis showed that most of the cultivated<br />

Fe(II)-oxidizers belong to four genera: Acidithiobacillus, Acidimicrobium,<br />

"Ferrimicrobium" and Leptospirillum. All analyzed Acidithiobacillus strains<br />

were identified as At. ferrooxidans. The Leptospirillum strains were affiliated<br />

with L. ferriphilum or L. ferrooxidans. The gram-positive strains related to<br />

Acidimicrobium or "Ferromicrobium" were phylogenetically more diverse and<br />

could be divided in three clusters. While several strains could be identified as<br />

syngeneic (16S rDNA) with "Ferrimicrobium acidiphilum", two other 16S<br />

rDNA clusters are distantly related and might represent new species or even<br />

new genera. In addition, one new Sulfobacillus strain and one new<br />

Alicyclobacillus strain could be identified. Furthermore several strains related<br />

to Acidiphilium acidophilum have been detected and form one 16S rDNA<br />

cluster. Research on the phylogeny, physiology and biogeography of the novel<br />

BGR-strains is in progress.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PO 38<br />

Hydrothermal sediments of the Bransfield Strait<br />

(Antarctica): Genetic diversity of microbial communities<br />

and anaerobic methane oxidation<br />

J. Süling *1 , R. Schmaljohann 1 , B. Weitzel 1 , M. Drews 1 , J.F. Imhoff 1<br />

1<br />

Marine Mikrobiologie, Leibniz-Institut für Meereswissenschaften, Kiel,<br />

Germany<br />

Sediments of Hook Ridge, Bransfield Strait (Antarctica), where diffuse<br />

hydrothermal venting released methane and reduced sulfur compounds as<br />

potential energy sources for specialized prokaryotes, were investigated.<br />

Geochemical parameters were analyzed, microbial activities were measured and<br />

the genetic diversity of microbial communities associated with these sediments<br />

was determined. The geochemical profiles in the top 20 cm indicated<br />

consumption of methane and a reduction of sulfate. Maximum activity of<br />

methane oxidation was detected in 4-6 cm sediment depth, where also highest<br />

rates of CO2 fixation were measured. 16S rDNA clone libraries of the microbial<br />

communities were established for archaea, eubacteria and sulfate-reducing<br />

bacteria (SRB) using group specific primers. Large portions of sequences<br />

belonging to sulfate-reducing bacteria were found among the eubacterial<br />

clones. The great majority of archaeal clone sequences from the sediment<br />

horizon with the highest rates of anaerobic methane oxidation formed a large,<br />

heterogeneous cluster. This cluster mainly consisted of euryarchaota known<br />

from other hydrothermal vent environments (deep-sea hydrothermal vent<br />

euryarchaeota, DHVE). Within the crenarchaeota clone sequences belonging to<br />

the group of MBGB/DSAG group (marine benthic group B/deep sea archaeal<br />

group) were the most numerous. Minor components of the microbial<br />

communitiy consist of members known to be of the ANME (anaerobic methane<br />

oxidizers) group within the euryarchaeota which are involved in the process of<br />

anaerobic oxidation of methane and their supposed eubacterial snytrophic<br />

partners of the Desulfobacterales (Desulfosarcina / Desulfococcus /<br />

Desulfobulbus).<br />

PO 39<br />

Oligotrophy in mesophilic ammonia-oxidizing<br />

Crenarchaeota<br />

W. Martens-Habbena *1 , D.A. Stahl 1<br />

1<br />

Civil and Environmental Engineering, University of Washington, Seattle,<br />

United States<br />

149<br />

Mesophilic Crenarchaeota have been found almost ubiquitously in seawater,<br />

freshwater, soil, and sediments by molecular surveys. More recently<br />

autotrophic ammonia oxidation has been discovered in mesophilic<br />

Crenarchaeota and genes for the putative ammonia monooxy-genase (Amo) are<br />

broadly distributed within this group [1,2,3]. Archaeal amo genes outnumber<br />

bacterial amo genes by several or<strong>der</strong>s of magnitude especially in oligotrophic<br />

environments, suggesting that Crenarchaeota could play a dominant role in<br />

nitrification. Despite their widespread occurrence very little is known about the<br />

lifestyle of these organisms. Here we report the physiology of ammonia<br />

oxidation in the first cultured representative of the mesophilic Crenarchaeota,<br />

Cand. Nitrosopumilus maritimus strain SCM1. Strain SCM1 grows with<br />

maximum growth rates of 0.027 h -1 and depletes ammonium below the<br />

detection limit of 10 nM. Ammonium concentrations above 2 mM increasingly<br />

inhibit growth. Ammonium is oxidized with a stoichiometry of 1.5 moles<br />

oxygen per 1 mole ammonium. Maximum activities were recorded over a wide<br />

concentration range of 10 µM up to 1000 µM ammonium. The half-saturation<br />

constant (Km) was among the smallest values of a microbial growth substrate<br />

(~130 nM total ammonium), ~100-fold smaller than of any investigated AOB.<br />

Even cells grown in batch culture exhibit extremely high substrate affinity<br />

(Vmax/Km ~169.1 L mg protein -1 h -1 ). These kinetic properties closely<br />

resemble ammonia oxidation kinetics in oligotrophic marine environments<br />

indicating that ammonia-oxidizing Crenarachaeota represent the missing<br />

oligotrophic ammonia oxidisers and indeed play a dominant role in the nitrogen<br />

cycle of marine and terrestrial environments.

150<br />

PO 40<br />

Isolation and identification of thermophilic bacteria from<br />

hot spring<br />

L. Mirzakhan *1 , Z. Mirzakhan 1<br />

1 Biology, Razibiotech, Tehran, Iran<br />

Regarding the fact that Thermopile Bacteria consist of special enzymes, have<br />

significant importance in researches and industries.<br />

The thermophilic bacteria were isolated from a shallow hot spring with 75°C in<br />

Ardabil located in northwest of Iran.<br />

The upper temperature limit for growth was 90˚C (optimally 55˚C) and<br />

optimum pH was between 6.0-6.5. A thermophil bacillus,facultative anaerobic,<br />

heterotrophic and gram-positive was isolated .<br />

The isolate grew on yeast extract, tryptone and peptone as well as on urea,<br />

starch, glucose, maltose, and with salt concentrations of up to 3%NaCl.<br />

DNA was purified by the method of phenol-chloroform extraction.The G+C<br />

content of the genomic DNA was 50 mol%.<br />

for recognition of this bacterium, molecular method of partial 16S ribosomal<br />

DNA sequence analysis was used.<br />

PO 41<br />

Assessment of the functional diversity of soil microbial<br />

communities in the German Biodiversity Exploratories by<br />

metagenomics<br />

H. Nacke *1 , C. Will 1 , R. Daniel 1<br />

1 Institut für Mikrobiologie und Genetik, Abteilung für Genomische und<br />

Angewandte Mikrobiolgie, Georg-August-Universität Göttingen, Göttingen,<br />

Germany<br />

The phylogenetic and functional microbial diversity present in soil samples<br />

<strong>der</strong>ived from grassland and forest areas of the three German Biodiversity<br />

Exploratories Schorfheide-Chorin, Hainich-Dün and Schwäbische Alb were<br />

analyzed. This research is part of an initiative to explore the dependence of<br />

diversity of soil microorganisms on vegetation type and land use intensity.<br />

The approach includes the isolation of whole genomic DNA from special<br />

sampling sites, including topsoil (A horizon) as well as subsoil (B horizon).<br />

Complex metagenomic small-insert and large-insert libraries were constructed<br />

from 32 sampling sites. The libraries were employed in comparative screenings<br />

for key microbial functions, such as cellulolytic and lipolytic activities. Several<br />

clones expressing cellulase- and esterase-activity were identified by functionbased<br />

screening. The recombinant vectors containing the targeted genes were<br />

sequenced. So far, representatives of the esterase superfamilies IV and V were<br />

detected. To generate starting material for enzyme characterization the esteraseencoding<br />

genes were placed un<strong>der</strong> control of T7 polymerase/promoter system.<br />

Subsequently the geneproducts were overproduced in Escherichia coli. The<br />

production of the proteins was verified by Western-Blot analysis. Currently,<br />

characterization of the esterases is performed.<br />

PO 42<br />

Detection of airborne bacteria in a German duck hatchery<br />

E. Martin *1 , S. Ernst 1 , U. Jäckel 1<br />

1<br />

Biologische Ar<strong>bei</strong>tsstoffe, Bundesanstalt für Ar<strong>bei</strong>tsschutz und Ar<strong>bei</strong>tsmedizin,<br />

Berlin, Berlin, Germany<br />

Exposures to bioaerosols at agricultural working places can cause a wide range<br />

of respiratory disor<strong>der</strong>s. In a preliminary study in a duck hatchery it has been<br />

assumed that breathing the with dust and microbes highly polluted air result in<br />

a decline in workers lung function during their working day. Despite of this<br />

acquaintance there were just rarely characterisations of the microbial diversity<br />

in bioaerosols at these working locations in general. Therefore we investigated<br />

the bacterial community in bioaerosol samples of the hatchery by both<br />

cultivation based and molecular methods. For isolation of abundant cultivable<br />

bacteria, six different media (Tryptone-soy-agar (TSA), Actinomyceteisolation-agar,<br />

MacConkey-agar, Middlebrock-agar, Glycerol-arginine-agar and<br />

Oat-flakes-agar) were used. Depending on the selectivity of the used media, the<br />

concentration of cultivable bacteria were at least one up to six dimensions<br />

lower than those observed by fluorescence microscopic quantification after<br />

DAPI staining. In total, 41 bacterial isolates were obtained and grouped<br />

according to similar cell- and colony morphology, Gram-staining and RFLPanalyses.<br />

For molecular approaches six 16S RNA-gene clone libraries were<br />

generated from DNA directly extracted from bioaerosols collected by personal<br />

samplers during the whole working day. Analyses of nearly 384 sequences<br />

showed a low bacterial diversity in bioaerosols from the investigated<br />

hatcheries. The main exposition seems to bacteria of the seven different genera:<br />

Acinetobacter, Achromobacter, Clostridium, Enterococcus, Klebsiella,<br />

Pseudomonas and Staphylococcus. Especially 16S rRNA-gene sequences<br />

closely related to Acinetobacter baumanii and Klebsiella pneumoniae are of<br />

interest because these bacterial species are known as causatives of pulmonary<br />

diseases.<br />

PO 43<br />

Screening for chitin degrading microorganisms in different<br />

marine habitats.<br />

T. Staufenberger *1 , H. Heindl 1 , A. Gärtner 1 , A. Labes 1 , J.F. Imhoff 1<br />

1 Kieler Wirkstoff-Zentrum am IFM-GEOMAR, IFM-GEOMAR, Kiel, Germany<br />

Chitin is one of the most abundant biopolymers found on earth as it is one of<br />

the main compounds in the exoskeleton of insects and crustaceae. Marine<br />

zooplankton produces billions of tons of chitin per year [1]. Thus, chitin<br />

degradation has to be widespread among marine primary degra<strong>der</strong>s. Otherwise<br />

the natural stock of carbon and nitrogen would be depleted completely in a very<br />

short time [2]. Yet an in silico analysis revealed that only 5 % of all known<br />

chitin degrading enzymes are <strong>der</strong>ived from marine organisms. By combining<br />

culture techniques and chitinase specific primers [3] we established a screening<br />

method to extend our knowledge of the phylogeny, distribution and in situ<br />

activity of marine chitin degrading microorganisms. These methods were used<br />

to describe chitin degrading microorganisms from different marine habitats.<br />

[1] Yu, C. et al. 1991. J Biol Chem 266: 24260-24267.<br />

[2] Johnstone, J. 1908. Conditions of Life in the Sea. University press.<br />

[3] Hobel, C.F. et al. 2005. Appl Environ Microbiol 71: 2771-2776.<br />

PO 44<br />

Analysis of microbial community composition on cathodes<br />

from sediment microbial fuel cells<br />

B. Breidenbach *1 , A. Cabezas 1 , M.W. Friedrich 2<br />

1<br />

Dept. of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology,<br />

Marburg, Germany<br />

2<br />

Faculty of Biology, University of Bremen, Bremen, Germany<br />

Sediment microbial fuel cells (SMFC) are electrochemical devices that generate<br />

electrical current from microbial oxidation of organic carbon. Recently, a novel<br />

SMFC was introduced which is fueled by rice root exudates as electron donor<br />

for the anode reaction [1]. Rice fields are a major source of methane. These<br />

SMFC might have the potential to decrease methane emissions by favouring<br />

electron flow to electrode coupling microorganisms instead of methanogens. So<br />

far, the diversity and function of microorganisms involved on the anode has<br />

been the major focus. However, microbial biofilms might play an important<br />

role also on (bio)cathodes by increasing the efficiency of MFCs.<br />

Here, we are investigating the microbial community present on cathodes from<br />

SMFCs fueled by root exudation. Bacteria-specific Terminal Restriction<br />

Fragment Length Polymorphism (T-RFLP) analysis of 16S rRNA revealed that<br />

closed circuit SMFCs cathodes have a more diverse microbial community than<br />

those from open circuits SMFCs (18 vs 8 TRFs). Moreover, samples from<br />

closed circuit SMFCs have unique TRFs (115bp) and differences in relative<br />

abundance for predominant TRFs were observed. Further cathode samples will<br />

be analyzed by T-RFLP and cloning/sequencing to gain more information about<br />

the microbial community present on cathodes.<br />

[1] De Schamphelaire et al. (2008) Microbial fuel cells generating electricity<br />

from rhizodeposits of rice plants. Env Sci & Tech 42 (8):3053-3058.<br />

PO 45<br />

Application of Live/Dead BacLight staining for analysis of<br />

microorganisms and bioaerosols<br />

K. Eisenbarth *1 , P. Kämpfer 1 , U. Jäckel 2 , K. Fallschissel 1<br />

1<br />

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen,<br />

Giessen, Germany<br />

2<br />

Gruppe 4.7 Biologische Ar<strong>bei</strong>tsstoffe, Bundesanstalt für Ar<strong>bei</strong>tsschutz und<br />

Ar<strong>bei</strong>tsmedizin, Berlin, Germany<br />

As yet standardized monitoring of airborne bacteria is based on cultivationdependent<br />

methods. Consequently, there is a high demand on the sampling<br />

system to maintain viability of microorganisms after their collection. Despite<br />

the high diversity of collection systems there are only few studies published, in<br />

which a species specific “biological collection efficiency” of a collection<br />

system is investigated. Here we report the results of an initial investigation of<br />

the suitability of the Live/Dead BacLight Bacterial Viability Kit for analysis of<br />

bioaerosol samples obtained after impingement sampling.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

The two-colour fluorescence assay differs into living and dead cells in one<br />

staining procedure, leading to green (living) and red (dead) fluorescent cells.<br />

Suitability was tested with reference and type strains of nine different species.<br />

Results revealed the suitability of Live/Dead BacLight staining for Bacillus<br />

subtilis DSM10 T , Corynebacterium glutamaticum DSM20300 T , Sphingomonas<br />

terrae IFO15098, Comamonas testosteroni DSM50244 and Staphylococcus<br />

aureus DSM20231. Living and dead cells of Rhodococcus erythropolis<br />

DSM43066, Micrococcus roseus DSM20447 T , Lactobacillus paracasei<br />

DSM5622 T and Pseudomonas putida DSM291 could not be differentiated with<br />

the Live/Dead BacLight staining. To study the effect of the sampling<br />

procedure, cells were added to AGI-30 impingers followed by collection of<br />

increasing volumes of cell-free air. Afterwards the proportion of living and<br />

dead cells was again determined by Live/Dead BacLight staining as well as by<br />

cultivation. Results showed clear differences in the biological sampling<br />

efficiency depending on examined species. While no lethal effect for<br />

Corynebacterium glutamicum was found, e.g. 9% of Bacillus subtilis cells died<br />

during sampling of 0,358 m 3 air.<br />

PO 46<br />

Risk assessment - airborne bacteria - in a German turkey<br />

stable<br />

K. Fallschissel *1 , K. Klug 2 , P. Kämpfer 1 , U. Jäckel 2<br />

1<br />

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen,<br />

Giessen, Germany<br />

2<br />

Gruppe 4.7 Biologische Ar<strong>bei</strong>tsstoffe, Bundesanstalt für Ar<strong>bei</strong>tsschutz und<br />

Ar<strong>bei</strong>tsmedizin (BAUA), Berlin, Germany<br />

The intensive livestock production with densely stocked and closed production<br />

buildings facilitates high concentrations of airborne microorganisms, their<br />

metabolic products and cell constituents. Exposure to those substances may<br />

affect the respiratory system leading to several respiratory health effects, e.g.<br />

asthma, asthma-like syndrome, chronic bronchitis as well as hypersensitivity<br />

pneumonitis. So far, the microbial communities in livestock stable’s air has not<br />

been studied in detail. For this reason, bioaerosol samples from two turkey<br />

stables were investigated by cultivation-based and molecular methods to<br />

determine the microbial concentrations and the compositions of bacterial<br />

communities. For isolation of abundant cultivable bacteria, TSA-agar,<br />

McConkey-agar and actinomycete-isolation-agar were used. Depending on the<br />

media, concentrations ranged between 8x10 4 and 8x10 5 CFU m -3 air. The<br />

corresponding cell number concentration after DAPI staining, in contrast were<br />

two magnitudes higher, showing the clear limitation of cultivation based<br />

approaches. Isolates were studied by 16S rRNA gene sequence analyses.<br />

Sequences of five isolates were found to be closely related to bacterial species<br />

belonging to risk group 2: Acinetobacter johnsonii, Aerococcus viridans and<br />

Pantoea agglomerans are known as causative agents for pulmonary diseases.<br />

These results show that organisms not only at high concentrations are found,<br />

but also species with a potential health risk can be detected in those livestock<br />

stables.<br />

PO 47<br />

Comparative Genomics of representative Members of the<br />

Roseobacter-Clade: The Genus Octadecabacter<br />

J. Vollmers *1 , S. Voget 1 , H. Liesegang 1 , B. Thorsten 2 , M. Simon 2 , R. Daniel 1<br />

1 Göttingen Genomics Laboratory, Georg-August-University, Göttingen,<br />

Germany<br />

2 Institute for Chemistry and Biology of the Marine Environment (ICBM), Carlvon-Ossietzky-University,<br />

Oldenburg, Germany<br />

The genus Octadecabacter comprises heterotrophic and psychrophilic bacteria<br />

belonging to the Roseobacter-clade [1]. Members of this genus were found in<br />

Arctic and Antarctic sea ice and seawater and appear to be indigeneous to the<br />

sea ice microbial communities of both poles. To date, two different species<br />

have been described: Octadecabacter arcticus and Octadecabacter antarcticus.<br />

The strains were isolated from the lower 20 cm of annual sea ice of the Arctic<br />

and the Antarctic, respectively.<br />

The genomes of O. arcticus 238 and O. antarcticus 307 were sequenced<br />

(https://moore.jcvi.org/moore/). To solve problems with misassembled regions<br />

caused by repetitive sequences and to close remaining gaps, PCR-based<br />

techniques on genomic DNA and fosmids were used. The sequencing results<br />

show that the genome of O. arcticus is organized in three replicons: a<br />

chromosome of 5.2 Mb and two plasmids of 160 kb and 120 kb. In contrast, the<br />

genome of O. antarcticus consists of one chromosome (4,98 Mb) and only one<br />

plasmid (62,8 kb). The genomes of both strains were compared with respect to<br />

differences caused by geographic separation.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

151<br />

[1] Gosink, J.J., Herwig, R.P., Staley J.T. (1997) Octadecabacter arcticus gen.<br />

nov.,sp. nov. and O. antarcticus, sp. nov., Nonpigmented, Psychrophilic Gas<br />

Vacuolate Bacteria from Polar Sea Ice and Water. System. Appl. Microbiol. 20,<br />

356-365<br />

PO 48<br />

An integrated approach to study ecology, cell morphology<br />

and the genome sequence of Magnetobacterium bavaricum<br />

M. Niebler *1 , L. Wei 2 , G. Wanner 3 , M. Kube 4 , P. Stief 5 , A. Beck 5 , N. Petersen 6 ,<br />

R. Reinhardt 5 , R. Amann 5 , C. Jogler 1 , D. Schüler 3<br />

1 Department Biologie I / AG Schüler, LMU, Planegg-Martinsried, Germany<br />

2 Institute of Geology and Geophysics, University of Beijing, Beijing, China<br />

3 Department Biology I, LMU, Planegg-Martinsried, Germany<br />

4 Institute for Molecular Genetics, Max Planck Institute, Berlin, Germany<br />

5 Institute for Marine Microbiology, Max Planck Institute, Bremen, Germany<br />

6 Department für Geo- und Umweltwissenschaften, LMU, München, Germany<br />

Magnetobacterium bavaricum is a gram-negative, rod-shaped bacterium<br />

belonging to the diverse group of magnetotactic bacteria (MTB). M. bavaricum<br />

occupies a distinct position in this group not only because of its size of up to<br />

10µm, a very thick cell wall and up to 1000 bullet-shaped magnetosomes but<br />

also because unlike other known MTB (belonging exclusively to the<br />

Proteobacteria) it could be phylogenetically linked to the Nitrospira phylum.<br />

With our work we wanted to analyse M. bavaricum with respect to ecology,<br />

cell biology and the genome sequence.<br />

Microscopic enumeration in combination with microsensor measurements<br />

revealed the presence of M. bavaricum below the oxic-anoxic transition zone<br />

implying an anaerobic or microaerophilic mode of life.<br />

For genome analyses we isolated MTB from the sediments using a magnetic<br />

strategy including a homemade magnetic trap. The enrichment of MTBs<br />

containing more than 10 7 M. bavaricum cells (about 40% of total amount of<br />

harvested cells) was used for DNA extraction. The subsequent construction of a<br />

metagenomic fosmid library resulted in > 4000 clones. Screening with a PCR<br />

specific for M. bavaricum 16S rDNA led to the identification of an rDNA<br />

operon-containing fosmid, which also harbours a gene encoding a putative type<br />

I RubisCO subunit which hints towards a chemolithoautotrophic lifestyle of M.<br />

bavaricum.<br />

Currently we are using an end sequence-based strategy to select fosmids<br />

containing M. bavaricum DNA, allowing full fosmid sequencing and the<br />

genome reconstruction of M. bavaricum in the near future.<br />

Based on the obtained genomic information, cultivation experiments are<br />

planned.<br />

PO 49<br />

Cloning and Characterization of Polyketide Synthase Gene<br />

from Forest Soils Using Metagenomic Analysis<br />

A. Latif *1 , I. Faisal 2 , F. Kurnia 3 , R. Lestari 3<br />

1 Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia<br />

2 Biotech Center, The Agency for the Assessment and Application of<br />

Technology, Jakarta, Indonesia<br />

3 Biology, University of Indonesia, Depok, Indonesia<br />

Polyketide synthase (PKS) are a family of enzymes complexes that produces<br />

polyketides, the most relevant classes of natural products. In this report we<br />

succeed in cloning and characterization of polyketide synthase gene from forest<br />

soil using metagenomic analysis. Eight locations of forest soil were collected<br />

from Halimun Mountain National Park, Indonesia and kept in -20oC. The<br />

genomic DNA from forest soil samples were extracted directly using CTAB<br />

and lysing matrix method. The PKS gene fragments were amplified using PCR<br />

with KS-AT domain primers designed from actinomycetes. Based on the size<br />

amplification products of the PCR about 1400bp in length confirmed the<br />

presence of PKS gene from various soil samples. Then the PCR product was<br />

recovered from the gel and cloned into pGEM-T Easy vector. PCR-RFLP<br />

analysis using MboI demonstrated the diversity of PKS gene in samples. The<br />

result of the present study indicated that metagenomic analysis is a powerful<br />

tool for exploring PKS gene diversity from soils.

152<br />

PO 50<br />

Poultry stable air: A newly-discovered source for<br />

Jeotgalicoccus spp.<br />

E. Martin 1 , U. Jäckel *1<br />

1<br />

Biologische Ar<strong>bei</strong>tsstoffe, Bundesanstalt für Ar<strong>bei</strong>tsschutz und Ar<strong>bei</strong>tsmedizin,<br />

Berlin, Berlin, Germany<br />

Previous investigations of bioaerosols from animal stables revealed enormous<br />

concentrations of airborne microorganisms to the extent of 10 10 cells per m 3 .<br />

These bioaerosols are interesting in two different aspects, medical and<br />

ecological. On the one hand an inhalation of microbial highly polluted air can<br />

cause worker’s lung diseases. Furthermore an inoculation of the closer<br />

surrounding soil with stable specific microorganisms is possible via air<br />

transport. Despite these acquaintances there were so far no detailed<br />

investigations carried out regarding the microbial communities in stables’ air.<br />

First investigations of duck stables’ bioaerosols revealed that in addition to<br />

Staphylococcus spp. and Streptococcus spp., 15% of 16S rRNA-genes in clone<br />

libraries were next related to Jeotgalicoccus psychrophilus and Jeotgalicoccus<br />

halotolerans. Until now these species were only recognised in jeotgal a<br />

traditional Korean food that is prepared with various kinds of seafood. For<br />

detailed investigation of these bacterial species we developed a group specific<br />

primer system targeting 16S rRNA-genes of both described strains. Specific<br />

PCR-products were gained from DNA directly extracted from poultry stables’<br />

bioaerosols and from investigated duck faeces showing the remarkable wide<br />

distribution of Jeotgalicoccus spp. in the poultry feeding industry. One<br />

potential source for these species is coquina which is used for feeding and from<br />

whom we could successfully isolate strain MK 7 (99.3% 16S rRNA-gene<br />

similarity to J. psychrophilus). As a consequence of our investigations, both<br />

from the ecological and medical point of view the increasing poultry farming<br />

should be regarded as an important source for complex and mainly unknown<br />

bacterial communities.<br />

PP 01<br />

Characteristics of Extended-spectrum beta-lactamases<br />

Enzymes in Gram-negative Bacilli from ICU Patients in<br />

Tabriz Hospitals<br />

Y. Hashemi Aghdam *1 , H. Mobaiyen 2 , M.R. Nahaie 2<br />

1 Student and Member of the scientific association of the Medical Faculty,<br />

Tabriz Islamic Azad University, Tabriz, Iran<br />

2 Department of Microbiology, Tabriz Islamic Azad University, Tabriz, Iran<br />

Aims- This study aimed to determine the prevalence of extended-spectrum<br />

beta-lactamases enzymes (ESBLs) producing Gram-negative bacilli (gr-B)<br />

isolated ICU patients and study the plasmid profiles and presence of relevant<br />

genes of the isolates.<br />

Methods- Patients of ICU wards from 5 hospitals of Tabriz were studied. gr-B<br />

was identified using conventional bacteriologic methods and confirmed by Hi<br />

Enterobacteriaceae Identification Kit. All of the isolated gr-B was tested for<br />

sensitivity against the third generation cephalosporins, cephamycin, quinolones,<br />

aminoglycosides, tetracycline and uridopenicillins by disc agar diffusion<br />

method. Double disc approximation test was used for screening of the isolates<br />

for ESBLs production. Combined test disc method and MIC determination by<br />

E-test were adopted for confirmation. Plasmid profiles of Esherichia coli and<br />

Klebsiella pneumoniae were determined by alkaline lysis procedure, while<br />

Kado and Liu technique was used for plasmid profiles of Pseudomonas<br />

aeruginosa. CTX-M-1 and CTX-M-2 was tested by Polymerase Chain<br />

Reaction.<br />

Results- ESBLs production was detected in E. coli (58.1%), K. pneumoniae<br />

(89.1%), P.aeruginosa (83.6%) and Acintobacter baumanii (82.4%) strains.<br />

72% of E.coli contained 1-5 plasmid(s) with 4.2-63 kb molecular weight.<br />

78.3% K.pneumoniae contained 1-4 plasmids with ~7-63 kb molecular mass.<br />

64.5% of P.aeruginosa harbored a single plasmid of 63 kb. amplicon of bla<br />

CTX-M-i presented in 17 E.coli and 29 K.pneumoniae isolates. None of the test<br />

isolates showed detectable band in bla CTX-M-Z group. P.aeruginosa isolates<br />

lacked CTX-M-1 and CTX-M-2 genes.<br />

Conclusion- K.pneumoniae was the most prevalent bacterium. Highest rate of<br />

resistance was showed against cefotaxime. Presence of CTX-M-l was<br />

confirmed in E. coli and K.pneumoniae isolates.<br />

PP 02<br />

Genomic fingerprints analysis of coagulase-positive and<br />

negative Staphylococci isolated from patients with<br />

bacteremia by rep-PCR method.<br />

M. Moosavian *1 , D. Darban 2<br />

1 Department of Microbiology, School of Medicine, Ahvaz Jundishapur<br />

University of Medical Sciences, Ahvaz, Iran<br />

Abstract: Staphylococci are important organisms involved in many of<br />

infections, including bacterimia or septicemia. The objective of this survey was<br />

to determine of DNA fingerprint patterns of coagulase positive and negative<br />

staphylococcal strains in patients with bacteremia and detection of their<br />

relationships.<br />

Methods: In this cross-sectional study, staphylococcal positive blood cultures<br />

were collected from patients with bacteremia which hospitalized in four<br />

hospitals dependent to Ahvaz Jundishapur University of Medical Sciences. The<br />

patients with 2 positive cultures out of 3 samples were consi<strong>der</strong>ed for this<br />

study. Isolated staphylococci were studied more, for identification of their<br />

species by standard biochemical tests. DNA was extracted from bacterial cells<br />

and genomic fragments which inserted between repetitive ERIC elements were<br />

amplified by rep-PCR. Furthermore, relationship of staphylococcal strains was<br />

determined based on the similarities between DNA fingerprints by using<br />

Jaccards coefficient.<br />

Results: In this survey, 88 cases of bacteremia caused by coagulase positive<br />

Staphylococcus aureus (36 cases), and coagulase negative strains (52 cases),<br />

were studied. Rep-PCR of genomic DNA from staphylococcal isolates<br />

produced multiple fingerprints in sizes ranging between 600 and 2642 bp. Also,<br />

the frequencies of 2642 bp and 600 bp bands among isolated strains were<br />

87.5% and 61%, respectively. The fingerprint patterns of S. aureus (33 strains),<br />

S. epi<strong>der</strong>midis (32 strains) and S. lugdunensis (7strains) were 31, 30 and 7<br />

types, respectively.<br />

This study showed <strong>bei</strong>ng of the same & closely related patterns among<br />

staphylococcal strains which could be due to dissemination of epidemic strain<br />

in the studied hospitals.<br />

Conclusion: Detection of DNA fingerprint patterns of staphylococcal strains by<br />

rep-PCR and their comparison to other genotypic and phenotypic properties<br />

could be suitable method for future epidemiological studies.<br />

PP 03<br />

Microbilogical features of infective endocarditis in injection<br />

drug users: A trend toward more resistant organisms<br />

A. Tavanaii Sani 1 , M. Mojtabavi *2 , R. Boland Nazar 1<br />

1<br />

Department of Infectious Diseases, Mashhad University of Medical Science,<br />

Mashhad, Iran<br />

2<br />

Department of Infctious Diseses, Mashhad University of Medical Science,<br />

Mashhad, Iran<br />

Introduction: Endocarditis is one of the most important complications of<br />

intravenous drug use, which is associated with high mortality and morbidity.<br />

The aim of this study was to define the microbiological characteristics of<br />

infective endocarditis in injection drug users.<br />

Material and Method: 34 patients were admitted to a university hospital in a<br />

four-year period and analyzed prospectively.<br />

Results: 38 episodes of endocarditis in 34 patients were identified. All the<br />

patients were males with the average age of 30.4 +/- 7.1 years. Fever was the<br />

most common compliant in these patients (31 cases, 81.6%), followed by<br />

fatigue (16 cases, 42.1%), dyspnea (16 cases, 42.1%), cough (13 cases, 34.2%),<br />

sweats (13 cases, 34.2%), chills (12 cases, 31.5%), and hemoptysis (12 cases,<br />

31.5%). The average length of symptomatic period, before presentation, was<br />

13.2 days. The tricuspid valve was involved in 26 cases (74.3%), and the mitral<br />

valve, mitral and tricuspid valves, and mitral and aortic valve involved in<br />

11.4%, 11.4% and 2.9%, respectively. The blood cultures were negative in<br />

28.9% 0f cases; microorganisms identified in positive blood cultures included<br />

Staphylococcus aureus (77.8%, including MRSA in 33.3% and MSSA in 44.5%<br />

of cases), Enterococci (7.4%), Streptococcus pyogenes (7.4%) and Klebsiella<br />

pneumoniae (7.4%). Eight patients (21.1%) un<strong>der</strong>went surgery, and nine of<br />

them (23.7%) were died.<br />

Conclusion: Consi<strong>der</strong>ing the increasing frequency of methicillin-resistant<br />

Staphylococcus aureus (MRSA) in the pathogenesis of community-acquired<br />

infective endocarditis in intravenous drug users, it is suggested that an<br />

antimicrobial agent with acceptable activity against such organisms (like<br />

vancomycin) included in the empirical treatment of infective endocarditis in<br />

this group of patients.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PP 04<br />

G-Streptococcal IgG-Binding Molecules Have Different<br />

Impact on Opsonization by C1q<br />

D.P. Nitsche-Schmitz *1 , S. Reißmann 1 , H. Linge 2 , I. Sastalla 1 , I.M. Frick 2 , G.S.<br />

Chhatwal 1<br />

1<br />

Mikrobielle Pathogenität, Helmholtz-Zentrum für Infektionsforschung,<br />

Braunschweig, Germany<br />

2<br />

Dept. of Clinical Sciences, BMC, Lund University, Lund, Sweden<br />

Beta-hemolytic streptococci belonging to Lancefield group C and G (GCS,<br />

GGS) are human pathogens of emerging epidemiological importance. They<br />

cause pharyngitis and similar skin and soft tissue manifestations as group A<br />

streptococci, occasionally resulting in life threatening conditions such as sepsis<br />

and necrotizing fasciitis. Recent epidemiological data on diseases caused by<br />

GCS and GGS un<strong>der</strong>line that they are an emerging threat to human health.<br />

Among various virulence factors expressed by GCS and GGS isolates from<br />

human infections, M- and M-like proteins are consi<strong>der</strong>ed important because of<br />

their anti-phagocytic activity. In addition, protein G has been implicated in the<br />

accumulation of IgG on the bacterial surface through non-immune binding. The<br />

function of this interaction, however, is still not known.Using isogenic mutants,<br />

lacking protein G or the M-like protein FOG, respectively, we could show that<br />

FOG contributes substantially to IgG-binding. A detailed characterization of the<br />

interaction between IgG and FOG revealed its ability to bind the Fc-region of<br />

human IgG and its binding to the subclasses IgG1, IgG2, and IgG4. FOG was<br />

also found to bind IgG of several animal species. Surface plasmon resonance<br />

measurements indicate a high affinity to human IgG with a dissociation<br />

constant of 2.4 pM. It has long been speculated about anti-opsonic functions of<br />

streptococcal Fc-binding proteins. The presented data for the first time provide<br />

evidences and, furthermore, indicate functional differences between protein G<br />

and FOG. By obstructing the interaction between IgG and C1q, protein G<br />

prevented recognition by the classical pathway of the complement system. In<br />

contrast, IgG that was bound to FOG remained capable of binding C1q, an<br />

effect that may have important consequences in the pathogenesis of GGS<br />

infections.<br />

PP 05<br />

Staphylococcus aureus Panton-Valentine Leukocidin<br />

transcription is linked to the phage life cycle and to the host<br />

chromosomal background<br />

C. Wirtz *1 , C. Wolz 1 , C. Goerke 1<br />

1 Institut für Med. Mikrobiologie und Hygiene, Universitätsklinikum Tübingen,<br />

Tübingen, Germany<br />

Panton-Valentine Leukocidin (PVL) is a pore-forming toxin secreted by<br />

Staphylococcus aureus strains associated with diseases such as necrotizing<br />

pneumonia and skin and soft-tissue infections. Here we demonstrate, that the<br />

transcription of the phage-encoded luk-PV gene is dependent on two major<br />

determinants: the phage life cycle and the host chromosomal background.<br />

Mitomycin C induction of PVL-encoding prophages from different S. aureus<br />

strains led to an increase in the amount of luk-PV mRNA as a result of readthrough<br />

transcription from latent phage promoters and an increase in phage<br />

copy numbers. In one strain (USA300) harbouring a replication defective<br />

prophage a constant expression of luk-PV was observed. Additionally, luk-PV<br />

transcription is influenced by the S. aureus global virulence regulators agr and<br />

sae. A strong impact of the host background on prophage induction and<br />

replication was detected when analyzing PVL phages in different strains. For<br />

example, phage ΦSa2mw was greatly induced in its native host MW2 but to a<br />

consi<strong>der</strong>ably lesser extent in lysogens of 8325-4, RN6390 and ISP479c. This<br />

discrepancy was not due to the presence of a helper phage and was not linked to<br />

differences in the SOS response between the bacteria. These results suggest a<br />

complex interaction between phages and their bacterial host.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

153<br />

PP 06<br />

A Novel Marker Reveals Consi<strong>der</strong>able Contribution of the<br />

Anginosus Group to Group C and Group G Streptococcal<br />

Infections<br />

S. Reißmann 1 , C. Friedrichs 2 , R. Rajkumari 3 , A. Itzek 1 , A.C. Rodloff 2 , K.N.<br />

Bramadathan 3 , G.S. Chhatwal 1 , D.P. Nitsche-Schmitz *1<br />

1<br />

Mikrobielle Pathogenität, Helmholtz-Zentrum für Infektionsforschung,<br />

Braunschweig, Germany<br />

2<br />

Institut für Medizinische Mikrobiologie und Epidemiologie von<br />

Infektionskrankheiten, Universität Leipzig, Leipzig, Germany<br />

3<br />

Dept. of Clinical Microbiology, Christian Medical College, Vellore, India<br />

Streptococci of Lancefield group C and G (GCS, GGS) cause a spectrum of<br />

diseases, which is similar to that caused by S. pyogenes, a group A<br />

streptococcus. The predominant species in human infections with GCS and<br />

GGS is S. dysgalactiae equisimilis. However, there are indications for a<br />

significant contribution of the anginosus group in human GCS and GGS<br />

infections, for instance in acute pharyngitis. Streptococci of the anginosus<br />

group (S. anginosus, S. constellatus, S. intermedius), which were formerly also<br />

referred to as S. milleri, are associated with purulent infections and severe<br />

abscess formation in the deep neck and in inner organs. These species exhibit a<br />

certain phenotypic as well as immunogenic diversity. Although the majority of<br />

isolates is non-beta-hemolytic, there are beta-hemolytic strains of each of the<br />

three species. If they carry a group antigen it belongs to group A, C, G, or F.<br />

During routine diagnostic that is normally restricted to determination of the<br />

type of hemolysis and of the Lancefield group, it is very likely that betahemolytic<br />

strains of the anginosus group may be erroneously typed as S.<br />

pyogenes or S. dysgalactiae equisimilis, respectively. Identification to the<br />

species level is rarely carried out and consequently our insight into the true<br />

epidemiology of infections with streptococci of the anginosus group remains<br />

restricted. The presented work describes a reliable marker-PCR for easy<br />

identification of anginosus isolates. Applying the marker we observe a<br />

consi<strong>der</strong>able epidemiological role of anginosus streptococci in clinical cases of<br />

group C- and group G streptococcal infections in Vellore (India); a finding that<br />

supports the notion of an un<strong>der</strong>rated pathogenic potential and clinical relevance<br />

of the anginosus group.<br />

PP 07<br />

Studying the Distribution of Virulence Factor Genes within<br />

the Oral Streptococci by Means of a DNA-Microarray<br />

A. Itzek 1 , C. Friedrichs 2 , S. Reißmann 1 , A.C. Rodloff 2 , G.S. Chhatwal 1 , D.P.<br />

Nitsche-Schmitz *1<br />

1<br />

Mikrobielle Pathogenität, Helmholtz-Zentrum für Infektionsforschung,<br />

Braunschweig, Germany<br />

2<br />

Institut für Medizinische Mikrobiologie und Epidemiologie von<br />

Infektionskrankheiten, Universität Leipzig, Leipzig, Germany<br />

Oral streptococcus is an umbrella term for streptococci of different species that<br />

colonize the human oral cavity. Many of these species are consi<strong>der</strong>ed as<br />

naturally competent and it is assumed that the interspecies exchange of genetic<br />

material is high. Although <strong>bei</strong>ng commensals oral streptococci bare a<br />

consi<strong>der</strong>able potential to cause severe diseases like bacterial endocarditis. The<br />

virulence mechanisms employed by oral streptococci are, however, only<br />

partially explored. Targeted research on the pathogenesis of oral streptococci<br />

requires a comprehensive view over the distribution of known and potential<br />

virulence factors within the oral streptococci. Aim of the presented work was to<br />

study the distribution of known and hypothetical surface-located streptococcal<br />

virulence factors within clinical isolates of oral streptococci. A DNAmicroarray<br />

was custom-designed to detect known and hypothetical surfacelocated<br />

virulence factors of different streptococcal species.The conducted<br />

DNA-microarray analysis of 50 clinical isolates revealed the distribution of<br />

virulence factor genes within different species of oral streptococci, thereby<br />

shedding light on the extend of their lateral transfer. Continuation of the study<br />

will allow to assess the importance of gene transfer in conversion of commensal<br />

streptococci into pathogens.

154<br />

PP 08<br />

Selectivity of the diarylquinoline TMC207 towards<br />

mycobacterial ATP synthase as compared to its eukaryotic<br />

homologue<br />

A.C. Haagsma *1 , R. Abdillahi Ibrahim 1 , K. Vergauwen 2 , K. Andries 2 , A. Koul 2 ,<br />

H. Lill 1 , D. Bald 1<br />

1<br />

Structural Biology, Department of Molecular Cell Biology, VU University<br />

Amsterdam, Amsterdam, Netherlands<br />

2<br />

Pharmaceutical Research and Development, Johnson & Johnson, Beerse,<br />

Belgium<br />

The diarylquinoline TMC207 efficiently kills Mycobacterium tuberculosis by<br />

specifically inhibiting ATP synthase [1,2]. However, in mitochondria of all<br />

eukaryotic cells a homologous ATP synthase is present. For determination of<br />

TMC207-mediated potential toxicity, the impact of this inhibitor on<br />

mitochondrial targets needs to be known. In the present study we show that the<br />

concentration of TMC207 needed to inhibit human mitochondrial ATP<br />

synthase (IC50 >200 microM) exceeded more than 20,000-fold the<br />

concentration needed to inhibit the mycobacterial ATP synthase (IC50 = 10<br />

nM) [3]. Furthermore, oxygen consumption and ATP synthesis in<br />

mitochondria isolated from mouse liver and bovine heart displayed only very<br />

low sensitivity for TMC207. These results suggest that TMC207 is a very<br />

specific inhibitor of the mycobacterial ATP synthase and may not elicit ATP<br />

synthesis-related toxicity in mammalian cells [3]. This target selectivity makes<br />

TMC207 the first ATP synthase inhibitor in clinical development with the<br />

potential to treat a bacterial infection. Thus, in spite of ATP synthase <strong>bei</strong>ng<br />

highly conserved between prokaryotes and eukaryotes, this enzyme may still<br />

qualify as an attractive antibiotic target.<br />

We will also present results from recent experiments on the role of ATP<br />

synthase in ATP production as opposed to a function in maintenance of the<br />

membrane potential.<br />

[1] Andries et al. (2005) Science 307, 223-7<br />

[2] Koul et al. (2007) Nat. Chem. Biol. 3, 323-4<br />

[3] Haagsma et al., submitted<br />

PP 09<br />

The role of the Cpx two-component system in the invasion<br />

of Salmonella typhimurium – In search of the missing link<br />

between CpxA and HilA<br />

V.S. Müller *1 , T.F. Meyer 2 , S. Hunke 1<br />

1 Institut für Biologie, Humboldt Universität zu Berlin, Berlin, Germany<br />

2 Molekulare Biologie, MPI-IB, Berlin, Germany<br />

The Cpx two-component system plays a crucial role in the envelope stress<br />

response, cell division, biofilm formation and pathogenesis of the food borne<br />

gram-negative pathogen S. typhimurium.<br />

In terms of invasion the precise sensing of activating external signals like pH<br />

shift, increasing osmolarity or misfolded pili subunits is essential for the<br />

pathogen to assign its location, to time the production of virulence factors and<br />

to defend it against host cell response. The Cpx-system consists of the<br />

membrane-anchored sensor kinase CpxA, the response regulator CpxR and the<br />

periplasmatic inhibiting protein CpxP. Un<strong>der</strong> SPI- or mild acetic conditions the<br />

expression of the master regulator of invasive genes HilA is controlled by<br />

CpxA independent of its cognate transcriptional regulator CpxR.<br />

By using "Strep-protein-interaction-experiment (SPINE)" we will elucidate the<br />

"interactome" of CpxA un<strong>der</strong> these growth conditions by in vivo cross-linking<br />

with formaldehyde. The aim is to identify possible new regulatory interaction<br />

partners of CpxA or other unknown crosstalk networks between different<br />

signaling pathways that lead to the activation of HilA by CpxA in the absence<br />

of CpxR. If we are successful in identifying a new interaction partner we will<br />

verify its role in invasion by knockout studies as well as by reconstituting and<br />

characterizing the signaling cascade in proteoliposomes in vitro.<br />

PP 10<br />

Prevalence of Virulence Genes from E. coli O157:H7<br />

Strains with Multiplex PCR among Children Un<strong>der</strong> 5 Years<br />

Old in Marvdasht (Iran)<br />

M. Kargar *1 , M. Homayoon 1 , R. Yaghobi 1 , M. Baghernezhad 1 , R. Ardalan 1<br />

1 Microbiology, Jahrom Azad University, Jahrom, Iran<br />

Introduction and objective: Enterohemorrhagic Escherichia coli (EHEC) strains<br />

are the most common enteric pathogens which cause the hemorrhagic colitis,<br />

hemolytic-uremic syndrome and especially renal failure in children. The<br />

purpose of this study is to survey the prevalence sever diarrhea arising from this<br />

bacteria in children un<strong>der</strong> 5 years old in Marvdasht.<br />

Materials and methods: In this study stool samples of children from four<br />

original areas in Marvdasht are collected and after enrichment in two culture<br />

media ECB, TSB in temperature 37°C, sorbitol fermentation on CT-SMAC<br />

evaluated. Then in sorbitol negative bacteria with the use of specific<br />

biochemical tests E.coli identified. In the next step their β-glucoronidase<br />

activity has been tested on specific chromogenic media.Then with the use of<br />

specific antisera the isolation of bacteria has been confirmed. Finally with<br />

multiplex PCR method presence of virulence genes stx1, stx2, eaeA and hly has<br />

been tested.<br />

Results: Out of 615 children (278girls, 337boys), from 7 children E.coli<br />

O157:H7 isolated (1.14%). A significant difference was seen between isolated<br />

bacteria from age groups 18-23 months and other age groups (P=0.004) and<br />

only 1 case had the stx1 and eaeA genes (0.16%) and none of them had stx2<br />

and hly genes.<br />

Conclusion: Regarding severity of E.coli O157:H7 pathogenesis, low infectious<br />

dose and lack of routin assay for detection of this bacteria in clinical laboratory,<br />

further and completed studies on diagnosis and genotyping of this E.coli<br />

O157:H7 strain has been recommended.<br />

PP 11<br />

Island or no island: phylogeny of GimA involved in<br />

pathogenesis of newborn meningitis caused by E. coli<br />

T. Homeier *1 , L.H. Wieler 1 , C. Ewers 1<br />

1 Institute of Microbiology and Epizootics, Veterinary Faculty, Free University<br />

Berlin, Berlin, Germany<br />

Newborn meningitic E. coli (NMEC) are the most important bacterial cause of<br />

newborn meinigitis. NMEC belong to the group of extraintestinal pathogenic E.<br />

coli (ExPEC). A major factor contributing to the transfer of the brain blood<br />

barrier, a crucial step in pathogenesis, is encoded by ibeA which is located on<br />

the putative genomic island of meningitic E. coli (GimA). In the present study<br />

we analyzed the distribution of GimA in 357 ExPEC strains by MLST,<br />

enabling a phylogenetic analysis. With the exception of a few, all strains could<br />

be assigned to two MLST based clusters. One cluster of closely related strains<br />

included all GimA + strains, while a second cluster containing strains with a<br />

greater diversity exclusively harbored strains lacking GimA. By sequence<br />

analysis of the ibeA gene of 35 ExPEC strains we identified 12 different ibeA<br />

alleles with a rigorous association to the phylogenetic background of the<br />

strains, i.e. strains with an identical sequence type (ST) harbored identical ibeA<br />

sequences.<br />

A detailed analysis (Dn/Ds ratio) of the ibeA sequences of strains belonging to<br />

ST Complex 95 (n=23) did not indicate any positive selection of this gene.<br />

Thus, the evolution of ibeA seems to be exclusively driven by purifying<br />

selection. These findings suggest GimA not to be a genetic island. Although<br />

some criteria for genetic islands are partially fulfilled, GimA rather seems to be<br />

part of the bacterial chromosome of a certain phlyogenetic group of ExPEC,<br />

having evolved analogous the core genome.<br />

PP 12<br />

Aconitase B and its Role in the Pathogenicity of<br />

Xanthomonas campestris pv. vesicatoria<br />

J. Kirchberg *1 , B. Thiemer 1 , D. Büttner 2 , G. Sawers 1<br />

1 Institute of Microbiology, Martin-Luther University, Halle (Saale), Germany<br />

2 Institute of Genetics, Martin-Luther University Halle-Wittenberg, Halle<br />

(Saale), Germany<br />

Aconitase (Acn) is an [4Fe-4S] protein that has two different functions<br />

depending on the cellular iron status. When iron is plentiful the enzyme<br />

possesses a labile [4Fe-4S]-cluster and is functional in the tricarboxylic acid<br />

cycle (TCA). If iron becomes limiting aconitase loses the [4Fe-4S] cluster and<br />

adopts a regulatory function and is referred to as an iron-responsive protein<br />

(IRP). Un<strong>der</strong> these conditions it is able to bind specifically to particular<br />

mRNAs where it has a post-transcriptional role in regulating gene expression.<br />

Aconitases are important factors in controlling the superoxide stress response<br />

and consequently can have a role in pathogenesis. Xanthomonas campestris pv.<br />

vesicatoria is an obligate aerobic ? proteobacterium that causes bacterial spot<br />

disease on pepper and tomato plants. The genome of X. campestris pv.<br />

vesicatoria encodes three aconitases. Two of the aconitases belong to the AcnA<br />

family while the third is a member of the AcnB family. In an attempt to<br />

determine whether the Acn enzymes of X. campestris pv. vesicatoria might be<br />

required for infection we created two independent acnB deletion mutants. Both<br />

mutants exhibited delayed appearance of disease symptoms and reduced growth<br />

in pepper plants. Notably the mutants were unaffected in their growth in in vitro<br />

cultures, indicating that AcnB is important specifically for virulence and<br />

growth in planta.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PP 13<br />

Essential Cell Division Proteins as Antibiotic Targets<br />

A. Rabenau 1 , B. Kutscher 1 , J. Bandow *1<br />

1 Biologie <strong>der</strong> Mikroorganismen, Ruhr-Universität Bochum, Bochum, Germany<br />

Antibiotic-resistant bacteria present a rising challenge no longer just in<br />

nosocomial but also in community-acquired infections. Thanks to the<br />

extraordinary adaptability of bacteria, resistance mechanisms against novel<br />

antibiotics are reported usually within the first few years of FDA approval<br />

necessitating a continuous quest for novel antibiotics. Classical antibiotics<br />

target essential cellular functions such as RNA, DNA, protein, or cell wall<br />

biosynthesis. However, there are a number of other vital cellular functions,<br />

which so far have not been fully evaluated as for their potential to yield new<br />

antibiotic targets. Cell division is such a cellular process, which only recently<br />

has come into focus as attractive antibacterial target [1], despite the fact that<br />

many genes encoding cell division proteins are essential and highly conserved<br />

among bacteria. The most significant progress has been reported for the Z ring<br />

forming protein FtsZ, for which inhibitors have tested effective in an infection<br />

model. In the absence of available inhibitors for many of the other cell division<br />

proteins we are using conditional mutants with repressible cell division genes to<br />

study the bacterial response to loss of function.<br />

[1] D. J. Haydon et al., Science, 321: 1673-5 (2008)<br />

PP 14<br />

Involvement of the agr-based Quorum sensing for virulence<br />

expression in Listeria monocytogenes EGDe<br />

M. Waidmann *1 , I.R. Monk 2 , C. Hill 2 , P. Walther 3 , B.J. Eikmanns 1 , C.U.<br />

Riedel 1<br />

1 Institute of Mikrobiology und Biotechnology, Universität Ulm, Ulm, Germany<br />

2 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland<br />

3 Central Facilit y of Electron Microscopy, Universität Ulm, Ulm, Germany<br />

The establishment of the intracellular lifecycle of Listeria monocytogenes<br />

depends on the activity of several virulence factors. An operon homologous to<br />

the staphylococcal accessory gene regulator (agr) locus which encodes a<br />

peptide quorum sensing system has been identified in L. monocytogenes and<br />

analysed for its role in virulence.<br />

A clean deletion in the agrD gene, encoding for the putative QS peptide,<br />

showed an attenuated virulence in Balb/c mice. Furthermore, the invasion of<br />

confluent monolayers of the enterocyte-like cell line Caco-2 was reduced 4fold.<br />

By contrast, the uptake by activated THP-1 monocytes was not affected by<br />

the agrD deletion. This prompted us to analyze the expression of internalin A<br />

(InlA), a virulence factor responsible for the internalization of L.<br />

monocytogenes into a variety of non-professional phagocytes including Caco-2.<br />

SDS-PAGE and Western blot revealed a decreased expression of InlA in the<br />

mutant cell wall. Moreover, reporter gene assays revealed a markedly decreased<br />

expression of inlA in vitro. These reporter gene assays also showed an impaired<br />

expression of additional virulence factors required for intracellular<br />

pathogenesis. We used electron microscopy to detect alterations in the<br />

intracellular location and bacterial enumeration in randomly chosen<br />

micrographs showed the ∆agrD-strain more often trapped in compartments<br />

looking similar to the recently described spacious Listeria-containing<br />

phagosomes (SLAPs). The decreased activity of the hlyA promoter and the<br />

formation of these SLAPs suggests an impaired expression of Listeriolysin O.<br />

This indicates that in L. monocytogenes EGDe, AgrD-dependent QS is involved<br />

in the regulation of virulence gene expression.<br />

PP 15<br />

Effect of the repeat region of protein A (SpA) of<br />

Staphylococcus aureus on the induction of proinflammatory<br />

responses<br />

S. Brüning 1 , C. Neumann *1 , C. Baum 1 , B.C. Kahl 1<br />

1 Institut of Medical Microbiology, University Hospital Münster, Münster,<br />

Germany<br />

Staphylococcus aureus is a major human pathogen causing a wide spectrum of<br />

diseases from minor disor<strong>der</strong>s to severe life-threatening infections. The<br />

bacterium produces a large number of virulence factors, which are required for<br />

the induction of infection. Among them protein A (spa) is a complex virulence<br />

factor with several, highly diverse functions. SpA interferes with opsonization<br />

by binding to the Fc region of IgG, thereby attenuating phagocytosis. In the<br />

lung, SpA acts as a staphylococcal proinflammatory factor by activating a<br />

receptor for tumor-necrosis factor α (TNFR1), which induces a TNF-α-like<br />

response. Next to the IgG binding domains, SpA possesses a polymorphic<br />

region consisting of variable numbers of 21 – 27 bp repeats, which is used for<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

molecular typing. Recently, it was shown that during persistence of S. aureus in<br />

the lung of cystic fibrosis patients, mutations occurred within the polymorphic<br />

region mostly due to deletions of repeats. We hypothesize, that the induction of<br />

a proinflammatory response is associated to the number of repeats of the<br />

polymorphic region. To investigate this, we recombinantly expressed SpAs<br />

with variable numbers of repeats (n = 0-11) in E. coli. These recombinant<br />

proteins will be used to perform ELISA and FACS assays to study the<br />

proinflammary response of epithelial cells and monocytes. Furthermore, we<br />

will heterologously express the constructs in Staphylococcus carnosus TM300,<br />

an apathogenic coagulase-negative strain, that lacks protein A and most of the<br />

virulence factors of S. aureus. This will allow us to investigate the<br />

proinflammatory effect of increasing repeat numbers of SpA on epithelial cells<br />

and monocytes.<br />

PP 16<br />

Identification of virulence markers and pathogenic effects<br />

of Aeromonas culicicola/veronii<br />

A. Rangrez *1 , H. Kumar 2 , Y. Shouche 2 , E. Grohmann 1<br />

1 Department of Environmental microbiology, Technical university of Berlin,<br />

Berlin, Germany<br />

2 Molecular Biology Unit, National Centre for Cell Science, Pune, India<br />

155<br />

Aeromonas culicicola/veronii is a Gram-negative, rod shaped, hemolytic<br />

bacterium isolated for the first time from mosquito mid-gut but subsequently<br />

detected in other insects and water also. On the basis of previous studies on<br />

Aeromonas, an attempt was made to identify and characterize virulence factors<br />

in A. culicicola/veronii. It was found that A. culicicola/veronii harbor three<br />

uncharacterized plasmids and partial sequencing of one of the plasmids<br />

revealed the presence of a type IV secretion (TFSS) system. An operon coding<br />

for TFSS was completely sequenced and further characterization is un<strong>der</strong><br />

process. The main focus is on characterization of the relaxase by in vitro<br />

cleavage assay and exact localization of oriT region by run-off DNA synthesis<br />

assay. Antibiotic susceptibility test showed resistance of A. culicicola/veronii to<br />

many antibiotics (carbenicillin, metronidazole, cephoxitin etc) which was then<br />

correlated with the presence of multidrug efflux protein (MEP). We could also<br />

determine the presence of a type III secretion system (TTSS) by PCR and<br />

hybridization studies using the ascV gene (one of the important TTSS genes) as<br />

a probe. Cytotoxic effect of culture supernatant was tested on mammalian Vero,<br />

CHO and Hep2 cells and the adhesion property of the bacterium was tested on<br />

Hep2 cells by Confocal Laser Scanning Microscopy. We also checked the<br />

virulence property of this bacterium on Hep2 cells using FACS analysis and<br />

found that the bacterium leads to apoptosis or necrosis. Further characterization<br />

of secretion systems identified would reveal the probable role of the same in<br />

virulence.<br />

PP 17<br />

Murein biosynthesis in staphylococci<br />

S. Heinrich *1 , D. Kühner 1 , M. Schumacher 2 , U. Bertsche 1<br />

1 Mikrobielle Genetik, Universität Tübingen, Tübingen, Germany<br />

2 Friedrich-Miescher-Laboratorium für biologische Ar<strong>bei</strong>tsgruppen in <strong>der</strong> Max-<br />

Planck-Gesellschaft, MPI Tübingen, Tübingen, Germany<br />

The murein or peptidoglycan sacculus is the stress bearing layer of bacterial<br />

cells, which consists of glycan strands cross-linked by peptides. Lots of clinical<br />

resistant bacteria like vancomycin and methicillin resistant Staphylococcus<br />

aureus (VRSA and MRSA) strains show major changes in murein biosynthesis,<br />

a major target for antibacterial treatment. During growth and division of<br />

coccoid cells like S. aureus the murein network has to be divided without losing<br />

its strength. So far little is known about murein biosynthesis and the roles of the<br />

involved proteins in coccoid Gram-positive bacteria.<br />

In our studies we elucidate the activities and interactions of the proteins during<br />

murein biosynthesis in staphylococci. The involved murein synthases are<br />

mainly penicillin-binding proteins (PBPs), the main murein hydrolase is the<br />

major autolysin Atl. Functional interactions between the PBPs have already<br />

been proposed. We want to search for involved protein-protein interactions by<br />

different in vivo and in vitro methods. Our model organism is S. carnosus, an<br />

apathogenic staphylococcus lacking immunoglobulin-binding proteins that<br />

would interfere with our experiments. We are currently purifying all known<br />

PBPs auf S. carnosus as well as PBP2A of S. aureus that is responsible for<br />

methicillin resistance. Protein-protein interactions will be investigated by in<br />

vivo chemical cross-linking followed by co-immunoprecipitation with specific<br />

antibodies [Bertsche et al., 2006] as well as with other established methods. An<br />

in vitro murein synthesis assay established for E. coli [Bertsche et al., 2005]<br />

will be applied to analyse the enzymatic reactions of individual PBPs and of<br />

different PBPs in combination.

156<br />

PP 18<br />

Secretome analysis of the human pathogenic fungus<br />

Aspergillus fumigatus<br />

D. Wartenberg *1 , O. Kniemeyer 1 , E. Shelest 1 , T. Heinekamp 1 , J. Teutsch<strong>bei</strong>n 1 ,<br />

R. Winkler 2 , A.A. Brakhage 1<br />

1 Leibniz Institute for Natural Product Research and Infection Biology e.V.,<br />

Hans-Knöll-Institute, Jena, Friedrich Schiller University of Jena, Jena,<br />

Germany<br />

2 Tecnologico de Monterrey, Departamento de Biotecnologia e Ing. de<br />

Alimentos, Monterrey, Mexico<br />

In many cases, secreted proteins of pathogenic microorganisms play an<br />

important role for its virulence. For Aspergillus fumigatus, the most prevalent<br />

airborne-pathogenic fungus, only little information about secreted proteins and<br />

their contribution to virulence is available. For this reason we started a largescale<br />

approach to identify the secretome of Aspergillus fumigatus un<strong>der</strong> in vitro<br />

conditions.<br />

We analysed the secretome using a bioinformatic as well as a proteomic<br />

approach. We were interested in the proteins that are secreted during growth on<br />

a highly utilisable substrate. Thus, A. fumigatus was grown in Aspergillus<br />

Minimal Medium (AMM) with 1% glucose as single carbon and energy source<br />

at 37°C as shaking culture. The incubation was stopped at three different time<br />

points (24, 48 and 72 hours). The extracellular proteins were precipitated from<br />

the supernatant using 10% trichloracetic acid and separated by 2-D gel<br />

electrophoresis. Protein spots were excised, tryptically digested and identified<br />

by MALDI-TOF-MS/MS.<br />

In addition we carried out immunoproteomic analysis using human sera of<br />

patients with probable invasive aspergillosis to elucidate immunogenic<br />

extracellular proteins of A. fumigatus.<br />

About 80 different proteins could be detected from which approximately 45%<br />

were predicted as secreted proteins by bioinformatic analysis. Proteases and<br />

proteins involved in cell wall metabolism were most abundant.<br />

PP 19<br />

Molecular mechanisms involved in Staphylococcus<br />

epi<strong>der</strong>midis biofilm detachment<br />

I. Bleiziffer *1 , F. Götz 2 , G. Peters 1 , C. Heilmann 1<br />

1 Institute of Medical Microbiology, University Hospital Münster, Münster,<br />

Germany<br />

2 Institute of Microbial Genetics, University of Tübingen, Tübingen, Germany<br />

Staphylococcus epi<strong>der</strong>midis, an important cause of nosocomial infection, is a<br />

common inhabitant of human skin and mucous membranes. Its ability to form<br />

biofilms on surfaces is consi<strong>der</strong>ed its major pathogenicity factor. Following<br />

formation and maturation of the biofilm, bacterial cells can detach from the<br />

biofilm and seed to new sites. The aim of this study is to characterize the<br />

molecular mechanisms involved in S. epi<strong>der</strong>midis biofilm detachment that is<br />

thought to play a major role in the development of sepsis.<br />

We performed transposon (Tn917) mutagenesis of the clinical isolate S.<br />

epi<strong>der</strong>midis O-47 and screened 5000 mutants for altered detachment behaviour<br />

by using a semi-quantitative biofilm formation assay. For the screening, the<br />

biofilms of the mutants were quantified in duplicates after 24h, 72h, 144h, and<br />

168h of growth in microtiter plates.<br />

Five mutants with different detachment behaviour were detected that can be<br />

divided into two classes. Biofilms of class1 mutants (mut1, mut2, and mut5)<br />

were more persistent than the biofilms formed by the wild type when grown in<br />

microtiter plates. In contrast, biofilms produced by class2 mutants (mut3 and<br />

mut4) detached significantly earlier than wild-type biofilms. Arbitrary PCR<br />

revealed that the transposon is inserted in genes of the urea cycle in mut2 and<br />

mut5 and between two genes of the glyoxylat cycle in mut4.<br />

In conclusion we identified at least three different genetic loci putatively<br />

involved in S. epi<strong>der</strong>midis biofilm detachment or its regulation. Further analysis<br />

are necessary to characterize the un<strong>der</strong>lying mechanisms.<br />

PP 20<br />

The global redox sensing regulator Rex: Transcriptomic<br />

analysis of a Staphylococcus aureus mutant<br />

J. Seggewiß *1 , G. San<strong>der</strong> 1 , R.A. Proctor 2 , G. Peters 1 , K. Becker 2 , C. von Eiff 1<br />

1 Institute of Medical Microbiology, University of Muenster, Muenster, Germany<br />

2 Department of Medical Microbiology and Immunology, University of<br />

Wisconsin, School of Medicine and Public Health, Madison, United States<br />

Staphylococcus aureus small-colony variants (SCVs) are a naturally occurring,<br />

slow-growing subpopulation with distinctive phenotypic characteristics and<br />

pathogenic traits. Many reports support a pathogenic role for SCVs in patients<br />

with persistent and/or recurrent infections. Multiple features of SCVs are<br />

similar to anaerobically grown S. aureus. Sequence analyses led to the<br />

hypothesis that a regulator protein called Rex (redox sensing regulator) senses<br />

oxygen limitation by responding to high levels of NADH. In this study, fullgenome<br />

DNA microarrays were used to analyze the transcriptome of a ∆rex<br />

mutant. Comparing the expression profile of the ∆rex mutant to that of its<br />

parental strain at different time points, the accessory gene regulator (agr), genes<br />

for capsular polysaccharide synthesis (cap5A, cap5B, cap5D, cap8E), a gene of<br />

a fibronectin-binding protein homologue, and genes of different Na+/H+<br />

antiporters were found to be significantly down-regulated. In contrast, genes for<br />

a cell division and morphogenesis-related protein (scdA), superoxide dismutase<br />

(sodM), the immunodominant antigen B (isaB), L-lactate dehydrogenase (lctE),<br />

a formate acetyltransferase activating enzyme (SA0219) and for the alcoholacetaldehyde<br />

dehydrogenase (adhE) were shown to be significantly upregulated.<br />

The large overlap between the Rex regulon and the transcriptome<br />

analyzed in a hemB mutant displaying the phenotype of clinical SCVs suggest<br />

that at least part of the SCV phenotype may arise from changes in Rex<br />

regulation of the anaerobic regulon.<br />

PP 21<br />

Characterization of D- and L-serine-deaminases from<br />

Staphylococcus saprophyticus<br />

M. Korte *1 , L. Marlinghaus 1 , S.G. Gatermann 1 , T. Sakinc 1<br />

1 Institut für Hygiene und Medizinische Mikrobiologie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

Staphylococcus saprophyticus is the only species of the staphylococci that is<br />

uropathogenic. Several surface factors that contribute to virulence have already<br />

been identified. In addition, it has been suggested that certain metabolic<br />

activities contribute to virulence, mainly the uncommon ability to degrade Dserine.<br />

This amino acid is present in relatively high concentrations in human<br />

urine and is toxic to several non-uropathogenic bacteria. Uropathogenic E. coli<br />

(UPEC) express a D-serine- deaminase (DsdA), which converts D-serine to<br />

pyruvate and ammonia. Therefore they are able to detoxify D-serine and use it<br />

as a nutrient source. Also the genome of S. saprophyticus contains a gene<br />

coding for a D-serine-deaminase. It was shown that S. saprophyticus grows in<br />

media supplemented with D-serine whereas other staphylococci, lacking the<br />

gene for the D-serine deaminase, like S. aureus, S. epi<strong>der</strong>minis or S. carnosus,<br />

do not. The dsdA gene was cloned in S. carnosus and enabled this strain to<br />

grow in media with D-serine. Interestingly, a dsdA-Knock-out-mutant of S.<br />

saprophyticus has the ability to grow with D-serine. Therefore there must be<br />

another enzyme which can detoxify D-serine. In S. saprophyticus, two genes<br />

for putative L-serine-deaminases exist. In E. coli, a L-serine-deaminase also<br />

utilizes D-serine.<br />

Our aim is to elucidate the D-serine metabolism and its relevance for the<br />

physiology of virulence of S. saprophyticus. For this purpose a biochemical<br />

characterization of the D- and L-serine-deaminases is essential, therefore we<br />

expressed the enzymes in E. coli as His-tag fusionproteins.<br />

PP 22<br />

How are cytoplasmic proteins translocated in staphylococci<br />

through the cytoplasmic membrane?<br />

L. Pasztor *1 , S. Haase 1 , A. Resch 1 , A.K. Ziebandt 1 , F. Götz 1<br />

1 Microbial Genetics, University Tuebingen, Tuebingen, Germany<br />

The virulence and persistence of Staphylococcus aureus largely depends on<br />

secreted proteins. It is therefore of great interest to analyze which proteins are<br />

found in the culture supernatant of the bacteria. When we analyzed the secreted<br />

proteins of Staphylococcus aureus SA113 by 2D-PAGE and ESI-MS/MS we<br />

found a lot of typical cytoplasmic proteins like glycerinaldehyd-3-phosphatdehydrogenase<br />

(GAP-DH) or elongation factor EF-Tu in the supernatant. After<br />

48 hours growth we found even more cytoplasmic proteins. The question is<br />

how these proteins that do not contain a signal peptide (SP) are released to the<br />

medium: by simple cell lysis or by a special SP-independent translocation<br />

system? Cell lysis can be caused by prophage-induction or autolysins.<br />

However, corresponding mutants where all prophages were eliminated or where<br />

the major autolysin gene was deleted showed the same high content of<br />

cytoplasmic proteins than the wild type. However, a mutant affected in one of<br />

the competence genes (com) revealed a significant decrease of cytoplasmic<br />

proteins in the external milieu. While cytoplasmic proteins were decreased in<br />

the com-mutant specific Sec-dependent proteins like lipase or the gammahemolysin<br />

were increased compared to the wild type. This work shows for the<br />

first time that in staphylococci cytoplasmic proteins are not only released by<br />

simple cell lysis but also by the competence system. Furthermore, the Comsystem<br />

is not only involved in secretion of cytoplasmic proteins, it also<br />

enhances the secretion of some Sec-dependent exo-proteins.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PP 23<br />

A small toxin gene tisB is involved in the formation of<br />

multidrug tolerant persister cells in response to a DNA<br />

damaging antibiotic<br />

T. Dörr *1 , M. Vulic 1 , K. Lewis 1<br />

1<br />

Antimicrobial Discovery Center, Northeastern University, Boston, United<br />

States<br />

Bacterial populations produce persister cells that are resistant to killing by all<br />

antibiotics currently in use, a phenomenon known as multidrug tolerance<br />

(MDT). Persisters are phenotypic variants of the wild type and are largely<br />

responsible for MDT of biofilms and stationary populations. We have<br />

previously shown that expression of toxin –antitoxin (TA) modules is<br />

upregulated in isolated persisters and that artificial overexpression of toxins can<br />

induce the persister state. In the present study, we focused on a TA module<br />

tisAB in Escherichia coli that is inducible by the SOS response and thus<br />

possibly induced by DNA damaging antibiotics, providing a link between<br />

environmental stress and persister formation.<br />

Using promoter(tisAB)-gfp fusions we show that tisAB promoter activity is<br />

induced 1000fold by bactericidal concentrations of ciprofloxacin, a<br />

fluoroquinolone antibiotic inducing DNA damage.<br />

A knockout of the tisAB locus leads to a 10- to 100-fold decrease of persisters<br />

in exponential growth phase in response to ciprofloxacin while a knockout of<br />

the corresponding antitioxin istr-1 leads to a 10 to 100fold increase of<br />

persisters. Similarly, a knockout of the tisAB locus in the clinical isolate E. coli<br />

0157:H7 Sakai lead to a 10fold decrease of persisters in colony biofilms<br />

challenged with ciprofloxacin indicating potential clinical relevance of this<br />

locus.<br />

Inducible overexpression of TisB from a low copy number vector increased<br />

tolerance against ampicillin, ciprofloxacin and streptomycin 100- to 1000-fold<br />

relative to the empty vector control.<br />

Combined, these results indicate that DNA damaging antibiotics induce<br />

persister formation by activating the SOS responsive TisB toxin.<br />

PP 24<br />

Adaptation of Pseudomonas aeruginosa to various<br />

conditions includes tRNA-dependent formation of alanylphosphatidylglycerol<br />

S. Klein *1 , C. Lorenzo 2 , S. Jäger 1 , J.M. Walther 1 , S. Storbeck 1 , T. Piekarski 1 ,<br />

B.J. Tindall 3 , V. Wray 4 , M. Nimtz 4 , J. Moser 1<br />

1 Institute of Microbiology, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

2 Physiology of Microorganisms, Ruhr-Universität Bochum, Bochum, Germany<br />

3 Microbiology, German Collection of Microorganisms and Cell Cultures<br />

(DSMZ), Braunschweig, Germany<br />

4 Department of Structural Biology, Helmholtz Centre for Infection Research<br />

(HZI), Braunschweig, Germany<br />

The opportunistic bacterium Pseudomonas aeruginosa synthesizes significant<br />

amounts of an additional phospholipid, identified as 2’ alanylphosphatidylglycerol<br />

(A-PG), when exposed to acidic growth conditions. At pH<br />

5.3 A-PG contributed up to 6% to the overall lipid content of the bacterium.<br />

Sequence analysis of P. aeruginosa revealed open reading frame PA0920<br />

showing 34% sequence identity to a protein from Staphylococcus aureus<br />

involved in tRNA-dependent formation of lysyl-phosphatidylglycerol. The P.<br />

aeruginosa deletion mutant ΔPA0920 failed to synthesize A-PG. Heterologous<br />

overproduction of PA0920 in Escherichia coli resulted in the formation of<br />

significant amounts of A-PG, otherwise not synthesized by E. coli.<br />

Consequently, the protein encoded by PA0920 was named A-PG synthase. The<br />

enzyme was identified as an integral component of the inner membrane. The<br />

protein was partially purified by detergent solubilization and subjected to an in<br />

vitro activity assay. tRNA Ala -dependent catalysis was demonstrated.<br />

Transcriptional analysis of the corresponding gene in P. aeruginosa using lacZ<br />

reporter gene fusion un<strong>der</strong> various pH conditions indicated a 4.4-fold acidactivated<br />

transcription. A phenotype microarray analysis was used to identify<br />

further conditions for A-PG function.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PP 25<br />

Host-specific colonization of enteropathogenic Yersinia spp.<br />

F. Uliczka *1 , T. Stolz 2 , P. Dersch 3<br />

1<br />

Institut für Mikrobiologie, Technische Universität, Braunschweig, Germany<br />

2<br />

Molecular Infection Biology, Helmholtz Centre for Infection Research,<br />

Braunschweig, Germany<br />

Yersinia enterocolitica and Yersinia pseudotuberculosis are responsible for up<br />

to 6.000 – 7.000 cases of gastrointestinal diseases in Germany per year. Most<br />

infections are caused by the virulent Y. enterocolitica serotypes O:3, O:5,27,<br />

O:8 and O:9 and Y. pseudotuberculosis Typ I, Typ III. Slaugthered pigs are<br />

known to be the most important reservoirs of human Yersinia infections, and<br />

outbreaks of Yersinioses are most frequently associated with consumption of<br />

pork. Although both enteropathogenic Yersinia species can efficiently colonize<br />

the porcine intestinal tract, pigs usually develop no disease symptoms and<br />

remain clinically healthy. To gain more information about the individual<br />

virulence determinants that contribute to host-specificity, we investigate the<br />

ability of clinical isolates to infect different animal and human cells in vitro.<br />

We show that the invasiveness of clinical Y. enterocolitica isolates is serotype<br />

dependent. Only isolates of serotype O:5,27, O:8 and O:9 can efficiently adhere<br />

to and invade in human and porcine epithelial cell lines.<br />

The analysis of the Y. enterocolitica and Y. pseudotuberculosis genomes further<br />

revealed that both Yersinia species encode additional putative adhesion factors,<br />

which might be crucial for host- and tissue-specific interactions. To investigate<br />

the influence of these putative colonization determinants, we overexpressed<br />

these factors in Escherichia coli and demonstrate that some of them mediate<br />

adhesion to or invasion into human and porcine epithelial cell lines.<br />

Furthermore, the expression of the relevant genes in both Yersinia species<br />

un<strong>der</strong> different environmental conditions could be determined.<br />

PP 26<br />

Temperature and growth phase dependent regulation of the<br />

global virulence regulator RovA in Yersinia<br />

pseudotuberculosis<br />

K. Herbst *1 , P. Dersch 2<br />

1 Institut für Mikrobiologie, TU Braunschweig, Braunschweig, Germany<br />

2 Molecular Infectionbiology, Helmholtz-Zentrum für Infektionsforschung<br />

GmbH, Braunschweig, Germany<br />

Yersinia pseudotuberculosis is a food-borne human pathogen which causes a<br />

variety of intestinal and extraintestinal diseases. During the infection process<br />

the bacteria invade through the M-cells of the epithelial layer of the ileum and<br />

colonize the Peyer`s patches. These step is primarily mediated through the outer<br />

membrane protein invasin.<br />

Expression of the inv gene is regulated by the global transcription regulator<br />

RovA. RovA is subject to autoregulation and regulation via a complex network,<br />

including the LysR-Type regulator RovM, the Csr-System and the nucleoidassociated<br />

proteins H-NS and YmoA. Recent studies revealed, that RovA<br />

regulation is strongly dependent on temperature and growth phase and occurs<br />

on a post-transcriptional level. We could show, that RovA is a thermosensor<br />

and changes its conformation by a temperature upshift from 25°C to 37°C,<br />

abolishing the DNA-binding capacity of the protein. Furthermore we found out,<br />

that RovA is degraded by the bacterial ATP-dependent proteases Lon and ClpP<br />

at 37°C in exponential growth phase. The Lon recognition sequence whitin the<br />

virulence regulator is located in the vicinity of the central DNA-binding<br />

domain, indicating that proteolysis is in direct competition with temperature<br />

dependent DNA-binding of the protein. This makes RovA the first regulatory<br />

protein where temperature sensing is linked to regulatory proteolysis.<br />

PP 27<br />

Evaluation of real-time PCR for rapid quantification of<br />

mycobacteria in metal working fluids (MWF)<br />

D. Rasche 1 , K. Fallschissel 1 , J. Schäfer 2 , N. Lod<strong>der</strong>s *1 , P. Kämpfer 1<br />

1<br />

Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen,<br />

Giessen, Germany<br />

2<br />

Gruppe 4.7 Biologische Ar<strong>bei</strong>tsstoffe, Bundesanstalt für Ar<strong>bei</strong>tsschutz und<br />

Ar<strong>bei</strong>tsmedizin, Berlin, Berlin, Germany<br />

157<br />

In recent years, the detected cases of respiratory diseases associated with metal<br />

working fluids (MWF) have increased, assuming mycobacteria as causative<br />

agents. Since symptoms occur after long-time exposure to high mycobacterial<br />

load, continuous monitoring of these organisms is necessary.<br />

In this study, quantification was performed by real-time PCR with a<br />

Mycobacterium-specific primer system described by Khan and Yadav (2004).<br />

The suitability of the method was tested for eight strains belonging to different

158<br />

Mycobacterium species. Six DNA-extraction protocols were evaluated and the<br />

DNA-extraction efficiency was calculated comprising total cell count<br />

determined by SYBRGreenI-staining after formaldehyde-treatment.<br />

GenElute Plant Genomic Miniprep-Kit showed the best results with pure<br />

cultures, whereas direct extraction of mycobacterial DNA from MWF was most<br />

successful using the Bactozol-Kit modified by Khan and Yadav (2004) with<br />

an initial mechanical cell lysis.<br />

Quantification of mycobacterial hsp genes from bacterial DNA extracted from<br />

MWF by real-time PCR showed different recovery rates depending on the<br />

species used as quantification standard. Best results were obtained with<br />

Mycobacterium abscessus (DSM 44196) as standard. Therefore, it was applied<br />

for quantification of mycobacteria in MWF. The concentration of hsp genetargets<br />

found in MWF ranged between 1.15x10 2 and 3.48x10 3 targets ml -1 .<br />

Because the DNA-extraction efficiency of the analysed mycobacteria was about<br />

10%, concentrations in the analysed MWF should be tenfold higher.<br />

The results show that real-time PCR, as described in this study, is a suitable<br />

method for quantification of mycobacteria in MWF. Nevertheless, it is crucial<br />

to develop an improved DNA-extraction method for obtaining mycobacterial<br />

DNA from MWF.<br />

PP 28<br />

Effect of antibiotic stress on the transposition frequency of<br />

IS256r in Staphylococcus aureus<br />

M. Nagel *1 , G. Bierbaum 1<br />

1 Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),<br />

University of Bonn, Bonn, Germany<br />

Infections with methicillin resistant Staphylococcus aureus (MRSA) have often<br />

been treated with vancomycin in the last three decades, however therapeutic<br />

failure caused by intermediate resistance against vancomycin is emerging and is<br />

caused by the accumulation of mutations, each of which confers an incremental<br />

increase in resistance. Interestingly, the presence of antibiotics themselves<br />

creates an environmental stress, that may lead to an induction of mutational<br />

mechanisms in bacteria. In this context, the bacterial SOS response, which is<br />

induced by double strand breaks and failure of DNA replication, seems to be<br />

involved. In E. coli, the SOS response is known to trigger delocalisation of IS<br />

elements and to favour expression of error-prone polymerases. Both<br />

mechanisms may contribute to the development of antibiotic resistance in S.<br />

aureus.<br />

We have studied the resistance mechanisms of an intermediately vancomycin<br />

resistant S. aureus (VISA) strain pair. In the strain, that displays higher<br />

resistance, a part of the decreased susceptibility to vancomycin is caused by<br />

insertion of IS256 into the gene tcaA. In or<strong>der</strong> to test whether the presence of<br />

antibiotics leads to the mobilisation of IS elements in S. aureus, a test system<br />

that measures the transposition frequency of IS256 is employed (Valle et al.,<br />

2007). This system comprises an IS256 element that is tagged with an<br />

erythromycin marker, named IS256r, and its non-transposable mutant.<br />

Treatment with subinhibitory concentrations of clinically relevant antibiotics<br />

(ciprofloxacin and vancomycin) resulted in an up to 6-fold increased<br />

transposition frequency of IS256r in S. aureus RN1-HG in the presence of<br />

ciprofloxacin.<br />

In conclusion, the transposition of IS256 is involved in resistance development,<br />

and there seems to be a correlation between antibiotic stress and mobilisation of<br />

this IS element.<br />

PP 29<br />

A novel subtilisin-like autotransporter: SprS of<br />

Pseudomonas aeruginosa<br />

S. Serci *1 , F. Rosenau 1 , S. Wilhelm 1<br />

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University<br />

Duesseldorf at Forschungszentrum Juelich, Juelich, Germany<br />

P. aeruginosa is a Gram-negative bacterium, which has become increasingly<br />

recognized as an emerging opportunistic pathogen of clinical relevance.<br />

In many Gram-negative organisms autotransporter proteins with a diverse array<br />

of N-terminal functional domains have been reported (e.g., Bordetella<br />

pertussis, Heamophilus influenza and Neisseria meningitidis). These proteins<br />

typically reveal virulence-associated functions such as adhesion, serum<br />

resistance, cytotoxicity and proteolysis. Here we introduce a novel<br />

autotransporter of P. aeruginosa which we have called SprS. The deduced<br />

amino acid sequence showed homology to several subtilisin-like<br />

autotransporter proteins of pathogenic bacteria like SphB1 from Bordetella<br />

pertussis and NalP from Neisseria meningitides. These proteins are known to<br />

be involved in the proteolytic processing of secreted proteins like the precursor<br />

FHA. Since P. aeruginosa PAO1 also possesses FHA homologues, we<br />

compared the agglutination activity of the wild type to the sprS-negative strain,<br />

in or<strong>der</strong> to see whether SprS might be involved in FHA processing. Previous<br />

studies of the secretome have shown that the sprS-negative strain produces less<br />

virulence associated proteases than the wild type. Therefore we have been<br />

analyzing the promoter activity of SprS and a second subtilase in the sprSnegative<br />

strain. We have shown that both protease genes are up regulated in the<br />

sprS-negative strain. This phenotype can be complemented by addition of sprS<br />

in trans. As a consequence this is an indication of coordinated regulation of<br />

proteases in a network like manner.<br />

PP 30<br />

HPr kinase/phosphorylase and PTS proteins: their role in<br />

Neisseria meningitidis virulence<br />

J. Deutscher *1 , M. Larribe 2 , A.E. Deghmane 2 , A. Mazé 1 , J. Nait-Abdallah 1 ,<br />

M.K. Taha 2 , S. Poncet 1<br />

1 Microbiologie et Genetique Moleculaire, CNRS/INRA/AgroParisTech,<br />

Thiverval-Grignon, France<br />

2 Laboratoire de Neisseria, Centre de Reference des Meningocoques, Institut<br />

Pasteur, Paris, France<br />

Neisseria meningitidis is a commensal bacterium of the human nasopharynx<br />

which, for reasons barely un<strong>der</strong>stood, can become virulent and thereby lead to<br />

septicaemia and meningitis. During the infection process, N. meningitidis is<br />

exposed to various environmental conditions and adaptation to environmental<br />

changes, including sugar availability, is a crucial factor for N. meningitidis<br />

pathogenicity.<br />

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) ensures both<br />

carbohydrate transport and regulation of fundamental cellular processes in<br />

numerous bacteria. In several proteobacteria, genes coding for HPr<br />

kinase/phosphorylase (HprK/P) and an incomplete PTS lacking EIIB and EIIC<br />

components and therefore unable to catalyze sugar transport are located next to<br />

genes involved in regulation of virulence. It was therefore tempting to assume<br />

that PTS proteins might also play a role in pathogenesis.<br />

RT-PCR experiments established that the pts genes in a <strong>der</strong>ivative of the<br />

serogroupe C N. meningitidis strain 8013 are organized in an operon with yhbJ,<br />

which is usually co-localised with the sigma-54-encoding rpoN gene. In vivo<br />

and in vitro experiments established that the proteins of the incomplete N.<br />

meningitidis PTS form a phosphoenolpyruvate-dependent phosphorylation<br />

cascade and that HprK/P can phosphorylate HPr at Ser-46 by using ATP.<br />

Mutations affecting HprK/P and some of the PTS protein-encoding genes had<br />

an influence on N. meningitidis virulence, particularly on cell adhesion,<br />

apoptosis and bacteraemia in mice, probably by altering capsular<br />

polysaccharide production. Most striking was the inverse relation between cell<br />

adhesion and the amount of capsular polysaccharide in the ptsH, hprK and ptsI<br />

mutants. Our results therefore establish that there is a link between HprK/P,<br />

PTS-proteins and virulence in N. meningitidis.<br />

PP 31<br />

Characterisation of the Staphylococcus epi<strong>der</strong>midis<br />

autolysin AtlE<br />

M. Schlag *1 , R. Biswas 2 , S. Zoll 3 , T. Albrecht 1 , R. Gaupp 1 , W. Yu 1 , T. Stehle 3 ,<br />

F. Götz 1<br />

1 Dept. of Microbial Genetics, University of Tübingen, Tübingen, Germany<br />

2 Microbiology and Hygiene Department, University of Tübingen, Tübingen,<br />

Germany<br />

3 Interdisciplinary Institute of Biochemistry, University of Tübingen, Tübingen,<br />

Germany<br />

Peptidoglycan (PG) hydrolases or autolysins are a group of enzymes that<br />

catalyze the cleavage of bacterial murein at specific sites during cell separation<br />

and growth. Staphylococcus epi<strong>der</strong>midis produces two major PG hydrolases:<br />

The major autolysin AtlE, and the autolysin/adhesion protein Aaa. The major<br />

autolysin AtlE is a bifunctional protein that consists of an N-terminal N-acetyl-<br />

L-alanine amidase (Ami) and a C-terminal endo-β-N-acetylglucosaminidase<br />

(GL) linked by three internal repeat domains (R1, 2, 3). Proteolytic processing<br />

of the atlE gene product generates an propeptide with unknown function and<br />

the two extracellular cell wall lytic enzymes (62 kDa Ami-R1,2 and 51 kDa R3-<br />

GL) that can be found in the culture supernatant of S. epi<strong>der</strong>midis. The major<br />

autolysins of S. aureus (AtlA) and S. epi<strong>der</strong>midis (AtlE) are similar in their<br />

domain organisation, and AtlE can compensate the deletion of the major<br />

autolysin in S. aureus. We have purified the amidase domain and the amidase<br />

(Ami) associated repeat domains (R1,2) for X-ray analysis from E. coli. Here<br />

we present the results from the structure analysis of the S. epi<strong>der</strong>midis amidase<br />

domain. We are also interested in the function of the repeat regions. For that we<br />

have studied PG binding and lytic properties of the S. epi<strong>der</strong>midis amidase with<br />

isolated peptidoglycan, heat killed and living cells from different<br />

staphylococcal mutants. Our results show that binding of PG and directing the<br />

amidase towards its site of action is mediated by the repeat domains dependent<br />

of the presence and distribution of teichoic acids in the cell wall.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PP 32<br />

Evaluation of glycosomal peroxins as potential targets for<br />

drug design against African sleeping sickness<br />

J. Wolf *1 , S. Hennig 2 , W. Schliebs 1 , R. Erdmann 1<br />

1<br />

Physiologische Chemie, Abteilung Systembiochemie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

2<br />

Institut für Molekulare Physiologie, MPI Dortmund, Dortmung, Germany<br />

The glycosome is an essential organelle for the parasitic Trypanosomatidae<br />

causing Chagas disease and African sleeping sickness. Therefore, it could<br />

provide a potential target for drug design. Previous studies demonstrated the<br />

mechanisms un<strong>der</strong>lying biogenesis of peroxisomes and of glycosomes are<br />

almost identical. In both organelles the membrane protein Pex14 provides a<br />

docking site for the soluble receptor Pex5 carrying enzymes destined for<br />

peroxisomal import. It was shown that pentapeptide repeats with the consensus<br />

sequence WxxxF/Y within the Pex5 sequence mediate binding to Pex14. In<br />

or<strong>der</strong> to define glycosome-specific drug targets without affecting biosynthesis<br />

of the human organelle, we investigated the ligand-specificity of recombinant<br />

Pex14 from both organisms using fluorescence titration analysis and in vitro<br />

complex formation.<br />

Although the basic characteristics of the Pex5-Pex14 interaction are conserved<br />

between man and kinetoplastids, we could detect species-specific sequence<br />

preferences in peptide recognition. To screen in a more systematically approach<br />

for high-affinity peptides which block the peptide binding sites of TbPex14<br />

without harming protein import in human cells we are using combinatorial<br />

peptide libraries of various WxxxF/Y motifs immobilized on nitrocellulose and<br />

incubate them with affinity-purified peroxins from both organisms. The<br />

efficiency of binding can be estimated by immunological detection of bound<br />

Pex14. With this approach, we hope to detect a peptide which binds<br />

trypanosomal Pex14 with high affinity without blocking the binding site of<br />

human Pex14.<br />

PP 33<br />

Localization of collagen binding and hydrophobic domain<br />

of SdrI<br />

B. Kleine 1 , S. Neumann *1 , S.G. Gatermann 1 , T. Sakinc 1<br />

1<br />

Institut für Hygiene und Medizinische Mikrobiologie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

Staphylococcus saprophyticus is a gram positive and coagulase negative<br />

pathogenic bacterium causes urinary tract infections in young women. It is<br />

hydrophob and able to bind fibronectin, laminin and collagen and is involved in<br />

hemagglutination of sheep erythrocytes. Some of its surface proteins have been<br />

characterized in the early past. SdrI, the serine-aspartate repeat protein, contains<br />

the longest SD repeat region described so far (854 aa) and the LPXTG-motif<br />

for cell wall anchoring. It is a member of the MSCRAMM protein family and<br />

shows a typical ABB domain structure. SdrI mediates collagen binding and<br />

additionally hydrophobicity. A sdrI knock out mutant shows decreased collagen<br />

binding and hydrophobicity in vitro. Cell surface hydrophobicity has been<br />

implicatd in the pathogenesis of infections with S. saprophyticus.<br />

To localize the protein domains for collagen binding and also hydrophobicity<br />

within SdrI we complemented the knock out mutant with different<br />

constructions of SdrI missing the A domain, the B domain, the AB domains and<br />

the BB domains. These were tested in vivo using ELISA with collagen type I<br />

coated microtiter plates and showed still collagen binding compared to the wild<br />

type. So the last possible functional region in the SdrI protein is the SD repeat<br />

region. It is necessary to get a mutant with SdrI missing the SD repeat region<br />

and checking this bacterium for collagen binding. Futhermore all<br />

complemented strains should be tested in BATH (Bacteria Adhesion To<br />

Hydrocarbonate) -tests.<br />

PP 34<br />

Isolation and characterization of small colony variants of<br />

Pseudomonas aeruginosa grown un<strong>der</strong> simulated urinary<br />

tract conditions<br />

K. Heinemann 1 , M. Sauer 2 , K. Zapf 2 , R. Krull 2 , D. Jahn 1 , P. Tielen *1<br />

1 Institute of Microbiology, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

2 Institute of Biochemical Engineering, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause<br />

severe acute and chronic infections, especially in immunocompromised<br />

patients. Common infections caused by P. aeruginosa are lung infections,<br />

wound infections, conjunctivitis and urinary tract infections, mostly in<br />

association with indwelling catheters.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

We developed a biofilm reactor mimicking urethral catheter infections using an<br />

artificial urine medium (AUM) with a continuous flow rate of 1.4 l per day. P.<br />

aeruginosa isolates from acute urinary tract infections (UTIs) and catheterassociated<br />

urinary tract infections (CAUTIs) were incubated un<strong>der</strong> these<br />

conditions in comparison to the lab strain PA14. After an incubation time of<br />

seven days we observed small colony variants (SCVs) in a concentration up to<br />

10 % of the whole population. A general characterization of the obtained SCVs<br />

showed global changes in phenotypic patterns in relation to the parent strains, e.<br />

g. increased antibiotic resistances, increased biofilm formation, decreased<br />

motilities and increased cell surface hydrophobicity. Moreover, several SCVs<br />

showed an enhanced cell aggregation.<br />

Interestingly, SCVs were also isolated from chronic CAUTIs, which indicate<br />

that phenotypic variations to SCVs might be a common strategy of P.<br />

aeruginosa to cause persistent infections in the urinary tact.<br />

PP 35<br />

Mallard ducks: a non-point source of pathogenic<br />

Escherichia coli?<br />

S. Guenther 1 , L.H. Wieler 1 , P. Schierack 2 , C. Ewers *1<br />

1 Institute for Microbiology and Epizootics, Freie Universität Berlin, Berlin,<br />

Germany<br />

2 Fachbereich Bio-, Chemie- und Verfahrenstechnik, Fachhochschule Lausitz,<br />

Senfenberg, Germany<br />

The risk of mallard ducks, representing one of the most abundant waterfowl<br />

species worldwide, as potential source of spreading pathogenic Escherichia coli<br />

via their fecal deposits to various environmental sources has not been addressed<br />

so far. In this study mallard duck fecal samples (n = 175) were processed for E.<br />

coli and unique clones, defined by macrorestriction analyses, were<br />

subsequently investigated for their virulence genotype and phylogenetic<br />

background. In addition, selected clones were characterized by Multi locus<br />

sequence typing and in vivo infection experiments.<br />

Of 60 clones identified among 142 E. coli isolated from the 175 samples, 15<br />

(25%) were recovered from multiple individuals (up to 23 per clone). None of<br />

the clones harbored Shiga toxin 1 or 2 genes (stx1, stx2) and only rarely other<br />

virulence-associated genes (VAGs) specifying intestinal pathogenic E. coli. In<br />

contrast, the clones possessed consi<strong>der</strong>able numbers of VAGs (up to 30) linked<br />

to the group of extraintestinal pathogenic E. coli (ExPEC). Their in vivo<br />

pathogenicity was confirmed in chicken infection experiments. Moreover,<br />

selected clones were assigned to sequence types (STs) <strong>bei</strong>ng most prominent<br />

for human ExPEC strains, including ST95 and ST73.<br />

Due to these findings, mallard ducks have to be consi<strong>der</strong>ed an important<br />

reservoir of zoonotic E. coli strains, thus serving as a substantial non-point<br />

source especially of strains capable of causing extraintestinal diseases.<br />

PP 36<br />

Detection of Saccharopolyspora rectivirgula in bioaerosols<br />

by PCR<br />

J. Schäfer *1 , M. Hippauf 2 , P. Kämpfer 1 , U. Jäckel 2<br />

1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen,<br />

Gießen, Germany<br />

2 Bundesanstalt für Ar<strong>bei</strong>tsschutz und Ar<strong>bei</strong>tsmedizin, BAuA, Berlin, Germany<br />

159<br />

Saccharopolyspora rectivirgula is described as the causative agent of exogen<br />

allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of<br />

dispensed spores in air. High concentrations of spores of Saccharopolyspora<br />

rectivirgula were found in different environments like agricultural production,<br />

compost facilities, mushroom cultivation or rooms with technical air<br />

moistening. Because of the medical relevance of Saccharopolyspora<br />

rectivirgula, a standardised detection process is necessary. Cultivation<br />

independent methods allow the analysis of viable and nonviable bacteria as<br />

well as cell particles, which may also cause allergic reactions. Therefore we<br />

designed a PCR primer system targeting the 16S rRNA-gene of the type strain<br />

S. rectivirgula DSM 43747 T . Furthermore we investigated seven different S.<br />

rectivirgula strains deposit at the DSMZ (Deutsche Sammlung für<br />

Mikroorganismen und Zellkulturen). Despite the optimization of a PCR<br />

protocol we could only detect the type strain (43747 T ) and strain DSM 43114.<br />

As a consequence, we analysed the 16S rRNA gene sequences of all other<br />

deposited strains. Because of unexpected “background” sequences, we used a<br />

cloning approach to gain detailed informations. The analyses revealed sequence<br />

differences at the target position both, between and within the strains, which<br />

may be explained by different 16S rRNA operons. Based on these informations<br />

we designed a new primer system for the detection of Saccharopolyspora<br />

rectivirgula in bioaerosol samples from different working places e.g. in poultry<br />

feeding industry and compost facilities.

160<br />

PP 37<br />

A novel gene cluster controlling the fatty acid composition<br />

of lipoteichoic acid and glycolipids in Staphylococcus aureus<br />

N. Göhring *1 , I. Fedtke 1 , D. Ma<strong>der</strong> 1 , A. Peschel 1<br />

1 Medizinische Mikrobiology und Hygiene, Universitätsklinikum Tübingen,<br />

Tübingen, Germany<br />

Lipoteichoic acid (LTA) is a component of the envelope of Gram-positive<br />

bacteria. This glycerol-phosphate surface polymer is embedded in the cell<br />

membrane via a glycolipid-anchor (diglucosyl-diacylglycerol). However, the<br />

physiological roles have remained controversial because of the lack of LTAdeficient<br />

mutants. Recently the ltaS gene has been characterised to play a role<br />

in the elongation-process of the 1,3-linked glycerol-phosphate subunits of the<br />

LTA backbone. Here we describe a novel gene cluster which seems to be<br />

involved in LTA biosynthesis. Analysis of column-purified LTA revealed<br />

different binding characteristics compared to wild type LTA. This might be due<br />

to an altered structure in the mutant, e.g. in the fatty acid composition of the<br />

anchor. Moreover, microanalysis-data point towards a role of the ltaB gene in<br />

fatty acid biosynthesis.<br />

PP 38<br />

The putative ATPase ORF5 encoded by the conjugative<br />

resistance plasmid pIP501<br />

E.K. Çelik *1 , M.Y. Abajy 2 , E. Grohmann 3 , W. Keller 4<br />

1<br />

Institute of Mol. Biosciences/Structure Biology; Environmental<br />

Microbiology/Genetics, Karl-Franzens-University Graz; Technische<br />

Universität Berlin, Graz, Austria<br />

2<br />

Environmental Microbiology/Genetics, Technische Universität Berlin, Berlin,<br />

Germany<br />

3<br />

Institute of Mol. Biosciences/Structure Biology, Karl-Franzens-University<br />

Graz, Graz, Austria<br />

Conjugative plasmid transfer is a very important mechanism for bacteria to<br />

deliver and acquire genetic information. Our enterococcal model plasmid<br />

pIP501 has a very broad host range for conjugative transfer among a wide<br />

variety of Gram-positive (G+) bacteria and Gram-negative (G-) Escherichia<br />

coli. Many type IV secretion systems (T4SSs) have been discovered in G- and<br />

G+ bacteria, they deliver protein and/or DNA to the host cells. T4SSs are found<br />

in many bacterial pathogens like Legionella pneumophila (dot/icm-system),<br />

Helicobactor pylori (cag-system) and Bordetella pertussis (ptl-system). The<br />

pIP501-encoded type IV homologues are an ATPase (ORF5 homologue to<br />

VirB4 from A. tumefaciens), a coupling protein (ORF10 homologue to VirD4)<br />

and a lytic transglycosylase (ORF7 homologue to VirB1). The system of<br />

Agrobacterium is the best studied T4SS until now.<br />

One focus of our work lies on ORF5 encoded by pIP501, we want to find out<br />

which role it plays for the assembly of a type IV like secretion system in G+<br />

bacteria. ORF5 could deliver energy by hydrolysis of ATP and therefore<br />

facilitate the energy consuming conjugation process.<br />

Yeast two-hybrid studies and pull-down assays showed that ORF5 interacts<br />

with itself. To verify this observation size exclusion chromatography of ORF5<br />

will be performed to verify the formation of multimeric structures. ORF5 has<br />

been purified as 7xHis-fusion and showed ATP-binding activity in vitro. In<br />

vitro ATP-hydrolysis activity will be tested. Immuno-TEM of cryo-sections of<br />

E. faecalis cells harbouring pIP501 with gold-labelled anti-ORF5 antibodies<br />

will be performed to detect its sub-cellular localization in the cell.<br />

PP 39<br />

Protein secretion mechanisms in S. aureus: a proteomic<br />

approach<br />

T. Winter *1 , M.J.J. Sibbald 2 , H. Antelmann 1 , M. Hecker 1 , J.M. van Dijl 2 , S.<br />

Engelmann 1<br />

1 Institute for Microbiology/ Department of Microbial Physiology, Ernst-Moritz-<br />

Arndt-University Greifswald, Greifswald, Germany<br />

2 Department of Medical Microbiology/, University Medical Centre Groningen,<br />

Groningen, Netherlands<br />

The Gram-positive bacterium S. aureus normally colonizes the anterior nares of<br />

at least one third of the human population. Extracellular and surface associated<br />

proteins constitute a reservoir of virulence factors. Thus, protein export<br />

mechanisms may also be important for the virulence of pathogenic bacteria<br />

such as S. aureus. In Bacillus subtilis, the model organism of Gram-positive<br />

bacteria, protein export mechanisms have been well studied. While the<br />

functionality of these pathways is poorly characterized in S. aureus. This work<br />

focuses on the comparative secretome analysis of S. aureus wild-type strains<br />

and isogenic mutants with defects in various secretory components and their<br />

impact on protein secretion.<br />

The most commonly used pathway for protein secretion is the Sec- pathway. In<br />

S. aureus, components of a secondary accessory secA2-secY2 pathway are<br />

present. This secA2-secY2 pathway was shown to contribute to the export of<br />

cytoplasmic and cell wall proteins in other pathogen Gram-positives.<br />

Extracellular proteome analyses of a secG, and secGY2 showed that the amount<br />

of several proteins with type I signal peptides was significantly decreased (e.g.<br />

LipA, Geh, Hlb and HlY). The decreased amount of these proteins was not<br />

accompanied by a decreased transcription level.<br />

Additionally, the role of the folding catalyst PrsA in secretion of extracellular<br />

proteins was characterized.<br />

PP 40<br />

Dose-dependent effects of the adenylate cyclase toxin<br />

(CyaA) of Burdetella pertussis on phagocytosis and the<br />

oxidative burst<br />

A. Khosravani *1<br />

1 Microbiology, Yasuj University of Medical Sciences, Yasuj, Iran<br />

Background and aim: Adenylate cyclase toxin (CyaA) toxin is an important<br />

virulence factor of Bordetella pertussis, the causative agent of whooping cough,<br />

and a potential component of acellular pertussis vaccine.<br />

Material & method: The work involved the production of two purified forms of<br />

CyaA with different enzymic and invasive properties. These were: the native<br />

enzymatically-active, invasive toxin (CyaA), an invasive <strong>der</strong>ivative lacking AC<br />

enzymic activity (CyaA*). These were expressed in E. coli BL21/DE3 as<br />

recombinant proteins. After purification by a combination of chromatographic<br />

methods (Q-and Butyl-Sepharose) their properties were investigated by several<br />

assays. The AC enzymic activity was assayed by a conductimetric method.<br />

CyaA had a high level of enzymic activity but that of CyaA* was very low. The<br />

cytotoxic properties of CyaA* towards J774.2 cells was 100-fold less than that<br />

of CyaA,<br />

Result: Different concentrations of CyaA and CyaA* were used to investigate<br />

dose-dependent effects of the toxins on phagocytosis and the oxidative burst in<br />

U937 human monoblastic cells, J774.2 mouse macrophage-like cells and fresh<br />

human granulocyte cells (whole blood used). Significant effects were seen with<br />

CyaA on both phagocytosis and oxidative burst, but CyaA* did not have a<br />

significant effect on either.<br />

Conclusion: The results of the study showed that both enzymatic and invasive<br />

functions are required for the cytotoxic effects of adenylate cyclase toxin.<br />

PP 41<br />

Degradation of HipB of the HipBA Toxin/Antitoxin by the<br />

ATP Dependent Protease Lon<br />

S. Hansen *1 , M. Vulic 1 , K. Lewis 1<br />

1 Biology, Northeastern University, Boston, United States<br />

Bacterial populations produce a subpopulation of non-growing persister cells<br />

that make up to 10-6-10-4 of the population in exponentially growing cultures,<br />

and ~10-2 in stationary phase. Persisters are phenotypic variants of the wild<br />

type that are tolerant to killing by antibiotics. Persisters contribute to the<br />

antibiotic recalcitrance of biofilm infections. Biofilms are implicated in many<br />

bacterial infections, including those of indwelling devices, dental infections,<br />

endocarditis and cystic fibrosis. Un<strong>der</strong>standing the mechanism of persister<br />

formation/maintenance is likely to lead to new effective therapies to treat<br />

biofilm infections.<br />

The first identified high-persistence allele in Escherichia coli, hipA7 increased<br />

the frequency of persistence by 10,000 fold. E. coli hipA is co-transcribed with<br />

a smaller upstream gene, hipB. HipB is an 88 residue protein that represses the<br />

hipBA operon by cooperative binding to four operators upstream of hipBA.<br />

HipB is a labile protein. HipA is highly toxic in the absense of HipB. Here we<br />

show that HipB is degraded by the ATP-dependent protease Lon and suggest<br />

that degradation of HipB occurs via its C-terminus. Un<strong>der</strong>standing the<br />

regulation of HipB will help to elucidate the role of HipB and HipA in persister<br />

formation.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PP 42<br />

T-RFLP-based mcrA gene analysis of human methanogenic<br />

archaea and association with oral pathogens<br />

H.P. Horz *1 , M. Vianna 2 , G. Conrads 1<br />

1<br />

Lehr- und Forschungsgebiet Orale Mikrobiologie und Immunologie, Klinik für<br />

Zahnerhaltung und Parodontologie und Präventive Zahnheilkunde, Institut für<br />

Medizinische Mikrobioloige, Universitätsklinikum RWTH Aachen, Aachen,<br />

Germany<br />

2<br />

Eastman Dental Institute for Oral Health Care Sciences, University College<br />

London, London, United Kingdom<br />

Methanobrevibacter oralis has recently been implicated with periodontitis and<br />

dental root canal infections. This finding hints at the intriguing possibility that<br />

some archaea may have the potential to invade primary sterile anatomic sites<br />

and to contribute to the pathogenesis of microbial diseases. In the current study<br />

we performed terminal restriction fragment length polymorphism (T-RFLP)<br />

analysis based on the mcrA gene which encodes for the methyl coenzyme-M<br />

reductase, to assess the overall diversity of methanogens in 102 periodontal and<br />

32 endodontic samples. In addition, quantification based on real-time PCR of<br />

methanogens and recognized endodontic pathogens, including Porphyromonas<br />

gingivalis, Prevotella intermedia, Tannerella forsythia, Treponema spp. and<br />

Synergistes spp. as well as the total bacterial load was performed. In 50% of<br />

periodontal samples and 25% of endodontic samples archaeal DNA was<br />

detected. T-RFLP and comparative sequence analysis of the mcrA gene and 16S<br />

rRNA gene revealed the existence of a second methanogenic phylotype in both<br />

periodontal and endodontic infections. The mean proportion of methanogens<br />

and Synergistes spp. ranged from 0.5 to 1.0% of the total microbial community<br />

in infected root canals. This value was exceeded only by Treponema spp. with a<br />

mean proportion of 10%, while the mean proportions of the other endodontic<br />

pathogens was below 0.1%. Within the set of endodontic samples no<br />

association was found among bacterial species however a positive association<br />

between methanogenic archaea and Synergistes spp. was found, which is in<br />

accordance with previous findings from an anaerobic sludge digester. Given<br />

these data, and the reported implication of Synergistes spp with soft tissue<br />

infections and peritonitis, our data un<strong>der</strong>score the possibility that methanogenic<br />

archaea may be supportive of true pathogenic organisms involved in anaerobic<br />

infections.<br />

PP 43<br />

A regulatory network controls the expression of the in vivo<br />

expressed HreP protease of Yersinia enterocolitica<br />

K. Wagner *1 , J. Schilling 1 , S. Fälker 1 , G. Heusipp 1<br />

1 ZMBE, Institut für Infektiologie, Westf. Wilhelms-Universität Münster,<br />

Münster, Germany<br />

HreP was previously identified as an in vivo expressed protease important for<br />

full virulence of the human pathogen Yersinia enterocolitica. So far, no in vitro<br />

conditions are known that lead to hreP expression un<strong>der</strong> laboratory conditions.<br />

We established a transposon screen for regulators of hreP transcription in Y.<br />

enterocolitica and identified three genes, termed pypA, pypB, and pypC (protein<br />

involved in regulation of Yersinia hreP expression A, B, C) which induce hreP<br />

transcription after overproduction. All pyp genes have a low GC% content<br />

reminiscent of horizontal gene transfer. PypA is an inner membrane protein<br />

with no significant similarity, while PypB and PypC have DNA binding<br />

domains typical for transcriptional regulators. We show that all Pyp proteins are<br />

able to activate hreP independent of each other. Furthermore, we could<br />

demonstrate that PypB and PypC directly bind to the hreP promoter region.<br />

Additionally, pypB and pypC are autoregulated and regulate each other. Our<br />

data also indicate that the histone-like nucleoid-structuring protein H-NS<br />

represses transcription of hreP in a temperature-dependent manner. In summary<br />

we were able to identify a new regulatory network controlling virulence<br />

functions of Y. enterocolitica.<br />

PP 44<br />

A heterotrimeric G protein (BCG1) regulates virulence in<br />

the grey mould fungus Botrytis cinerea<br />

J. Schumacher *1 , B. Tudzynski 1<br />

1 Institut für Botanik, Westf. Wilhelms-Universität, Münster, Germany<br />

Botrytis cinerea is a necrotrophic pathogen, causing serious pre- and postharvest<br />

diseases in more than 230 dicotyledonous plant species: infection<br />

results in cell death, damage of plant tissue and eventually in the „grey mould“<br />

of the crop. The fungus uses sophisticated mechanisms to kill and to<br />

decompose plant tissue, e.g., by the secretion of cell wall-degrading enzymes,<br />

the biosynthesis of phytotoxic secondary metabolites, the secretion of oxalic<br />

acid, the expression of proteins exhibiting phytotoxic activities, and by the<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

stimulation of an oxidative burst due to the accumulation of reactive oxygen<br />

species (ROS).<br />

Conserved signal transduction pathways, i.e., the cAMP-dependent and several<br />

MAP kinase pathways, have been shown to be important for cell function<br />

during morphogenesis, differentiation and distinct stages of infection. The<br />

Galpha subunit BCG1 of a heterotrimeric G protein is crucial for the<br />

progression of infection: knock-out mutants penetrate the host tissue, but are<br />

not able to complete the infection cycle. Based on present data, it has been<br />

concluded that BCG1 affects virulence by regulating at least two signalling<br />

cascades, the cAMP cascade and the Ca 2+ /calcineurin pathway. cAMPdependent<br />

signal transduction is mainly involved in regulation of carbon<br />

source-dependent growth characteristics, including colony morphology,<br />

biomass accumulation, and conidial germination, and is moreover essential<br />

during the advanced stages of infection. The Ca 2+ -regulated calcineurin<br />

phosphatase is crucial for normal growth and pathogenicity, and the<br />

transcription factor BcCRZ1, a calcineurin target, is in general required to<br />

respond to stress conditions and for full virulence.<br />

PP 45<br />

Identification of virulence factors of Paenibacillus larvae<br />

E. García-González *1 , J. Nachtigall 2 , J. Vater 2 , R. Süssmuth 2 , E. Genersch 3 , R.<br />

Borriss 1<br />

1 Bacterial Genetics, Humboldt University, Berlin, Germany<br />

2 Institut für Chemie, Technische Universität Berlin, Berlin, Germany<br />

3 Diagnostik und Molekularbiologie, Län<strong>der</strong>institut für Bienenkunde Hohen<br />

Neuendorf e.V, Berlin, Germany<br />

American foulbrood (AFB) is consi<strong>der</strong>ed as the most contagious and<br />

destructive infectious disease in the honeybee, caused by the bacterium<br />

Paenibacillus larvae. AFB is worldwide distributed. This disease leads to huge<br />

losses not only in apicultural economy but also in pollination rates, since Apis<br />

mellifera is the primary commercial pollinator of agricultural crops in North<br />

America and the most widely used actively managed pollinator in the world<br />

(Delaplane and Mayer, 2000; Kearnes et al., 1998; McGregor, 1976).<br />

Some reports have been written about prevention and control of the disease, but<br />

it is not sufficient to suppress the spread of this disease due to the little<br />

knowledge about the virulence factors of the bacteria and its process of<br />

infection.<br />

The main aim of this work is to identify the virulence factors of Paenibacillus<br />

larvae and the mechanisms involved in the host-pathogen interactions, and to<br />

elucidate the molecular pathogenesis of AFB in honeybee larvae.<br />

Two genotypes of Paenibacillus larvae (ERIC I and ERIC II), which have<br />

important phenotypic differences such as pathogenicity, are used as model<br />

systems in this work. These strains show interesting antibiotical activities,<br />

which may be involved in colonization in the midgut of honeybee; surfactin and<br />

fengycin were detected by MALDI TOF MS; some unknown active compounds<br />

should be further characterized. So far no antimicrobial activity has been<br />

described for this bacterium, which may have a virulence role in infecting<br />

young bee larvae. At the moment sequencing of the giant gene clusters<br />

involved in nonribosomal biosynthesis of the antimicrobial polyketides and<br />

lipopeptides is in progress. Elucidation of the genetic structure combined with<br />

chemical analysis will deepen un<strong>der</strong>standing of their possible role in<br />

colonization of P. larvae of the honeybee larvae midgut.<br />

PQ 01<br />

Function of the cytochrome bc1 complex in Gluconobacter<br />

oxydans<br />

T. Hanke *1 , S. Bringer 2 , H. Sahm 3 , M. Bott 2<br />

1 Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, Jülich,<br />

Germany<br />

161<br />

The aerobic bacterium Gluconobacter oxydans is known for low growth yields<br />

which are likely due to an inefficient respiratory chain. Membrane-bound<br />

dehydrogenases containing prosthetic groups, like pyrroloquinoline quinone,<br />

flavines or cytochromes, enable G. oxydans to oxidize sugars and sugar<br />

alcohols in two or more steps in the periplasm. The electrons supplied by<br />

substrate oxidation are transferred to the ubichinone pool of the respiratory<br />

chain. In parallel, substrates are catabolised via the pentose phosphate and the<br />

Entner-Doudoroff pathways, the two functional cytoplasmic pathways for sugar<br />

degradation in G. oxydans. [1].<br />

The genomic data reveal a simple structure of the respiratory chain of G.<br />

oxydans consisting of a non-proton-pumping NADH: ubiquinone<br />

oxidoreductase and two quinol oxidases [1]). An open question is the function<br />

of cytochrome c and the cytochrome bc1 complex in the absence of a<br />

cytochrome c oxidase. In the present work the cytochrome bc1 complex was<br />

inactivated by marker-free deletion of the gene for the cytochrome subunit as

162<br />

well as for all three genes of the operon. Investigation of growth parameters and<br />

oxidation activities of the deletion mutant performed at different growth<br />

conditions showed a lower growth rate but almost the same end-OD in<br />

comparison to the wild type. The cytochrome bc1 complex is important when<br />

cells are grown at a pH of 4. The deletion mutant is able to grow with<br />

gluconate. Further work for elucidation of the functions of cytochrome c and<br />

the cytochrome bc1 complex in G. oxydans is in progress.<br />

[1] Prust C, Hoffmeister M, Liesegang H, Wiezer A, Fricke WF, Ehrenreich A,<br />

Gottschalk G, Deppenmeier U (2005) Complete genome sequence of the acetic<br />

acid bacterium Gluconobacter oxydans. Nature Biotechnol 23:195-200<br />

PQ 02<br />

Using Saccharomyces cerevisiae for the organelle-targeted<br />

production of an antiporter-like subunit of complex I<br />

(NADH dehydrogenase)<br />

W. Steffen 1 , A.C. Gemperli 1 , J. Steuber *1<br />

1 Biochemisches Institut, Universität Zürich, Zürich, Switzerland<br />

Facile genetic manipulation, and similarity to higher eukaryotes in the cell<br />

structure, has established the yeast Saccharomyces cerevisiae as a versatile<br />

workhorse. The respiratory NADH dehydrogenase (complex I) contributes to<br />

the formation of electrochemical gradients in bacteria, plastids and<br />

mitochondria, but is absent from S. cerevisiae. Subunit NuoL of complex I<br />

(NDH I) from E. coli is a 60 kDa, transmembrane protein which catalyzed Na +<br />

and K + transport [1], in accord with its proposed function in cation<br />

translocation. The mitochondrial homolog, subunit ND5, represents a hot spot<br />

for mutations associated with MELAS disor<strong>der</strong> (mitochondrial<br />

encephalomyopathy, lactic acidosis and stroke-like episodes). Wild type nd5<br />

and mutated nd5 encoding for the E145V variant observed in MELAS patients<br />

were expressed in S. cerevisiae. Subcellular localizations of ND5 variants<br />

targeted to the ER or the inner mitochondrial membrane were confirmed by<br />

immunofluorescence. S. cerevisiae producing wild type ND5 in the presence of<br />

Li + or K + exhibited diminished salt resistance of ER-resident ND5 at 100 mM<br />

LiCl and of mitochondrially targeted ND5 at 800 mM KCl. In contrast, S.<br />

cerevisiae producing the E145V variant of ND5 exhibited salt-resistant growth<br />

un<strong>der</strong> these conditions. The intracellular cation concentrations in yeast cells<br />

were perturbed by ND5 in an organelle- and sequence-specific manner, most<br />

likely due to the dissipation of cation gradients by ND5.<br />

[1] Gemperli, A. C., Schaffitzel, C., Jakob, C., Steuber, J. (2007). „Transport of<br />

Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH<br />

dehydrogenase I produced in Saccharomyces cerevisiae.“ Arch Microbiol<br />

188(5): 509-21.<br />

PQ 03<br />

The cytochrome b6f complex from Thermosynechococcus<br />

elongatus - Characterization by ESI-MS and EPR involving<br />

the new subunit PetP<br />

D. Gomolla *1 , F. Baymann 2 , C. Lüer 1 , S. Rexroth 1 , M. Rögner 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

2 BIP, CNRS, Marseille, France<br />

The cytochrome b6f complex of oxygenic photosynthesis from the<br />

thermophilic cyanobacterium Thermosynechococcus elongatus connects the<br />

electron transport between photosystem 1 and 2. Each monomer of the dimeric<br />

integral membrane complex consists of four major subunit (17-34 kDa) and<br />

four small subunits with molecular masses ranging from 3-7 kDa.<br />

Recently, we identified an additional 7.2 kDa subunit for the cyt b6f complex of<br />

Synechocystis sp. PCC 6803, which we named PetP [1]. In or<strong>der</strong> to investigate<br />

structure-function relationships in a stable and fully active complex - including<br />

PetP - we have developed a new strategy for the purification of the cyt b6f<br />

complex from T. elongatus, involving His-tagged cyt f and two<br />

chromatographic steps. By this we could isolate an active dimeric complex<br />

containing all subunits as well as the 7.2 kDa protein (in analogy to S. 6803).<br />

ESI-MS analysis revealed that this protein is encoded by the open reading<br />

frame tlr0524 – with high homology to PetP (ORF ssr2998) of S. 6803.<br />

Crystal structure analysis of cyt b6f from the thermophilic cyanobacterium<br />

Mastigocladus laminosus [2] and the green alga Chlamydomonas reinhardtii<br />

[3] showed an additional redox cofactor heme ci. This cofactor located in the Qi<br />

pocket of the complex close to heme bH is covalently linked to the cyt b6 protein<br />

by a single thioether bond to a cystein residue. Preliminary EPR analysis of<br />

heme ci from T. elongatus in presence and absence of the inhibitor NQNO<br />

showed similar spectra as obtained with the cyt b6f complex from C.<br />

reinhardtii.<br />

[1] Volkmer T., Schnei<strong>der</strong> D., Bernát G., Kirchhoff H., Wenk S-O., Rögner M.,<br />

J Biol Chem, 2007, 282(6):3730-7<br />

[2] Kurisu G., Zhang H., Smith J.L., Cramer W.A., Science, 2003, 302, 1009-<br />

1014<br />

[3] Stroebel D., Choquet Y., Popot J-L., Picot D., Nature, 2003, 426, 413-418<br />

PQ 04<br />

The crystal structure of C176A mutated [Fe]-hydrogenase<br />

suggests an acyl-iron ligation in the active site iron complex<br />

T. Hiromoto 1 , K. Ataka 2 , O. Pilak 1 , S. Vogt 1 , M.S. Stagni 3 , W. Meyer-Klaucke 3 ,<br />

E. Warkentin 4 , R.K. Thauer 1 , S. Shima *1 , U. Ermler 4<br />

1<br />

Biochemistry, Max-Planck-Institute for Terrestrial Microbiology, Marburg,<br />

Germany<br />

2<br />

Chemistry, Bielefeld University, Bielefeld, Germany<br />

3<br />

EMBL, DESY, Hamburg, Germany<br />

4<br />

Molecular Membrane Biology, Max-Planck-Institute for Biophysics, Frankfurt,<br />

Germany<br />

[Fe]-hydrogenase is one of three types of enzymes known to activate H2.<br />

Crystal structure analysis recently revealed that its active site iron is ligated<br />

square-pyramidally by Cys176-sulfur, two CO, an „unknown“ ligand and the<br />

sp 2 -hybridized nitrogen of a unique iron-guanylylpyridinol- cofactor [1]. We<br />

report here on the structure of the C176A mutated enzyme crystallized in the<br />

presence of dithiothreitol (DTT). It suggests an iron center octahedrally<br />

coordinated by one DTT-sulfur and one DTT-oxygen, two CO, the 2pyridinol’s<br />

nitrogen and the 2-pyridinol’s 6-formylmethyl group in an acyl-iron<br />

ligation. This result led to a re-interpretation of the iron ligation in the wild<br />

type.<br />

[1] Shima, S., Pilak, O., Vogt, S., Schick, M., Stagni, M.S., Meyer-Klaucke,<br />

W., Warkentin, E., Thauer, R.K. and Ermler, U. (2008). The crystal structure of<br />

[Fe]-hydrogenase reveals the geometry of the active site. Science 321, 572-575.<br />

PQ 05<br />

Insights into the puf operon of a purple bacterium with<br />

unusual spectral properties<br />

O.L. Rücker *1 , K. Sichau 1 , J. Overmann 1<br />

1 Mikrobiologie Department Biologie I, Ludwig Maximilians Universität<br />

München, München, Germany<br />

A recently isolated species of the photosynthetic purple sulfur bacteria,<br />

provisionally called strain 970, exhibits unusual spectral properties. The<br />

bacterium has a single antenna complex of the light harvesting 1 (LH1) type<br />

containing bacteriochlorophyll a, with a Qy absorption band at the wavelength<br />

of 963nm, which is the most red-shifted absorption band described so far for<br />

antenna complexes containing bacteriochlorophyll a as the chromophore.<br />

Previously described structures of LH1 show that the Qy absorption is<br />

determined by the pigment-protein interaction between bacteriochlorophyll and<br />

the α- and β-polypeptides of the light harvesting complex. In this project the<br />

sequence of the puf operon, which consists of the genes coding for reaction<br />

center and LH1 polypeptides, was determined. Cloning and several inverse<br />

PCR experiments revealed the existence of two pairs of pufA and pufB genes.<br />

Based on sequence analysis, pufBA shows some particular similarities to the<br />

homolog found in another bacterium showing a red-shifted Qy absorption band,<br />

the purple sulfur bacterium Thermochromatium tepidum. These insights show<br />

that strain 970 is a unique subject for the study of antenna and reaction center<br />

function, and has the potential to reveal new facts about pigment-protein<br />

interactions in photosynthetic antenna complexes.<br />

PQ 06<br />

Dynamic protein protein interaction of Dark Operative<br />

Protochlorophyllide Oxidoreductase (DPOR)<br />

D. Wätzlich *1 , S. Virus 1 , U. Frank 1 , M. Bröcker 1 , D. Jahn 1 , J. Moser 1<br />

1 Institut für Mikrobiologie, TU Braunschweig, Braunschweig, Germany<br />

During (bacterio-)chlorophyll biosynthesis the conversion of<br />

protochlorophyllide (PChlide) to chlorophyllide (Chlide) is an important<br />

regulatory step. The reaction is catalyzed by a light dependent<br />

protochlorophyllide oxidoreductase (LPOR) in angiosperms, while nonflowering<br />

plants (gymnosperms), algae and photosynthetic bacteria possess a<br />

dark operative protochlorophyllide oxidoreductase (DPOR). DPOR shows<br />

significant homology to the nitrogenase system and consists of the<br />

heterotetrameric complex (NB)2 and the dimeric L2 protein which are both<br />

essential for DPOR activity. For the conversion of PChlide to Chlide, the<br />

enzyme requires ATP as cofactor in the presence of a reducing agent.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

To investigate the interaction between (NB)2 and L2 of the DPOR, we cloned<br />

the corresponding genes from different organisms (Chlorobaculum tepidum,<br />

Prochlorococcus marinus and Thermosynechococcus elongatus). After<br />

showing activity for the recombinantly produced enzymes, we combined the<br />

individual subunits of the different organisms. Interestingly, the heterologous<br />

DPOR was enzymatically active which might indicate that residues involved in<br />

the protein protein interaction between NB and L are highly conserved. An<br />

overall of ten amino acid residues in these conserved regions were<br />

mutagenized. Particular mutants showed drastically reduced activities. Thus,<br />

we assume that the corresponding amino acids are involved in the proposed<br />

protein protein interaction.<br />

PQ 07<br />

Analysis of the hydrogen metabolism of a<br />

metallophosphoesterase-deficient Chlamydomonas<br />

reinhardtii mutant strain un<strong>der</strong> normal growth condition<br />

T. Rühle *1 , A. Hemschemeier 1 , A. Melis 2 , T. Happe 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

2 Department of Plant and Microbial Biology, University of California,<br />

Berkeley, United States<br />

Chlamydomonas reinhardtii (C. reinhardtii) wild type cultures exhibit a four to<br />

seven fold higher photosynthesis rate than respiration rate un<strong>der</strong> normal growth<br />

condition. As a consequence, cultures kept in the light stay aerobic. Hydrogen<br />

production does not take place because the oxygen-sensitive [FeFe]hydrogenases<br />

are inhibited and their gene expression is suppressed in the<br />

presence of oxygen.<br />

In regard to a photobiological hydrogen production un<strong>der</strong> normal growth<br />

condition a screening procedure was developed to identify C. reinhardtii<br />

mutants with an attenuated photosynthesis respiration capacity ratio (P/R ratio).<br />

Out of 9000 mutants generated by insertional mutagenesis 4 mutants with<br />

varying P/R ratios from 0 to 3 were isolated. For one mutant strain (apr1) the<br />

wild type phenotype could be restored by complementation with a gene coding<br />

for a bacterial-like metallophosphoesterase [1]. To test the effect of the<br />

imbalanced P/R ratio of apr1 (P/R = 1,5) on the hydrogen metabolism un<strong>der</strong><br />

normal growth condition, cultures were sealed and exposed to medium light<br />

conditions. It could be demonstrated that apr1 cultures established anaerobiosis<br />

and induced [FeFe]-hydrogenase synthesis although in vivo hydrogen<br />

production rates were close to zero. However, inhibitor studies proved that<br />

photosynthetic electron transport was predominantly directed to the Calvin-<br />

Benson cycle rather than to the hydrogen metabolism in the apr1 mutant strain<br />

[2].<br />

As a conclusion, the control between carbohydrate degradation and<br />

accumulation has to be taken into account for long-term hydrogen production<br />

un<strong>der</strong> normal growth condition.<br />

[1] Andreeva AV, Kutuzov MA: Widespread presence of "bacterial-like" PPP<br />

phosphatases in eukaryotes. BMC Evol Biol 2004, 4:47.<br />

[2] Rühle T, Hemschemeier A, Melis A, Happe T: A novel screening protocol<br />

for the isolation of hydrogen producing Chlamydomonas reinhardtii strains.<br />

BMC Plant Biol 2008, 8:107.<br />

PQ 08<br />

Molecular insights into evolution and mechanisms of<br />

binding, release and electron transfer between plastocyanin<br />

and photosystem I<br />

S. Kuhlgert *1 , F. Drepper 2 , H. Kirchhoff 3 , M. Hippler 1<br />

1 Institute of Biochemistry and Biotechnology of Plants, Westfälische Wilhelms-<br />

Universität, Münster, Germany<br />

2 Institute of Biochemistry, Albert-Ludwigs-Universität, Freiburg, Germany<br />

3 Institute of Botany, Westfälische Wilhlems-Universität, Münster, Germany<br />

In eukaryotic photosynthetic organisms the reduction of oxidized P700 of<br />

photosystem I (PSI) is facilitated by the copper containing electron donor<br />

plastocyanin (pc). Previous studies have shown that the oxidizing side of PSI is<br />

formed by the N-terminal domain of PsaF as well as residues of PsaA and PsaB<br />

[1-4].<br />

Here we present the Chlamydomonas chloroplast D612H/E613H mutant, in<br />

which two acidic PsaB residues, important for rapid unbinding of pc from PSI<br />

after electron donation, have been replaced by two histidines. This mutant is<br />

light sensitive un<strong>der</strong> photoautotrophic conditions. In single flash absorption<br />

measurements we determined the dissociation constant for binding and electron<br />

transfer between pc and the modified PSI. Our data revealed that the KD value<br />

for binding and electron transfer between pc and PSI is about three to four<br />

times lower for the mutant as compared to wild type. Furthermore the second<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

or<strong>der</strong> rate constant for electron transfer between pc and PSI is twofold higher in<br />

the mutant in respect to wild type. These data support the observations earlier<br />

made for the E613N mutant [3] and point to the fact that unbinding (Koff) of pc<br />

from the double His mutant PSI might also be slowed down.<br />

For the in vivo characterization of electron transfer in the mutant, the<br />

equilibration constant (KE) between cytochrome f and P700 was determined<br />

using difference absorption spectroscopy [5]. The comparison of the KE value<br />

determined for mutant D612H/E613H with those of wild type, strain E613N<br />

and a PsaF-deficient mutant revealed that the D612H/E613H mutation had the<br />

most dramatic effect on KE, which will be further discussed on the poster.<br />

[1] Hippler, M. et al. Biochemistry 36 (1997), pp. 6343-9.<br />

[2] Sommer, F. et al. J Biol Chem 279 (2004), pp. 20009-17.<br />

[3] Sommer, F. et al. J Biol Chem 277 (2002), pp. 6573-81.<br />

[4] Hippler, M. et al. Proc Natl Acad Sci U S A 95 (1998), pp. 7339-44.<br />

[5] Kirchhoff, H. et al. Biochim Biophys Acta 1659 (2004), pp. 63-72.<br />

PQ 09<br />

Functional characterization of stress-induced lightharvesting<br />

proteins in C. reinhardtii.<br />

E. Ostendorf *1 , G. Peers 2 , A. Busch 1 , B. Naumann 1 , H. Kirchhoff 3 , K.K.<br />

Niyogi 2 , M. Hippler 1<br />

1<br />

Institute of Biochemistry and Biotechnology of Plants, Westfälische Wilhelms-<br />

Universität, Münster, Germany<br />

2<br />

Department of Plant and Microbial Biology, University of California,<br />

Berkeley, United States<br />

3<br />

Institute of Botany, Westfälische Wilhelms-Universität, Münster, Germany<br />

In addition to light harvesting and transfer of excitation energy to the<br />

photosynthetic reaction centres, light-harvesting proteins (LHC) also play an<br />

important role in light energy dissipation. Gene expression studies and<br />

microarray analyses demonstrated that the expressions of LhcSR genes, a gene<br />

family of four genes in Chlamydomonas, were induced un<strong>der</strong> high light stress,<br />

phosphorus- and sulfur-deficiency[1-3]. Protein expression studies revealed that<br />

the C. reinhardtii homolog of the LI818 group, LhcSR3, is strongly induced<br />

un<strong>der</strong> iron-deprivation and light stress[4]. LhcSR proteins represent an ancient<br />

group of LHCs, present in many of the major groups of algae, and even<br />

bryophytes[5]. To further examine the function of LhcSR3, we generated and<br />

functionally characterized RNAi mutant strains. Interestingly, LhcSR3deficient<br />

strains are highly light-sensitive, suggesting a protective role of<br />

LhcSR3 in light-stress adaptation. We also analyzed a Chlamydomonas npq4<br />

mutant, which is deficient in lhcsr3.1 and -3.2 genes[6]. Our data confirm that<br />

LhcSR proteins contribute to non-photochemical quenching[6]. Additionally<br />

our data reveal that LhcSR proteins are involved in (i) stabilization of PSI-<br />

LHCI and PSII-LHCII supercomplexes, (ii) regulation of antenna sizes and (iii)<br />

remodelling of the photosynthetic machinery in response to iron-deficiency.<br />

[1] Im et al. Photosynth Res. 2003;75(2):111-25.<br />

[2] Moseley et al. Eukaryot Cell. 2006 Jan;5(1):26-44.<br />

[3] Zhang et al. Eukaryot Cell. 2004 Oct;3(5):1331-48.<br />

[4] Naumann et al. Proteomics. 2007 Nov;7(21):3964-79.<br />

[5] Koziol et al. Plant Physiol. 2007;143:1802-16.<br />

[6] Peers et al., submitted.<br />

163<br />

PQ 10<br />

The CO ligand of the active site of [NiFe]-hydrogenases is<br />

<strong>der</strong>ived from the cellular metabolism<br />

I. Bürstel *1 , P. Hummel 2 , N. Wisitruangsakul 2 , I. Zegber 2 , B. Friedrich 1 , O.<br />

Lenz 1<br />

1<br />

Institut für Biologie / Mikrobiologie, Humboldt-Universität zu Berlin, Berlin,<br />

Germany<br />

2<br />

Max-Volmer Laboratorium für Physikalische Chemie, Technische Universität<br />

Berlin, Berlin, Germany<br />

The utilization and production of molecular hydrogen is a common trait in the<br />

microbial world and is catalyzed by complex metalloproteins, called<br />

hydrogenases. One major subclass of hydrogenase possesses an active site<br />

which is composed of one nickel and one iron atom that are coordinated by four<br />

cysteine residues. During catalysis the nickel alters its oxidation state, while the<br />

iron remains in the low-spin Fe II form. The low-spin state of the iron is<br />

conserved by ligandation of three diatomic ligands, two cyanides and one<br />

carbon monoxide. In or<strong>der</strong> to prevent toxic effects, assembly of the CN - and CO<br />

ligand is mediated by a sophisticated maturation machinery that is composed of<br />

at least six auxiliary proteins [1].

164<br />

It has been shown that the CN - ligands are <strong>der</strong>ived from carbamoyl phosphate<br />

while the CO ligand has a different origin [2-4]. However, the source of the CO<br />

ligand remained unclear.<br />

13 C labelling experiments combined with Fourier transform infrared<br />

spectroscopy revealed for the regulatory [NiFe]-hydrogenase of Ralstonia<br />

eutropha that externally added carbon monoxide is incorporated into the Ni-Fe<br />

active site if provided in ample concentrations. However, CO at atmospheric<br />

concentration of approximately 0.1 ppmv is not sufficient for synthesis of<br />

active hydrogenase. From these results we conclude that the precursor of the<br />

CO ligand is <strong>der</strong>ived from the metabolism of the cell.<br />

[1] Böck et al. 2006 Adv. Microb. Physiol. 51:1-71.<br />

[2] Reissmann et al. 2003 Science 299:1067-1070.<br />

[3] Lenz et al. 2007 FEBS Lett. 58:3322-3326.<br />

[4] Forzi et al. 2007 FEBS Lett. 58:3331-3337.<br />

PQ 11<br />

Radical Mechanism of Phycoerythrobilin Synthase<br />

A. Busch *1 , E. Hofmann 2 , N. Frankenberg-Dinkel 1<br />

1<br />

Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany<br />

2<br />

Biophysics, Department of Biology and Biotechnology, Ruhr-University<br />

Bochum, Bochum, Germany<br />

Phycoerythrobilin (PEB) is an open chain tetrapyrrole (phycobilin) which<br />

functions as a chromphore in the light harvesting structures of cyanobacteria,<br />

the phycobilisomes (PBS). There, the pink-colored pigment is attached to<br />

phycobiliproteins and helps transferring light energy to photosystem II.<br />

The biosynthesis of phycobilins starts with the oxidative cleavage of heme<br />

yielding biliverdin IXα (BV). This reaction is catalyzed by hemeoxygenases.<br />

BV is then the substrate of a novel class of radical enzymes, the ferredoxindependent<br />

bilin reductases (FDBRs). The FDBRs do not possess any metal or<br />

organic cofactors and utilize ferredoxin as the electron donor. In cyanobacteria,<br />

the two major pigments phycocyanobilin (PCB) and PEB are synthesized via<br />

two independent reactions involving members of the FDBR family.<br />

PCB:ferredoxin oxidoreducatse (PcyA) catalyzes the formal four-electron<br />

reduction of BV to PCB and 15, 16-dihydrobiliverdin:ferredoxin<br />

oxidoreductase (PebA) and PEB:ferredoxin oxidoreductase (PebB) the<br />

sequential two-electron reduction of BV to PEB. Just recently another FDBR<br />

involved in PEB biosynthesis was discovered. Interestingly, PEB synthase<br />

(PebS) identified in the cyanophage P-SSM2 directly catalyzes the fourelectron<br />

reduction of BV to PEB. The PebS mechanism seems to be exclusive<br />

to phage since all cyanobacteria known so far utilize two enzymes yielding<br />

PEB in two sequential reductions. Surprisingly, P-SSM2 has been found to<br />

solely infect strains of the genus Prochlorococcus, which are one of the major<br />

primary producers in the oceans and do not possess PBS.<br />

Here we show that PebS acts via a radical mechanism as shown for other bilin<br />

reductases suggesting a general mechanism for FDBRs. Mutant analyses<br />

support the mechanism that was proposed based on the crystal structure of<br />

substrate bound PebS.<br />

PR 01<br />

Autophosphorylation of essential phosphosugar mutases in<br />

Bacillus subtilis<br />

N. Pietack *1 , M. Arnold 1 , K. Gronau 2 , D. Becher 2 , M. Hecker 2 , J. Stülke 1<br />

1 Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

2 Institute of Microbiology, University of Greifswald, Greifswald, Germany<br />

The modification of proteins is a ubiquitous mechanism to regulate cellular<br />

processes in all domains of life. Protein phosphorylation is one of the most<br />

important and best studied forms of modification and has for long time been<br />

assumed to be unique to eukaryotes. Recent studies indicated that this<br />

modification does also occur in bacteria. In B. subtilis 78 proteins are<br />

phosphorylated on Ser-/Thr and Tyr residues [1]. In most cases the function as<br />

well as the origin of these phosphorylations are unknown.<br />

Proteins are phosphorylated by specific protein kinases or via autokinase<br />

activity. The activity of specific kinases, like the HPr kinase is regulated by<br />

intermediates [2]. The autophosphorylation of kinases, like PrkC, is only<br />

dependent on the presence of metal ions. In contrast this protein kinase activity<br />

is regulated by specific stimuli (3). Furthermore it is unknown if ATP serves as<br />

the only phosphate donor for Ser-/Thr and Tyr phosphorylations.<br />

Nearly all glycolytic enzymes of B. subtilis are phosphorylated. Experiments<br />

with crude extracts, radiolabeled ATP and glycolytic intermediates showed<br />

significant changes in the phosphoproteome. Further analysis indicated that<br />

two essential phosphosugar mutases, the phosphoglycerate mutase (Pgm) and<br />

the glucosamine mutase (GlmM) do perform autophosphorylation. This<br />

autokinase activity is strictly dependent on manganese. In this work we<br />

analysed the effect of the autophosphorylations on the activity of these proteins<br />

and their essential function.<br />

[1] Macek et al., 2007. Mol Cell Proteomics 6: 697-707<br />

[2] Deutscher et al., 2006. MMBR 70: 939-1031<br />

[3] Shah et al., 2008. Cell 135: 486-496<br />

PR 02<br />

Influence of nitrogen on the life style of Rhodopirellula<br />

baltica<br />

C. Frank *1 , P. Langhammer 1 , B. Fuchs 2 , J. Har<strong>der</strong> 1<br />

1 Department of Microbiology, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

2 Department of Molecular Ecology, Max Planck Institut for Marine<br />

Microbiology, Bremen, Germany<br />

Bacteria of the genus of Rhodopirellula are aerobic, sessile-living bacteria<br />

belonging to the phylum of Planctomycetaceae. This benthic lifestyle requires<br />

for dispersal a planktonic, free-living life phase. The next generation of cells<br />

start their life with a large flagellum, swimming to new habitats. Upon arrival,<br />

the flagellum is lost and holdfast substances are produced to attach to a surface,<br />

e.g. a grain of sand or a marine snow particle. It is currently unknown which<br />

environmental signals cause the induction of the sessile lifestyle.<br />

We investigated the possibility to culture the planctomycete Rhodopirellula<br />

baltica strain SH1(T) in continuously chemostat cultures. In the presence of a<br />

large excess of glucose, the cells formed in continuous culture a<br />

macroscopically free-living community. Attachment of the cells to the walls of<br />

the chemostat was induced with an increase of the ammonium supply.<br />

Quantitative measurements showed a shift from an ammonium limited to an<br />

ammonium saturated population. Flow cytometry revealed the presence of<br />

attached-living cells in cell aggregates in both growth situations. However, a<br />

sufficient ammonium supply caused a decrease of single cells (swimming) and<br />

an increase of cell aggregates. Our observations suggest that the carbon to<br />

nitrogen ratio of the substrate influences the regulatory circuits that govern the<br />

decision to switch between benthic and planktonic life.<br />

PR 03<br />

Compatible solute degradation in halophilic Bacteria:<br />

Ectoine catabolism in Halomonas elongata DSM 2581 T<br />

K. Schwibbert *1 , G. Heidrich 1 , G. Lentzen 2 , H. Seitz 3 , H.J. Kunte 1<br />

1 FG IV.1, Bundesanstalt für Materialforschung und -prüfung (BAM), Berlin,<br />

Germany<br />

2 Forschung & Entwicklung, bitop AG, Witten, Germany<br />

3 Functional Protein Analysis, Max-Planck-Institut für Molekulare Genetik,<br />

Berlin, Germany<br />

Ectoine is the main compatible solute of the halophilic bacterium Halomonas<br />

elongata. H. elongata can utilize ectoine as carbon and nitrogen source. The<br />

ectoine degradation pathway proceeds via N-acetyl diamino (N-Ac-DABA)<br />

butyric acid to diamino butyric acid by the means of the enzymes ectoine<br />

hydrolase and N-Ac-DABA acylase. The enzymes are encoded by two adjacent<br />

ORFs named doeA and doeB. RT-PCR experiments showed that doeAB is<br />

transcribed together along with a third ORF named doeX. The transcriptional<br />

initiation site of the doeABX operon was mapped by rapid amplifying of cDNA<br />

ends (RACE). Inspection of the DNA sequence upstream of the initiation site<br />

revealed the presence of a putative –10 and –35 sequences that resembles the<br />

consensus sequence of sigma 70 dependent promoters. The newly identified<br />

doeX locus is coding for a putative protein with a calculated molecular mass of<br />

17.9 kDa. The deduced amino acid sequence of DoeX shows a high degree of<br />

identity to transcriptional regulator proteins of the AsnC/Lrp family.<br />

Recombinant expression of doeX in Escherichia coli led to a protein with an<br />

apparent molecular mass of 18 kDa. Electrophoretic mobility shift assays<br />

(EMSA) proved that DoeX is indeed a DNA-binding protein with significant<br />

binding affinity to the promoter region of doeABX. The influence of DoeX on<br />

doeAB transcription was analyzed by quantitative RT-PCR (qRT-PCR).<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PR 04<br />

Functional inactivation of Cap0129 of Clostridium<br />

acetobutylicum: a protein associated with granulose<br />

granules<br />

D. Lehmann *1 , R.J. Fischer 1<br />

1 Institute of Biological Sciences / Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

The cell cycle of the strict anaerobic Gram-positive Clostridium acetobutylicum<br />

is characterized by some interesting metabolic and morphological<br />

differentiations. For example cells are able to produce the polysaccharide<br />

granulose in form of unsoluble granules which are expected to serve as energy<br />

source during sporulation.<br />

Analyses of purified granulose granules revealed two proteins to be associated<br />

to the granules: Cap0129 with two carbohydrate binding modules which is<br />

encoded by the megaplasmid (pSol1) and glycogen synthase (GlgA). Here we<br />

present results dealing with the functional inactivation of the cap0129 gene<br />

based on the integration of a group II intron using the ClosTron-System [1].<br />

Molecular analyses (PCRs, Southern Blots, Northern Blots) clearly proved the<br />

correct integration of the intron into the cap0129 gene and strongly indicate<br />

functional inactivation of protein production. However, the mutant-cells were<br />

still able to produce granulose granules similar in shape and size to the wild<br />

type. Preliminary data might indicate a reduced amount of granules in the cells,<br />

but, contradictory the same effect seemed to be occur in wild type cells in<br />

which the amount of Cap0129 protein is increased (plasmid based homologous<br />

overexpression).<br />

[1] Heap, Pennington, Cartman, Carter, Minton, (2007) J. Microbiol. Methods,<br />

70(3): 452-64<br />

PR 05<br />

Characterization of a GlcNAc/MurNAc kinase of<br />

Clostridium acetobutylicum<br />

J. Schlei<strong>der</strong> *1 , C. Mayer 1<br />

1 Molekulare Mikrobiologie, Universität Konstanz, Konstanz, Germany<br />

The murein (peptidoglycan) sacculus is the essential exoskeleton-like structure<br />

of most bacteria. It is necessary to protect the cell from lysis due to the high<br />

intracellular turgor. N-acetylmuramic acid (MurNAc) and N-acetylglucosamine<br />

(GlcNAc) are the sugar components of the murein, which are steadily reutilized<br />

(recycled). In Escherichia coli lytic transglycosylases release an anhydro form<br />

of MurNAc (anhydroMurNAc) that can serve either as carbon source or as<br />

precursor for new murein synthesis. For this reason anhydroMurNAc and<br />

GlcNAc are phosphorylated by the kinases AnmK and NagK, respectively,<br />

yielding N-acetylmuramic acid-6-phosphate (MurNAc-6P) and Nacetylglucosamine-6-phosphate<br />

(GlcNAc-6P). Furthermore the MurNAc-6P<br />

etherase MurQ catalyzes the conversion of MurNAc-6P to GlcNAc-6P and Dlactate.<br />

So far, cell wall recycling in Gram-positive bacteria has not been well<br />

investigated. We identified a novel kinase of the Gram-positive, non-pathogen<br />

bacterium Clostridium acetobutylicum. MurK of Clostridium phosphorylates<br />

both, GlcNAc and MurNAc. It has no similarity to AnmK and NagK of E. coli<br />

and, in contrast to AnmK, it is unable to phosphorylate anhydroMurNAc.<br />

Biochemical characterizations of MurK revealed identical Km values for<br />

MurNAc and GlcNAc but a 1.5-fold higher vmax of the latter. This enzyme was<br />

used to detect for the first time muramic acid in the cell wall of chlamydia,<br />

which has been reported previously to lack murein.<br />

PR 06<br />

DsrEFH, DsrC and TusA: potential sulfur transferases in<br />

the phototrophic sulfur oxidizer Allochromatium vinosum<br />

Y. Stockdreher *1 , N. Dobler 1 , F. Grimm 1 , C. Dahl 1<br />

1 Institut für Mikrobiologie und Biotechnologie, Universität Bonn, Bonn,<br />

Germany<br />

Globules of polymeric sulfur are formed as a transient product by many photo-<br />

and chemotrophic sulfur-oxidizing bacteria. A ΔdsrE mutant experiment<br />

proved that the cytoplasmic protein DsrEFH is essential for sulfur globule<br />

degradation in the purple sulfur bacterium Allochromatium vinosum.<br />

Complementation of dsrEFH in trans fully restored the wild type phenotype.<br />

Active sites, harboring conserved cysteine residues, were identified in DsrE and<br />

DsrH. Cys78 of DsrE corresponds to the active site cysteine of Escherichia coli<br />

TusD. In E. coli, the proteins TusA, TusBCD and TusE are parts of a sulfur<br />

relay system involved in thiouridine biosynthesis [1]. Accordingly, DsrEFH<br />

interacts with DsrC, a TusE homologue encoded in the same operon [2]. Here<br />

we show that Cys78 of DsrE is strictly required for interaction with DsrC in<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

vitro while the active site Cys20 of DsrH is dispensable for that reaction. On<br />

the other hand, complementation experiments proved both active site cysteines<br />

to be essential for sulfur oxidation in A. vinosum. We furthermore identified a<br />

tusA homologue in A.vinosum and demonstrate specific interaction of AvTusA<br />

with DsrEFH in vitro. This interaction is not only strictly dependent on Cys78<br />

of DsrE but also requires the presence of conserved Cys15 of AvTusA.<br />

Although studies on the role of AvTusA in vivo are pending, our results point at<br />

the occurrence of sulfur transfer reactions during sulfur oxidation in A. vinosum<br />

and possibly also in other sulfur-oxidizing bacteria.<br />

[1] Ikeuchi et al. (2006) Mol. Cell. 21:97-108<br />

[2] Cort et al. (2008) J. Mol. Biol. 382, 692-707<br />

PR 07<br />

Online-Monitoring of physiological fitness of Lb. plantarum<br />

during fermentation and identification of optimal harvest<br />

points<br />

A. Schulz *1 , D. Knorr 1<br />

1 Department of Food Biotechnology and Food Process Engineering, TU Berlin,<br />

Berlin, Germany<br />

Many functional microorganism cultures which are involved in food production<br />

un<strong>der</strong>go a preservation process to prolong shelf life and optimise their<br />

applicability (e.g. deep frozen or freeze-dried starter cultures). Unfavourably,<br />

freezing and dehydration can cause severe losses of culturability.<br />

The survival rate depends on strain properties, process parameters, as well as<br />

presence or absence of protective or inhibitory substances. Moreover, intrinsic<br />

factors which can be summarised by the term „physiological fitness of<br />

microorganisms“ determine the resistance against stress. The energetic status of<br />

membranes and metabolic activity are two of these factors.<br />

The physiological fitness and thus the potential stress resistance is subject to<br />

change during fermentation, depending on the age of the culture, environmental<br />

and physical parameters. The measurement of optical density, pH, temperature<br />

and release or uptake of certain metabolites or nutritive substances is either<br />

time-consuming or not alone sufficient to describe these intrinsic changes.<br />

However, an online monitoring system containing flowcytometry and<br />

EloTrace ® , an electrooptical measurement device will be shown to capture and<br />

visualize aspects of the physiological fitness with a very short time lag. Thus,<br />

the exact and timely identification of optimal points for harvesting and<br />

preservation is feasible and leads to maximal survival rates.<br />

PR 08<br />

A view on the protein turnover in stress situations - A<br />

quantitative analysis using Pulse Chase in Corynebacterium<br />

glutamicum<br />

C. Trötschel *1 , D. Wolff 1 , M. Rögner 1 , A. Poetsch 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

We are focusing on the membrane as well as cytoplasmic proteome of C.<br />

glutamicum using shotgun proteomics and the nLC-ESI-MS/MS technology to<br />

quantify the turnover rates of proteins in general and that are especially<br />

involved in the physiological adaptation of the bacterium to different stresses.<br />

Proteins are subject to continuing changes in terms of their synthesis and<br />

degradation, named turnover. At the current state there exist only a rare number<br />

of publications for this topic, e.g. iron-starved Mycobacterium [1]. In contrast<br />

we want to give a global view on the protein abundance and turnover rates for<br />

much more proteins and additionally in response to biotechnologically relevant<br />

stress factors, such as heat, and salt stress (see also presentation of B. Fränzel).<br />

To un<strong>der</strong>stand the adaptation to different stress factors in fermentation<br />

processes we are now characterizing the proteomes un<strong>der</strong> the well-known heat<br />

stress [2].<br />

In our approaches we are investigating the dynamic of proteins by using Pulse<br />

Chase experiments. Thereby we transfer the C. glutamicum cells which reached<br />

the mid-exponentiell growth phase from a minimal medium that contains 15 N<br />

nitrogen to a medium with 14 N nitrogen. The 14 N will be incorporated into the<br />

newly synthesized proteins, so that a discrimination between the existing and<br />

the “new” protein via the 14 N/ 15 N ratio is possible by using mass spectrometry<br />

and the quantification software ProRata [3] as well as Census [4]. As a proof of<br />

principle we found very high turnover rates for the heat-induced DnaK and<br />

GroEL proteins.<br />

[1] Rao, P. K. et al. (2008) Anal Chem 80 (18): pp. 6860-6869<br />

[2] Barreiro, C. et al. (2005) J Bacteriol 187 (3): pp. 884-889<br />

[3] Pan, C. et al. (2006) Anal Chem 78 (20): pp. 7121-7131<br />

[4] Park, S. K. et al. (2008) Nat Methods 5 (4): pp. 319-322<br />

165

166<br />

PR 09<br />

Overproduction and characterization of DsrL: an [FeS]flavoprotein<br />

from Allochromatium vinosum<br />

L. Kammler *1 , F. Grein 1 , I.A.C. Pereira 2 , C. Dahl 1<br />

1 Institut für Mikrobiologie und Biotechnologie, Rheinische Friedrich-Wilhelms-<br />

Universität, Bonn, Germany<br />

2 Instituto de Tecnologia Quimica e Biologica, UniVersidade NoVa de Lisboa,<br />

Oeiras, Portugal<br />

The protein DsrL from the purple sulfur bacterium Allochromatium vinosum is<br />

indispensable for the degradation of sulfur globules formed as intermediates<br />

during the oxidation of reduced sulfur compounds [1]. On the basis of its amino<br />

acid sequence, DsrL is predicted to contain FAD and at least three iron-sulfur<br />

clusters. However, while recombinant DsrL clearly contained FAD, iron-sulfur<br />

clusters were not present even when the E. coli isc (iron sulfur cluster) gene<br />

cluster was co-expressed. The latter strategy has been successfully applied, e.g.<br />

for the production of several [FeS]-cluster-loaded reporter ferredoxins in E. coli<br />

[2]. Here, we show that in vitro reconstitution in the presence of 3.2 mM DTT,<br />

0.5 mM FeCl3 and 0.5 mM Na2S un<strong>der</strong> anoxic conditions finally led to insertion<br />

of [FeS] clusters into DsrL. Iron-sulfur cluster assembly further improved when<br />

the sulfur-transmitting cysteine desulfurase IscS from E. coli, FeCl2 in 10 fold<br />

molar excess instead of FeCl3 and 2mM cysteine were added to the reaction<br />

mixtures. Iron-sulfur cluster formation was followed by UV-Vis spectroscopy<br />

showing a strong increase of absorption at 420 nm over time. Macroscopically,<br />

the yellow FAD-containing protein solution obtained a brownish color.<br />

Chemical analysis proved the presence of at least 10 iron atoms per protein<br />

monomer. EPR spectroscopy so far confirmed the presence of [4Fe4S] 2+/1+<br />

clusters. Reconstituted DsrL exhibits NAD(P)H:acceptor oxidoreductase<br />

activity with a strong preference for NADH over NADPH.<br />

[1] Lübbe Y. et al (2006) FEMS Lett. 261: 194-202<br />

[2] Takahashi Y & Nakamura M. (1999) J. Biochem. 126: 917-926<br />

PR 10<br />

Complex organization and regulation of the genes encoding<br />

the Entner-Doudoroff pathway in Ralstonia eutropha H16<br />

B. Kusian *1 , A. Näther 1 , V. Halacheva 1 , R. Budinova 1 , A. Dimitrova 1 , S.<br />

Peykov 1 , G. Stahlhut 1 , B. Bowien 1<br />

1 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen,<br />

Göttingen, Germany<br />

Ralstonia eutropha H16 is one of the most extensively studied facultatively<br />

chemoautotrophic bacteria. Heterotrophic growth of the organism is supported<br />

by diverse organic compounds with a preference for organic acids. Fructose and<br />

N-acetylglucosamine (NAG) are the only sugars, gluconate, 2-ketogluconate<br />

and glucosaminate the only sugar acids utilized. Glucose is additionally<br />

metabolized by G + mutants. These substrates are solely degraded through the<br />

Entner-Doudoroff (ED) pathway. Most of the ED-related genes are located<br />

within five independent clusters/operons: A0310nagFECAB-zwf1,<br />

frcRACB-zwf2-pgi2-frcK, edd2zwf3-pgl-glk-B2563-B2562, eddRedd1gntK<br />

and B1213gal-kdgK-eda. Gene deletion mutagenesis revealed a<br />

differential involvement of these genes in the utilization of the substrates. It<br />

was found that the glucose-6-phosphate dehydrogenase genes zwf1 and zwf2<br />

play a major role for growth on NAG and fructose, respectively, whereas pgl<br />

(phosphogluconolactonase) was essential for the breakdown of both sugars.<br />

Inactivation of edd1 (phosphogluconolactonase) affected growth on gluconate<br />

as well as fructose but not on NAG. A defect in gntK (gluconokinase)<br />

specifically prevented the mutant from growing on gluconate while glk<br />

(glucokinase) was shown to be essential for glucose utilization by a G + strain.<br />

The growth phenotypes of the various mutants in combination with enzyme<br />

activities and transcriptional data obtained suggested a complex regulation<br />

governing the expression of the ED-encoding operons in R. eutropha H16.<br />

FrcR and EddR were identified as specific regulators of their cognate operons,<br />

whereas a two-component signal transduction system, FrcST, appeared to<br />

control fructose and NAG utilization in a more general fashion.<br />

PR 11<br />

Oxaloacetate decarboxylase of Corynebacterium glutamicum<br />

S. Klaffl *1 , B.J. Eikmanns 1<br />

1 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

Enzymes catalysing the irreversible decarboxylation of oxaloacetate fall into<br />

two different classes: i) the membrane-bound sodium-dependent oxaloacetate<br />

decarboxylase and ii) the soluble divalent-cation-dependent oxaloacetate<br />

decarboxylase. The former type of enzyme was subject to extensive studies, the<br />

encoding genes were identified and regulation, structure, function, and catalytic<br />

mechanism of several of these enzymes have been elucidated [1]. Although<br />

some decarboxylases of the latter type have been characterized, respective<br />

genes have not been identified and their function have not been analysed so far.<br />

Corynebacterium glutamicum is widely used for the large scale manufacture of<br />

L-lysine and L-glutamate. Oxaloacetate decarboxylase of this organism belongs<br />

to the soluble type [2] and is part of the so called PEP-pyruvate-oxaloacetate<br />

node. Since oxaloacetate is the precursor molecule in L-lysine biosynthesis, the<br />

question of the significance of the decarboxylation for fermentation processes<br />

arises. Therefore, oxaloacetate decarboxylase from C. glutamicum was isolated<br />

and purified, and the corresponding odx gene was identified, overexpressed and<br />

functionally analysed. Overexpression studies revealed that high oxaloacetate<br />

decarboxylase activity redirects carbon flux towards pyruvate. However,<br />

although overexpression of the odx gene in L-lysine-producing strains of C.<br />

glutamicum led to lower L-lysine accumulation, inactivation of the odx gene<br />

did not improve the L-lysine production. The ongoing biochemical study of the<br />

enzyme might reveal new insights into the physiological role of the soluble<br />

divalent-cation-dependent oxaloacetate decarboxylases.<br />

[1] Buckel, W. (2001) Biochim. Biophys. Acta 1505, 15-27<br />

[2] Jetten M.S. and Sinskey, A.J. (1995) Ant. Van Leeuwenhoek 67, 221-227<br />

PR 12<br />

Kinetics of the Sulfur oxidizing (Sox) Enzyme System from<br />

Paracoccus pantotrophus<br />

C. Friedrich 1 , A. Quentmeier *1 , R. Wichmann 1<br />

1 Fakultät Bio- und Chemieingenieurwesen, Technische Universität Dortmund,<br />

Dortmund, Germany<br />

Kinetics of the periplasmic sulfur-oxidizing (Sox) enzyme system of<br />

Paracoccus pantotrophus are described. The sulfur substrate is covalently<br />

bound to the central SoxYZ complex by the heme enzyme SoxXA, oxidized by<br />

the molybdoprotein cytochrome complex SoxCD, and sulfate is released by<br />

thioesterase SoxB. This system reduces horse heart cytochrome c with<br />

thiosulfate, sulfide, and sulfite as substrates [1, 2]. The oxidation rates were<br />

independent on the concentrations of the sulfur substrates and of horse heart<br />

cytochrome c. SoxYZ serves as substrate for the other three Sox proteins, and<br />

the rates increase with protein concentrations fitting well with the Michaelis-<br />

Menten model while the data did not fit with the control-of-flux-model [3]. The<br />

temperature-dependent thiosulfate oxidizing activity indicated an activation<br />

energy Ea of 72 kJ/mol below 16°C and 46 kJ/mol above suggesting a<br />

conformational change of a Sox protein. Differences in temperature optima<br />

with SoxCD (35°C) and without (45°C) suggested the intermediate complex<br />

with SoxCD to be temperature-sensitive as supported by its low temperature<br />

tolerance. The activity declined from pH 5.5 to pH 7.5 suggesting<br />

deprotonation of the active site possibly Arg-247 of SoxA of the SoxXA<br />

complex, the partner of SoxYZ in the initial reaction.<br />

[1] Friedrich et al. (2001) Appl. Environ. Microbiol. 67, 2873–2881.<br />

[2] Quentmeier et al. (2007) Biochemistry 46, 10990-10998<br />

[3] Kacser H. and J. A. Burns (1995) Biochem. Soc. Trans. 23, 341-66.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PR 13<br />

New aerobic aromatic metabolism – the cases of benzoate<br />

and phenylacetate<br />

R. Teufel *1 , L. Rather 1 , V. Mascaraque 2 , J. Perera 2 , W. Eisenreich 3 , W. Ismail 1 ,<br />

G. Fuchs 1<br />

1<br />

Institut für Biologie II – Mikrobiologie, Albert-Ludwigs-Universität Freiburg,<br />

Freiburg, Germany<br />

2<br />

Departamento de Bioquímica y Biología Molecular, I, Facultad de Ciencias<br />

Biológicas, Universidad Complutense de Madrid, Madrid, Spain<br />

3<br />

Institut für Organische Chemie und Biochemie, Technische Universität<br />

München, München, Germany<br />

In the aerobic metabolism of aromatic compounds, oxygenases catalyze ring<br />

hydroxylation and oxygen-dependent C-C cleavage. New pathways for the<br />

aerobic metabolism of benzoate and phenylacetate use CoA-thioesters as<br />

intermediates and surprisingly a non-oxygenolytic C-C cleavage, as studied in<br />

Azoarcus sp.(benzoate) and Pseudomonas sp.(phenylacetate) [3]. In both cases<br />

the genes are organized in large operons [1,2]. In a first step CoA-thioesters of<br />

the aromatic acids are formed by ATP-dependent CoA-ligases. Benzoyl-CoA<br />

and phenylacetyl-CoA are further converted into non-aromatic dihydrodiols by<br />

novel types of dioxygenases/reductases. The following non-oxygenolytic ring<br />

cleavage is catalyzed by unique enzymes of the enoyl-CoA-hydratase<br />

superfamily [4]. The open chain intermediates are further processed via<br />

modified beta-oxidation leading to acetyl-CoA and succinyl-CoA. This<br />

contribution represents the state of the art of this new type of aerobic aromatic<br />

metabolism.<br />

[1] Gescher, J. et al. (2002) J Bacteriol. 184(22): 6301-15<br />

[2] Alonso, S. et al.(2003) Gene 319:71-83<br />

[3] Zaar, A. et al. (2004) Mol Microbiol 54(1):223-38.<br />

[4] Gescher, J. et al. (2005) Mol Microbiol 56(6):1586-600.<br />

PR 14<br />

Granulose Granules and a Glycogen Synthase (glgA)<br />

Defect Mutant of Clostridium acetobutylicum ATCC 824<br />

H. Janssen *1 , M. Zechlau 1 , R.J. Fischer 1<br />

1 Institute of Biological Sciences / Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

The cell cycle of the strict anaerobe Gram positive bacterium Clostridium<br />

acetobutylicum is characterized by some interesting metabolic and<br />

morphological differentiations. One feature is the synthesis of granulose<br />

granules within the cells shortly before the sporulation is initiated.<br />

It is known that this particles are formed by an slightly branched polysaccharide<br />

of α-D-glucose. However, there is only little knowledge as well about their<br />

ultra structure as about the synthesis machinery.<br />

In this study, we present data concerning the purification of granulose granules<br />

and their analysis via SDS-PAGE. Furthermore, we were able to create a<br />

functional granulose negative mutant of Clostridium acetobutylicum ATCC 824<br />

by the integration of group II intron (LlltrB) using the clostron system (1) into<br />

the gene glgA (cac2239), which is expected to code for a granulose synthase.<br />

[1] Heap J. T., O. J. Pennington, S. T. Cartman, G. P. Carter, N. P. Minton.<br />

(2007) J. Microbiol. Methods. 70(3): 452-64<br />

PR 15<br />

Physiological characterization of Streptomyces coelicolor<br />

strains containing deletions in essential nitrogen<br />

metabolism genes<br />

E. Waldvogel *1 , J. Grimpo *1 , W. Wohlleben 1 , J. Reuther 1<br />

1 Microbiology/ Biotechnology, Eberhard Karls University, Tübingen, Germany<br />

Streptomycetes are Gram positive soil bacteria, which produce around 60 % of<br />

commercial available antibiotics. A better un<strong>der</strong>standing of the primary and in<br />

particular the nitrogen metabolism in the model organism Streptomyces<br />

coelicolor might help to develop a strain with an enhanced antibiotic<br />

production.<br />

We constructed several mutant strains of Streptomyces coelicolor M145 with<br />

deletions of essential genes involved in nitrogen metabolism: glnA, glnII, gdhA,<br />

glnK and amtB/glnK.<br />

S. coelicolor contains two different glutamine synthetase genes: glnA and glnII<br />

encode the glutamine synthetases GSI and GSII, respectively. Glutamine<br />

synthetases are responsible for nitrogen assimilation from ammonium into<br />

glutamine un<strong>der</strong> low nitrogen conditions. GSI is thought to be the housekeeping<br />

GS, whereas GSII is present only in the late growth phase and could have a<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

specific role in secondary metabolism or differentiation. Un<strong>der</strong> high nitrogen<br />

conditions ammonium is assimilated by the glutamate dehydrogenase (gene<br />

product of gdhA).<br />

glnK encoding a PII signal protein is in many bacteria involved in<br />

transcriptional and posttranslational regulation of nitrogen metabolism. In S.<br />

coelicolorglnK is located in an operon together with amtB (encoding an<br />

ammonium transporter) and glnD (encoding an adenylyltransferase). The<br />

regulatory function of S. coelicolorGlnK is yet unclear. In contrast to other<br />

bacteria, e.g. Escherichia coli, GlnK is not involved in posttranslational<br />

modulation of glutamine synthetase activity.<br />

Growth curve experiments were performed to characterize these mutant strains<br />

physiologically. Biomass, antibiotic production and the consumption of<br />

glucose and ammonium were estimated. Furthermore we characterized the<br />

intracellular amino acid pool and determined glutamine synthetase - and<br />

glutamate dehydrogenase activity.<br />

PR 16<br />

Recombinant SoxYZ and SoxF are active in the<br />

reconsituted Sox-Enzyme System of Paracoccus<br />

pantotrophus<br />

D. Rother *1 , A. Quentmeier 1 , C.G. Friedrich 1<br />

1 Lehrstuhl Technische Mikrobiologie/Fachbereich Bio- und<br />

Chemieingenieurwesen, Technische Universität Dortmund, Dortmund,<br />

Germany<br />

The periplasmic sulfur-oxidizing (Sox) enzyme-system of the facultatively<br />

chemotrophic alpha-proteobacterium Paracoccus pantotrophus GB17 is<br />

composed of four proteins SoxXA, SoxB, SoxCD and SoxYZ, the central<br />

protein of the system. Together these proteins reduce horse heart cytochrome c<br />

with thiosulfate and sulfur substrates in vitro [1]. SoxF reactivates SoxYZ when<br />

inactivated by reduction e.g. by tris(2-carboxyethyl) phosphine (TCEP) [2]. The<br />

soxYZ and soxF genes were amplified by PCR and cloned into the expression<br />

vector pQE30. The recombinant SoxY and SoxF were produced as cellular<br />

His6-tagged proteins in Escherichia coli. His6-SoxY formed with SoxZ in E.<br />

coli the His6-SoxYZ complex which was purified with Ni-NTA-agarose. His6-<br />

SoxF was purified with Ni-NTA-agarose followed by resource Q anion<br />

exchange chromatography. The protein biochemical properties of His6-SoxYZ<br />

like activation by sulfide and inactivation by TCEP were similar to that of<br />

SoxYZ of the wild type as was the specific thiosulfate oxidizing activity in<br />

vitro. His6-SoxF like SoxF had sulfide dehydrogenase activity in vitro.<br />

Moreover, His6-SoxF reactivated SoxYZ inactivated by TCEP. Thus,<br />

recombinant SoxYZ and SoxF exhibited principal biochemical properties<br />

identical to those of the wild type proteins.<br />

[1] Friedrich et al. (2001) Appl. Environ. Microbiol. 67, 2873–2882.<br />

[2] Quentmeier et al. (2008) FEBS Letters 582, 3701-3704.<br />

PR 17<br />

The role of the glyoxylic acid cyle and the aco- and acu<br />

genes during growth of Bacillus licheniformis on C-2<br />

Substrates or amino acids<br />

M. Bauch *1 , M. Schwarzer 2 , J. Bongaerts 3 , S. Evers 3 , W. Liebl 1 , A. Ehrenreich 1<br />

1 Institut für Mikrobiologie, TU München, Freising, Germany<br />

2 Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen,<br />

Göttingen, Germany<br />

3 Global R&D Laundry and Home Care Biotechnology, Henkel AG & Co.<br />

KGaA, Düsseldorf, Germany<br />

167<br />

Bacillus licheniformis DSM13 is an organism of great industrial importance<br />

that becomes increasingly accessible to methods of molecular biology.<br />

Using our developed method for marker-less deletions we deleted the genes of<br />

the glyoxylate operon and the acu- and the aco-operon for the investigation of<br />

the acetate metabolism of B. licheniformis MW3 (AG Meinhardt, Münster). We<br />

could show the glyoxylate cycle and the acu-operon to be involved into acetate<br />

and amino acid metabolism and the aco-operon to be involved in acetate<br />

metabolism and acetoine degradation.<br />

For further metabolic studies and to define the essential genes for fermentation<br />

processes with B. licheniformis we are working on the establishment of a<br />

method for a mariner transposon mutagenesis.<br />

While microbiological research has mainly focused on the degradation of<br />

substrates like sugars or single amino acids, little is known on the much more<br />

common case of growth on proteins or mixtures of amino acids. To investigate<br />

growth of B. licheniformis on these compounds, we first determined which<br />

amino acids could be used as carbon and/or nitrogen source and then<br />

determined the or<strong>der</strong> of their depletion from mixtures. In this context we

168<br />

created a defined medium that imitates the amino acid composition of soybean<br />

protein. To define genes characteristic for growth on amino acids or mixtures of<br />

them, we used DNA microarray based on oligonucleotides representing all<br />

genes of B. licheniformis.<br />

PR 18<br />

Interrelations between respiratory and light-driven energy<br />

conservation in Dinoroseobacter shibae<br />

J. Holert *1 , S. Hahnke 1 , H. Cypionka 1<br />

1 Institute for Chemistry and Biology of the Marine Environment, ICBM,<br />

University of Oldenburg, Oldenburg, Germany<br />

The marine bacterium Dinoroseobacter shibae belongs to the aerobic<br />

anoxygenic phototrophic bacteria (AAPB), which are able to use light as an<br />

energy source un<strong>der</strong> oxic conditions without forming oxygen.<br />

In the present study we have investigated the influence of light on the adenylate<br />

content and the energy charge (EC) in D. shibae. A modified<br />

Luciferin/Luciferase assay was used for the simultaneous analysis of ATP,<br />

ADP and AMP in a single assay. Washed cell suspensions of D. shibae, preincubated<br />

un<strong>der</strong> anoxic conditions, multiplied their ATP content within 40<br />

seconds from about 0.6 nmol (mg dm) -1 to about 14.8 nmol (mg dm) -1 upon<br />

addition of oxygen in the dark. The AMP content decreased stoichiometrically<br />

while the EC increased from 0.05 to 0.9. When the cells were illuminated, they<br />

developed nearly the same ATP generation rate and ATP yield as dark cells.<br />

However, an influence of light was proven regarding the respiration rate of D.<br />

shibae, which was significantly slowed down upon illumination. Furthermore,<br />

light-driven proton translocation in washed cell suspensions of D. shibae could<br />

be demonstrated.<br />

Therefore, we conclude that respiratory and light-driven electron transfer and<br />

consequently proton translocation both contribute both to the rapid ATP<br />

generation in D. shibae. Light can be used as an alternative energy source, thus<br />

saving electron donors for respiration.<br />

PR 19<br />

The accompanying organism of a marine Beggiatoa culture<br />

A. Bachmann *1 , V. Bondarev 1 , H.N. Schulz-Vogt 1<br />

1 Ecophysiology Group, Department of Microbiology, Max Planck Institute for<br />

Marine Microbiology, Bremen, Germany<br />

Beggiatoa are colorless, sufide-oxidizing gammaproteobacteria living at the<br />

transition between oxygen and sulfide. Big marine Beggiatoa are able to use<br />

nitrate in addition to oxygen as electron acceptor and oxidize sulfide in anoxic<br />

sediments. In our lab we are cultivating a marine nitrate-storing Beggiatoa<br />

strain. To our knowledge, this Beggiatoa strain is the only culture of a larger<br />

vacuolated sulfur bacterium. Until now, however, it was not possible to achieve<br />

a pure culture of the Beggiatoa. There is one accompanying organism left in the<br />

culture. Therefore, this accompanying organism was isolated into pure culture<br />

and is investigated in detail. The 16S-rRNA-gene of this accompanying<br />

organism is similar to the one of Pseudovibrio denitrificans, a heterotrophic and<br />

denitrifying alphaproteobacterium. Interestingly, the cultivated organism is able<br />

to grow on rich media, as Pseudovibrio denitrificans, but on the other hand<br />

growth also occurs un<strong>der</strong> extremely oligotrophic conditions. It is growing in<br />

pure artificial seawater without fixed nitrogen and with an organic carbon<br />

content of < 1 mg carbon per liter. When we measure organic carbon in<br />

inoculated cultures and sterile controls we observe a decrease of < 0.1 mg<br />

carbon per liter in the inoculated samples. Un<strong>der</strong> these oligotrophic conditions,<br />

the alphaproteobacteria achieve cell counts of approximately 10 5 cells/ml.<br />

Therefore, it is of great interest to know whether the decrease of carbon is due<br />

to assimilation into biomass or if the present carbon is enough to be used as<br />

electron donor because no other possible electron donor is determined yet.<br />

PR 20<br />

Physiology of a facultative oligotrophic bacterium<br />

V. Bondarev *1 , A. Bachmann 1 , H.N. Schulz-Vogt 1<br />

1 Ecophysiology Group, Department of Microbiology, Max Planck Institute for<br />

Marine Microbiology, Bremen, Germany<br />

Pseudovibrio denitrificans has been described in the year 2004 as a<br />

heterotrophic, facultatively anaerobic marine bacterium capable of<br />

denitrification and fermentation. In our lab an α-proteobacterium has been<br />

isolated and identified as P. denitrificans via 16S rRNA analysis. Although the<br />

similarity of the 16S rRNA gene is > 99,5 %, the physiology of the isolate<br />

seems to differ from the described P. denitrificans species. The new isolate has<br />

been verified to be capable of denitrification, fermentation as well as aerobic<br />

growth with polypeptone and yeast extract. However, in contrast to the already<br />

described P. denitrificans strain, no aerobic growth with sugars or fatty acids<br />

could be observed in a defined medium, indicating the deficiency of a trace<br />

element or another supplement. Intriguingly, the addition of diverse trace<br />

elements as well as vitamins did not affect the growth and makes it still unclear,<br />

why glucose, which is used during fermentation, cannot be metabolized without<br />

the addition of yeast extract un<strong>der</strong> aerobic growth conditions. Determining the<br />

factors that are needed for aerobic growth in a defined medium of the new<br />

isolate is the main goal of this study. Additionally, the newly isolated αproteobacterium<br />

has been shown to be capable of oligotrophic growth in a<br />

medium with less than 1 mg carbon per liter. Investigating the nutrition and<br />

supplement requirements of this bacterium un<strong>der</strong> different growth conditions<br />

might shed more light on the poorly un<strong>der</strong>stood physiology of facultative<br />

oligotrophic bacteria.<br />

PR 21<br />

Autotrophic 3-hydroxypropionate cycle in Chloroflexus<br />

aurantiacus: finally completed<br />

J. Zarzycki *1 , G. Fuchs 1<br />

1 Institut für Biologie II / Abt. Mikrobiologie, Albert-Ludwigs-Universität<br />

Freiburg, Freiburg, Germany<br />

Chloroflexus aurantiacus, a thermophilic green non-sulfur bacterium, can grow<br />

photoautotrophically un<strong>der</strong> anaerobic conditions in the presence of H2 or H2S.<br />

Twenty years ago a new autotrophic CO2 fixation cycle for C. auranticus was<br />

proposed in which acetyl-CoA and 3-hydroxypropionate are intermediates [5].<br />

The primary CO2 fixation product of this 3-hydroxypropionate cycle is<br />

glyoxylate [6]. The assimilation of glyoxylate into cell carbon compounds was<br />

at issue. A second cycle was proposed [3, 4] in which glyoxylate and<br />

propionyl-CoA are converted to acetyl-CoA and pyruvate [1, 2, 7]. We show<br />

that this second cycle requires only three additional enzymes. The glyoxylate<br />

condensation with propionyl-CoA to β-methylmalyl-CoA is catalyzed by Lmalyl-CoA<br />

lyase, followed by dehydration to mesaconyl-C1-CoA by a specific<br />

hydratase. Intramolecular CoA transfer yields mesaconyl-C4-CoA. This is<br />

hydrated to L-citramalyl-CoA which is cleaved into pyruvate and acetyl-CoA<br />

by L-malyl-CoA lyase.<br />

[1] Friedmann et al. 2007. J Bacteriol 189:2906<br />

[2] Friedmann et al. 2006. J Bacteriol 188:6460<br />

[3] Herter et al. 2002a. J Bacteriol 184:5999<br />

[4] Herter et al. 2002b. J Biol Chem 277:20277<br />

[5] Holo and Grace. 1989. Arch. Microbiol. 151:252<br />

[6] Strauss and Fuchs. 1993. Eur J Biochem 215:633<br />

[7] Zarzycki et al. 2008. J Bacteriol 190:1366<br />

PR 22<br />

Comprehensive analysis of pH homeostasis in<br />

Corynebacterium glutamicum on the bioenergetic,<br />

transcriptome, as well as proteome level revealed a<br />

functional link between pH response, oxidative stress, iron<br />

homeostasis and methionine synthesis<br />

I. Ochrombel *1 , M. Follmann 1 , R. Krämer 1 , C. Trötschel 2 , A. Poetsch 2 , C.<br />

Rückert 3 , A. Hüser 3 , M. Persicke 3 , J. Kalinowski 3 , K. Marin 1<br />

1 Institute of Biochemistry, University of Cologne, Cologne, Germany<br />

2 Institute of Plant Biochemistry, University of Bochum, Bochum, Germany<br />

3 Institute for Genome Research and Systems Biology, University of Bielefeld,<br />

Bielefeld, Germany<br />

The Gram-positive soil bacterium Corynebacterium glutamicum is used for<br />

industrial fermentation processes and thereby exposed to different stress<br />

conditions, like low pH. We determined the capacity of C. glutamicum for pH<br />

homeostasis and were interested in identification of limitations for growth<br />

un<strong>der</strong> acidic conditions. It was shown that the maintenance of the proton<br />

motive force was not impaired over a broad range of external pH values. In<br />

or<strong>der</strong> to address physiological targets for pH stress we applied transcriptome<br />

and proteome analyses. The alignment of the differential gene expression and<br />

protein pattern with known regulatory modules revealed the occurrence of<br />

secondary stresses during pH response of C. glutamicum. Among them are the<br />

induction of oxidative stress and the activation of the iron starvation response at<br />

low pH. As a consequence metabolome studies revealed that metabolic<br />

pathways like the TCA, NAD synthesis and methionine and cysteine pathways<br />

are affected. In the latter case the pool size of the effector S-adenosylhomocysteine<br />

was found to be increased triggering McbR the master regulator<br />

of the methionine and cysteine synthesis. However, in spite of the activation of<br />

the McbR controlled pathway the methionine pool was slightly reduced which<br />

is in agreement with detection of low activities of particular enzymes at low<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

pH. On the other hand cysteine accumulation was observed and addition of<br />

cysteine to C. glutamicum cultures at low pH was found to be toxic. In<br />

summary, we could identify limitations for growth at low pH for C. glutamicum<br />

by a comprehensive multi level analysis.<br />

PR 23<br />

Assembly of the scaffold protein PspA from Escherichia coli<br />

H. Osadnik *1 , T. Brüser 1<br />

1 Institute of Biology / Microbiology, University of Halle-Wittenberg, Halle,<br />

Germany<br />

The phage shock protein A (PspA) is a stress-inducible membrane-associated<br />

protein of Escherichia coli that is produced in response to various<br />

environmental changes like heat shock or osmotic upshift. It is thought to be<br />

involved in membrane stabilization, preventing the dissipation of the PMF. It is<br />

prevalent in bacteria while occurring also in archea and eukaryotic chloroplasts.<br />

Recently, we were able to show that PspA can form large scaffold-like<br />

structures that can be preserved when no detergent is used during purification<br />

[1]. These new structural insights into the PspA complexes raise fundamental<br />

questions such as: How do PspA monomers assemble to form highly or<strong>der</strong>ed<br />

networks? How stable are these scaffolds? Do PspA-scaffolds assemble<br />

spontaneously? What are the minimal PspA assemblies that together interact to<br />

form larger networks? Using a broad range of biochemical and biophysical<br />

approaches, we are investigating these important questions and will present our<br />

latest results.<br />

[1] Standar, K., Mehner, D, Osadnik, H., Berthelmann, F., Hause, G., Lünsdorf,<br />

H., and Brüser, T. (2008) PspA can form large scaffolds in Escherichia coli.<br />

FEBS Lett. 582, 3585-3589<br />

PR 24<br />

Analysis of an extracellular biofilm inducing factor in<br />

Burkhol<strong>der</strong>ia glumae PG1<br />

J. Knorr *1 , S. Isenhardt 1 , S. Wilhelm 1 , K.E. Jäger 1 , F. Rosenau 1<br />

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University<br />

Duesseldorf, Juelich, Germany<br />

Burkhol<strong>der</strong>ia glumae PG1, formally known as Pseudomonas glumae, is a<br />

phytopathogenic β-proteobacteria with biotechnological potential. These<br />

bacteria have the type II secretion system Gsp (general secretory pathway)<br />

which is homologous to the Xcp-system of the human pathogenic Pseudomonas<br />

aeruginosa.<br />

Here we constructed two Gsp-mutants, by inserting a cassette in the gspD-gene<br />

and in the gspDEF-operon and studied their effects on various phenotypes of B.<br />

glumae. These studies showed that PG1 wild type was able to form biofilms in<br />

minimal medium, whereas both mutants were not able to form it un<strong>der</strong> the same<br />

conditions. The biofilm formation of the mutants is rescued by addition of spent<br />

cell-free medium of wild type. Initial analysis of extracellular components of<br />

the wild type showed that the inducing factor has a molecular mass of more<br />

than 50 kDa. We purified the inducing factor by size exclusion chromatography<br />

and found an apparent size of 290 kDa indicating the biofilm inducing factor<br />

<strong>bei</strong>ng a protein complex. The protein(s) involved were further analysed by mass<br />

spectrometry.<br />

PR 25<br />

Proline metabolism in the mo<strong>der</strong>ately halophilic bacterium<br />

Halobacillus halophilus<br />

S. Köcher *1 , M. Tausendschön 1 , S. Saum 1 , V. Müller 1<br />

1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,<br />

Goethe University Frankfurt, Frankfurt, Germany<br />

The dominant osmolyte in the mo<strong>der</strong>ately halophilic bacterium Halobacillus<br />

halophilus grown at very high salinities is proline [1]. However, at the end of<br />

exponential growth, intracellular proline disappears (and ectoine is produced<br />

instead) demonstrating the ability to degrade proline [2]. Proline supplied to the<br />

medium is taken by the cells but not used as an osmolyte, instead it is oxidized<br />

to carbon dioxide. Growth experiments revealed that H. halophilus can use<br />

proline as sole carbon and energy source. Uptake is maximal at high salinities,<br />

but not driven by hyperosmolarity across the membrane. Genes encoding the<br />

putative proline transporter as well as the degradation pathway(s) were<br />

identified on the chromosome. Interestingy, two open reading frames encoding<br />

a putative proline-dehydrogenase (prodh1 and prodh2) and a putative Δ 1 -<br />

pyrroline-5-carboxylate-dehydrogenase (p5cdh1 and p5cdh2) could be<br />

identified. When cells were grown with proline as sole carbon and energy<br />

source the transcript levels of prodh2 and p5cdh2 increased. Transcription of<br />

these genes was independent of the salinity of the medium.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

169<br />

[1] Saum S.H. and Müller V. 2007. Salinity-dependent switching of osmolyte<br />

strategies in a mo<strong>der</strong>ately halophilic bacterium: glutamate induces proline<br />

biosynthesis in Halobacillus halophilus. J. Bacteriol. 189: 6968–6975<br />

[2] Saum S.H. and Müller V. 2008. Growth phase-dependent switch in<br />

osmolyte strategy in a mo<strong>der</strong>ate halophile: ectoine is minor osmolyte but major<br />

stationary phase solute in Halobacillus halophilus. Environ. Microbiol. 10:<br />

716-726<br />

PR 26<br />

Maltose metabolism of Corynebacterium glutamicum:<br />

Identification of a metabolic pathway widespread in Gramnegatives<br />

in a Gram-positive bacterium<br />

R. Kempkes 1 , A. Henrich 1 , L. Both 1 , G.M. Seibold *1<br />

1 Institute of Biochemistry, University of Cologne, Cologne, Germany<br />

Corynebacterium glutamicum is a Gram-positive soil bacterium employed in<br />

the production of various amino acids and serves as model organism for<br />

subor<strong>der</strong> Corynebacterianeae, which comprises species such as<br />

Corynebacterium diphtheriae and Mycobacterium tuberculosis. Intracellular<br />

maltose and maltodextrins are substrates for trehalose synthesis, a prerequisite<br />

for the production of mycolates, major and structurally important constituents<br />

of the cell envelope of Corynebacterianeae. In contrast to most other bacterial<br />

model organisms maltose metabolism has been scarcely investigated in C.<br />

glutamicum and other members of the subor<strong>der</strong>.<br />

We analysed cell extracts of C. glutamicum for the presence of enzymes<br />

involved in maltose metabolism in other bacteria and found activities of 4-αglucanotransferase<br />

(MalQ), maltodextrin phosphorylase (MalP), glucokinase<br />

(Glk) and α–phosphoglucomutase (α–Pgm) and no activities of maltose<br />

hydrolase, maltose phosphorylase and β-Pgm, all three known to be involved in<br />

maltose utilisation of Gram-positives. Further analysis revealed that C.<br />

glutamicum accomplishes maltose uptake by a high affinity ABC-transport<br />

system. We identified the genes encoding MalQ and MalP by sequence<br />

comparisons in the genome sequence of C. glutamicum and constructed the<br />

deletion mutants C. glutamicum ΔmalQ and C. glutamicum ΔmalP. Both<br />

mutants showed impaired growth when cultivated with maltose as sole source<br />

of carbon and energy.<br />

Based on these findings we conclude that C. glutamicum metabolises maltose<br />

via a pathway involving maltodextrin and glucose formation by MalQ, glucose<br />

phosphorylation by Glk and maltodextrin degradation via the reactions of MalP<br />

and α-Pgm, a pathway hitherto known to be present in Gram-negative rather<br />

than in Gram-positive bacteria.<br />

PR 27<br />

Effect of oxygen availability on catabolic gene expression of<br />

aerobic and anaerobic toluene degrading bacteria<br />

P. Martinez *1 , C. Müller 1 , M. Buffing 1 , U. Kappelmeyer 1 , I. Nijenhuis 1 , H.J.<br />

Heipieper 1<br />

1 Department of Environmental Biotechnology, Helmholtz Centre for<br />

Environmental Research-UFZ, Leipzig, Germany<br />

Bacteria in the environment are constantly exposed to oxygen variations and<br />

gradients as they occur, e.g., in aquifers, in microbial mats and the plant root<br />

and the rhizosphere. Microorganisms living in polluted sites have the ability to<br />

process these input signals in or<strong>der</strong> to develop adaptive responses to survive<br />

fluctuations of external conditions.<br />

The relative expression of catabolic genes un<strong>der</strong> oscillating oxygen conditions<br />

was studied in two toluene degrading bacteria capable of aerobic toluene<br />

degradation, Pseudomonas putida mt-2 and an anaerobic toluene degra<strong>der</strong>,<br />

Thauera aromatica K172. The central catabolic genes targeted were xylM and<br />

xylE for P. putida and bssA and bcrA for T. aromatica; the quantification was<br />

done using real-time PCR. A decrease in the expression level of xylM and xylE<br />

was observed un<strong>der</strong> oxygen limiting conditions when P. putida mt-2 was grown<br />

on toluene as carbon source. Thus, oxygen is needed as a kind of co-inducer for<br />

the expression of the catabolic genes of the TOL plasmid. P. putida mt-2 was<br />

able to modulate the expression of its catabolic genes according to the oxygen<br />

availability in the media. During anoxic periods these bacteria decrease the<br />

growth rate and the expression of catabolic genes to a level which allow them<br />

to recover the activity when oxygen is present again in the medium. The<br />

addition of oxygen to T. aromatica K172 cultures grown with toluene as the<br />

carbon source immediately caused a repression of bssA and bcrA expression. In<br />

mixed binary cultures of P. putida and T. aromatica, submitted to anoxic/oxic<br />

cycles, a regulation of catabolic genes depending on the presence of oxygen<br />

was observed. After two oxic cycles T. aromatica showed an up-regulation of<br />

catabolic genes once oxygen was depleted by P. putida.

170<br />

PR 28<br />

Bifunctional fructose 1,6-bisphosphate<br />

aldolase/phosphatase, the ancestral glucogenic enzyme<br />

R.F. Say *1 , G. Fuchs 1<br />

1<br />

Institut für Biologie II, Mikrobiologie, Albert-Ludwigs-Universität Freiburg,<br />

Freiburg i. Br., Germany<br />

Most archaeal groups and deeply branching bacterial lineages harbour<br />

thermophilic organisms with a chemolithoautotrophic metabolism. They live at<br />

the high temperatures of their volcanic habitats at the expense of inorganic<br />

substances, often un<strong>der</strong> anoxic conditions. Such features reflect the conditions<br />

of primordial life [1]. These autotrophic organisms use different carbon dioxide<br />

fixation mechanisms generating acetyl-CoA, from which gluconeogenesis must<br />

start [2,3].<br />

We show that virtually all archaeal groups as well as the deeply branching<br />

bacterial lineages (Aquificales, Thermoanaerobacterales, Thermotogales,<br />

Chloroflexales-Dehalococcoidetes, and the Deinococcus-Thermus group)<br />

contain a fructose 1,6-bisphosphate (FBP) aldolase/phosphatase with both FBP<br />

aldolase and phosphatase activity; normal FBP aldolases [4,5] are mostly<br />

missing. The bifunctionality of FBP aldolase/phosphatase ensures that heatlabile<br />

triosephosphates are quickly removed and trapped in stable fructose 6phosphate.<br />

We propose that this unidirectional FBP aldolase/phosphatase, which is highly<br />

conserved, represents the ancestral glucogenic enzyme.<br />

[1] Wächtershäuser (2007) Chem Biodivers 4:584-602.<br />

[2] Berg et al. (2007) Science 318:1782-6.<br />

[3] Huber et al. (2008) PNAS 105:7851-6.<br />

[4] Altekar et al. (1988) Orig Life Evol Biosph 18:59-64.<br />

[5] Lorentzen et al. (2004) Biochem Soc Trans 32:259-63.<br />

PR 29<br />

Response of UV-evolved Bacillus subtilis cells to different<br />

environmental stressors<br />

M. Wassmann *1 , R. Moeller 1 , G. Reitz 1 , P. Rettberg 1<br />

1 Institute of Aerospace Medicine, Radiation Biology Department, German<br />

Aerospace Center (DLR), Cologne, Germany<br />

In a precursory study for the space experiment ADAPT (Molecular adaptation<br />

strategies of microorganisms to different space and planetary UV climate<br />

conditions), approximately 700 generations of B. subtilis had been periodically<br />

exposed to UV radiation. Cells evolved un<strong>der</strong> UV stress were 3-fold more<br />

resistant to UV-C compared to the ancestral and equally evolved but not UVirradiated<br />

populations. Spores of both cell types respond similar to UV<br />

irradiation and exhibit ancestor UV survival characteristics. UV-evolved cells<br />

were also more resistant to ionizing radiation than their non-UV exposed<br />

evolved relatives and ancestor, whereas no changes in the spore survival after<br />

ionizing radiation exposure of all three populations were detectable.<br />

To get more information on the changes taken place during the adaptation<br />

phase on the molecular level, several physiological parameters, i.e the response<br />

to UV-A and UV-B irradiation, dry and wet heat exposure, desiccation and<br />

peroxides treatment were determined.<br />

Current investigations on the molecular mechanisms, e.g. transcriptional<br />

profiling, will allow un<strong>der</strong>standing changes on the adaptation level.<br />

PR 30<br />

Clostridium ljungdahlii - insight in the metabolism of a<br />

homoacetogenic Clostridium<br />

C. Held *1 , M. Köpke 2 , A. Wiezer 3 , A. Ehrenreich 1 , W. Liebl 1 , H. Liesegang 3 , R.<br />

Daniel 3 , G. Gottschalk 2<br />

1 Institut für Mikrobiologie, TU München, Freising, Germany<br />

2 Institute of Microbiology and Biotechnology, Universität Ulm, Ulm, Germany<br />

3 Göttingen Genomics Laboratory, Institute of Microbiology and Genetics,<br />

Georg-August Universität Göttingen, Göttingen, Germany<br />

Clostridium ljungdahlii is an acetogenic bacterium that was isolated for its<br />

ability to produce ethanol autotrophically from syngas. It is of industrial interest<br />

because syngas is an inexpensive substrate that can be easily generated by<br />

gasification of coal, biomass or municipal waste.<br />

The genome sequence of C. ljungdahlii with a size of 4.6 million base pairs was<br />

recently determined. We analyzed the genome with special regard to genes<br />

possibly involved in the Wood-Ljungdahl pathway and in energy conservation<br />

during autotrophic growth.<br />

During autotrophic growth no net-gain of ATP seems to be possible by<br />

substrate level phosphorylation as the ATP generated by acetate kinase has to<br />

be used for formate activation. In contrast to Moorella thermoacetica, we could<br />

not identify genes for cytochromes. Instead, genes similar to an rnf-complex are<br />

present as has been described for the Na + -dependent homoacetogen<br />

Acetobacterium woodii. As C. ljungdahlii grows autotrophically in the absence<br />

of Na + we propose that the rnf-complex is involved in the generation of a<br />

proton gradient that could be used for ATP synthesis by electron transport<br />

phosphorylation.<br />

For further investigations of the energy metabolism of C. ljungdahlii, we<br />

designed a DNA-microarray containing 4166 oligonucleotides covering 98.5%<br />

of ORFs identified on the genome to compare expressed genes un<strong>der</strong> different<br />

growth conditions. Experiments are on the way.<br />

PS 01<br />

RsiD-dependent regulation in Bacillus subtilis<br />

C. Diethmaier *1 , S. Hübner 1 , N. Pietack 1 , J. Stülke 1<br />

1 Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

The Gram-positive soil bacterium Bacillus subtilis can live its life in different<br />

ways - either as unicellular individual or as a member of a biofilm community.<br />

The regulatory network to switch between these different lifestyles is very<br />

complex. We have identified the rsiD gene, encoding a regulator of Sigma D<br />

activity. A one-dimensional proteome analysis of a rsiD mutant revealed a very<br />

strong overexpression of flagellin, the product of the hag gene. Reporter<br />

analyses of the hag promoter confirmed the increased promoter activity in a<br />

rsiD mutant strain. RsiD might control hag expression at the level of<br />

transcription initiation or at the level of transcript stability. The latter possibility<br />

was excluded by Northern blot analyses suggesting that RsiD is directly or<br />

indirectly involved in the control of transcription initiation. A deletion analysis<br />

of the hag promoter region showed that rsiD directly affects the conserved core<br />

promoter region. However, RsiD does not bind directly to the promoter<br />

suggesting that the effect is indirect. We did therefore consi<strong>der</strong> the possibility<br />

the RsiD might control the activity of the sigma factor σ D . The expression of<br />

other σ D -dependent genes was studied by qRT-PCR. The results demonstrate a<br />

global effect of RsiD on the σ D -regulon in B. subtilis. Moreover, the rsiD<br />

mutant was unable to form biofilms, a σ D -dependent phenotype. Bacterial-Two-<br />

Hybrid analyses excluded a direct interaction between RsiD and SigD or its<br />

antisigma factor FlgM.<br />

Interestingly the gene downstream of rsiD called spoVS has exactly the same<br />

phenotypes as the rsiD-mutant. Northern blot analyses excluded a RsiDdependent<br />

spoVS expression. Therefore the direct interaction between RsiD and<br />

SpoVS are investigated in a Bacterial-Two-Hybrid analysis. So the mechanism<br />

of the RsiD-dependent regulation is subject to further investigations.<br />

PS 02<br />

Mutants of the bacterial PPM phosphatase tPphA reveal<br />

novel residues important for substrate recognition and<br />

enzymatic function<br />

J. Su *1 , C. Schlicker 2 , K. Forchhammer 1<br />

1<br />

Lehrstuhl für Mikrobiologie/Organismische Interaktionen, Eberhard-Karls-<br />

Universität Tübingen, Tübingen, Germany<br />

2<br />

Department of Physiological Chemistry, Ruhr-University Bochum, Bochum,<br />

Germany<br />

Protein phosphatases of the PPM family are widely distributed in bacteria and<br />

play important regulatory roles. The crystal structure of the phospho-PII (PII-P)<br />

phosphatase from the thermophilic cyanobacterium Thermosynechococcus<br />

elongatus, (tPphA) was resolved recently. The enzyme has 3 metals in the<br />

catalytic center and displays a unique flap-subdomain that controls access to the<br />

catalytic site. The role of the third metal in PPM phosphatases has not been<br />

resolved to date. 17 site-directed mutants of tPphA were generated, purified and<br />

characterized enzymatically by using three kinds of substrates - pNPP,<br />

phospho-peptides and the PII-P protein. Mutants D119A and D193A of the Asp<br />

residues that coordinate the third metal abolish activity towards all substrates,<br />

indicating that the third metal is essential for catalytic activity. The mutant<br />

H39A indicates that H39 plays a key role in phosphoprotein recognition. To<br />

directly analyze the binding between tPphA and PII in vitro, a combined crosslink<br />

and pull down assay was designed. Whereas the enzymatic activity of<br />

tPphA is inhibited by Ca 2+ ions, Ca 2+ favors the co-purification of PII-P with<br />

tPphA. All mutants of the Asp-residues involved in coordinating the three metal<br />

ions where impaired both in enzymatic activity and binding of PII. With the<br />

exception of H39, all mutants impaired in enzymatic activity also show reduced<br />

binding of PII-P. The effector molecules ATP, ADP and 2OG greatly decrease<br />

the specific binding of PII-P to tPphA, implying that these molecules affect the<br />

conformation of PII thereby antagonizing tPphA binding.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PS 03<br />

Heterogeneity in quorum sensing regulated<br />

bioluminescence of Vibrio harveyi<br />

C. Anetzberger *1 , T. Pirch 1 , K. Jung 1<br />

1 Department Biologie I, Institut für Mikrobiologie, Ludwig-Maximilians-<br />

Universität, Planegg-Martinsried, Germany<br />

Quorum-sensing (QS) refers to the ability of bacterial populations to<br />

collectively activate gene expression. In or<strong>der</strong> to un<strong>der</strong>stand how single cells<br />

behave within a QS-activated community, we determined the time-dependent<br />

QS-induced bioluminescence of Vibrio harveyi cells, one of the best<br />

characterized model organisms in QS. Unexpectedly, even at high cell densities<br />

only 69% of the cells of a wild-type population produced bioluminescence (QSactive<br />

cells), 25% of the cells remained dark (QS-inactive), and 6% of the cells<br />

were dead. Moreover, light intensities greatly varied from cell to cell at high<br />

population density. Addition of extra autoinducer to a bright liquid culture of V.<br />

harveyi increased the percentage of bright cells up to 99% suggesting that V.<br />

harveyi produces and/or keeps the autoinducers at non-saturating<br />

concentrations. In contrast, all living cells of a constitutive QS-active mutant<br />

(ΔluxO) produced light. We also found that wild-type cells had a high tendency<br />

to aggregate in liquid culture, whereas mutant cells remained apart. Moreover,<br />

the capability of the ΔluxO mutant to produce biofilm was significantly reduced<br />

compared to the wild-type. These data indicate that even a QS committed<br />

population of V. harveyi takes advantage of heterogeneity, and extend the<br />

current view of QS regulated uniformity.<br />

PS 04<br />

Regulation of the pstSCAB operon in Corynebacterium<br />

glutamicum by the regulators of acetate metabolism RamB<br />

and GlxR<br />

M. Panhorst *1 , U. Sorger-Herrmann 2 , V.F. Wendisch 1<br />

1 Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms<br />

University Muenster, Muenster, Germany<br />

2 Institute of Biotechnology 1, Research Center Juelich, Juelich, Germany<br />

The Gram-positive Corynebacterium glutamicum is widely used for large-scale<br />

biotechnological production of amino acids. Phosphorus, an essential nutrient<br />

for all cells, constitutes about 2% of the cell dry weight of C. glutamicum [1].<br />

When phosphate and energy are abundant, C. glutamicum accumulates<br />

cytosolic or granular polyphosphate [2], which is synthesized by polyphosphate<br />

kinase [3]. Upon phosphate starvation, the psi genes of the phosphate starvation<br />

stimulon are induced [4] with the two-component regulatory system PhoS-<br />

PhoR <strong>bei</strong>ng important for the immediate response to phosphate limitation [5,<br />

6]. The fact that the pstSCAB operon encoding an ABC transporter for high<br />

affinity phosphate uptake is partially induced in a ΔphoRS mutant indicated<br />

additional regulator(s) and DNA-affinity chromatography revealed that the<br />

carbon regulators GlxR and RamB bind to the pstS promoter. GlxR was shown<br />

to bind to the pstS promoter in vitro in a cAMP dependent manner.<br />

Overexpression of glxR retards growth when phosphate is in excess, but<br />

enhances growth when phosphate is scarce. RamB was shown to bind in vitro<br />

to two partially conserved 13 bp RamB binding motifs. On acetate the<br />

induction of psi genes was less pronounced in the WT, but <strong>der</strong>epressed in the<br />

ΔramB mutant. Thus, expression of pstSCAB in C. glutamicum is subject to<br />

carbon control by the regulators of acetate metabolism RamB and GlxR.<br />

[1] Wendisch VF & Bott M (2008) In Corynebacteria: Genomics and molecular<br />

biology (Burkovski A., ed.), Horizon Scientific Press, Norwich, UK, pp. 203-<br />

216.<br />

[2] Klauth et al. (2006) Appl Microbiol Biotechnol 72: 1099-1106.<br />

[3] Lindner et al. (2007) Appl Environ Microbiol 73: 5026-5033.<br />

[4] Ishige T et al. (2003) J Bacteriol 185: 4519-4529.<br />

[5] Kočan M et al. (2006) J Bacteriol 188: 724-732.<br />

[6] Schaaf S & Bott M (2007) J Bacteriol 189: 5002-5011.<br />

PS 05<br />

Analysis of molybdenum-regulated promoters in<br />

Rhodobacter capsulatus<br />

A. Müller *1 , B. Masepohl 1<br />

1 LS Biologie <strong>der</strong> Mikroorganismen, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

The phototrophic α-proteobacterium Rhodobacter capsulatus synthesizes two<br />

molybdenum-dependent regulators, MopA and MopB, which exhibit 52 %<br />

sequence identity to each other. Either MopA or MopB is sufficient to repress<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

transcription of the anfA gene coding for the transcriptional activator of ironnitrogenase<br />

genes. In addition to its role as a repressor, MopA acts as<br />

transcription activator of the mop gene coding for a Mo-binding protein of<br />

unknown function. MopA and MopB bind to conserved palindromic promoter<br />

elements, so-called Mo-boxes, and binding of the regulators to their target<br />

promoters is enhanced by molybdenum.<br />

As a basis for fine-structure analysis of Mo-repressed and Mo-activated<br />

promoters, variants of the anfA-Mo-box and the mop-Mo-box were constructed<br />

by site-directed mutagenesis. Binding of purified MopA and MopB proteins to<br />

these promoter variants was analyzed by DNA mobility shift assays. The<br />

influence of Mo-box mutations on gene transcription was examined with R.<br />

capsulatus wild-type, mopA, mopB, and mopA-mopB mutant strains carrying<br />

appropriate lacZ reporter gene fusions. The results obtained in these studies<br />

may be summarized as follows. (i) The anfA- and mop-Mo-boxes are essential<br />

and sufficient for regulator binding. (ii) Mutations in the Mo-repressed anfA<br />

promoter influence regulation by MopA and MopB to comparable extents. (iii)<br />

Specific mutations in the mop-Mo-box do not influence MopA binding but<br />

abolish mop gene activation.<br />

PS 06<br />

Regulation of copper tolerance genes in Rhodobacter<br />

capsulatus<br />

C. Rademacher *1 , B. Masepohl 1<br />

1 LS Biologie <strong>der</strong> Mikroorganismen, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

Copper is an essential cofactor of many enzymes involved in photosynthesis,<br />

respiration, and iron transport. At the same time, copper is highly toxic to cells<br />

since free Cu(I) ions promote formation of reactive oxygen species. To<br />

maintain suitable copper concentrations within cells, bacteria have evolved<br />

homeostasis systems controlling uptake, export, and detoxification of copper.<br />

The phototrophic α-proteobacterium R. capsulatus synthesizes a multicopper<br />

oxidase, CutO, in response to elevated copper concentrations. Like Escherichia<br />

coli CueO and other multicopper oxidases, CutO exhibits phenoloxidase<br />

activity.<br />

The cutO gene is flanked by two genes, orf635 and cutR. Orf635 might be<br />

specific for R. capsulatus and close relatives as implicated by database<br />

searches. In contrast, CutR belongs to a widely distributed, but hardly<br />

characterized protein family implicated in resistance towards different cations.<br />

Orf635, CutO, and CutR confer copper tolerance as demonstrated by mutational<br />

analysis. The respective mutants exhibit wild-type-like tolerance towards silver,<br />

nickel, and zinc, suggesting that Orf635, CutO, and CutR are not involved in<br />

general metal homeostasis but are copper-specific. Metal determination of<br />

recombinant proteins revealed stoichiometric amounts of copper for CutO and<br />

CutR, thus un<strong>der</strong>lining a role of these proteins in copper homeostasis. Orf635<br />

apparently did not contain copper.<br />

Reverse transcriptase PCR analysis suggests that orf635, cutO, and cutR form a<br />

tri-cistronic operon. However, while transcription of cutO-lacZ and cutR-lacZ<br />

fusions is clearly copper-induced, expression of orf635 seemingly is not<br />

influenced by copper.<br />

PS 07<br />

Quantitative analysis of the Kdp system un<strong>der</strong> K + limitation<br />

in Escherichia coli<br />

K. Zigann *1 , S. Gayer 2 , A. Kremling 2 , R. Heermann 1 , K. Jung 1<br />

1 Department Biologie I, Mikrobiologie, Ludwig-Maximilians-Universität<br />

München, Planegg-Martinsried, Germany<br />

2 MPI für Dynamik komplexer technischer Systeme, Max-Planck-Institut,<br />

Magdeburg, Germany<br />

171<br />

Potassium (K + ), the dominant intracellular cation, serves important functions in<br />

bacteria. It plays a major role in the maintenance of turgor. Bacteria have<br />

evolved several K + uptake systems to adjust the intracellular K + concentration.<br />

Un<strong>der</strong> conditions of severe K + limitation or osmotic upshift synthesis of the<br />

high affinity K + uptake system KdpFABC is induced in Escherichia coli. This<br />

system serves as an emergency system to scavenge K + when the other<br />

transporters cannot keep up with the cells requirement for K + . Expression of<br />

kdp is transcriptionally regulated by the sensor kinase/response regulator<br />

system KdpD/KdpE.<br />

A mathematical model has been generated describing the signal transduction<br />

cascade of the Kdp system. It comprises two modules: i) the KdpD/KdpE<br />

phosphorylation cascade and ii) the kdpFABC transcription and translation. To<br />

evaluate the existing model the time dependent induction of kdpFABC<br />

transcription, KdpFABC synthesis and the K + balance were quantitatively<br />

analyzed in the E. coli strains MG1655 (wild-type) and MG1655kdpA4<br />

(KdpFABC-inactive mutant) upon an shift to K + limitation. In the wild-type,

172<br />

kdpFABC expression was strongly induced within the first minutes, followed<br />

by a down-regulation and a second increase after 60 min. This time course can<br />

be attributed to feed-back regulation by the KdpD/KdpE signaling cascade. On<br />

the contrary, the number of kdpFABC transcripts in the KdpFABC-inactive<br />

mutant followed a hyperbolic time course and was three-fold higher compared<br />

to the wild-type. At the protein level, in both, the wild-type and the mutant a<br />

hyperbolic increase of KdpFABC molecules was observed. However, the<br />

number of KdpFABC molecules per cell was significantly lower than expected<br />

from the transcript levels. Thus, a second feed-back regulation was identified<br />

that influences the stability of KdpFABC.<br />

PS 08<br />

Phenotypic characterization of cbrAB and ntrBC deletion<br />

mutants of Pseudomonas putida KT2440<br />

C. Kistler 1 , T. Friedrich 1 , A. Waldhuber 1 , A. Stück 1 , H. Jung *1<br />

1 Mikrobiologie, Biozentrum <strong>der</strong> LMU München, Planegg-Martinsried,<br />

Germany<br />

CbrA/CbrB represents a signal transduction system containing a sensor kinase<br />

(CbrA) with a N-terminal domain similar to members of the sodium/solute<br />

symporter family (e.g., PutP, PanF) and a C-terminal domain sharing homology<br />

to histidine kinases (e.g., NtrB) [1;2]. In Pseudomonas aeruginosa CbrA/CbrB<br />

is crucial for the utilization of arginine and histidine [2;3]. Stimuli and target<br />

genes of CbrA/CbrB are not known. We investigate the role of CbrA/CbrB in<br />

the soil bacterium P. putida KT2440. Previously we demonstrated that CbrA<br />

has autokinase activity and transfers the phosphoryl group to CbrB. A transport<br />

activity is not found for CbrA. Here we describe a comprehensive phenotypic<br />

analysis of cbrAB, ntrBC and cbrAB/ntrBC deletion mutants of P. putida<br />

KT2440. The cbrAB mutant exhibits severe defects upon growth on several<br />

amino acids and poylamines as carbon or carbon and nitrogen sources, as well<br />

as on citric acid cycle intermediates. The utilization of amino acids and<br />

polyamines as nitrogen sources is only slightly affected by cbrAB deletion.<br />

Growth of the ntrBC mutant is impaired un<strong>der</strong> conditions of nitrogen limitation.<br />

Gene expression analyses reveal a significant impact of CbrA/CbrB on amino<br />

acid uptake and degradation and central metabolic pathways (e.g., glyoxylate<br />

cycle). While direct target genes have not been identified yet, the results<br />

indicate that CbrA/CbrB plays an important role in regulating the carbon and<br />

nitrogen metabolism of P. putida KT2440.<br />

[1] Jung, H. (2002) FEBS Lett. 529, 73-77<br />

[2] Nishijyo, T., Haas, D., and Itoh, Y. (2001) Mol. Microbiol. 40, 917-931<br />

[3] Li, W. and Lu, C. D. (2007) J. Bacteriol. 189, 5413-5420<br />

PS 09<br />

The RSP2889 gene of Rhodobacter sphaeroides encodes a<br />

CueR homolog, a regulator of copper resistance<br />

V. Peuser *1 , J. Glaeser 1 , G. Klug 1<br />

1 Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität<br />

Gießen, Gießen, Germany<br />

Copper (Cu) is an essential trace element required by organisms from bacteria<br />

to humans. Due to its redox chemistry copper ions can act as cofactors for<br />

redox-active proteins. Copper proteins are involved in biological processes like<br />

respiration, iron transport and oxidative stress protection. On the other hand<br />

copper is highly toxic to cells even at low concentrations since it contributes to<br />

the generation of reactive oxygen species (ROS) including the highly reactive<br />

hydroxyl radical. In or<strong>der</strong> to maintain appropriate copper levels in the cell the<br />

uptake of copper, its distribution within the cell and its removal need to be well<br />

balanced.<br />

The RSP2889 gene product of the alphaproteobacterium Rhodobacter<br />

sphaeroides shows high similarity to CueR (Cu efflux regulator) of E. coli or<br />

Alphaproteobacteria (e. g. Rhizobium leguminosarum). CueR of E. coli is a<br />

copper-sensing regulatory factor that induces the expression of copA which<br />

encodes a copper translocating ATPase.<br />

On the R. sphaeroides chromosome the cueR gene is localized next to the genes<br />

for a putative Cu transporting P-type ATPase (RSP2890) and for a putative Cuchaperone<br />

(RSP2891). A ΔcueR mutant was significantly more sensitive to<br />

CuSO4 than the wild type. To confirm a similar function of the RSP2889 gene<br />

product of R. sphaeroides as for the transcriptional regulator CueR of other<br />

bacteria, we expressed the protein in a Strep-tagged version in E. coli. The<br />

isolated protein specifically binds to DNA fragments that carry the promoter<br />

regions of gene RSP2890 or RSP2891.<br />

PS 10<br />

Proteins involved in acetone formation of Clostridium<br />

acetobutylicum<br />

B. Schiel *1 , S. Brehm 1 , P. Dürre 1<br />

1 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

Clostridium acetobutylicum is a Gram-positive, endospore-forming, anaerobic<br />

soil bacterium. The importance of this bacterium is due to its ability to naturally<br />

produce solvents, namely butanol and acetone.<br />

The acetoacetate decarboxylase (Adc) catalyzes the last step of the acetone<br />

formation, converting acetoacetate to acetone. The respective operon (adc), is<br />

controlled by the global transcription regulator Spo0A. Destruction of the<br />

Spo0A-binding sites in the adc promoter region did lower, but not completely<br />

abolish the expression of the adc gene. This indicates that at least one more<br />

transcription factor is acting besides the Spo0A protein. AdcR, which was<br />

isolated by DNA affinity chromatography, represents such a protein. RT-PCR<br />

experiments proved an operon structure of the adcR gene and the downstream<br />

gene, adcS. The appropriate transcription start was determined by primer<br />

extension analysis. The promoter region of the adcR/S operon contains a<br />

potential reverse Spo0A-binding site and a potential CodY-binding site. Cody<br />

is known as pleiotropic transcription regulator in Gram-positive bacteria. It has<br />

also been shown to repress butanol formation in C. acetobutylicum<br />

(accompanying poster, Standfest et al.). Spo0A, AdcR, AdcS, and CodY were<br />

purified as his-tag fusion proteins and used in gel retardation assays using the<br />

promoter region of the adc operon as well as the promoter region of the adcR/S<br />

operon as probes. Sense, antisense, and knock-out (Heap et al., 2007) mutants<br />

of the genes adcR and adcS are established, and the respective mutants were<br />

characterized concerning growth and product spectrum. The data indicate a role<br />

of AdcR as a repressor and AdcS as a positive regulator of the formation of<br />

acetone. The protein CodY seems to play a role in the transcription of the<br />

adcR/S operon.<br />

PS 11<br />

The regulatory duties of the abridged phosphotransferase<br />

system (PTS Ntr ) of Pseudomonas putida<br />

K. Pflüger-Grau *1 , M. Chavarría 1 , U. Sauer 2 , V. de Lorenzo 1<br />

1 Systems Biology Program, Centro Nacional de Biotecnología (CSIC), Madrid,<br />

Spain<br />

2 Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland<br />

The activity of most individual bacterial promoters is submitted to various<br />

layers of control, so that expression of specific genes is coordinated to the<br />

overall physiology of the cells. One prevalent physiological sensor is the<br />

phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS). The<br />

genome of the soil bacterium Pseudomonas putida KT2440 encodes only 5<br />

recognizable proteins belonging to two classes of PTS systems: [i] a classical<br />

system for fructose intake (PTS Fru ) and [ii] a metabolic nitrogen-sensing PTS<br />

(PTS Ntr ) encoded by ptsP(EI Ntr ), ptsO(NPr), and ptsN(EIIA Ntr ). Recently, we<br />

detected a direct protein-protein interaction of PtsN with the pyruvate<br />

dehydrogenase (PDH) complex leading to down-regulation of the PDH activity,<br />

thereby altering the acetyl-CoA pool. Current experiments are set to analyze<br />

this interaction in or<strong>der</strong> to determine the influence of the phosphorylation state<br />

of PtsN on PDH regulation. Additionally, to explore the gross functional depth<br />

of PTS Ntr on the cell physiology, we analyzed the metabolic fluxes of isogenic<br />

strains bearing non-polar directed mutations in each of the corresponding pts<br />

genes. This analysis revealed that PTS Ntr controls the connection of pyruvate to<br />

the TCA cycle by inhibiting the pyruvate shunt, which bypasses malate<br />

dehydrogenase in the TCA cycle. Surprisingly, the examined metabolic effects<br />

of the PTS Ntr could be traced to the sole presence/absence of PtsN, regardless of<br />

its phosphorylation state. These results, together with the observation that the<br />

PTS Ntr influences expression of the TOL biodegradation pathway, biofilm<br />

formation, and the intracellular accumulation of polyhydroxyalkanoates,<br />

provide evidence for a central regulatory function of the PtsN(EIIA Ntr ) protein<br />

of P. putida.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PS 12<br />

Vertical Regulation, a New Layer of Transcriptional<br />

Regulation upon Protocatechuate Degradation by<br />

Acinetobacter baylyiy<br />

F. Bleichrodt *1 , U. Gerischer 1<br />

1 Institute for Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

The soil bacterium Acinetobacter baylyi is able to degrade a wide range of<br />

aromatic compounds via the β-ketoadipate pathway. To cope with<br />

environmental changes most efficiently, this pathway un<strong>der</strong>lies complex<br />

regulatory networks, which act at different levels (Rita Fischer, Fenja S.<br />

Bleichrodt and Ulrike C. Gerischer, 2008. Aromatic degradative pathways in<br />

Acinetobacter baylyi un<strong>der</strong>lie carbon catabolite repression. Microbiology, 154,<br />

3095-3103). In this study we focus on the regulation of the protocatechuate<br />

branch of the pathway. Protocatechuate is a metabolite of vanillate or<br />

hydroxycinnamate catabolim (e.g. ferulate or caffeate). The required enzymes<br />

are encoded by the van or the hca genes. With transcriptional fusions of the<br />

luciferase reporter gene to these genes, it was possible to show that the presence<br />

of protocatechuate results in decreased expression of the hca or van operon. To<br />

evaluate if the well studied IclR-type transcriptional regulator of the pca operon<br />

PcaU is involved in this mechanism, we deleted this gene. In addition we<br />

searched for binding motifs at the respective operons. We could show, that<br />

PcaU plays a role in the mechanism of vertical regulation.<br />

PS 13<br />

Elucidation of the target genes of the YehU/YehTsensorkinase/response<br />

regulator system in Escherichia coli<br />

L. Fried *1 , T. Kraxenberger 1 , C. Guggenberger 1 , K. Jung 1<br />

1 Institut für Mikrobiologie / AG Prof. K. Jung, Ludwig-Maximilians-Universität<br />

München, Department Biologie I, Planegg-Martinsried, Germany<br />

Two-component systems (TCS) are the predominant signal transduction<br />

systems in prokaryots and consist of at least two elements: a membraneintegrated<br />

sensor kinase (SK) with histidine kinase activity which senses a<br />

stimulus and transduces it in a cellular signal, and a response regulator (RR)<br />

with DNA-binding activity. Whereas most TCS in Escherichia coli are well<br />

characterized, little is known about the SK YehU and the corresponding RR<br />

YehT. YehU is anchored with six transmembrane domains in the membrane. It<br />

has a GAF domain, and the input domain is structurally similar to the input<br />

domain of LytS, a potential sensor for murein subunits in Gram-positive<br />

bacteria. YehT possesses a CheY homologous regulator domain, and a LytR<br />

DNA-binding domain. The effect of the chromosomal deletion of yehUT on the<br />

physiology and the proteome of E. coli MG1655 was investigated un<strong>der</strong><br />

microaerobic growth conditions at 30°C. The phenotypical characterization of<br />

the mutant revealed altertions in cell motility, aggregation, and biofilm<br />

formation. Comparative proteome analyses of the wild-type and the mutant<br />

identified a set of differently produced proteins. These proteins are involved in<br />

regulation (HscA, IbpA, TdcF, YtfE), metabolism (AnsB, TdcB, TdcE, YfiD),<br />

and cell motility (CheZ, FliC). Transcriptional analysis corroborated that most<br />

of these genes are un<strong>der</strong> direct control of the TCS YehU/YehT. Deletion of<br />

yehUT also abolished expression of the neighbouring genes yehR, yehS and<br />

osmF as analyzed by Northern blot hybridization. The current model of the<br />

YehU/YehT-dependent regulatory network was extended.<br />

PS 14<br />

The two-component regulatory system CiaRH in<br />

Streptococcus pneumoniae: Characterization of novel alleles<br />

of the histidine kinase gene ciaH<br />

M. Müller *1 , R. Hakenbeck 1 , B. Reinhold 1<br />

1 Department of Microbiology, University of Kaiserslautern, Kaiserslautern,<br />

Germany<br />

The two-component regulatory system CiaRH of Streptococcus pneumoniae<br />

has been implicated in β-lactam resistance, maintenance of cell integrity,<br />

competence, and virulence. The ciaRH genes were originally identified in<br />

spontaneous mutants of S. pneumoniae showing resistance to the β-lactam<br />

antibiotic cefotaxime. A mutation in the histidine kinase gene ciaH (ciaH306)<br />

changing threonine at position 230 to prolin in the CiaH protein increased βlactam<br />

resistance and prevented development of spontaneous genetic<br />

competence. Subsequent gene expression analyses showed that the kinase<br />

mutation resulted in enhanced regulation of 15 promoters that are directly<br />

controlled by the response regulator CiaR.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

In several mutational screens using cefotaxime as well as piperacillin aimed at<br />

identifying novel β-lactam resistance determinants mutations in ciaH were<br />

isolated that differed from the known ciaH306 allele. Since these strains<br />

frequently harbored mutations in genes other than ciaH, the impact of altered<br />

CiaH could not be assessed unambiguously in this genetic background. To<br />

analyze their influence on CiaR-mediated gene expression the novel alleles<br />

were introduced into the wild type and gene expression initiated from CiaRactivated<br />

promoters was measured. The new ciaH alleles, ciaH102, 103, 202,<br />

208, 305, and 408 enhanced promoter activities between three- to fourteenfold<br />

compared to the wild type. Thus, activation by some of the novel alleles<br />

exceeded the sixfold stimulation by ciaH306.<br />

Accordingly, spontaneous genetic competence was also repressed. The<br />

characterization of these new ciaH alleles will provide further insight into the<br />

function of the histidine kinase CiaH.<br />

PS 15<br />

New insights into the RNA-degradosome of Bacillus subtilis<br />

M. Lehnik-Habrink *1 , H. Pförtner 1 , J. Stülke 1<br />

1 Dept. of General Microbiology, University of Göttingen, Göttingen, Germany<br />

Many cellular functions are carried out by macromolecular complexes like<br />

ribosomes, replisomes and proteasomes. Another prominent example is the<br />

RNA turnover by a multiprotein complex. This protein assembly called<br />

degradosome or exosome is conserved in all domains of life [1]. In E.coli, it<br />

consists of the essential RNase E, polynucleotide phosphorylase, the RNA<br />

helicase RhlB, and the glycolytic enzyme enolase [2].<br />

Recently a putative degradosome-like complex was identified in the gram<br />

positive model organism Bacillus subtilis [3]. By investigating interactions of<br />

glycolytic enzymes it was found that the enolase is associated with the essential<br />

protein Rny. This protein was further shown to be involved in the processing of<br />

the gapA-operon. A direct interaction with the polynucleotide phosphorylase<br />

was demonstrated, too.<br />

Assuming a degradosome composition similar to that in other organisms the<br />

involved helicase remains mysterious. B. subtilis encodes four potential RNA<br />

helicases. Therefore these helicases were analyzed and the impact of single<br />

components of the Bacillus degradosome in the gapA-processing event was<br />

investigated.<br />

[1] Carpousis et al. (1999) Trends Genet 15, 24-28<br />

[2] Carpousis et al. (1994) Cell, 76, 889-900<br />

[3] Commichau et al. (2008) submitted<br />

PS 16<br />

Deletion of ptsN in Escherichia coli LJ110 correlates with<br />

higher acetate formation and slower growth<br />

S. Jahn *1 , K. Bettenbrock 1<br />

1 Systems Biology of Global Control and Specific Regulations, Max Planck<br />

Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany<br />

173<br />

For many years un<strong>der</strong>standing of the metabolism of Escherichia coli has been<br />

one of the main topics in microbiology research. Many details have been<br />

elucidated about the regulation of nitrogen and carbon metabolism but little is<br />

known about their coordination. Probably the nitrogen metabolic<br />

phosphotransferase system PTS Ntr is a component of this tuning. It consists of<br />

enzyme I Ntr (EI Ntr encoded by ptsP), NPr (encoded by ptsO) and enzyme II Ntr<br />

(EIIA Ntr encoded by ptsN). On the one hand the genes ptsO and ptsN are<br />

located on the same operon as rpoN, the nitrogen dependent sigma factor. On<br />

the other hand the PTS Ntr consists of the phosphoryl transfer chain, PEP -> EI Ntr<br />

-> NPr -> EIIA Ntr , similar to the carbohydrate PTS. Both phosphoryl transfer<br />

chains can exchange phosphate groups.<br />

To analyse the function of the PTS Ntr we investigated growth rates and acetate<br />

formation of Escherichia coli LJ110 and of <strong>der</strong>ivatives mutated in the PTS Ntr<br />

un<strong>der</strong> different carbon conditions in batch experiments. Our results showed a<br />

higher acetate yield in strains deleted in ptsN. To provided our observation we<br />

used real time PCR for analyzing expression of genes connected to acetate<br />

formation.

174<br />

PS 17<br />

Intramembrane proteolysis: Rhomboid proteases in<br />

Corynebacterium glutamicum<br />

A. Lünenschloß *1 , C. Trötschel 1 , D. Schlüsener 2 , A. Poetsch 1<br />

1 Lehrstuhl Biochemie <strong>der</strong> Pflanzen, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

2 GENEART AG, GENEART Europe, Regensburg, Germany<br />

Proteolysis is an important process designating the cleavage of cytosolic<br />

proteins as well as integral membrane proteins. Thereby either misfolded or<br />

misassembled proteins are degraded or proteins are selectively cleaved to<br />

become a new functional state which is called „Regulated Intramembrane<br />

Proteolysis“ (RIP) [1]. Rhomboid proteases are intramembrane serine proteases<br />

that are found in eukaryotes and prokaryotes. These enzymes contain six or<br />

seven transmembrane helices and a catalytic dyad of serine and histidine [2].<br />

While other intramembrane proteases release factors into the cytosol, it is<br />

presumed that rhomboids release cleavage products to the periplasm. One<br />

indication is the mediation of quorum sensing in Providencia stuartii by the<br />

rhomboid protease AarA [3].<br />

For our model organism Corynebacterium glutamicum two rhomboid proteases<br />

named Cg0049 and Cg2767 are predicted. Our current results for cells<br />

containing single and double deletions of cg0049 and cg2767 indicate that the<br />

rhomboids are not essential.<br />

Nevertheless we could show that the amount of Cg2767 increases obviously<br />

after heat stress. This leads to the speculation, that the rhomboid Cg2767 is<br />

involved in heat shock response. In future we want to clarify the physiological<br />

role of the rhomboid proteases in heat and other stresses in Corynebacterium<br />

glutamicum.<br />

[1] Brown, MS. et al. (2000) Cell 100(4): 391-8<br />

[2] Wang, Y. et al. (2006) Nature 444(7116): 179-80<br />

[3] Stevenson, LG. et al. (2007) Proc Natl Acad Sci U S A 104(3): 1003-8<br />

PS 18<br />

Detection of low external pH by the membrane-integrated<br />

transcriptional activator CadC of Escherichia coli<br />

I. Haneburger *1 , K. Jung 1<br />

1 Department I, Mikrobiologie, Ludwig-Maximilians-Universität, München,<br />

Germany<br />

The Cad system is one of the acid inducible amino acid decarboxylase systems<br />

involved in acid stress adaptation of E. coli. At conditions of low external pH<br />

and concomitantly available lysine, CadC activates the transcription of the<br />

cadBA operon. This polycistronic mRNA encodes the two other components of<br />

the Cad system: The lysine-decarboxylase CadA, which decarboxylates lysine<br />

un<strong>der</strong> consumption of a cytoplasmic proton, and the lysine-cadaverine<br />

antiporter CadB, which exports the produced cadaverine in exchange with the<br />

substrate lysine.<br />

We are interested in the molecular mechanism of stimulus perception and<br />

signal transduction by CadC, a representative of the ToxR-like proteins. It was<br />

shown that CadC is not a direct sensor for lysine, but senses this stimulus<br />

indirectly via an interaction with the lysine permease LysP [1]. To identify<br />

amino acids involved in pH-sensing, several aspartate, glutamate and histidine<br />

residues of the periplasmic domain were individually replaced by directed<br />

mutagenesis. Functional analyses of the resulting CadC <strong>der</strong>ivatives revealed<br />

that Asp471, Glu461 and Glu468 are involved in sensing of low pH. The<br />

exchange of Asp471 against Gln led to a pH-insensitive CadC <strong>der</strong>ivative. It is<br />

assumed that this <strong>der</strong>ivative mimics the protonated state, hence the active state<br />

of CadC at low pH. Currently, the mechanistic role of the other amino acids<br />

during pH detection is investigated.<br />

[1] Tetsch, L., Koller, C., Haneburger I. und Jung, K. (2008) The membraneintegrated<br />

transcriptional activator CadC of Escherichia coli senses lysine<br />

indirectly via the interaction with the lysine permease LysP, Mol. Microbiol.,<br />

67, 570-583<br />

PS 19<br />

Insights into the two-component signaling system<br />

YehU/YehT in Escherichia coli<br />

T. Kraxenberger *1 , L. Fried 1 , K. Jung 1<br />

1 Mikrobiologie, LMU München, München, Germany<br />

Two-component systems (TCS) are the major players in signal transduction in<br />

prokaryotes. The prototype of a TCS comprises a membrane-integrated sensor<br />

with histidine kinase (HK) activity and a response regulator (RR) with DNA-<br />

binding activity upon phosphotransfer. Whereas most TCS in Escherichia coli<br />

are well studied, little is known about the HK YehU and the corresponding RR<br />

YehT. YehU shares homology with LytS-YhcK like histidine kinases; YehT<br />

posseses a CheY-homologous regulator domain and a LytTR-like DNA binding<br />

domain.<br />

In this study we identified a set of genes regulated by YehU/YehT in E. coli.<br />

DNA fragments corresponding to the YehT-binding site were enriched by solid<br />

phase DNA-binding in vitro or after co-purification of in vivo cross-linked<br />

chromosomal DNA with His-tagged YehT. The specificity of the DNA-protein<br />

interaction was demonstrated by gel retardation experiments using purified<br />

YehT. The YehT-binding site was further narrowed by DNase I footprinting.<br />

Furthermore, differently produced proteins were identified in a yehUT deletion<br />

mutant by comparative proteome analyses. Transcriptional analysis and<br />

electromobility shift assays confirmed that most, but not all, of the<br />

corresponding genes are un<strong>der</strong> direct transcriptional control of YehU/YehT.<br />

Based on this information, reporter strains were constructed to identify the<br />

signal received by YehU. A model of the YehU/YehT-dependent regulatory<br />

network is presented.<br />

PS 20<br />

Involvement of RamA and/or RamB in control of sugar<br />

utilization pathways of Corynebacterium glutamicum<br />

M. Auchter *1 , A. Cramer 1 , P. Schwarz 1 , B.J. Eikmanns 1<br />

1 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

Recent investigations on acetate metabolism in Corynebacterium glutamicum<br />

led to the identification of two regulatory proteins, RamA and RamB. The<br />

former represents an activator, the latter a repressor of genes coding for key<br />

enzymes involved in corynebacterial acetate metabolism [1, 2]. DNA<br />

microarray experiments with RamA- and RamB-deficient mutants suggested<br />

that both regulators also control other genes, including those for some enzymes<br />

of sugar uptake, glycolysis, pentose phosphate pathway (PPP) and<br />

gluconeogenesis.<br />

Gel shift experiments with promoter regions of C. glutamicum genes encoding<br />

enzymes of sugar uptake (ptsG, ptsF, ptsS, ptsI, ptsH), glycolysis (pgi, pfkA),<br />

PPP (tkt) and gluconeogenesis (gapX, fbp) revealed binding of RamA to the<br />

ptsG, ptsH, pfkA, tkt, gapX and fbp promoters and of RamB to the ptsG and tkt<br />

promoter, indicating a direct influence of RamA and/or RamB on the<br />

expression of these genes and thus, on the activity of the respective pathways.<br />

Further analyses such as transcriptional fusions were carried out to clarify the<br />

functional role of RamA and RamB for expression control of genes found to be<br />

regulated in DNA microarray experiments as well as possess RamA and/or a<br />

RamB binding site/s in the respective promoter regions.<br />

[1] Cramer A., R. Gerstmeir, S. Schaffer, M. Bott, B. J. Eikmanns. 2006.<br />

Identification of RamA, a novel LuxR-type transcriptional regulator of genes<br />

involved in acetate metabolism of Corynebacterium glutamicum. J Bacteriol<br />

188:2554-67.<br />

[2] Gerstmeir R., A. Cramer, P. Dangel, S. Schaffer, B. J. Eikmanns. 2004.<br />

RamB, a novel transcriptional regulator of genes involved in acetate<br />

metabolism of Corynebacterium glutamicum. J Bacteriol 186:2798-809.<br />

PS 21<br />

CodY, a potential repressor of butanol formation in<br />

Clostridium acetobutylicum<br />

T. Standfest *1 , N. Nold 2 , B. Schiel 1 , P. Dürre 1<br />

1 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

2 Flad & Flad, Communication GmbH, Heroldsberg, Germany<br />

Clostridium acetobutylicum is a Gram-positive, spore-forming, anaerobic soil<br />

bacterium, which is very well known for its solvent formation. In this respect,<br />

the sol operon is the essential transcription unit as it comprises nearly all genes<br />

necessary for the formation of butanol. Besides the global transcription<br />

regulator Spo0A, other DNA-binding proteins, namely CcpA and CodY, seem<br />

to be involved in the regulation of the transcription of the sol operon. The<br />

respective proteins could be identified by DNA affinity chromatography. CodY<br />

protein was purified as his-tag fusion protein and gel retardation assays were<br />

carried out. Furthermore, this his-tag fusion protein was used to conduct<br />

footprint experiments to determine the exact binding site of the protein to the<br />

sol promoter region. A C. acetobutylicum knock-out mutant (Heap et al. 2007)<br />

of the codY gene was characterized concerning growth and product spectrum,<br />

and the results indicate a role of the CodY protein as a repressor of butanol<br />

production.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PS 22<br />

The role of RpoHI and RpoHII in the 1 O2 mediated stress<br />

response in Rhodobacter sphaeroides<br />

A.M. Nuss *1 , J. Glaeser 1 , J. Schwarz 1 , G. Klug 1<br />

1 Institut für Mikro- und Molekularbiologie, Justus-Liebig-Universität Giessen,<br />

Giessen, Germany<br />

In the photosynthetic Alphaproteobacterium Rhodobacter sphaeroides 1 O2 is a<br />

stress factor and acts as a signal for gene regulation. The 1 O2 response is<br />

regulated by the ECF sigma factor RpoE, but only few of the genes induced by<br />

1<br />

O2 exposure exhibit an RpoE target sequence. Only recently we demonstrated<br />

that most of the 1 O2 induced genes directly depend on the alternative sigma<br />

factor RpoHII. Because the rpoHII gene exhibits an RpoE promoter, a sigma<br />

factor cascade exists. As RpoHII is one of two heat hock sigma factors besides<br />

RpoHI, we also investigated the relative expression of rpoHI. Interestingly, the<br />

expression of rpoHI is not only induced by heat, but also by 1 O2. RpoHI and<br />

RpoHII are both able to recognize heat inducible promoters, but RpoHII<br />

recognizes a set of promoters not recognized by RpoHI un<strong>der</strong> 1 O2 exposure.<br />

Here we elucidate the overlap of the RpoHI and RpoHII regulons. Therefore the<br />

rpoHI gene was deleted in the wild type and the rpoHII deletion mutant,<br />

yielding strain ΔrpoHI and ΔrpoHI/rpoHII. We tested the sensitivity of the<br />

mutants to 1 O2, heat and methylglyoxal. Soluble proteins of 1 O2 exposed<br />

ΔrpoHI and ΔrpoHI/rpoHII cultures were analyzed by 2D gel electrophoresis<br />

and compared with protein patterns of respective wild type and ΔrpoHII<br />

cultures. For detailed informations about the promoter specificity of RpoHI and<br />

RpoHII the 5’ends of selected mRNAs were mapped by 5’RACE. Both factors<br />

seem to regulate the heat and 1 O2 response, but we propose that RpoHI is more<br />

important for the heat stress response, whereas RpoHII is a major player of the<br />

1<br />

O2 stress response.<br />

PS 23<br />

Comprehensive overview on hyperosmotic conditions in<br />

Corynebactrium glutamicum<br />

B. Fränzel *1 , C. Trötschel 2 , A. Poetsch 2 , D. Wolters 1<br />

1 Analytical Chemistry, Ruhr University, Bochum, Germany<br />

2 Plant Biochemistry, Ruhr University, Bochum, Germany<br />

The soil bacterium, Corynebacterium glutamicum, is Gram-positive, aerobic<br />

and non-pathogen. It is of great biotechnological interest because of its ability<br />

to enrich L-glutamate and other amino acids in the extracellular lumen, e.g.<br />

lysine. Furthermore, C. glutamicum belongs to the group of mycobacteria,<br />

which also comprises the pathogen bacteria Corynebacterium diphtheriae,<br />

Mycobacterium tuberculosis, and Mycobacterium leprae. To improve the<br />

commercial growth process the impact on several growth conditions that might<br />

occur during growth have to be studied. We focus on the impact of<br />

hyperosmotic conditions on the proteome that emerge while synthesized amino<br />

acids are extracellularly accumulated. As a soil bacterium, C. glutamicum has<br />

to cope with different osmotic conditions. Therefore, it strongly depends on<br />

effective adaptation mechanisms against both hypo and hyper osmotic stress.<br />

Our main purpose was to study the regulation of the membrane proteome.<br />

Recent Dotblot experiments showed in which way the subsequent osmo carriers<br />

are regulated. ProP, BetP, LcoP, and EctP are instantly up-regulated on the<br />

level of transcription after hyperosmotic stress. It has also been shown that<br />

these transmembrane proteins are up-regulated by the two-component system<br />

MtrAB. The cells can embark on a strategy, which relies on synthesis and<br />

uptake of compatible solutes such as trehalose, betaine and ectoine, and amino<br />

acids and their <strong>der</strong>ivates e.g. proline. A significant up-regulation of the proline<br />

synthesis pathway, promoted by ProA, ProB, and ProC was already reported.<br />

Here, we present a comprehensive and coherent analysis of hyperosmotic<br />

conditions in C. glutamicum. Proteome and transcriptome data have been<br />

investigated in detail, focussing on the membrane proteome, but not neglecting<br />

the soluble and secreted proteins. For relative protein quantification we applied<br />

an already published stable isotope labelling strategy.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PS 24<br />

Characterization of a ferric uptake regulator (Fur) from<br />

the magnetotactic bacterium Magnetospirillum<br />

gryphiswaldense suggests a specific role in<br />

biomineralisation<br />

R. Uebe *1 , D. Schüler 1<br />

1 Department Biologie I - Bereich Mikrobiologie, Ludwig-Maximilians-<br />

Universität München, München, Germany<br />

The biomineralization of magnetosomes, which are membrane-enclosed<br />

crystals of the ferrimagnetic mineral magnetite (Fe3O4), involves the uptake of<br />

large amounts of iron, its intracellular sequestration and crystallization. Due to<br />

iron toxicity there is a strong need for magnetotactic bacteria to sustain a strict<br />

iron homeostasis. However, it is not clear how iron biomineralization is<br />

regulated and integrated with the biochemical iron requirement. Therefore we<br />

started to investigate components of general iron metabolism and their<br />

contribution to magnetite biomineralization in the magnetic model organism M.<br />

gryphiswaldense MSR-1.<br />

Using the Fur titration assay we identified putative Fur binding sites in the<br />

promoter region of the iron regulated mamGFDC and mms6 operons, whose<br />

gene products control magnetite biosynthesis.<br />

Genome analyses revealed five ORFs which code for proteins belonging to<br />

different subfamilies of the Fur superfamily, the best characterized and most<br />

abundant metal-responsive regulator in bacteria. While three proteins are<br />

related to the Irr subfamily, only one protein belongs to the Zur and Fur/Mur<br />

subfamily, respectivly. None of these five proteins complemented a fur null<br />

mutation in Escherichia coli. Transcriptional analysis in MSR-1 showed that<br />

fur is transcribed. Therefore we constructed an unmarked, in-frame deletion<br />

mutant. Preliminary results indicate that the deletion strain is impaired in<br />

magnetosome maturation, but not in growth un<strong>der</strong> various metal and oxygen<br />

concentrations. Further analyses will include growth and iron uptake<br />

experiments, transcriptional reporter gene fusions as well as proteomic<br />

approaches.<br />

In summary, our results suggest that Fur is involved in regulation of<br />

magnetosome formation, but apparently not in the general iron metabolism.<br />

PS 25<br />

The O2-Sensor NreB of Staphylococcus carnosus contains a<br />

PAS domain with an oxygen sensitive [4Fe-4S] 2+ cluster<br />

F. Reinhart *1 , M. Müllner 1 , E. Bill 2 , G. Unden 1<br />

1 Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg<br />

Universität, Mainz, Germany<br />

2 Institut für Bioanorganische Chemie, Max Planck Institut, Mülheim/Ruhr,<br />

Germany<br />

175<br />

Nitrate and nitrite reduction by S. carnosus is repressed by O2. The NreBC twocomponent<br />

system is responsible for the O2-dependent regulation of the nitrate<br />

and nitrite reductase genes [1]. The NreBC system consists of the cytosolic<br />

sensor histidine kinase NreB and the response regulator NreC. The sensor<br />

domain of NreB is a PAS domain with similarities to the HemeB containing<br />

PAS domain of FixL [2]. NreB contains four essential cysteine residues<br />

(Cys59-X2-Cys62-X11-Cys74-X2-Cys77).<br />

Mössbauer spectroscopy revealed that anaerobically purified NreB contains a<br />

[4Fe-4S] 2+ cluster, which is coordinated by the four cysteine residues.<br />

Exchange of each Cys residue against serine leads to a complete loss of nitrite<br />

and nitrate reduction by the bacteria. By exposure of [4Fe-4S] 2+ NreB to<br />

oxygen, [2Fe-2S] 2+ NreB and finally apoNreB lacking a FeS-cluster is formed<br />

[3].<br />

The loss of the [4Fe-4S] 2+ cluster is related to the loss of kinase activity of<br />

NreB. ApoNreB can be differentiated from FeS containing forms of NreB in<br />

vivo and in vitro by a changed accessibility of thiol residues [4]. ApoNreB is<br />

the major form of NreB in aerobically growing bacteria.<br />

[1] Kamps A., Achebach S., Fedtke I., Unden G. and Götz F. (2004) Mol<br />

Microbiol 52, 713-723<br />

[2] Gilles-Gonzalez MA. and Gonzalez G. (2004) J Appl Physiol 96, 774-783<br />

[3] Müllner M., Mienert B., Bill E. and Unden G. (<strong>2009</strong>) Biochemistry, in prin<br />

[4] Reinhart F., Achebach S., Koch S. and Unden G. (2008) J. Bac. 190, 879-<br />

886

176<br />

PS 26<br />

The cytoplasmic PAS domain of the histidine kinase DcuS:<br />

potential role in signal transduction<br />

P. Dünnwald *1 , M. Etzkorn 2 , M. Baldus 2 , G. Unden 1<br />

1 Insitut für Mikrobiologie und Weinforschung, AG Unden, Johannes<br />

Gutenberg-Universität Mainz, Mainz, Germany<br />

2 Institut für Biophysikalische Chemie, AG Festkörper-NMR-Spektroskopie,<br />

Max-Planck Institut, Göttingen, Germany<br />

Bacteria contain membrane integral sensors for response to changing<br />

environmental conditions. Many of the sensors are two-component systems<br />

consisting of a sensor histidine kinase and a response regulator that triggers the<br />

cellular response [1]. The sensor kinases are typically membrane integral.<br />

DcuS, the C4-dicarboxylate sensor of E. coli is a periplasmic sensing histidine<br />

kinase [2]. DcuS is a multidomain protein consisting of a periplasmic PASP<br />

(Per-Arnt-Sim) domain, two transmembrane helices, a cytoplasmic PASC and<br />

the C-terminal transmitter or kinase domain. PAS domains are the most<br />

common domains in bacterial sensor proteins [3]. A large number of PAS<br />

domains have been identified. Many of the PASC domains contain no cofactor<br />

and their function is unknown.<br />

A combination of mutagenesis and solid-state NMR (ssNMR) experiments with<br />

structural modelling [4] was used to study the structure and function of a<br />

membrane embedded construct of DcuS and of the cytoplasmic PASC domain.<br />

The experiments suggest a role for PASC in signal transduction.<br />

[1] Mascher T., Helmann J. D., Unden G. (2006) Microbiol Mol Biol Rev 70:<br />

910-938<br />

[2] Zientz E., Bongaerts J., Unden G. (1998) J. Bacteriol 180: 5421-5425<br />

[3] Taylor B. L., Zhulin I. B. (1999) Microbiol Mol Biol Rev 63: 479-506<br />

[4] Etzkorn et al. (2008) Nature Struct & Mol Biol 15:1031-1039<br />

PS 27<br />

The impact of truncated and full-length TetR-like<br />

regulators on kirromycin production in different<br />

Streptomyces strains<br />

E. Pross *1 , W. Wohlleben 1 , T. Weber 1<br />

1 Institut für Mikrobiologie/Biotechnologie, Universität Tübingen, Tübingen,<br />

Germany<br />

The protein biosynthesis inhibitor kirromycin is produced by several<br />

actinomycetes. In previous work the kirromycin biosynthetic gene cluster of<br />

Streptomyces collinus Tü 365 was isolated and sequenced.<br />

The existence of closely related biosynthetic gene clusters in other producer<br />

strains of kirromycin or kirromycin <strong>der</strong>ivatives was confirmed via PCR using<br />

specific primers for different genes and sequencing of deduced PCR products.<br />

HPLC analyses of three kirromycin producing strains from the „Tübinger<br />

Stammsammlung“ revealed noticeable differences in the produced amounts of<br />

kirromycin. Strain Streptomyces spec. Tü 1062 produced the lowest kirromycin<br />

amount (2,6 mg/l), whereas the strains Streptomyces spec. Tü 2431 (up to 60<br />

mg/l) and Streptomyces collinus Tü 365 (39 mg/l) showed higher production.<br />

The gene clusters were checked for genes coding for putative regulatory<br />

proteins. In the strain Tü 1062 a gene encoding a putative TetR-like repressor<br />

was identified. A genetic region with striking similarity in the DNA sequence<br />

was also found in the two other strains. Interestingly, the open reading frame of<br />

the putative regulator gene in Tü 2431 encoded a TGA stop codon after eight<br />

amino acids. In contrast, in strain Tü 365 two open reading frames were found,<br />

one resembling the N-terminal part of a putative TetR-like repressor including<br />

the DNA-binding domain, the other one resembling the C-terminal part,<br />

including dimerization and ligand binding domain.<br />

Gene-replacement of both putative regulator genes kirRIIRI in Streptomyces<br />

collinus Tü 365 resulted in a twofold kirromycin overproduction proving the<br />

remaining functionality of the splitted TetR-like repressor.<br />

PS 28<br />

An ECF sigma factor system of C. acetobutylicum<br />

M. Zechlau *1 , S. Barthel 1 , R.J. Fischer 1 , H. Bahl 1<br />

1 Institute of Biological Sciences / Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

Extracytoplasmic function (ECF) sigma factors and their corresponding antisigma<br />

factors are involved in the transcriptional regulation of multiple cellular<br />

processes such as sporulation or envelope stress responses. The genome of<br />

Clostridium acetobutylicum reveals six ECF sigma factor systems of unknown<br />

function. The deduced amino acid sequences of the overlapping open reading<br />

frames cac3267 (sigma factor) and cac3266 (anti-sigma factor) show<br />

similarities to their respective homologues in the Streptomyces σ R system. SigR<br />

and the anti-sigma factor RsrA have previously been characterized as regulators<br />

of redox dependent transcription. To analyze the role of CAC3267/3266 in C.<br />

acetobutylicum, we constructed strains with altered expression of these genes.<br />

Deletion mutants of the cac3265/3266/3267 operon reveal reduced survival<br />

after O2 exposure but an increased transcript level of the Hsp21, a rubredoxin<br />

dependent peroxidase. Knock out mutants of the anti-sigma factor (cac3266)<br />

are less sensitive to oxygen and Northern hybrization experiments show<br />

increased transcript levels of the gene encoding the oxidative stress repressor<br />

PerR, leading to a reduced expression of the Hsp21. Further results on the<br />

regulatory role of this novel sigma factor system will be presented.<br />

PS 29<br />

Oxidative stress response in Clostridium acetobutylicum<br />

M. Scheel *1 , O. Riebe 1 , F. Hillmann 1 , R.J. Fischer 1 , H. Bahl 1<br />

1 Institute of Biological Sciences / Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

The reaction of microbial organisms to changing environmental conditions or<br />

stress factors does often result in genetic responses. Such genetic regulation<br />

mechanisms are for instance induced by oxidative stress. Clostridia belong to<br />

those bacteria that are consi<strong>der</strong>ed as obligate anaerobe. In consequence survival<br />

of such organisms is impaired by oxidative stress e.g. in the form of O2 or<br />

H2O2. Nevertheless, recent studies revealed that Clostridia are able to cope with<br />

microoxic conditions indicating protection mechanisms to defend against<br />

oxidative stress.<br />

Recently we were able to identify the genes responsible for O2, O2 - and H2O2<br />

detoxification in Clostridium acetobutylicum by in vivo and in vitro assays on<br />

the respective proteins. Analyses of the promoter sequences led to the<br />

identification of PerR, the responsible regulator of the oxidative stress response.<br />

Its mutation showed a high tolerance to oxidative stress as a result of the<br />

upregulation of oxidative stress genes [1]. Another protein that is consi<strong>der</strong>ed to<br />

be involved in the regulation of the defence against oxidative stress in<br />

C. acetobutylicum is SoxR which is encoded by the open reading frame<br />

cac2451. To confirm this function for SoxR we manipulated the soxR gene and<br />

analyzed the mutants with regard to the reaction to oxidative stress. Here, we<br />

present first results respecting the regulation of the oxidative stress response in<br />

C. acetobutylicum.<br />

[1] Hillmann F., R.J. Fischer, F. Saint-Prix, L. Girbal, H. Bahl, (2008).<br />

Molecular Microbiology, 68 [4], 848-860<br />

PS 30<br />

Expanding the Rcs membrane stress signaling system in E.<br />

coli<br />

G. Raja Venkatesh *1 , F. Kembou 1 , A. Paukner 1 , K. Schnetz 1<br />

1 Institute for Genetics, University of Cologne, Cologne, Germany<br />

The enterobacterial Rcs multicomponent signal transduction system is induced<br />

by membrane stress and important for biofilm development and pathogenicity.<br />

The response regulator RcsB acts either as homodimer or as heterodimer in<br />

association with RcsA and regulates genes important for motility, capsule<br />

synthesis and various stress responses. Many of these genes are repressed by<br />

the global regulator H-NS. We identified BglJ and YjjQ as two new partners of<br />

RcsB. YjjQ is known to be important for infection of chicken by avian<br />

pathogenic E. coli, while we show here that BglJ in association with RcsB<br />

activates the H-NS repressed bgl operon. Further, expression of the co-encoded<br />

yjjQ and bglJ genes is also repressed by H-NS. Our results expand the Rcs<br />

system and implicate a pleiotropic role of Rcs in modulating repression by H-<br />

NS and in controlling genes for stress responses and virulence.<br />

PS 31<br />

Osmoregulation of the Escherichia coli prou operon at a<br />

post-transcriptional level<br />

K. Kavalchuk *1 , S. Madhusudan 1 , K. Schnetz 1<br />

1 Institute for Genetics, University of Cologne, Cologne, Germany<br />

Escherichia coli, as other bacteria, can survive extreme changes of the<br />

environment like variations in temperature, osmolarity and acidity. The proU<br />

operon is one of the systems helping E. coli cells to survive un<strong>der</strong> osmotic<br />

stress conditions encoding proteins for uptake of the osmoprotectants glycine<br />

betaine and proline into the cell. Expression of the operon is elevated several<br />

hundred times by hyper-osmotic stress and remains high in hyperosmotic<br />

medium. Osmoregulation of the proU operon does presumably not involve<br />

specific regulators. However, it was shown that the global regulatory protein H<br />

NS represses proU at the level of transcription initiation with high specificity.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

In addition, proU expression is affected by unspecific factors like the potassium<br />

glutamate concentration, other nucleoid-associated proteins (e.g. HU), and cold<br />

shock. However, up to date the mechanism of proU osmoregulation is not clear.<br />

Our recent experimental data suggest that osmoregulation of proU at the<br />

transcriptional level occurs at the promoter and an indirect modulation of<br />

repression by H-NS, and that in addition expression of proU is regulated at a<br />

post-transcriptional level.<br />

PS 32<br />

Aconitase AcoA of Aspergillus nidulans, regulation at the<br />

atomic level?<br />

C. Maerker *1<br />

1 Mikrobiologie, Leibniz Universität Hannover, Hannover, Germany<br />

We study the propionate-degradation pathway of the filamentous fungus A.<br />

nidulans.<br />

In contrast to mammals, which use the methylmalonyl-CoA pathway for<br />

degradation, several microorganisms degrade propionate via the methylcitrate<br />

cycle [1].<br />

One of the key enzymes of this cycle is the methylaconitase, which we have<br />

shown to be the main aconitase (AcoA) of the citric acid cycle, too.<br />

This enzyme bears an interesting regulatory feature: the fully reduced 4Fe-4S<br />

cluster of this enzyme shows a constant ratio of aconitase and methylaconitase<br />

activity, whereby the oxidized 3Fe-4S cluster only shows methylaconitase<br />

activity. Therefore, the ratio of both activities indicates the oxidative state of<br />

the enzyme AcoA. We tested wild type cultures of A. nidulans, grown on<br />

different carbon sources for both enzymes. The different ratios implies the<br />

relevance of this regulatory mechanism in vivo.<br />

[1] Tabuchi, T., and Hara, S. (1974) Production of 2-methylisocitric acid from<br />

n-Paraffins by mutants of Candida lipolytica. Agr Biol Chem 38, 1105-1106<br />

PS 33<br />

Antagonistic Regulation by H-NS and the LysR-Type<br />

Transcription Factor LeuO: Binding of LeuO to the<br />

Regulatory Region of the yjjP-yjjQ-bglJ locus in E.coli<br />

B. Blissenbach *1 , T. Stratmann 1 , K. Schnetz 1<br />

1 Institute for Genetics, University of Cologne, Cologne, Germany<br />

The nucleoid-associated protein H-NS binds weakly specific to a degenerate<br />

AT-rich DNA sequence motif and forms oligomeric nucleoprotein complexes<br />

on the DNA, which silences transcription when located close to a promoter.<br />

Repression by H-NS can be relieved by various mechanisms including binding<br />

of a specific transcription factor. LeuO is one transcription factor which<br />

relieves repression by H NS at several loci either by competing with H-NS for<br />

binding or by forming a barrier which prevents spreading of H-NS complex to<br />

the promoter. We investigated antagonistic regulation of the Escherichia coli<br />

yjjQ-bglJ operon by H-NS and LeuO. The yjjQ-bglJ operon encodes LuxR-type<br />

transcription factors, with YjjQ <strong>bei</strong>ng an important for virulence of avian<br />

pathogenic Escherichia coli and BglJ counteracting H-NS mediated silencing<br />

of the bgl (aryl-β,D-glucoside) operon. Genetic analysis and binding studies<br />

suggest that H-NS binds to three regions within the extended regulatory region.<br />

For LeuO likewise three binding sites were identified and characterized by<br />

DNA footprinting. These LeuO-sites overlap with the putative H-NS specific<br />

binding sites suggesting that LeuO competes with H-NS for binding. Taken<br />

together the data suggest that anti-silencing of the yjjQ-bglJ operon by LeuO is<br />

mediated by restructuring of the nucleoprotein complex formed by H-NS.<br />

PS 34<br />

Growth inhibition of Sporomusa ovata by vitamin B12<br />

A. Hiergeist *1 , E. Stupperich 1<br />

1 Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany<br />

The anaerobic eubacterium Sporomusa ovata produces acetic acid from<br />

methanol plus CO2. These methyl transfer reactions are catalyzed by corrinoiddependent<br />

enzymes. The involved corrinoid cofactors are p-cresolylcobamide<br />

and phenolylcobamide instead of vitamin B12. The phenolic moieties of these<br />

corrinoids are linked to ribose by unusual O-glycosidic bonds instead of the Nglycosidic<br />

bond in vitamin B12. Physiological experiments clearly revealed that<br />

Sporomusa ovata is able to synthesize vitamin B12 from the B12 precursor 5,6dimethylbenzimidazole.<br />

Surprisingly, increasing B12 concentrations within the<br />

cells caused a strong growth inhibition. This is the first report on vitamin B12 as<br />

the inhibitor of corrinoid-dependent reactions. Two tentative inhibition<br />

mechanisms were investigated: (I) Gene expression by B12 riboswitches and (II)<br />

inhibition by improper cofactor-binding to corrinoid-proteins.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

A B12 riboswitch-regulated cobT gene was identified and its gene product was<br />

biochemically characterized. CobT is the potential candidate for the O- and Nglycosidic<br />

bond-formations of p-cresolylcobamide and vitamin B12 corrinoidbiosyntheses<br />

in Sporomusa ovata. In addition, the complete operon of the<br />

acetyl-CoA synthase of the homoacetogenic bacterium was investigated. The<br />

operon is not controlled by a B12 riboswitch although it encodes a corrinoiddependent<br />

subunit. Thus, the corrinoid-dependent acetyl-CoA synthase was<br />

also inhibited by vitamin B12 formation because this inhibition was reverted by<br />

the growth substrate pyruvate as the precursor of acetyl CoA.<br />

PS 35<br />

S-Adenosylmethionine (SAM) Riboswitches in Sporomusa<br />

ovata<br />

C. Gallinger *1 , E. Stupperich 1<br />

1 Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany<br />

Methylation reactions are fundamental biochemical reactions that are essential<br />

for the survival of pro - and eukaryotic cells. The homoacetogenic bacterium<br />

Sporomusa ovata utilizes a number of methyl donators and hence methyl<br />

transfer reactions in its energy metabolism. Genes encoding these proteins<br />

might be controlled by SAM - r B12 - riboswitches. Here we report about the<br />

analysis of SAM regulated genes in the chromosome of this homoacetogenic<br />

bacterium.<br />

Four SAM regulated genes were identified and completely sequenced. The<br />

enzymes encoded in these regions are the S-adenosylmethionine synthetase 1<br />

(metK), methionine import ATP-binding protein (metN), pyruvate<br />

flavodoxin/ferredoxin oxidoreductase domain protein (porN) and the<br />

cystathionine gamma synthetase (metB).<br />

All of these genes show high identities to homologous genes in other<br />

Firmicutes, which are closely related to Sporomusa ovata. One of these genes,<br />

metK, was investigated in detail. The gene encodes the S-adenosyl methionine<br />

synthetase (E.C. 2.5.1.6).<br />

We analysed this gene and its gene product, due to its tentative role in the<br />

nucleic acid metabolism of the bacterium. The gene expression was quantified<br />

by qRT-PCR. In addition the gene was heterologously expressed as histidinetagged<br />

protein in E. coli and antibodies were raised against MetK protein in<br />

mice.<br />

PS 36<br />

Expression of the high affinity K + transport system Kdp is<br />

regulated by the EIIA Ntr protein of the "nitrogen PTS" in<br />

Escherichia coli.<br />

D. Lüttmann *1 , R. Heermann 2 , A. Hillmann 1 , B. Zimmer 1 , K. Jung 2 , B. Görke 2<br />

1 Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen,<br />

Göttingen, Germany<br />

2 Department Biologie I Mikrobiologie, Ludwig-Maximilians Universität<br />

München, München, Germany<br />

In E. coli the phosphotransferase system (PTS) plays a central role in sugar<br />

transport and signal transduction. The homologous EI Ntr , NPr and EIIA Ntr<br />

proteins also form a phosphoryl-group transfer chain working in parallel to the<br />

canonical PTS [1]. However, a final phosphoryl-group acceptor is lacking.<br />

Thus, it is unlikely that this system has a sugar transport function. Recently, it<br />

was shown that EIIA Ntr inhibits K + uptake by directly interacting with the TrkA<br />

component of the low affinity K + transporter Trk [2]. Un<strong>der</strong> low potassium<br />

concentrations, K + is taken up by the high affinity K + transporter KdpFABC.<br />

Expression of the kdp genes is controlled by the KdpD/KdpE two-component<br />

system in response to low intracellular K + concentrations [3]. Here we show<br />

that EIIA Ntr regulates expression of the kdp operon. According to our data, dephosphorylated<br />

EIIA Ntr stimulates transcription initiation at the kdp promoter in<br />

a TrkA independent manner. Our data suggest that EIIA Ntr regulates the activity<br />

of the two-component system controlling transcription of the kdp operon.<br />

[1] Zimmer B., Hillmann A., and Görke B., FEMS Microbiol Lett. 2008<br />

;286:96-102.;<br />

[2] Lee C.R., Cho S.H., Yoon M.J. Peterkofsky A. and Seok Y.J., Proc. Natl.<br />

Acad. Sci. 2007; 104:4124-9.;<br />

[3] Heermann R., Fohrmann A., Altendorf H. and Jung K., J. Biol. Chem.<br />

2003 ;47:839-48.<br />

177

178<br />

PS 37<br />

Regulatory mechanisms involved in the expression of lysP<br />

encoding the specific lysine permease of Escherichia coli<br />

J. Ruiz *1 , K. Jung 1<br />

1 Department Biologie I, Mikrobiologie, Ludwig-Maximilians-Universität<br />

München, München, Germany<br />

Enteric bacteria initiate stress responses that allow them to survive in acidic<br />

environments, such as in the stomach or during fermentation. Escherichia coli<br />

resists acid challenges by induction of genes encoding various amino acid<br />

decarboxylases. One of these acid-resistance systems, the Cad-system,<br />

comprises the lysine decarboxylase CadA and the lysine/cadaverine antiporter<br />

CadB. Expression of the cadBA operon is regulated by the membraneintegrated<br />

transcriptional activator CadC at low external pH and the<br />

simultaneous presence of exogenous lysine. These two stimuli are<br />

independently sensed. Whereas the pH signal is sensed by CadC, perception of<br />

the lysine signal is dependent on LysP, the specific lysine permease. In absence<br />

of lysine, LysP prevents activation of CadC by interaction with its<br />

transmembrane domain. To better un<strong>der</strong>stand the role of LysP and lysine in<br />

acid-stress adaptation, the aim of this work was to study the factors involved in<br />

the transcriptional regulation of lysP. For that purpose we constructed a reporter<br />

strain with a transcriptional fusion of lysP::lacZ in the chromosome and<br />

measured the β-galactosidase activity un<strong>der</strong> different growth conditions. In<br />

agreement with previous results, there is a lysine-mediated repression of lysP<br />

expression. However, besides the lysine concentration, the level of lysP<br />

expression seems also to depend on the pH and the oxygen availability. The<br />

role of putative regulators proteins of lysP expression is currently un<strong>der</strong> study.<br />

PS 38<br />

Towards un<strong>der</strong>standing the nitrogen signal transduction<br />

for nif gene expression in Klebsiella pneumoniae<br />

J. Glöer *1 , R.A. Schmitz-Streit 1<br />

1 Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität, Kiel,<br />

Germany<br />

In the diazotroph Klebsiella pneumoniae, the nitrogen sensory protein GlnK<br />

mediates the cellular nitrogen status towards the NifL ⁄ NifA system that<br />

regulates transcription of the nitrogen fixation genes in response to ammonium<br />

and molecular oxygen. To identify amino acids of GlnK essential for this signal<br />

transduction by protein–protein interaction, we performed random point<br />

mutagenesis by PCR amplification un<strong>der</strong> conditions of reduced Taq polymerase<br />

fidelity. Three thousand two hundred mutated glnK genes were screened to<br />

identify those that would no longer complement a K. pneumoniae ΔglnK strain<br />

for growth un<strong>der</strong> nitrogen fixing conditions. Twenty-four candidates resulting<br />

in a Nif - phenotype were identified, carrying 1–11 amino acid changes in<br />

GlnK. Based on these findings, as well as structural data, several single<br />

mutations were introduced into glnK by site-directed mutagenesis, and the Nif<br />

phenotype and the respective effects on NifA-mediated nif gene induction was<br />

monitored in K. pneumoniae using a chromosomal nifK´–´lacZ fusion. Single<br />

amino acid changes resulting in significant nif gene inhibition un<strong>der</strong> nitrogen<br />

limiting conditions were located within the highly conserved T-loop (A43G,<br />

A49T and N54D), the body of the protein (G87V and K79E) and in the Cterminal<br />

region (I100M, R103S, E106Q and D108G). Complex formation<br />

analyses between GlnK (wild-type or <strong>der</strong>ivatives) and NifL or NifA in response<br />

to 2-oxoglutarate indicated that: (a) besides the T-loop, the C-terminal region of<br />

GlnK is essential for the interaction with NifL and NifA and (b) GlnK binds<br />

both proteins in the absence of 2-oxoglutarate, whereas, in the presence of 2oxoglutarate,<br />

NifA is released but NifL remains bound to GlnK.<br />

PS 39<br />

A cryptochrome-like protein is involved in the regulation of<br />

photosynthesis genes in Rhodobacter sphaeroides<br />

A.K. Hendrischk 1 , S. Frühwirth *1 , J. Moldt 2 , A. Jäger 1 , G. Klug 1<br />

1 Institute for Molecular Biology and Microbiology, Justus-Liebig-University<br />

Giessen, Giessen, Germany<br />

2 Institute for Plantphysiology and Photobiology, Phillips-University Marburg,<br />

Marburg, Germany<br />

Rhodobacter sphaeroides is a phototrophic α-proteobacterium. To prevent the<br />

production of harmful reactive oxygen species, the formation of photosynthetic<br />

apparatus has to be tightly regulated. Blue light receptors belonging to the<br />

cryptochrome/photolyase family are widely distributed throughout all<br />

kingdoms of life. Here we present the identification of a cryptochrome-like<br />

protein (CryB) in R. sphaeroides. The protein was purified in Rhodobacter and<br />

E. coli and it was shown that it lacks the C-terminal extension, necessary for<br />

protein interaction, but is also not related to Cry-DASH proteins. HPLC<br />

analysis showed that CryB binds FAD as a chromophore and a blue light<br />

dependent photocycle of the protein was analyzed by absorbance spectroscopy.<br />

Deletion of cryB in R. sphaeroides led to altered spectra of whole cells,<br />

indicating lower amounts or a different composition of photosynthetic<br />

complexes. Suprisingly a complementation by a plasmid containing cryB<br />

increased this effect even more. However, this was also observed when the<br />

plasmid was transferred into the wildtype. We also quantified puc- (encoding<br />

proteins of the light harvesting complex II) and puf-mRNA (encoding proteins<br />

of the LH I and the reaction centre) levels by Northern Blot analysis to confirm<br />

these observations. Furthermore, realtime RT-PCR experiments are carried out<br />

to quantify the gene expression of photosynthesis genes in the cryB-deletion<br />

mutant in detail.<br />

PS 40<br />

A molecular switch in osmoregulation: BetP from<br />

Corynebacterium glutamicum<br />

V. Ott *1 , J. Koch 2 , K. Spaete 3 , C. Ziegler 4 , S. Morbach 1 , R. Kraemer 1<br />

1 Institute of Biochemistry, University of Cologne, Cologne, Germany<br />

2 Institute of Biochemistry, Goethe University Frankfurt/Main, Frankfurt/Main,<br />

Germany<br />

3 Institute of Medical Biochemistry, University of Cologne, Cologne, Germany<br />

4 Department of Structural Biology, Max-Planck-Institute of Biophysics,<br />

Frankfurt/Main, Germany<br />

Osmoregulation is one of the most important homeostatic mechanisms of<br />

bacteria to respond to changes in their environment. Un<strong>der</strong> hyperosmotic stress<br />

conditions the soil bacterium Corynebacterium glutamicum accumulates high<br />

amounts of compatible solutes by synthesis or uptake, thus counteracting<br />

osmotically induced water efflux. The most efficient uptake system for<br />

compatible solutes is the secondary transporter BetP. This carrier responds to<br />

osmotic stress by tight regulation at the level of activity. Investigations in<br />

proteoliposomes showed that BetP harbors osmosensory and osmoregulatory<br />

properties and that the activation of BetP depends solely on increased internal<br />

K + concentrations. Recent data suggest that K + acts as a specific BetP stimulus<br />

in vivo, too. Characterization of C-terminal truncated protein variants implied<br />

that the C-terminal domain of the transporter is the regulatory domain and the<br />

putative potassium-sensor within the protein. Site-directed scanning<br />

mutagenesis in this putative α-helical regulator domain revealed that the<br />

conformation of this domain is critical for the regulation of the transporter. As<br />

different lipid surroundings modulated the regulatory behaviour of both, the<br />

wildtype protein and C-terminal BetP-variants, interactions of the C-terminal<br />

BetP-domain with membrane lipids might be directly involved in the regulatory<br />

mechanism. These interactions were proven by surface-plasmon-resonance<br />

spectroscopic studies. Interestingly, further biochemical interaction studies<br />

indicated that also intramolecular interactions of the regulatory BetP-domain<br />

are crucial for the activation of the transporter. These current findings can now<br />

be combined in a detailed working model describing the C-domain as a<br />

molecular switch to regulate the transport activity of BetP.<br />

[1] Ott et al. (2008) Biochemistry.<br />

PS 41<br />

Characterization of a potential heme regulator in<br />

Pseudomonas aeruginosa<br />

J.C. Lorenzo Fajardo *1 , K. Weichbrodt 1 , A. Harms 2 , K. Barkowits 1 , B.<br />

Schubert 1 , M. Schobert 2 , M. Scheer 2 , N. Frankenberg-Dinkel 1<br />

1 Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany<br />

2 Institute for Microbiology, Technical University Braunschweig, Braunschweig,<br />

Germany<br />

For microorganisms like Pseudomonas aeruginosa not only sensing and<br />

adapting to external changes but also the sensing and homeostasis of internal<br />

crucial yet potential cytotoxic compounds is important. One way to sense<br />

internal signals is the use of LysR-type transcriptional regulators (LTTRs)<br />

which combine sensing and transcriptional regulation of multiple genes via a<br />

coinducer binding domain and a helix-turn-helix motif, respectively. Here we<br />

describe the characterization of such an LTTR which was named HemR due to<br />

its high homology to the heme dependent regulator HbrL from Rhodobacter<br />

capsulatus. Spectroscopic experiments, using recombinant produced HemR<br />

showed that neither heme nor the heme degradation product biliverdin-IXα is<br />

able to bind to HemR. However, β-Gal. activity assays comparing the wild type<br />

and a hemR deletion mutant showed a clear involvement of HemR in<br />

maintaining heme homeostasis by regulating the expression of the heme<br />

oxygenase bphO and the expression of hemA, the first enzyme in heme<br />

biosynthesis. Interestingly, the results point towards a discrimination between<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

internal and external heme sources. Experiments testing this hypothesis are<br />

un<strong>der</strong>way and will partially be presented.<br />

Furthermore, a global transcriptomic analysis using commercially available<br />

DNA microarrays showed a more general involvement of HemR in distinct<br />

cellular functions like energy metabolism, cell to cell communication and host<br />

microbe interaction. Although not related on the first sight, many of the<br />

regulated genes are somehow involved in iron availability or their<br />

corresponding proteins make use of a heme cofactor. From these data, a<br />

possible HemR dependent regulon will be discussed.<br />

PS 42<br />

The universal stress protein UspC scaffolds the<br />

KdpD/KdpE signaling cascade of Escherichia coli un<strong>der</strong> salt<br />

stress<br />

R. Heermann 1 , A. Weber 1 , B. Mayer 1 , M. Ott 1 , E. Hauser 1 , G. Gabriel *1 , T.<br />

Pirch 1 , K. Jung 1<br />

1 Department Biologie I, Abteilung Mikrobiologie, Ludwig-Maximilians-<br />

Universität München, München, Germany<br />

In response to K + limitation or salt stress the sensor kinase KdpD<br />

phosphorylates the response regulator KdpE that in turn induces the kdpFABC<br />

operon encoding the high affinity K + -uptake system KdpFABC. Un<strong>der</strong> K + -<br />

limiting conditions the Kdp-system helps to restore the optimal intracellular K +<br />

concentration, while in response to salt stress K + is accumulated far above the<br />

normal content. The kinase activity of KdpD is inhibited at high concentrations<br />

of K + , so it has been puzzling how kdpFABC expression is activated in response<br />

to salt stress. The Escherichia coli genome encodes three single domain<br />

proteins of the universal stress protein subfamily (Usp), UspA, UspC and<br />

UspD. Their copy number is increased upon exposing the cells to starvation,<br />

mo<strong>der</strong>ate heat, oxidative or osmotic stress. It was found that UspC stimulates<br />

the in vitro reconstructed signaling cascade resulting in phosphorylation of<br />

KdpE at a K + concentration that would otherwise attenuate signal transmission.<br />

In agreement, in a ΔuspC mutant KdpFABC production was unchanged un<strong>der</strong><br />

K + limitation but significantly down-regulated when cells were exposed to salt<br />

stress. Surface plasmon resonance (SPR) and further biochemical studies<br />

revealed that UspC specifically interacts with a Usp domain of the UspA<br />

subfamily in the stimulus perceiving N-terminal domain of KdpD. Furthermore,<br />

UspC stabilized the KdpD/KdpE~P/DNA complex and is therefore believed to<br />

act as a scaffolding protein of the KdpD/KdpE signaling cascade. This study<br />

describes the stimulation of a bacterial two-component system un<strong>der</strong> distinct<br />

stress conditions by a scaffolding protein, and thereby highlights a new role of<br />

universal stress proteins.<br />

PS 43<br />

In vitro and in vivo analysis of MtrB of Corynebacterium<br />

glutamicum: Searching for the sensor domain<br />

P. Reihlen *1 , N. Möker 1 , S. Morbach 2<br />

1 Institut für Biochemie, Universität zu Köln, Köln, Germany<br />

2 Lehrstuhl für Mikrobiologie, Friedrich-Alexan<strong>der</strong>-Universität, Erlangen,<br />

Germany<br />

The most common type of a bacterial sensory and signal transduction system is<br />

the two component system. This system typically consists of a membranebound<br />

histidine sensor kinase and a cytoplasmic response regulator acting as a<br />

transcription factor. In vivo the two component system MtrBA mediates the<br />

expression of osmoregulated genes e.g. proP and betP (encoding uptake<br />

systems of compatible solutes) in response to increased medium osmolarity.<br />

The expression is induced in a dose-dependent manner suggesting that MtrB is<br />

able to detect a stimulus related to hyperosmotic stress conditions. MtrB<br />

reconstituted in liposomes was shown to autophosphorylate if incubated with<br />

specific stimuli like glucose, glutamate or glycine betaine.<br />

To identify the sensing domain different regions of MtrB were either deleted or<br />

swapped with the corresponding regions of DcuS, a fumarate sensing histidine<br />

kinase from Escherichia coli. These MtrB <strong>der</strong>ivatives were reconstituted in<br />

liposomes and tested for autophosphorylation if incubated with MtrB specific<br />

stimuli like glucose or glycine betaine. In a second approach the MtrB<br />

<strong>der</strong>ivatives were tested in vivo for complementation using the expression of<br />

proP and betP un<strong>der</strong> hyperosmotic stress as an indicator for functionality.<br />

Surprisingly, the ΔmtrB strain was not suitable as a background for the in vivo<br />

expression analysis, since proP and betP were regulated identical as in wild<br />

type cells. This suggested cross talk/regulation of MtrA with a second two<br />

component system. By double deletions of mtrB and other histidine kinase<br />

genes the cross talk/regulation causing kinase could be identified.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PS 44<br />

Identification of SO0577 as putative cognate sensor kinase<br />

to the global regulator ArcA in Shewanella oneidensis MR-1<br />

J. Lassak *1 , K. Thormann 2<br />

1 Max-Planck-Institut für terrestrische Mikrobiologie / Ökophysiologie,<br />

Philipps-Universität, Marburg, Germany<br />

2 Max-Planck-Institut für terrestrische Mikrobiologie / Ökophysiologie,<br />

Marburg, Germany<br />

Shewanella oneidensis MR-1 is well known for its respiratory diversity. Un<strong>der</strong><br />

anaerobic conditions the organism is capable of using a wide range of<br />

alternative electron acceptors, including a number of metal oxides. Thus, S.<br />

oneidensis MR-1 has a huge potential for bioremediation processes.<br />

One of the dominant regulators involved in adaptation of S. oneidensis MR-1 to<br />

anaerobic conditions is the ArcA response regulator. Recent studies unveiled<br />

HptA as a phosphodonor to ArcA. However, a cognate sensor kinase to ArcA<br />

so far remains unknown.<br />

Focusing on orphan hybrid sensor kinases lacking an hpt-domain we identified<br />

SO0577 as a potential candidate to represent ArcB. To proof our hypothesis, we<br />

generated in-frame deletions in S00577, hptA and arcA as well as the<br />

corresponding double and triple knock-outs. Phenotypic analysis revealed<br />

similar phenotypes regarding growth and motility.<br />

The Arc system has been shown before to strongly influence structure and<br />

dynamics of community formation. Biofilm analysis in static and<br />

hydrodynamic systems displayed that all mutants were similarly unable to<br />

develop the typical three-dimensional structures. Accordingly, analysis of<br />

transcriptomic changes in the SO0577 knock-out strain exhibited striking<br />

similarities to those of an arcA-mutant. Among the genes regulated<br />

differentially, crucial factors for community formation were identified, such as<br />

mxdABCD, aggA, and csgAB.<br />

Based on this we conclude that we have identified a cognate sensor kinase for<br />

ArcA, a major factor in regulation of cell physiology and community<br />

formation.<br />

PS 46<br />

Transcriptional regulation of acetyl-CoA synthetase in P.<br />

aeruginosa ATCC 17933<br />

V. Khodaverdi *1 , U. Kretzschmar 1<br />

1 Inst f. Biotechnologie, FG Angew Biochemie, TU Berlin, Berlin, Germany<br />

Pseudomonas aeruginosa ATCC 17933 is characterized by its ability to<br />

aerobically oxidize ethanol via acetaldehyde to acetate. Acetate is then<br />

activated to acetyl-CoA by an AMP-forming acetyl-CoA synthetase (ACS).<br />

ACS is encoded by the gene acsA. To investigate the transcriptional response to<br />

specific carbon sources, we grew cells carrying the acsA::lacZ fusion in<br />

different media and monitored their β-galactosidase activity. The promotor<br />

activity of acsA varied 50-fold depending on the nature of the carbon source.<br />

These results correlate with the respective enzymatic activities of ACS [1].<br />

P. aeruginosa ATCC 17933 shows diauxic behaviour when growing on the<br />

mixed substrates ethanol plus 5 mM succinate [2]. Ethanol is used only when<br />

succinate is exhausted. This effect is abolished in the P. aeruginosa crc - mutant<br />

VK9. Crc is responsible for the catabolite repression control.<br />

Transcription of acsA is regulated by the carbon source and subjected to<br />

catabolite repression by Crc.<br />

[1] Kretzschmar et al. (2001) Microbiology 147: 2671-2677<br />

[2] Kretzschmar et al. (2008) Arch of Microbiol 190: 151-158<br />

179<br />

PS 47<br />

Deletion mutants reveal additional limbs of the salt stress<br />

regulatory network in Methanosarcina mazei Gö1<br />

K. Schlegel *1 , B. Meyer 1 , R. Saum 1 , V. Müller 1<br />

1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,<br />

Goethe University Frankfurt, Frankfurt, Germany<br />

In their natural habitat methanogenic archaea have to cope with changing salt<br />

concentrations in the environment. Their answer to salt stress is the<br />

accumulation of compatible solutes, either by de novo synthesis (α-glutamate or<br />

N ε -acetyl-β-lysine) or uptake (glycine betaine) [1]. Yet, little is known about<br />

Na + homeostasis and the structural adaptation of the cell surface. To address the<br />

role of Na + /H + antiporters, primary Na + pumps and surface layer proteins and<br />

their export systems in salt tolerance we have deleted the encoding genes from<br />

the genome of Methanosarcina mazei Gö1. The genotypes of the mutants were<br />

verified by Southern blot analysis. Interestingly, some of these mutants are

180<br />

impaired for growth at high salt demonstrating the presence of previously<br />

unrecognized limbs of the salt stress regulon of M. mazei Gö1.<br />

[1] Spanheimer, R., and Müller, V. (2008). The molecular basis of salt<br />

adaptation in Methanosarcina mazei Gö1. Arch. Microbiol. 190, 271-279.<br />

PS 48<br />

The essential YycFGHI system of Staphylococcus aureus:<br />

Establishment of an in vitro test system<br />

M. Türck *1 , G. Bierbaum 1<br />

1 Institute of Medical Microbiology, Immunology & Parasitology (IMMIP),<br />

University of Bonn, Bonn, Germany<br />

YycFG (VicRK/WalRK) is a two-component regulatory system (TCS) that is<br />

involved in regulation of envelope synthesis in Gram-positive bacteria with low<br />

G+C content. YycG (VicK/WalK) is a sensor-histidine kinase and YycF<br />

(VicR/WalR) represents the appropriate response regulator of this essential<br />

system.<br />

It has been described that truncated YycG (VicK/WalK) orthologs of Bacillus<br />

subtilis and Staphylococcus aureus can un<strong>der</strong>go autophosphorylation in vitro<br />

and, furthermore, in a subsequent step are able to transfer a phosphoryl group to<br />

their appropriate response regulator YycF (VicR/WalR). However, in all of<br />

these assays an important part of YycG (VicK/WalK), comprising the two Nterminal<br />

transmembrane domains and the extracytoplasmic loop between them,<br />

was missing in the recombinant proteins. In general, and in case of B. subtilis in<br />

particular, it has been speculated that the transmembrane and extracellular<br />

domains might play an important role in signal recognition and in regulation of<br />

the YycG (VicK/WalK) kinase activity, respectively.<br />

With regard to the possible importance of these protein domains and their<br />

contribution to the full functionality in protein-protein interactions between<br />

YycG (VicK/WalK) and the accessory membrane proteins YycH/YycI, we used<br />

the complete proteins to establish an in vitro phosphorylation setup for further<br />

interaction studies.<br />

We demonstrated in vitro the autophosphorylation activity of the entire YycG<br />

(VicK/WalK) histidine-kinase and the subsequent phosphorylation of the YycF<br />

(VicR/WalR) response regulator. Additionally, we showed the influence of the<br />

presence of the auxiliary YycH and YycI proteins on the phosphorylation<br />

activities of the YycG kinase.<br />

PS 49<br />

Different roles of the ribosome modulation factor in<br />

Pseudomonads<br />

A. Steen *1 , A. Wesche 1 , N. Bös 1 , M. Schobert 1<br />

1<br />

Institut für Mikrobiologie, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

The bacterial stringent response is a ppGpp-mediated cellular adaptation to<br />

nutrient limitation, such as amino acid or carbon starvation. In the Gramnegative<br />

Pseudomonas group, synthesis of ppGpp is dependent on the proteins<br />

RelA and SpoT. We are interested in comparing the stringent response regulons<br />

of the opportunistic pathogen Pseudomonas aeruginosa recently defined in our<br />

lab, to that of its non-pathogenic relative Pseudomonas putida. For that<br />

purpose, we constructed a P. putida relA spoT double mutant strain and<br />

monitored its behaviour un<strong>der</strong> various growth conditions.<br />

The rmf gene, encoding the ribosome modulation factor, was found upregulated<br />

in the P. aeruginosa stringent response regulon. In Escherichia coli, the Rmf<br />

protein was shown to mediate the dimerization of 70S ribosomes, leading to an<br />

increased tolerance during stationary phase as well as acid and heat stress. To<br />

our surprise, a P. aeruginosa PAO1 rmf mutant strain showed slightly increased<br />

survival rates during acid and heat stress. As the Rmf binding site on the<br />

ribosome is also targeted by various antibiotics, we investigated if the ribosome<br />

modulation factor may have a function in antibiotic tolerance in P. aeruginosa.<br />

We found that the PAO1 rmf mutant strain showed reduced survival rates in<br />

mature biofilms incubated on tobramycin compared to the wild-type. Moreover,<br />

deletion of the rmf gene leads to an increased formation of small, slow growing<br />

colonies un<strong>der</strong> these conditions. These results indicate, that the P. aeruginosa<br />

Rmf protein plays an important role in antibiotic tolerance but contributes only<br />

mo<strong>der</strong>ately to acid, heat and stationary phase survival.<br />

PS 50<br />

Membrane-mediated c-di-GMP turnover in the<br />

opportunistic pathogen Pseudomonas aeruginosa<br />

M. Entian *1 , N. Frankenberg-Dinkel 1<br />

1 Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany<br />

In or<strong>der</strong> to cope with different environmental conditions bacteria have evolved<br />

a variety of different regulatory systems. One of these are two component<br />

systems, which are typically composed of a sensor histidine kinase and a<br />

corresponding response regulator. In addition other regulatory elements can be<br />

found which sense environmental stimuli and convert them into internal<br />

signals. Novel elements of such a system are the so-called GGDEF- and EALdomains.<br />

Both domains are involved in the turnover of the novel second<br />

messenger c-di-GMP: GGDEF-domains harbour diguanylate cyclase activity<br />

and build up c-di-GMP from two molecules of GTP, while EAL- domains act<br />

as phosphodiesterases and break down c-di-GMP into two molecules of pGpG.<br />

Here we present a combination of genetic and biochemical analyses to<br />

investigate the function of two novel membrane sensor proteins in<br />

Pseudomonas aeruginosa. The gene products of PA1727 and PA3311 consist<br />

of an N-terminal membrane sensor domain called MHYT and a C-terminal<br />

cytoplasmic GGDEF- and EAL-domain. Different variants of both proteins<br />

were constructed, recombinantly produced in E. coli, purified and tested for<br />

their catalytic activity. In vitro enzymatic activity assays showed that PA1727<br />

has diguanylate cyclase activity; whereas PA3311 is active but has a long<br />

turnover time. Furthermore, a PA3311 deletion mutant was constructed and<br />

analysed with regard to its phenotype to gain insights into the function of this<br />

novel sensor protein in P. aeruginosa.<br />

PS 51<br />

The role of the FliZ regulator in the coordination of motility<br />

and curli expression in Escherichia coli<br />

C. Pesavento *1 , R. Hengge 1<br />

1 Institut für Biologie - Mikrobiologie / AG Hengge, Freie Universität Berlin,<br />

Berlin, Germany<br />

The flagellar class II gene product FliZ plays a pivotal role in the coordination<br />

of flagellum-mediated motility and curli-mediated adhesion in Escherichia coli<br />

during entry into stationary phase. FliZ acts as an inhibitor of RpoS activity,<br />

and during the post-exponential growth phase gives motility temporarily<br />

priority over the entire general stress response (which includes RpoS-dependent<br />

curli fimbriae expression). This FliZ-mediated regulation is integrated with a<br />

second mode of inverse regulation of motility and curli expression that operates<br />

with the signalling molecule c-di-GMP, which is antagonistically controlled by<br />

RpoS-dependent GGDEF proteins and YhjH, a c-di-GMP specific<br />

phosphodiesterase and flagellar class III gene product [1].<br />

We further investigated the role of FliZ in the regulation of curli expression and<br />

motility and found FliZ to play an important role in the precise timing of gene<br />

expression during entry into stationary phase. Amongst the genes involved in<br />

the expression of curli fimbriae, the RpoS-dependent genes mlrA, which<br />

encodes a MerR-like transcriptional regulator and yciR, encoding a c-di-GMPspecific<br />

phospodiesterase, exhibit earlier expression in a fliZ mutant. In the<br />

wildtype background, YciR and the diguanylate cyclase YdaM antagonistically<br />

control curli expression, but do not affect motility [1]. However, in a fliZ<br />

mutant background, elevated yciR expression in post-exponential phase already<br />

can partially suppress the non-motility phenotype of a yhjH mutant. This<br />

demonstrates the important role of FliZ in coordinating the precise expression<br />

and physiological functions of regulators that influence curli-expression and<br />

motility. In addition, new insights into the molecular mode of action of FliZ in<br />

the regulation of RpoS activity will be presented.<br />

[1] Pesavento C, Becker G, Sommerfeldt N, Possling A, Tschowri N, Mehlis A,<br />

Hengge R. Genes Dev 2008, 22: 2434-46<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PS 52<br />

Transcriptome analysis of Bacillus subtilis and<br />

definition of the SigB regulon using a random forest class<br />

prediction algorithm<br />

P. Nannapaneni *1 , F. Hertwig 2 , S. Praveen Kumar 1 , S. van Hijum 3 , L. Steil *1 , M.<br />

Hecker 4 , U. Völker 1<br />

1 Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Ernst-<br />

Moritz-Arndt-Universität Greifswald, Greifswald, Germany<br />

2 Lund Strategic Research Center for Stem Cell Biology and Cell Therapy,<br />

University of Lund, Lund, Sweden<br />

3 NIZO food research BV, NIZO, Ede, Netherlands<br />

4 Institut für Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-<br />

Universität Greifswald, Greifswald, Germany<br />

In Bacillus subtilis the alternative sigma factor SigB controls a large set of<br />

genes in response to a whole variety of environmental challenges like heat,<br />

cold, osmotic, ethanol and oxidative stress as well as a series of nutrient<br />

limitations such as those for glucose, phosphate or oxygen. Even if the structure<br />

of the regulon has been studied by a number of groups using transcriptomic and<br />

proteomic approaches the degree of overlap between the results was<br />

astonishingly low [1,2,3,4].<br />

Here we present an approach to define the SigB-regulon using the random<br />

forest class prediction algorithm [5] based on gene expression data on a subset<br />

of 300 potential members of the SigB-regulon and 50 control genes, whole<br />

genome expression data from literature and promoter sequence information.<br />

The gene expression data of the candidate genes and controls were examined in<br />

a time dependent fashion for a whole array of inducing conditions in the<br />

wildtype and the sigB-mutant. Genomic features like promoter information are<br />

incorporated to support the experimental data and to distinguish subclasses of<br />

the SigB-regulon.<br />

[1] Price CW et al. Mol Microbiol. 2001 Aug; 41(4):757-774.<br />

[2] Helmann JD et al. J Bacteriol. 2001 Dec; 183(24):7318-7328.<br />

[3] Petersohn A et al. J Bacteriol. 2001 Oct; 183(19):5617-5631.<br />

[4] Bernhardt J et al. Microbiology. 1997 Mar; 143(Pt 3):999-1017.<br />

[5] Breiman L: Random Forests. Machine Learning 2001; 45:5-32.<<br />

PS 53<br />

Pseudomonas aeruginosa and its phytochrome operon:<br />

more than a photoreceptor?<br />

K. Barkovits *1 , N. Frankenberg-Dinkel 1<br />

1 Physiology of Microorganisms, Ruhr-University Bochum, Bochum, Germany<br />

Adaptation to changing lighting conditions is of particular importance for<br />

phototrophic organisms and is mediated by photoreceptors. One of the first<br />

photoreceptors, a red/far-red light reversible protein called phytochrome, was<br />

detected in plants. However, phytochromes were also newly discovered in nonphototrophic<br />

bacteria, whereas the function in these organisms is obscure.<br />

Biochemical analyses have established that the genes bphO and bphP from the<br />

opportunistic pathogen Pseudomonas aeruginosa encode the two necessary<br />

phytochrome components: BphO, a heme oxygenase that produces the<br />

chromophore biliverdin IXα, and bphP encoding the apo-phytochrome.<br />

Transcriptional analyses established that both genes form a bicistronic operon.<br />

Expression of the bphOP operon was shown to be induced at increasing cell<br />

density in the stationary phase, indicating an involvement of the P. aeruginosa<br />

quorum sensing (QS) system and/or the stationary phase alternative sigma<br />

factor RpoS. While a direct involvement of the QS system could be excluded,<br />

the dependence of bphOP expression on RpoS was clearly demonstrated.<br />

Phenotypic analyses of chromosomal knock-out mutants had no obvious<br />

growth defect un<strong>der</strong> various conditions but showed decreased heat tolerance in<br />

the stationary phase indicating a potential protective role of the BphO reaction<br />

product biliverdin IXα. In addition, increased levels of pyocyanin and the<br />

Pseudomonas quinolone signal (PQS) were detected in the ΔbphO strain<br />

pointing towards an additional function of BphO besides providing the<br />

chromophore for BphP and that both proteins are likely to fulfil a task in the<br />

stationary phase. Furthermore, global transcriptional studies showed an effect<br />

on the energy metabolism of P. aeruginosa dependent on the absence of BphO<br />

or BphP.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PS 54<br />

Transcription of reductive dehalogenase homologous genes<br />

of Dehalococcoides sp. strain CBDB1 in the presence of<br />

1,2,3- or 1,2,4-trichlorobenzene<br />

A. Wagner *1 , L. Adrian 1 , U. Lechner 2<br />

1 Inst. für Biotechnologie/FB Angewandte Biochemie, TU Berlin, Berlin,<br />

Germany<br />

2 Institut für Biologie/Mikrobiologie, Martin-Luther Universität Halle, Halle,<br />

Germany<br />

Microbial reductive dehalogenation is the only known biodegradation process<br />

for highly chlorinated aromatic compounds including chlorobenzenes un<strong>der</strong><br />

anaerobic conditions. The chlorinated compounds can serve as electron<br />

acceptors in respiratory dehalogenation. Bacteria of the class<br />

Dehalococcoidetes strictly depend on respiratory dehalogenation.<br />

Dehalococcoides sp. strain CBDB1 grows with 1,2,3- and 1,2,4trichlorobenzene<br />

(TCB) and contains 32 reductive dehalogenase homologues<br />

(rdh) genes in the genome, which are closely associated with regulatorencoding<br />

genes. The aim of this study was to analyze, if transcription of<br />

specific rdhA genes was up-regulated in response to 1,2,3- and/or 1,2,4-TrCB.<br />

A method based on reverse transcription (RT) and terminal restriction fragment<br />

length polymorphism (t-RFLP) was established to detect and differentiate<br />

transcripts of the 32 rdhA genes of strain CBDB1. Ten rdhA transcripts were<br />

selected for further analysis by quantitative PCR (qPCR). RT-t-RFLP and<br />

qPCR analyses revealed the up-regulation of all 32 rdhA genes in the presence<br />

of both trichlorobenzenes. However, transcript levels differed by or<strong>der</strong>s of<br />

magnitude between individual rdhA genes. Enhanced transcription of<br />

cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TrCB, while<br />

transcription of cbdbA1624 was strongly induced by 1,2,4-TrCB. CbrA, coding<br />

for a recently identified trichlorobenzene dehalogenase [1], possessed the<br />

highest transcript level of all analyzed rdhA genes after induction with 1,2,3-<br />

and 1,2,4-TrCB (22 transcript copies per gene copy). Therefore, the cbrA gene<br />

or transcript seems to be a suitable biomarker for the detection of a natural<br />

potential for chlorobenzene dechlorination in the environment.<br />

[1] Adrian et al. (2007) Appl. Environ. Microbiol. 73, 7717-7724<br />

PS 55<br />

In vivo activation of Tetracycline repressor by Cre/lox<br />

mediated gene assembly<br />

R. Bertram *1 , M. Kolb 1 , W. Hillen 2<br />

1 Lehrbereich Mikrobielle Genetik, Universität Tübingen, Tübingen, Germany<br />

2 Lehrstuhl für Mikrobiologie, Universität Erlangen-Nürnberg, Erlangen,<br />

Germany<br />

Tetracycline repressor (TetR) bears an unstructured loop region between<br />

helices α8 and α9, which is mo<strong>der</strong>ately permissive to amino acid exchanges and<br />

length variations. Recognition sites for the site-specific recombinases Flp (FRT)<br />

or Cre (lox) were inserted in-frame into tetR, substituting some of this loop’s<br />

codons. A number of the deduced TetR variants displayed efficient regulation<br />

in vivo, thus allowing the establishment of a new mode of TetR activation on<br />

the genetic level. Chromosomally encoded tetR in Bacillus subtilis was<br />

disrupted and inactivated by insertion of a lox66-aphAIII-lox71 kanamycin<br />

resistance cassette. Marker excision by Cre recombinase led to the assembly of<br />

a novel tetR allele. The encoded regulator, termed TetR lox72/1 , is distinguished<br />

from wt-TetR by a slightly elongated and altered α8 to α9 loop only, harboring<br />

an amino acid stretch encoded by lox72. Despite decreased intracellular protein<br />

amounts, TetR lox72/1 displayed efficient in vivo activity in B. subtilis and E. coli,<br />

indistinguishable from that of wt-TetR.<br />

PS 56<br />

New architectures for Tet-ON and Tet-OFF regulation in<br />

Staphylococcus aureus<br />

E. Stary 1 , E. Tichy 1 , M. Kolb 1 , R. Bertram *1<br />

1 Lehrbereich Mikrobielle Genetik, Universität Tübingen, Tübingen, Germany<br />

181<br />

Inducible gene expression systems are valuable tools for deciphering genefunction<br />

relationships. Tetracycline dependent settings exploit the regulator<br />

TetR and its cognate DNA sequence tetO to control transcription initiation of<br />

target genes. The mode of action of Tet-ON regulation involves TetR binding<br />

to tetO until administration of an inducer such as anhydrotetracycline (ATc)<br />

causes TetR detachment from tetO and thus target gene expression. While well<br />

established in Gram negative bacteria, adaptations of promoter elements have<br />

paved the way for tet-regulation also in firmicutes. We here describe novel

182<br />

architectures applicable to Staphylococcus aureus. tetR, driven by a strong<br />

synthetic promoter was integrated into a non-essential chromosomal locus. A<br />

second strain was constructed harbouring revtetR, the deduced protein of which<br />

binds to tetO in the presence of ATc only (Tet-OFF regulation). gfpmut2,<br />

driven by a promoter with two tetO sequences, was also ectopically integrated<br />

into the two strains’ chromosomes. In both cases, ATc dose-dependent<br />

fluorescence was observed. The tetR + strain was also useful for downregulating<br />

S. aureus genes in trans. This was achieved by ATc dependent expression of<br />

episomally encoded antisense (AS)-RNA. As tested with the essential fabI<br />

gene, growth retardation was observed upon induction of TetR. We further aim<br />

to conduct mutageneses of the S. aureus (rev)tetR + strains using transposon-like<br />

sequences carrying tet-regulatable outward-facing promoters. Thereby,<br />

essential genes or those involved in virulence traits may stochastically be<br />

placed un<strong>der</strong> tet-control and could thus be analyzed by regulated expression.<br />

PS 57<br />

Protein-protein-interaction in regulation of nitrogen<br />

metabolism of Streptomyces coelicolor<br />

M. Nentwich *1 , W. Wohlleben 1 , J. Reuther 1<br />

1 Fakultät für Biologie, Lehrstuhl für Mikrobiologie/Biotechnologie, Eberhard<br />

Karls Universität Tübingen, Tübingen, Germany<br />

In the antibiotic producer Streptomyces coelicolor nitrogen metabolism is<br />

controlled on the transcriptional and posttranslational level. Glutamine<br />

synthetases (GS) are key enzymes in nitrogen assimilation and convert<br />

glutamate and ammonia to glutamine un<strong>der</strong> ATP consumption. Un<strong>der</strong> lownitrogen<br />

conditions this reaction is essential for ammonium assimilation.<br />

Expression and activity of the GS enzymes are strictly regulated in bacteria.<br />

In the genomic sequence of S. coelicolor five genes encoding putative GS<br />

enzymes were identified. The glutamine synthetase I (GSI) is encoded by the<br />

glnA gene and shows high sequence similarity to other prokaryotic GS<br />

enzymes. The activity of GSI is regulated by the adenylyl transferase GlnE via<br />

adenylylation. The regulation of GlnE in S. coelicolor is not known so far. For<br />

identification of possible interaction partners of GlnE a genomic library is<br />

screened using a bacterial two-hybrid system. For the generation of the library,<br />

fragments of partially digested genomic DNA of S. coelicolor were ligated in<br />

the bacterial two-hybrid vector. Furthermore, the interaction of GlnE with<br />

proteins involved in nitrogen metabolism is investigated using the bacterial<br />

two-hybrid system.<br />

In S. coelicolor three additional glnA-type genes glnA2, glnA3 and glnA4 were<br />

identified. No GS enzyme activity could be shown so far for the gene products<br />

and the physiological functions remain to be elucidated. In or<strong>der</strong> to get more<br />

information about possible functions, protein-protein-interaction with other<br />

proteins involved in nitrogen metabolism is analysed using the bacterial twohybrid<br />

system.<br />

PS 58<br />

Nitrogen control in Corynebacterium glutamicum:<br />

Interaction of the global repressor AmtR with its target<br />

DNA and the PII-type protein GlnK-AMP<br />

K. Hasselt *1 , N. Jessberger 1 , D. Muhl 1 , M. Sevvana 2 , Y. Muller 2 , A. Burkovski 1<br />

1 Mikrobiologie, Friedrich-Alexan<strong>der</strong>-Universität Erlangen-Nürnberg,<br />

Erlangen, Germany<br />

2 Biotechnik, Friedrich-Alexan<strong>der</strong>-Universität Erlangen-Nürnberg, Erlangen,<br />

Germany<br />

Nitrogen is an essential nutrient for bacteria since it is necessary for example<br />

for the synthesis of nucleic acids and amino acids. In or<strong>der</strong> to provide optimal<br />

nitrogen supply un<strong>der</strong> different nitrogen conditions bacteria developed several<br />

regulation systems. The nitrogen control in C. glutamicum is <strong>bei</strong>ng governed by<br />

the TetR-type regulator AmtR [1]. Bioinformatical analyses showed that at<br />

least 36 genes are un<strong>der</strong> the control of AmtR [2]. In contrast to TetR, AmtR<br />

does not interact with a small effector molecule such as an antibiotic, but with a<br />

protein complex consisting of an adenylylated GlnK trimer [2]. For a better<br />

un<strong>der</strong>standing of the mechanisms of nitrogen regulation in C. glutamicum, the<br />

interaction of AmtR with the PII-type protein GlnK and with its target DNA<br />

was analyzed. Therefore, several amino acid exchanges were carried out in<br />

AmtR as well as in GlnK. In or<strong>der</strong> to examine the influence of the introduced<br />

mutations on the DNA-protein interaction, RNA hybridisation and gel shift<br />

experiments were carried out. Additionally large scale purification and<br />

crystallization of AmtR was performed to unravel the crystal structure.<br />

[1] Jakoby, M., Nolden, L., Meier-Wagner, J., Krämer, R. and Burkovski, A.<br />

(2000) AmtR, a global repressor in the nitrogen regulation system of<br />

Corynebacterium glutamicum. Mol. Microbiol. 37, 964-977.<br />

[2] Beckers, G., Strösser, J., Hildebrandt, U., Kalinowski, J., Farwick, M.,<br />

Krämer, R. and Burkovski, A. (2005). Regulation of AmtR-controlled gene<br />

expression in Corynebacterium glutamicum. Mechanism and characterization<br />

of the AmtR regulon. Mol. Micobiol. 58, 580-595.<br />

PS 59<br />

Nitrogen control in Mycobacterium smegmatis<br />

N. Jeßberger *1 , J. Amon 1 , A. Burkovski 1<br />

1 Lehrstuhl für Mikrobiologie, Friedrich-Alexan<strong>der</strong>-Universität Erlangen-<br />

Nürnberg, Erlangen, Germany<br />

Based on sequence analysis, two homologs of actinobacterial regulators of<br />

nitrogen metabolism were found in the Mycobacterium smegmatis genome,<br />

namely GlnR [1,2] and AmtR [2]. Recently we identified GlnR as the<br />

transcriptional regulator of nitrogen metabolism [3], while the role of AmtR<br />

remains unclear. A glnR deletion mutant did no longer respond to nitrogen<br />

starvation on a transcriptional level. The wild type strongly expressed genes<br />

coding for ammonium assimilation and uptake systems in response to nitrogen<br />

starvation, while this could not be detected in the glnR deletion strain [3]. This<br />

is also supported by growth experiments using MSX to cause artificial nitrogen<br />

starvation.<br />

GlnR binding to its target genes was determined performing gel retardation<br />

experiments. GlnR binds to sequences upstream of glnA and amtB, not<br />

upstream of glnR [3]. Thus the expression of the glnR gene is not<br />

autoregulated.<br />

In or<strong>der</strong> to investigate DNA binding manners of GlnR in detail, competitive gel<br />

retardation experiments are in progress as well as comparative genomics and<br />

deletion mutant analysis to provide new insights into the role of AmtR in M.<br />

smegmatis and its close relatives.<br />

[1] Tiffert, Y., Supra, P., Wurm, R., Wohlleben, W., Wagner, R. & Reuther, J.<br />

(2008). The Streptomyces coelicolor GlnR regulon: identification of new GlnR<br />

targets and evidence for a central role of GlnR in nitrogen metabolism in<br />

actinomycetes. Mol Microbiol.<br />

[2] Amon, J., Titgemeyer, F. & Burkovski, A. (2008). A Genomic View on<br />

Nitrogen Metabolism and Nitrogen Control in Mycobacteria. J. Mol. Microbiol.<br />

Biotechnol.<br />

[3] Amon, J., Bräu, T., Grimrath, A., Hänßler, E., Hasselt, K., Höller, M.,<br />

Jeßberger, N., Ott, L., Szököl, J., Titgemeyer, F. & Burkovski, A. (2008).<br />

Nitrogen control in Mycobacterium smegmatis: Nitrogen-dependent expression<br />

of ammonium transport and assimilation proteins depends on OmpR-type<br />

regulator GlnR. J. Bacteriol.<br />

PS 60<br />

A Model for the DNA-binding of the global regulator AbrB<br />

to the phyC-region<br />

S. Neubauer *1 , O. Makarewicz 1<br />

1 Biologie / Bakteriengenetik, Humboldt Universität zu Berlin, Berlin, Germany<br />

AbrB is a global transcription regulator of the transition state in Bacillus<br />

species. It influences moreover than 100 genes. One of these genes is the<br />

phytase gene phyC [1]. Since the AbrB binding regions of approximately 40<br />

genes are known, no consensus sequence could be determined. It is assumed<br />

that AbrB interacts with DNA as a homoteramer preferring specific three<br />

dimensional structures of the DNA. AbrB contains two dimerisation interfaces<br />

at the N- and C-terminus. Thereby to dimers, which are connected via the Ntermini<br />

form a tetramer through their C-termini. This form seem to be stabilized<br />

by reduced Cys54 in the N-terminal domain [2], but former studies showed that<br />

the truncated AbrBN is able to form tetramers through disulfide bonds as well<br />

[3].<br />

In our studies we used the AbrB of Bacillus subtilis as well as B.<br />

amyloliquefaciens FZB45 and the truncated AbrBN that is identical for both<br />

strains. We could show that higher polymerisation forms of AbrB are detectable<br />

by different methods un<strong>der</strong> non reducing conditions. We identified two AbrB<br />

binding sites within the phytase region, each captures approximately 100 bp<br />

and they are separated by a window of 200 bp. Although AbrB binds to each<br />

site respectively the transcriptional repression depends on the presence of both<br />

sites [1].<br />

Here we present a new DNA-binding model for AbrB to the phytase promoter.<br />

We propose a loop structure of the phyC region bound to AbrB polymers.<br />

[1] Makarewicz, O., Neubauer, S., Preusse, C., Borriss, R. (2008) J.<br />

Bacteriology 190: 6467-6477<br />

[2] Fude, Y., Strauch, M. A. (2005) J. Bacteriology 187: 6354-6362<br />

[3] Ke, X., Strauch, M. A. (2005) J. Bacteriology 183: 4094–4098<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PS 61<br />

Characterization of a putative phenol efflux transporter<br />

from P. putida<br />

C. Lehmann 1 , G. Burchhardt *1<br />

1 Abteilung Genetik <strong>der</strong> Mikroorganismen, Ernst Moritz Arndt Universität<br />

Greifswald, Greifswald, Germany<br />

P. putida strain H is able to metabolize phenol via the meta pathway. The<br />

corresponding genes are localized on the mega plasmid pPGH1. Upstream of<br />

the phenol degrading gene cluster is located another gene cluster called<br />

phlSTUV which is also expressed un<strong>der</strong> a sigma54 dependant promoter using<br />

PhlR as activator of phl gene expression. Comparison analyses of the deduced<br />

aa sequences revealed homology to several transporters of the RND family. The<br />

corresponding genes were heterologous expressed by generating gene fusions<br />

to GFP or GST. Localization experiments showed that PhlU and PhlW are<br />

membrane or membrane associated proteins. Expression of this gene cluster<br />

was studied with different LacZ fusions. The phlT-lacZ reporter construct<br />

which was expressed from a broad host range vector in P. putida demonstrated<br />

that an additional promoter is responsible for phenol independent low level<br />

expression. High expression of phlS was observed when phenol is present<br />

without any other carbon source involved in carbon catabolite repression.<br />

Insertion mutagenesis of phlW was carried out. This mutant is still able to grow<br />

on phenol as carbon source, but has an increased sensitivity towards phenol.<br />

We propose that the phlSTUV gene cluster encodes an efflux system for phenol.<br />

PT 01<br />

Microviridins – cyanobacterial tricyclic depsipeptides of<br />

ribosomal origin<br />

N. Ziemert *1 , A.R. Weiz 1 , K. Ishida 2 , A. Liaimer 3 , C. Hertweck 2 , E. Dittmann 1<br />

1<br />

Molecular Ecology, Institute of Biology, Humboldt University, Berlin,<br />

Germany<br />

2<br />

Leibniz Institute for Natural Product Research and Infection Biology, Hans<br />

Knöll Institute, Jena, Germany<br />

3<br />

Plant Physiology and Microbiology Group, Institute for Biology, Tromsø<br />

University, Tromsø, Norway<br />

Various types of planctonic cyanobacteria such as Microcystis frequently form<br />

toxic blooms in freshwater lakes, thus leading to a dramatic decrease of<br />

biodiversity in these ecosystems. Some of the most remarkable cyanobacterial<br />

toxins belong to the microviridin familiy of tricyclic depsipeptides.<br />

Microviridins feature an unparalleled cage-like architecture and specifically<br />

inhibit various types of proteases. Microviridin J affects the molting process of<br />

Daphnia and is discussed as a feeding deterrent that may partly explain the<br />

enormous success of Microcystis species in the field. On the other hand,<br />

microviridin B has high therapeutic potential as an elastase inhibitor in the<br />

treatment of lung emphysema. Here we report that microviridins are<br />

synthesized from a ribosomal precursor peptide in Microcystis by a unique<br />

mechanism involving stand-alone ATP grasp type ligases for ω-ester and ωamide<br />

bond formation, as well as a specialized transporter peptidase. This<br />

strategy is widespread among bloom-forming Microcystis.<br />

PT 02<br />

Molecular tools for biotechnically relevant filamentous<br />

fungi: Development of an RNAi system<br />

D. Janus *1 , B. Hoff 1 , U. Kück 1<br />

1<br />

Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

Filamentous fungi and yeast have a strong industrial relevance for the<br />

production of antibiotics, vitamins, and enzymes. Some of the most important<br />

fungal secondary metabolites are the β-lactam antibiotics penicillin and<br />

cephalosporin C. These pharmaceuticals are predominantly applied against<br />

bacterial infections and thus have a world market of about 18 billion Euros per<br />

year. The two filamentous fungi, Penicillium chrysogenum and Acremonium<br />

chrysogenum are mainly used for the industrial production of these metabolites.<br />

Classical strain improvements use approaches including several rounds of UV-<br />

or chemical mutagenesis but in most cases the generated „high-producer“<br />

strains show a genetic instability which can finally lead to a decrease in<br />

productivity. In or<strong>der</strong> to perform a controlled genetic manipulation diverse<br />

approaches have been used to disrupt target genes by homologous<br />

recombination. But in most filamentous fungi, such as P. chrysogenum and A.<br />

chrysogenum, substitution of target genes occurs only at very low frequencies<br />

due to ectopic integration of transforming DNA. Therefore, RNA-silencing<br />

(RNA interference; RNAi) has become an attractive alternative to disruption<br />

experiments for the functional analysis of genes in filamentous fungi.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Here we present the development of an inducible RNAi system to down<br />

regulate gene expression in P. chrysogenum using the autofluorescent DsRed<br />

protein as phenotypic marker. Furthermore, the applicability of this system is<br />

demonstrated by silencing an endogenous gene which regulates fungal<br />

morphology. The RNA-silencing system presented here will be a powerful tool<br />

for metabolic engineering, strain improvement and genome-wide analysis of<br />

this biotechnically important filamentous fungus.<br />

PT 03<br />

Development of molecular-genetic methods for genetic<br />

modification of biotechnologically relevant fungi<br />

K. Kopke *1 , B. Hoff 1 , U. Kück 1<br />

1<br />

Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

The development of tools for genetic engineering of biotechnologically relevant<br />

filamentous fungi like Penicillium chrysogenum is highly valuable for research<br />

application and strain improvement. The classical strategy to realize targeted<br />

gene deletion for functional analyses is based on homologous recombination by<br />

using resistance markers. To circumvent the limited availability of only three<br />

efficient antibiotic resistance markers in P. chrysogenum, we have established a<br />

system for marker recycling. For this purpose, we have chosen the FLP/FRTrecombinase<br />

system from the yeast Saccharomyces cerevisiae [1]. The 45 kDa<br />

FLP protein, which is a member of the tyrosine recombinases, is able to<br />

mediate site-specific recombination between the two 34 bp long FLP<br />

recognition targets (FRTs). In a first approach, we have generated a<br />

nourseothricin resistance cassette flanked by the FRT sequences (FRT-nat1<br />

cassette) in direct repeat orientation and integrated this construct ectopically in<br />

a P. chrysogenum recipient strain. To get the FLP/FRT-recombinase system<br />

working, the FLP recombinase, un<strong>der</strong> the control of a constitutive promoter,<br />

was integrated in a subsequent step into a strain carrying the FRT-nat1 cassette.<br />

The corresponding transformants were analysed by PCR to verify successful<br />

recombination events.<br />

[1] Morschhäuser J, Michel S, Staib P (1999) Mol Microbiol 32: 547-556<br />

PT 04<br />

Characterization of the pyrroindomycin B biosynthetic<br />

gene cluster<br />

E.P. Patallo *1 , K.H. van Pée 1<br />

1 Biochemie, TU Dresden, Dresden, Germany<br />

183<br />

Pyrroindomycin B an antibiotic compound produced by Streptomyces<br />

rugosporus LL-42D005 is active against Gram-positive bacteria such as<br />

methicillin-resistant Staphylococcus aureus and vancomycin-resistant<br />

Enterococci strains [1, 2]. Three different parts constitute the final structure of<br />

pyrroindomycin B, an indolopyrrole moiety, a trisaccharide and a polyketide<br />

macro-ring system.<br />

From a cosmid library of Streptomyces rugosporus, a cosmid (cos12D4) was<br />

isolated containing a tryptophan 5-halogenase gene (pyrH). PyrH chlorinates<br />

tryptophan to yield 5-chlorotryptophan the first intermediate in the biosynthesis<br />

of a three ring structure [3]. Cos12D4 has been completely sequenced and<br />

revealed several ORFs which could be involved in the biosynthesis of the<br />

Pyrroindomycin B.<br />

To characterize the activity of some genes involved in the biosynthesis of the<br />

pyrroindomycin an apramycin resistance gene was inserted directly into these<br />

genes by using a PCR mediated system and a conjugative vector. These<br />

deletion mutants have been verified by Southern analysis and their accumulated<br />

products will be isolated and characterized.<br />

PT 05<br />

New Insights into the Biosynthetic Pathway of Thienodolin<br />

in Streptomyces albogriseolus<br />

D. Milbredt *1 , E.P. Patallo 1 , K.H. van Pée 1<br />

1 Biochemie, TU Dresden, Dresden, Germany<br />

The plant growth-regulating substance thienodolin is produced by Streptomyces<br />

albogriseolus. It is assumed that tryptophan is a precursor of thienodolin. For<br />

its formation, starting from tryptophan, regioselective chlorination in the 6position<br />

of the indole ring could take place, an activation of the α-carbon for<br />

incorporation of the single sulphur atom as well as the formation of an amide<br />

function from the carboxylic acid group of tryptophan.<br />

Recently, a tryptophan 6-halogenase gene has been isolated from the<br />

thienodolin producer. This tryptophan 6-halogenase catalyses the regioselective<br />

chlorination of tryptophan, probably the first step in thienodolin biosynthesis.

184<br />

Based on this assumption, the tryptophan 6-halogenase gene was used as a<br />

starting point for the isolation and sequencing of neighbouring DNA fragments.<br />

The whole biosynthetic cluster of thienodolin could thus be cloned. Analysing<br />

the proposed functions of the revealed ORFs resulted in two genes which are<br />

candidates for catalysing the second step in thienodolin biosynthesis, an amino<br />

transferase and an amido transferase reaction. The amino transferase could<br />

convert 6-chlorotryptophan into 6-chloro-indole-3-pyruvate which could be the<br />

substrate for an enzyme introducing the sulphur atom at the α-carbon followed<br />

by a ring closure reaction resulting in the formation of the thiophene ring. In<br />

this proposed pathway, the amido transferase, forming the amide group, would<br />

be the last enzyme in thienodolin biosynthesis. But it is also possible that the<br />

amido transferase catalyses the second step using 6-chloro-tryptophan as a<br />

substrate to form 6-chloro-3-(2-amino-(3-amidopropyl))indole.<br />

In or<strong>der</strong> to elucidate the biosynthetic pathway of thienodolin, a series of<br />

deletion mutants of Streptomyces albogriseolus was created and the<br />

accumulated metabolites were analyzed.<br />

PT 07<br />

The putative acyltransferase genes kirCI and kirCII<br />

E.M. Musiol *1 , T. Härtner 1 , W. Wohlleben 1 , T. Weber 1<br />

1 Microbiology/Biotechnology, University of Tuebingen, Tuebingen, Germany<br />

The antibiotic kirromycin was isolated in 1972 from Streptomyces collinus Tü<br />

365.<br />

The cluster encoding kirromycin biosynthesis comprises 26 genes spanning 82<br />

kb DNA. Sequence analysis revealed that the kirromycin skeleton is assembled<br />

by the large polyketide synthase (PKS)/non-ribosomal peptide synthetase<br />

(NRPS) enzymes KirAI-KirAVI and KirB (Weber et al, 2008 Chem. Biol. 15:<br />

175-188).<br />

KirAI-KirAV have a „trans-AT“-architecture, where acyltransferase (AT)<br />

domains are not encoded within the PKS genes. KirAVI is of the classical “cis-<br />

AT-type” with internal AT domains.<br />

Two separate AT genes, kirCI and kirCII, are present in the gene cluster that<br />

are presumed to take over the AT functionality in trans.<br />

All extension modules of the trans AT-type PKS (KirAI-KirAV), except<br />

module 5 of KirAII, have a predicted specificity for malonyl-CoA. The<br />

incorporation of the unusual exten<strong>der</strong> unit ethylmalonyl-CoA is postulated for<br />

module 5 of the kirromycin PKS/NRPS synthase.<br />

To test, how this specificity is accomplished, an in vitro ACP-loading assay is<br />

<strong>bei</strong>ng developed.<br />

Therefore, purified AT enzymes and ACPs are required. In a first approach, we<br />

focused on the ATs (KirCI and KirCII) and ACP of module 4 and 5 (ACP4 and<br />

ACP5).<br />

The expression of PKS or fatty acid synthase AT domains is often problematic.<br />

Due to this fact, several expression systems were tested.<br />

Whereas KirCI expressed in E. coli leads to formation of inclusion bodies, the<br />

expression of a soluble KirCII protein, using the pET52 expression system, was<br />

successful.<br />

Additionally the Strep/KirCI and StrepKirCII were expressed in Streptomyces<br />

lividans but both acyltransferases couldn’t be purified. Soluble ACP-proteins<br />

were obtained as Strep/His-fusion proteins using the<br />

pET52 E. coli-expression vector.<br />

PT 08<br />

Method development for the isolation of transcription<br />

factors from whole extract<br />

D. Löper *1 , B. Hoff 1 , M. Piotrowski 2 , U. Kück 1<br />

1<br />

Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

2<br />

Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, Bochum,<br />

Germany<br />

Primary and secondary metabolism in filamentous fungi is usually controlled<br />

by a network of transcription factors that act as activators or repressors on gene<br />

expression. In or<strong>der</strong> to isolate proteins that bind specifically to promoter<br />

sequences we have developed a reliable method using a Streptavidin<br />

MicroBeads kit and biotinylated DNA-Fragments combined with Q-TOF<br />

spectrometry.<br />

The filamentous fungus Acremonium chrysogenum is the main producer of<br />

cephalosprin C. The biosynthesis of this β-lactam antibiotic is catalyzed by at<br />

least seven enzymes, two of which are expandase / hydroxylase and<br />

acetyltransferase [1]. These proteins are encoded by the cefEF and cefG genes,<br />

whose expression is driven by a strong 939 bp promoter. Using the biotinylated<br />

cefEF/cefG-promoter DNA sequence a method development was performed for<br />

the isolation of DNA binding proteins. Using a Streptavidin MicroBeads kit<br />

following Q-TOF spectrometry we were able to isolate at least one promoter<br />

binding protein which is involved in the regulation of cephalosporin<br />

biosynthesis.<br />

[1] Schmitt EK, Hoff B, Kück U (2004) Adv Biochem Engin/Biotechnol 88: 1-<br />

43<br />

PT 09<br />

Enhancement of micro elements composition for endhanced<br />

bioemulsifier production by yeast strains isolated from local<br />

palm wine using taguchi experimental design<br />

O. Okoye *1 , I.S. Okonkwo 2<br />

1<br />

Research and Logistics, Ebonyi State Environmental Protection Agency,<br />

Abakaliki, Nigeria<br />

2<br />

Department of Industrial Physics, Enugu State University of Technology,<br />

Enugu, Nigeria<br />

Bioemulsifiers are surface active compounds produced by a wide range of<br />

microorganisms and they are of great industrial and commercial interests. In<br />

this study, an optimization of micro element composition was attempted as a<br />

main step towards enhancement of bioemulsifier production from some yeast<br />

strains isolated from a local palm wine from Nigeria. Taguchi experimental<br />

design was applied for the purpose of identifying optimal micro element<br />

composition in the medium (MSM). Of the five trace elements examined, Mg 2+ ,<br />

K + , Mn 2+ , and Fe 2+ were found to be more significant factors affecting<br />

bioemulsifier production by the yeast strain. In the absence of Mg 2+ or K + ,<br />

bioemulsifier yield decreased to 0.48 g/L, which was only 28% of the value<br />

obtained from the control run. When Mn 2+ and Fe 2+ were both absent, the<br />

production yield also dropped to about 0.57 g/L, approximately one-fourth of<br />

the control value. However, when only one of the two metal ions (Fe 2+ or Mn 2+ )<br />

was absent, the yeast strain was able to remain over 75% of original<br />

biosurfactants productivity, suggesting that some interactive correlations among<br />

the selected metal ions may involve. Taguchi method was thus applied to reveal<br />

the interactive effects of Mg 2+ , K + , Mn 2+ , Fe 2+ on bioemulsifier production.<br />

Theresults revealed that the interaction of Mg 2+ and K + reached significant<br />

level.<br />

PT 10<br />

Is the human Aryl hydrocarbon receptor involved in<br />

demelanization and UV-insensitivity of lesions of pityriasis<br />

versicolor?<br />

K. Zuther *1 , P. Mayser 2 , J. Schirawski 1<br />

1<br />

Organismische Interaktionen, Max-Planck-Institut für terrestrische<br />

Mikrobiologie, Marburg, Germany<br />

2<br />

Zentrum für Dermatologie und Andrologie, Universitätsklinikum Giessen,<br />

Giessen, Germany<br />

The saprophytic yeast Malassezia furfur is the causative agent of the human<br />

skin mycosis pityriasis versicolor (PV). PV is characterized by brownish or<br />

demelanized skin patches that show fluorescence un<strong>der</strong> UV light and are not<br />

hypersensitive in response to UV irradiation. Such symptoms could be<br />

explained by M. furfur’s capability to produce pigments and fluorochromes in<br />

the presence of tryptophan.<br />

Our previous genetic and biochemical approaches had shown that the only<br />

enzyme needed for synthesis of such indole compounds is the Trp<br />

aminotransferase Tam1 catalyzing Trp to indole pyruvate (IP) conversion. IP in<br />

turn reacts spontaneously in an aqueous environment to form a wide variety of<br />

coloured and fluorescent indole compounds. We are currently investigating<br />

whether these indole compounds indeed play a role in symptom development of<br />

pityriasis versicolor. While some of the indole compounds are known to have<br />

UV absorbing properties, they might also be involved in the UV-B stress<br />

response via the aryl hydrocarbon receptor. Since the known Trp photoproduct<br />

6-formylindolo[3,2-b]carbazole (FICZ) is structurally similar to some of the<br />

indole compounds and acts as a highly potent aryl hydrocarbon receptor (AhR)<br />

agonist in humans, we tested the effect of aqueous IP solution on the AhR by<br />

measuring typical AhR mediated upregulated parameters in immortalized<br />

keratinocytes (HaCaTs). Activation of the AhR could be responsible for the UV<br />

insensitivity and also for the demelanization associated with the disease.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PT 11<br />

AcmI and AcmL are aromatic C-methyltransferases<br />

catalysing the conversion of 3- hydroxy-kynurenine to 3hydroxy-4-methyl-kynurenine,<br />

a step in the biosynthesis of<br />

actinomycin in Streptomyces chrysomallus.<br />

I. Crnovcic *1 , U. Keller 1<br />

1 Institut für Chemie, Biochemie, Technische Universität Berlin, Berlin,<br />

Germany<br />

Cloning of actinomycin biosynthesis genes from Streptomyces chrysomallus<br />

revealed a gene cluster encoding actinomycin peptide assembly and genes<br />

responsible for biosynthesis of 4-methyl-3-hydroxyanthranilic acid (4-MHA)<br />

and genes for self resistence [1]. For the synthesis of 4-MHA we identified<br />

genes encoding tryptophan pyrrolase, kynureninformamidase and<br />

kynureninases all beeing involved in the degradation of tryptophan to 3hydroxyanthranilic<br />

acid. To address the issue of methylation of the benzene<br />

ring of 4-MHA we analyzed two further genes in the actinomycin gene clusters<br />

encoding putative O-methyltransferases. These genes, designated acmI and<br />

acmL were both expressed as HexaHis fusion protein in E. coli. After<br />

purification, proteins were assayed for their ability to methylate various<br />

potential precursors of 4-MHA such as tryptophan, N-formyl-kynurenine (f-<br />

Kyn), kynurenine (Kyn), 3-hydroxykynurenine (Hykyn) and 3hydroxyanthranilic<br />

acid (3-HA) with consumption of S-adenosyl-L-methionine<br />

(AdoMet). Both enzymes exclusively methylate Hykyn but not 3-HA in the 4position<br />

of the benzene ring yielding 4-methyl-3-hydroxykynurenine the<br />

structure of which was confirmed by its in vitro conversion to 4-MHA<br />

catalyzed by hydroxykynureninase from S. chrysomallus. The data clearly show<br />

that the methyl groups of the phenoxazinon chromophore of actinomycins are<br />

introduced into the benzene ring of tryptophan precursor at the stage of Hykyn<br />

and not of 3-HA as proposed previously [2]. A detailed characterization of the<br />

two 3-hydroxykynurenine-4-C-methyltransferases from S. chrysomallus will be<br />

presented.<br />

[1] Schauwecker, F., Pfennig,F., Grammel, N., Keller, U. (2000) Chem. Biol. 7,<br />

287-297<br />

[2] Jones, G.H. (1994) J.Biol.Chem. 268, 6831-6834<br />

PU 01<br />

Small RNAs from the halophilic archaeon Haloferax<br />

volcanii<br />

J. Straub *1 , M. Brenneis 1 , A. Jellen-Ritter 2 , R. Heyer 2 , B. Tjaden 3 , J. Soppa 1 , A.<br />

Marchfel<strong>der</strong> 2<br />

1<br />

Institute for Molecular Biosciences, Goethe-University, Frankfurt, Germany<br />

2<br />

Molecular Botany, University of Ulm, Ulm, Germany<br />

3<br />

Computer Science Department, Wellesley College, Wellesley, MA, United<br />

States<br />

To elucidate the role of small regulatory RNAs in archaea we identified small<br />

RNAs in the halophilic archaeon Haloferax volcanii using experimental and<br />

bioinformatic approaches. The computational approach identified 31 sRNAs in<br />

intergenic regions. In addition, 28 potential C/D box RNAs but no H/ACA box<br />

sRNAs were predicted.<br />

Using a specialised cDNA library we identified a total of 88 different<br />

previously uncharacterised sRNAs ranging in size from 130 to 460 nucleotides.<br />

21 of these sRNAs are located in intergenic regions and 3 of them are predicted<br />

to encode peptides. 18 sRNAs are in antisense orientation and 49 are in sense<br />

orientation to open reading frames. To characterise the expression of the<br />

sRNAs we performed northern blot analyses with RNA isolated from cells<br />

grown un<strong>der</strong> different conditions. Transcripts of most analyzed sRNA genes<br />

were detected and several gene were found to be differentially expressed.<br />

Furthermore, deletions of two sRNAs genes were constructed to analyse their<br />

biological functions in vivo. Both deletion strains were viable but knock out of<br />

the sRNA30 gene made the cell less resistant to higher temperatures and knock<br />

out of the sRNA63 gene resulted in a severe growth defect at low salt<br />

concentrations. In both cases proteome analyses showed clear differences<br />

between wild type and knock out strains. Taken together, it was shown that H.<br />

volcanii possesses a non-negligable number of small non-coding RNAs, that<br />

they are (differentially) expressed and that mutant construction is a powerful<br />

tool to unravel their biological functions.<br />

[1] Julia Straub, Mariam Brenneis, Angelika Jellen-Ritter, Ruth Heyer, Brian<br />

Tjaden, Jörg Soppa and Anita Marchfel<strong>der</strong>. Small RNAs in haloarchaea:<br />

Identification, differential expression and biological function. Submitted.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PU 02<br />

Control of Synechocystis heat shock gene expression by an<br />

RNA thermometer<br />

J.F. Kortmann *1 , S.D. Sczodrok 1 , F. Narberhaus 1<br />

1 Lehrstuhl für Biologie <strong>der</strong> Mikroorganismen, Ruhr-Universität Bochum,<br />

Bochum, Germany, Bochum, Germany<br />

In the unicellular cyanobacterium Synechocystis sp. PCC6803, the single small<br />

heat shock protein (sHsp) Hsp17 is essential for tolerance to elevated<br />

temperatures and during UV-B stress [1,2]. In good agreement with its function<br />

as molecular chaperone, Hsp17 interacts with a large number of denatured<br />

proteins [3]. Furthermore Hsp17 is potentially contributing to the preservation<br />

of thylakoid membrane integrity, facilitating photosynthesis un<strong>der</strong> heat stress<br />

conditions [4]. sHsps are ubiquitous and their biosynthesis is regulated on<br />

transcriptional and post-transcriptional level. In Salmonella, translation of the<br />

small heat shock messenger agsA is enabled by an RNA element located in the<br />

5´ untranslated region (UTR) [5]. RNA thermometers respond to changing<br />

temperatures. At physiological temperature, translation is blocked as a result of<br />

a masked ribosome binding site by imperfect base pairing. At elevated<br />

temperatures, the structure was shown to melt giving access to the ribosome to<br />

start translation [6].<br />

Analysis of the hsp17 5´ UTR reveal a novel class of cyanobacterial RNA<br />

thermometers. Structure probing experiments identify the temperature-induced<br />

changes in hsp17 RNA architecture. Melting of the ribosome binding site<br />

initiates from two sites in the 5´ UTR. Ribosome-RNA interaction toeprinting<br />

assays confirm these results in vitro.<br />

[1] LaRossa et al. (2002) J. Bacteriol., 184: 6845-6858<br />

[2] Horváth et al. (2008) J.Biol.Chem., 283: 22983-22991<br />

[3] Vierling et al. (2004) J.Biol. Chem., 279: 7566-7575<br />

[4] Vigh et al. (2002) Proc. Natl. Acad. Sci. USA., 99: 13504-13509<br />

[5] Waldminghaus et al. (2007) Mol. Microbiol., 65: 413-424<br />

[6] Narberhaus et al. (2006) FEMS Microbiol. Rev., 30: 3-16<br />

PU 03<br />

Regulation of the small noncoding RNA GlmY<br />

B. Reichenbach *1 , Y. Göpel 1 , B. Görke 1<br />

1 Institut für Mikrobiologie und Genetik, Allgemeine Mikrobiologie, Georg-<br />

August Universität Göttingen, Göttingen, Germany<br />

The small RNAs GlmY and GlmZ form a regulatory system for feedback<br />

regulation of glmS expression. GlmS catalyzes the biosynthesis of glucosamine-<br />

6-phosphate, which is an essential precursor for cell wall biosynthesis. When<br />

the product of the reaction, Glucosamine-6-phosphate (GlcN-6-P), is low in the<br />

cell [1, 2], translation of GlmS is activated by the GlmZ RNA through base<br />

pairing with the glmS messenger. The intracellular GlcN-6-P concentration is<br />

sensed upstream of GlmY or by GlmY itself. The activating signal is then<br />

transduced to GlmZ by accumulation of GlmY, which subsequently results in<br />

accumulation of the active, unprocessed form of GlmZ (1). While the<br />

mechanism for activation of glmS expression by the small RNA GlmZ has been<br />

elucidated [3], the mechanism which allows sensing of the GlcN-6-P signal by<br />

GlmY remains unknown. This work focuses on factors which govern the<br />

expression level of GlmY and its abundance in the cell.<br />

[1] Reichenbach, B., Maes, A., Kalamorz, F., Hajnsdorf, E. and B. Görke,<br />

2008, Nucleic Acids Res, 36(8):2570-80<br />

[2] Urban, J. and J. Vogel, 2008, PLOS Biol, 6(3):e64<br />

[3] Görke, B. and J. Vogel, 2008, Genes Dev, 22(21):2914-25<br />

185<br />

PU 04<br />

Selenocysteine Biosynthesis in the Archaeon Methanococcus<br />

maripaludis<br />

T. Stock *1 , S. Scheuffele 1 , M. Selzer 1 , C. Sattler 1 , M. Rother 1<br />

1 Institute of Molecular Biosciences/Molecular Microbiology & Bioenergetics,<br />

Goethe-University Frankfurt, Frankfurt (Main), Germany<br />

Selenocysteine, the 21 st proteinogenic amino acid, is encoded on the mRNA by<br />

the stop codon UGA, which in Bacteria is recoded by a quaternary complex of<br />

the selenocysteine specific translation factor SelB, GTP, selenocysteinyltRNA<br />

sec and a secondary structure on the mRNA downstream of the UGA<br />

codon (the SECIS element). Synthesis of selenocysteine in bacteria proceeds in<br />

a two-step mechanism. First, tRNA sec is misacylated with serine to ser-tRNA sec<br />

by seryl-tRNA synthetase. In a second step the misacylated tRNA is converted<br />

to sec-tRNA sec by selenocysteine synthase with selenomonophosphate as the<br />

selenium donor. Biochemical analyses showed that Archaea employ a pathway

186<br />

in sec-tRNA sec formation very similar to that found in Eukarya. As in Bacteria<br />

tRNA sec is mischarged with serine. In the next step the serylated tRNA is<br />

phosphorylated by phosphoseryl-tRNA sec kinase (PSTK) to O-PhosphoseryltRNA<br />

sec . In the following reaction the intermediate is converted into sectRNA<br />

sec by O-Phosphoseryl-tRNA sec :selenocysteine synthase (SepSecS). We<br />

investigated the pathway of sec-tRNA sec formation in the model archaeon<br />

Methanococcus maripaludis by genetic analysis to unravel the selective<br />

advantage of a three-step mechanism for sec-tRNA sec formation found in<br />

Archaea (and Eukarya) over the two-step mechanism present in Bacteria.<br />

PU 05<br />

Salmonella has recruited the conserved small RNA, SgrS, of<br />

the phosphosugar-stress pathway for control of a secreted<br />

virulence protein<br />

K. Papenfort *1 , D. Podkaminski 1 , L. Schulte 1 , S. Lucchini 2 , J.C.D. Hinton 2 , J.<br />

Vogel 1<br />

1<br />

RNA Biology Group, Max Planck Institute for Infection Biology, Berlin,<br />

Germany<br />

2<br />

Molecular Microbiology Group, Institute of Food Research, Norwich, United<br />

Kingdom<br />

The small regulatory RNA (sRNA) SgrS is known to counteract phosphosugarstress<br />

in Escherichia coli. The molecular mechanisms un<strong>der</strong>lying this response<br />

involve the post-transcriptional control of the major glucose transporter PtsG<br />

utilizing an antisense targeting mechanism. To gain insight into the functions of<br />

SgrS in the pathogenic bacterium Salmonella typhimurium we utilized a<br />

transcriptomic approach revealing several other mRNAs targeted by this sRNA.<br />

Besides regulating an additional sugar transport system we found that the<br />

Salmonella specific effector protein SopD is subjected to SgrS control.<br />

Consequently, when grown un<strong>der</strong> conditions known to facilitate Salmonella<br />

invasion SgrS becomes upregulated and therefore represses SopD expression.<br />

Biochemical and genetic experiments further revealed posttranscriptional<br />

regulation of sopD by SgrS and pinpointed to an interaction that is guided by a<br />

short conserved region located near the 3’end of SgrS. Compensatory base-pair<br />

exchanges exposed that a short stretch of 11 nts in coding region of SopD is<br />

sufficient to establish this interaction.<br />

Moreover, S. typhimurium harbours a duplication of this effector protein<br />

(SopD2) that was shown to be essential for virulence. Both proteins share high<br />

similarity at the nucleotide level but SopD2 abundance is not affected by SgrS<br />

expression. Employing a GFP-based two-plasmid reporter system we explored<br />

that a single nucleotide transistion (C-T) in the open reading frame of sopD2,<br />

resulting in G-U instead of a G-C base-pair, while keeping the same amino-acid<br />

composition, is sufficient to mediate SgrS dependent target discrimination. This<br />

is the first example of a small RNA that accomplishes target recognition at the<br />

single nucleotide level.<br />

PU 06<br />

Identification of regulatory RNAs in the marine<br />

cyanobacterium Prochlorococcus using different methodical<br />

approaches<br />

C. Steglich *1 , M. Futschik 2 , C. Sharma 3 , J. Mitschke 1 , D. Lindell 4 , S.W.<br />

Chisholm 5 , J. Vogel 3 , W.R. Hess 1<br />

1 Institute of Biology III / Genetics, University of Freiburg, Freiburg, Germany<br />

2 Centre for Molecular and Structural Biomedicine, University of Algarve, Faro,<br />

Portugal<br />

3 Institute for Infection Biology, Max Planck, Berlin, Germany<br />

4 Faculty of Biology, Technion, Haifa, Israel<br />

5 Department of Civil and Environmental Engineering, Massachusetts Institute<br />

of Technology, Cambridge, United States<br />

Small non-coding RNAs (ncRNAs) are functional RNA molecules, mostly<br />

without a protein-coding function, that have been found in all domains of life.<br />

In bacteria these functional RNA molecules range in size between 50 – 400 nt<br />

and frequently play a crucial role in regulatory networks particularly in<br />

response to environmental stress. Another class of regulatory RNAs are<br />

chromosomally encoded antisense RNAs (asRNAs). ncRNAs and asRNAs are<br />

known to control plasmid and viral replication, bacterial virulence and quorum<br />

sensing, yet, the function of many has remained unknown. Cis-encoded<br />

asRNAs are transcribed from the opposite strand of the same genomic locus as<br />

the target (m)RNA and feature 100% base complementarity. In contrast,<br />

ncRNAs that are mostly located in intergenic regions, act trans in a different<br />

genomic locus and exhibit only a short and imperfect base complementarity<br />

with their target transcripts.<br />

Our analysis of microarray expression data of intergenic regions, and of 454<br />

sequencing data together with a previous comparative genomics approach<br />

revealed the existence of more than 20 ncRNAs and approximately 100<br />

asRNAs in Prochlorococcus MED4. The relative number of ncRNAs in<br />

Prochlorococcus thus is comparable with those found in enterobacteria like E.<br />

coli, each with 1 – 2% of the genes coding for ncRNAs. Genome reduction in<br />

Prochlorococcus has particularly affected the number of genes coding for<br />

regulatory proteins, suggesting that regulation of gene expression through<br />

ncRNAs plays an important role in Prochlorococcus’ response to<br />

environmental cues. Some of these functional RNAs are likely to be involved in<br />

processes such as light stress adaptation, the response to phage infection or<br />

nitrogen starvation as inferred from their mode of regulation. Furthermore, the<br />

enrichment in ncRNA genes in genomic islands of Prochlorococcus suggests<br />

that these islands are an important vehicle for the acquisition of ncRNAs.<br />

PU 07<br />

Non-coding RNAs and antisense transcripts in<br />

Synechocystis<br />

I. Scholz *1 , J. Georg 1 , B. Voss 1 , C. Steglich 1 , C. Sharma 2 , J. Mitschke 1 , J.<br />

Vogel 2 , W. Hess 1<br />

1 Institute of Biology III / Genetics, University of Freiburg, Freiburg, Germany<br />

2 Institute for Infection Biology, Max-Planck, Berlin, Germany<br />

Cyanobacteria are oxygenic phototrophs which inhabit both terrestrial and<br />

aquatic environments. Although the genome of the unicellular model<br />

cyanobacterium Synechocystis PCC 6803 was sequenced as early as 1996, little<br />

is known about the presence and function of cis-encoded antisense RNAs<br />

(asRNAs) and non-coding RNAs (ncRNAs) in this organism. By means of<br />

bioinformatic predictions and a 105K tiling array (which covers about 1,200<br />

genes) we investigated the distribution of asRNAs, identifying 73 new ones in<br />

addition to 57 ncRNAs. Verification of this data was done manually by<br />

5´RACE-experiments and Northern blot analysis.<br />

Since the data obtained by this work seemed to be very promising we decided<br />

to sequence total RNA by 454-pyrosequencing. One set of total RNA was<br />

enriched for primary 5’ ends whereas a second RNA pool was taken as is. First<br />

analysis of this data suggests a strong correlation between information obtained<br />

by earlier bioinformatics and array-based analysis and pyrosequencing,<br />

especially for the more strongly accumulating asRNAs and ncRNAs.<br />

Intriguingly, sequencing data also gave a first hint for the presence of three very<br />

strongly transcribed regions in pSYSA, one of the seven plasmids of<br />

Synechocystis. All three regions turned out to consist entirely of Clustered<br />

Regularly Interspaced Short Palindromic Repeat elements, a poorly<br />

characterized class of genetic elements which are thought to protect the<br />

bacterium from infection by phages through a kind of prokaryotic RNA<br />

interference.<br />

This data gives the first overview on potentially regulatory RNA in the<br />

cyanobacterium Synechocystis. Un<strong>der</strong>standing their functions and biological<br />

importance will be the next task to accomplish.<br />

PU 08<br />

The Yersinia pseudotuberculosis Csr system is a key<br />

regulatory system for the control of virulence, motility and<br />

metabolism<br />

A.K. Heroven *1 , K. Böhme 1 , P. Dersch 1<br />

1 Molecular infection biology, Helmholtz Centre for Infection Research,<br />

Braunschweig, Germany<br />

The MarR-type regulator RovA of Y. pseudotuberculosis controls the<br />

expression of the crucial internalization factor invasin and other virulence genes<br />

in response to various environmental signals. Expression of rovA is repressed<br />

during growth in minimal medium by the LysR-type regulator RovM. Recent<br />

studies revealed that the carbon storage regulator system (Csr) affects rovA by<br />

regulating the synthesis of RovM. A CsrA-like RNA-binding protein indirectly<br />

activates RovM synthesis. Furthermore, the regulatory RNA CsrC was shown<br />

to control media-dependent expression of rovM. CsrC is highly induced in<br />

complex but not in minimal media. A second regulatory RNA, CsrB,<br />

participates in rovM and rovA regulation. In contrast to CsrC, CsrB synthesis is<br />

generally very low and activated by the response regulator UvrY. CsrC<br />

synthesis is dependent upon the presence of the nucleoid-associated protein<br />

YmoA.<br />

Further studies revealed that the Csr system must regulate additional factors<br />

besides rovM and RovM-dependent genes such as rovA. We found that loss of<br />

CsrA has a severe effect on the morphology, motility and general physiology of<br />

the bacterial cell. Future studies to identify additional Csr components and Csrdependent<br />

genes in Y. pseudotuberculosis will help us to define the role of this<br />

regulatory system for the physiology and virulence of this enteric pathogen.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PU 09<br />

Deep sequencing analysis of non-coding RNAs in<br />

Methanosarcina mazei Gö1<br />

D. Jäger *1 , J. Thomsen 1 , C. Ehlers 1 , C. Sharma 2 , J. Vogel 2 , R.A. Schmitz-Streit 1<br />

1 Institut für Allgemeine Mikrobiologie, Christian-Albrechts Universität Kiel,<br />

Kiel, Germany<br />

2 RNA Biologie, Max-Planck-Institut für Infektionsbiologie Berlin, Berlin,<br />

Germany<br />

Small non-coding RNAs (sRNAs) are an emerging field of research as their<br />

global impact in regulatory processes becomes more and more obvious. Noncoding<br />

RNAs have been identified in all three domains of life. In Eukarya and<br />

Bacteria functions have been assigned for many sRNAs. In Bacteria, most of<br />

the characterized sRNAs regulate several environmental stress-responses, such<br />

as oxygen-, envelope- or starvation-stress on the posttranscriptional-level. To<br />

get a deeper insight into potential regulatory roles of sRNAs in Archaea, we<br />

chose the methanogenic archaeon Methanosarcina mazei strain Gö1 as model<br />

system, which is able to fix molecular nitrogen. It is a perfect candidate as, due<br />

to its high ecological importance in biogenic methane production, many aspects<br />

of the organism’s adaptation to different stress situations are currently un<strong>der</strong><br />

investigation, e.g. limiting carbon sources (Hovey et al. 2005), nitrogen<br />

limitation (Weidenbach et al. 2008) and osmotic stress (Pflüger et al. 2007,<br />

Spanheimer & Müller, 2008).<br />

Here we present genome-wide identification of sRNAs in M. mazei , with the<br />

main focus on those potentially involved in nitrogen metabolism. cDNAs<br />

<strong>der</strong>ived from total-RNA of cells grown with ammonium or molecular nitrogen<br />

as sole nitrogen-source were analysed by massive parallel sequencing, resulting<br />

in 228.000 cDNA sequences in total. Approximately 200 non-coding RNAs<br />

were identified to be expressed from intergenic regions (IGRs) of the 4.1 Mbp<br />

genome of which 70 were further characterized by Northern-Blot analysis.<br />

PU 10<br />

Engineered riboswitches - an alternative means to control<br />

gene expression<br />

B. Suess *1 , J. Weigand 1<br />

1<br />

Institut für Molekulare Biowissenschaften, Goethe Universität, Frankfurt,<br />

Germany<br />

Riboswitches are genetic control elements that regulate gene expression in a<br />

small molecule-dependent way. They act on the mRNA in cis and respond to<br />

metabolites related to genes they control. Riboswitches consist solely of RNA<br />

and sense their ligand in a preformed binding pocket. Upon ligand binding they<br />

perform a conformational switch, resulting in altered gene expression. This<br />

direct RNA-ligand interaction ren<strong>der</strong>s the involvement of trans-acting protein<br />

factors unnessecary.<br />

Engineered riboswitches have been developed which regulate translation<br />

initiation by insertion of aptamer sequences into the 5’ untranslated region of an<br />

mRNA. These switches exploit the fact that upon small molecule binding the<br />

aptamer-ligand-complex interferes with scanning of the ribosome and thus<br />

inhibits translation of the downstream coding regions. The advantage of such<br />

synthetic RNA-regulators is that they can be designed to respond to any ligand,<br />

which is non-toxic and cell-permeable.<br />

Only a very small fraction of the in vitro selected aptamers show regulatory<br />

activity in vivo. Therefore, we built up a simple screening system to identify<br />

regulatory active aptamers, which are able to control translation initiation in<br />

yeast. We used this screen to isolate neomycin-dependent riboswitches from an<br />

in vitro selected pool of neomycin binding sequences. We identified 10<br />

aptamers which are able to regulate gene expression in vivo. From these 10<br />

motives, a consensus sequence emerged with similarities to the ribosomal<br />

decoding site which is the natural target of aminoglycosides. Interestingly,<br />

these aptamers are highly un<strong>der</strong>represented in the original, in vitro selected<br />

pool and show no sequence similarity to the consensus sequence of randomly<br />

picked aptamers.<br />

We characterized the most active neomycin binding riboswitch in detail by<br />

genetic and biochemical analyses and compared it to several non or less active<br />

variants to unravel the determinants important for regulatory activity.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PU 11<br />

Deep sequencing analysis of the primary transcriptome of<br />

the major human pathogen, Helicobacter pylori<br />

C.M. Sharma *1 , S. Hoffmann 2 , F. Darfeuille 3 , J. Reignier 3 , A. Sittka 1 , J.<br />

Hackermüller 4 , P. Stadler 2 , J. Vogel 1<br />

1 Max Planck Institute for Infection Biology, RNA Biology, Berlin, Germany<br />

2 Bioinformatics Group, Department of Computer Science and Interdisciplinary<br />

Center for Bioinformatics, University of Leipzig, Leipzig, Germany<br />

3 INSERM U386, IFR Pathologies Infectieuses et Cancers, Université Victor<br />

Segalen, Bordeaux, Bordeaux, France<br />

4 Fraunhofer Institute for Cell Therapy und Immunology, IZI, Leipzig, Germany<br />

The intense study of Helicobacter pylori, one of the most prevalent human<br />

pathogens, has contributed much to un<strong>der</strong>standing bacterial virulence<br />

mechanisms. The availability of the ~1.7 Mb H. pylori genome sequence has<br />

greatly facilitated these studies, including the discovery of proteins with<br />

important functions in gastric infections. In comparison, much less is known<br />

about the overall transcriptional organization and the noncoding regions of the<br />

H. pylori genome. We have analyzed the H. pylori transcriptome by sequencing<br />

a total of ~3.7 million cDNAs <strong>der</strong>ived from H. pylori grown un<strong>der</strong> standard<br />

laboratory and stress conditions, or in contact with eukaryotic cells. Differential<br />

analysis of primary and processed RNA species facilitated the identification of<br />

~800 transcription start sites of mRNAs across the H. pylori genome. Unlike<br />

many other model pathogens, Helicobacter species lack an Hfq protein<br />

normally required for the action of small noncoding RNA (sRNAs).<br />

Nonetheless, we identified >30 sRNAs expressed from this small genome,<br />

including the long missing ubiquitous 6S RNA and its associated pRNAs. The<br />

results of our analyses will improve the functional annotation of the H. pylori<br />

and related genomes, and the approach used here should facilitate the global<br />

transcriptome analysis of mixed pathogen-host populations<br />

PU 12<br />

In vivo expression and purification of small RNA-protein<br />

complexes<br />

R. Rie<strong>der</strong> *1 , N. Said 1 , R. Hurwitz 2 , J. Deckert 3 , H. Urlaub 3 , J. Vogel 1<br />

1 RNA Biology group, Max Planck Institute for Infection Biology, Berlin,<br />

Germany<br />

2 Protein Purification Facility, Max Planck Institute for Infection Biology,<br />

Berlin, Germany<br />

3 Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical<br />

Chemistry, Goettingen, Germany<br />

Small noncoding RNAs (sRNAs) are an emerging class of post-transcriptional<br />

regulators of bacterial gene expression. Although much has been learned about<br />

the molecular mechanisms of sRNA-mediated regulation, we generally know<br />

little about the nature and number of proteins bacterial sRNAs associate with<br />

throughout their life time as a regulator.<br />

We present an RNA-based affinity chromatographic approach that allows the<br />

purification of in vivo assembled sRNA-protein complexes. We developed a<br />

family of plasmids to express sRNAs with any of three widely used aptamer<br />

sequences (MS2, boxB, eIF4A), and tested how the aptamer tagging impacted<br />

on intracellular accumulation and target regulation of the Salmonella GcvB,<br />

InvR or RybB sRNAs. Preliminary evidence suggests that some of the tagged<br />

sRNAs are still functional un<strong>der</strong> wild-type conditions, e.g., a chromosomal<br />

copy of RybB-MS2 RNA is functional as an envelope stress-induced repressor<br />

of the major porin mRNAs. We further demonstrate that the common sRNAbinding<br />

protein, Hfq, co-purifies with MS2-tagged sRNAs of Salmonella. Our<br />

presented approach may facilitate the isolation of proteins along with in vivo<br />

expressed RNAs in a wide range of bacteria.<br />

PU 13<br />

Multiple effects of the Rcs phosphorelay system and the<br />

small RprA RNA in the δ S /CsgD/curli fimbriae control<br />

cascade in Escherichia coli<br />

S. Busse *1 , M. Pruteanu 1 , F. Mika 1 , R. Hengge 1<br />

1 Institut für Biologie – Mikrobiologie, Freie Universität Berlin, Berlin,<br />

Germany<br />

187<br />

Escherichia coli is able to switch between motile-planktonic and sessilemulticellular<br />

lifestyles. For the latter, which leads to biofilm formation,<br />

adhesive curli fimbriae play an essential role. Curli expression requires the<br />

RpoS subunit of RNA polymerase, the transcription factors MlrA and CsgD<br />

and the diguanylate cyclase YdaM, which operate in a complex feedforward<br />

cascade. Here we investigated the function of the Rcs – phosphorelay system in

188<br />

the control of curli fimbriae. The response regulator RcsB activates a large set<br />

of genes including capsule formation genes and the small RNA RprA, which in<br />

turn can activate RpoS expression by stabilizing rpoS mRNA and stimulating<br />

its translation [1]. Moreover, RcsB down-regulates curli formation [2, 3] and an<br />

RcsB binding site has been found in the promoter region of the csgD regulatory<br />

gene [2].<br />

In addition, we now show that the small RNA RprA is mainly responsible for<br />

the negative effect of the Rcs system on curli expression. In that respect, curli<br />

regulation is the rule rather than the exception, as our microarray analyses<br />

revealed that RcsB control of most genes is mediated by RprA (this may often<br />

be in a feed forward loop regulatory pattern, in which RcsB both directy and<br />

indirectly, i.e. via RprA, controls a target gene). Genetic analysis revealed that<br />

within the curli control cascade, targets for RprA regulation are YdaM and<br />

CsgD. Using bioinformatical tools, two possible binding sites for RprA were<br />

predicted in the 5` UTR of the csgDmRNA. Introducing deletions and point<br />

mutations in these regions showed both of them to be involved in the posttranscriptional<br />

control of CsgD expression.<br />

[1] Majdalani, N. & Gottesman, S. (2005). Annu. Rev. Microbiol. 599, 379-405.<br />

[2] Vianney, A., Jubelin, G., Renault, S., Dorel, C., Lejeune, P. & Lazzaroni, J.<br />

C. (2005). Microbiology 151, 2487-2497.<br />

[3] Tschowri, N., Busse, S. & Hengge, R. (2008). Genes Dev. (in revision).<br />

PU 14<br />

The conserved small regulatory RNA, SraH, controls<br />

expression of serine uptake genes in Salmonella<br />

typhimurium<br />

N. Said *1 , K. Papenfort 1 , T. Welsink 1 , S. Lucchini 2 , J. Hinton 2 , J. Vogel 1<br />

1 Max Planck Institute for Infection Biology, Berlin, Germany<br />

2 Institute of Food Research, Norwich, United Kingdom<br />

Since 2001 several screens have aimed at the identification of small regulatory<br />

RNAs (sRNAs) in enterobacterial species and especially in the model organism<br />

Escherichia coli. In this respect various methods have been used covering in<br />

silico, biochemical and genetic approaches. A survey of this analysis has<br />

revealed more close to 100 of these regulatory factors in E.coli [1], and many<br />

sRNA have been identified by more than one method. SraH (a.k.a. RyhA) is<br />

one such sRNA identified in parallel by genomic searches and microarray<br />

based detection of small transcripts [2,3]. Subsequent searches for conservation<br />

of SraH in other enterobacterial revealed its prominent conservation in<br />

pathogenic and non-pathogenic bacteria [1].<br />

To evaluate the role of this sRNA in the model pathogen Salmonella<br />

typhimurium we cloned SraH un<strong>der</strong> the control of the tighly controlled pBAD<br />

promotor. Pulse expression and whole genome microarrays were used to<br />

identify genes that were post-transcriptionally controlled by SraH. This analysis<br />

clearly revealed downregulation of the serine uptake gene sdaC and its cistronic<br />

partner gene sdaB by SraH.<br />

Validation of these results using quantitative real-time PCR as well as genetic<br />

approaches proved post-transcriptional regulation of sdaC, probably in an Hfq<br />

dependent manner targeting the primary coding of sdaC by a GU-rich element<br />

in the conserved 3’end of the SraH molecule.<br />

References:<br />

[1] Hershberg R., Altuvia S., Margalit H. (2003) A survey of small RNAencoding<br />

genes in Escherichia coli. NAR 31(7):1813-20<br />

[2] Argaman L, Hershberg R, Vogel J, Bejerano G, Wagner EG, Margalit H,<br />

Altuvia S. (2001) Novel small RNA-encoding genes in the intergenic regions of<br />

Escherichia coli. Curr Biol. 11(12):941-50.<br />

[3] Wassarman KM, Repoila F, Rosenow C, Storz G, Gottesman S. (2001)<br />

Identification of novel small RNAs using comparative genomics and<br />

microarrays. Genes Dev. 15(13):1637-51.<br />

PV 01<br />

Systems biology of Clostridium acetobutylicum – regulation<br />

of solventogenesis in an important butanol producer<br />

S. Noack *1 , H. Bahl 2 , C. Döring 3 , A. Ehrenreich 4 , R.J. Fischer 2 , P. Götz 5 , S.<br />

Haus 6 , H. Janssen 2 , D. Krause 4 , T. Millat 6 , M. Reuss 5 , O. Wolkenhauer 6 , P.<br />

Dürre 1<br />

1 Institute of Microbiology and Biotechnology, University of Ulm, Ulm,<br />

Germany<br />

2 Institute of Biological Sciences, Division of Microbiology, University of<br />

Rostock, Rostock, Germany<br />

3 Institute of Microbiology and Genetics, Department of Genomic and Applied<br />

Microbiology, University of Göttingen, Göttingen, Germany<br />

4 Department of Microbiology, Technical University of Munich, Munich,<br />

Germany<br />

5<br />

Institute of Biochemical Engineering, University of Stuttgart, Stuttgart,<br />

Germany<br />

6<br />

Department of Computer Science, University of Rostock, Rostock, Germany<br />

Microbially produced biofuels gain increasing importance due to limited<br />

resources of fossil fuels and CO2 increase by their combustion. Butanol proved<br />

to be a superior biofuel than ethanol, because of its higher energy content,<br />

lower water absorption, better blending ability, and use of existing<br />

infrastructure and conventional engines. An important microbial butanol<br />

producer is Clostridium acetobutylicum.<br />

The clostridial acetone-butanol production starts with the typical butyric acid<br />

pathway and then switches to production of acetone and butanol. Although the<br />

biochemical pathways leading to solvent formation in C. acetobutylicum are<br />

known, the regulation of these processes is only poorly un<strong>der</strong>stood. Objective<br />

of this work is to gain insight into the key regulatory events that occur during<br />

the transition between acidogenic, vegetative growth and the onset of solvent<br />

production and sporulation. For this purpose, continuous culture experiments<br />

with C. acetobutylicum were performed at pH 6,5 and pH 4,5. At pH 6,5 acids<br />

were formed, at pH 4,5 mainly solvents are produced. By transcriptome<br />

analyses from cells of both steady states, genes were found, which are up- and<br />

downregulated after their switch from acidogenic to solventogenic growth.<br />

Together with data concerning metabolism, significance of redox state,<br />

glycosylation, and reaction to stress obtained by a consortium of several<br />

European scientists (COSMIC), these results will be used to mathematically<br />

model the various interactions at the cellular level.<br />

PV 02<br />

Simulation-based Aerobic High-Cell Density Cultivation of<br />

Rhodospirillum rubrum<br />

L. Zeiger *1 , H. Grammel 1<br />

1 Redox Phenomena in Photosynthetic Bacteria, Max-Planck-Institut für<br />

Dynamik komplexer technischer Systeme, Magdeburg, Germany<br />

The biotechnological potential of facultative photosynthetic bacteria, such as<br />

Rhodospirillum rubrum has been shown in various fields. However, generally<br />

these processes are dependent on the input of light which so far complicates<br />

large-scale applications. The recent development of a new cultivation process,<br />

which allows the maximal expression of photosynthetic genes un<strong>der</strong> semiaerobic<br />

dark conditions in common bioreactors, offers a new possibility for<br />

exploring this potential. In the present study we establish a second crucial<br />

precondition - the maximal obtainable cell number in a bioreactor -, in or<strong>der</strong> to<br />

estimate the volumetric yields that can be expected in industrial applications. At<br />

this initial stage, we focus on aerobic growth since the highest growth rates are<br />

achieved un<strong>der</strong> this condition.<br />

High-cell density cultures (HCD) of R. rubrum could be used for the industrial<br />

production of hydrogen, polyhydroxyalkanoates, carotenoids and quinones, as a<br />

recombinant host for the expression of heterologous membrane proteins, or for<br />

the production of bacteriochlorophyll for photodynamic tumor therapy. Process<br />

control strategies were optimized by applying an unstructured model, which is<br />

based on mixed-substrate kinetics. To avoid the accumulation of inhibiting<br />

substrates during the fed-batch cultivation, an exponential feeding strategy,<br />

combined with pH-stat was applied. The maximum cell density obtained with<br />

this strategy was ~ 59 g dry weight per liter. A biochemical analysis of highcell<br />

density cultures showed interesting differences to batch-cultures, such as a<br />

large accumulation of protoporphyrin IX.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PV 03<br />

New facts of an old paradigm - The Escherichia coli lac<br />

operon<br />

A. Medger *1 , K. Bettenbrock 1<br />

1 Systems Biological Analysis of Local and Global Regulations, MPI for<br />

Dynamics of Complex Technical Systems, Magdeburg, Germany<br />

The regulation process of the Escherichia coli lac operon is a well established<br />

fact. Negative control via the lac repressor LacI and positive control via cAMP-<br />

CRP adjust the strength of transcription. Inducers like allolactose, IPTG or<br />

TMG inhibit LacI operator binding and therefore enhance lac operon<br />

expression. But most studies dealing with these inducers are based on averaged<br />

values of a whole population. This becomes improper when the inducer<br />

concentration is low. A homogenous population can split up in two<br />

subpopulations where cells either express the lac operon or not. Hence,<br />

investigation on single cell level is necessary. For this purpose we used GFP as<br />

a reporter gene. We fused the lac promoter to GFPmut3.1 in pAH162 and<br />

integrated the plasmid in the genome of E. coli LJ110. Single cell analysis was<br />

carried out with the help of fluorescence microscopy and flow cytometry. Here<br />

we present induction experiments with the non-metabolizable inducers TMG<br />

and IPTG.<br />

PV 04<br />

Changes in metabolism of Escherichia coli MG1655<br />

depending on oxygen<br />

S. Stagge *1 , S. Steinsiek 1 , S. Frixel 1 , K. Bettenbrock 1<br />

1 Systems Biological Analysis of Local and Global Regulations, MPI for<br />

Dynamics of Complex Technical Systems, Magdeburg, Germany<br />

As a facultative anaerobic bacterium Escherichia coli has several strategies to<br />

grow un<strong>der</strong> conditions with or without oxygen. The metabolism is adapted to<br />

the oxygen concentration un<strong>der</strong> the control of global regulatory proteins like<br />

FNR and ArcA/B. While FNR directly senses oxygen, ArcA is likely to<br />

respond to the intra- and extracellular redox state and oxygen. The redox state<br />

of a cell is influenced by the availability of oxygen but also by metabolic<br />

pathways. This means adaption of the metabolism through ArcA/B and FNR is<br />

depending on external as well as internal conditions. It is our aim to<br />

quantitatively un<strong>der</strong>stand the regulatory and metabolic networks and their<br />

changes mediated through ArcA and FNR. By using an integrative approach of<br />

experimental analysis for the characterization of E. coli MG1655 and for<br />

isogenic knock-out mutants we are measuring the mediated changes via<br />

detection of intracellular metabolite concentrations of glycolytic compounds,<br />

TCA-cycle molecules and redox ratio (NAD(P)H2) applying anion-exchange<br />

chromatography and enzymatic analysis. In first experiments an sdhC knockout<br />

mutant obtained an inverse and more oxidized redox ratio along with lower<br />

overall concentrations of adenine nucleotides compared to the wildtype. This<br />

indicates an expected reduced flux through the TCA-cycle along with lesser<br />

ability to generate NAD(P)H2. Furthermore we expect the energy charge to be<br />

reduced in case of the mutant due to the changed flux, when comparing the<br />

wildtype and the mutant un<strong>der</strong> oxygen concentrations ranging from 0% to<br />

100% aerobiosis.<br />

PV 05<br />

Changes in the Proteome of Pseudomonas puitda upon<br />

overexpression of a heterologous lipase<br />

H. Funken *1 , K.E. Jäger 1 , S. Wilhelm 1 , F. Rosenau 1<br />

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University<br />

Duesseldorf, Juelich, Germany<br />

Protein expression is essential for the industrial production of biocatalysts and<br />

pharmaceutically relevant proteins. Here, we report the results of a study we<br />

have carried out, in the european „SYSMO“framework, to un<strong>der</strong>stand the<br />

molecular consequences of expression-induced stress in the biotechnologically<br />

relevant host strain Pseudomonas putida KT2440. As an example for<br />

overexpression the lipase operon lipAH from Pseudomonas aeruginosa.<br />

However, heterologous protein production always poses physiological stress on<br />

the bacterial host cells which can exert a significant influence on the yield of<br />

functional protein.<br />

The stress response of P. putida was investigated by determination of growth,<br />

lipase protein expression and activity, and analysis of the whole cell proteome.<br />

Analysis of 2D gels using the „delta 2D“-software (DECODON, Germany)<br />

revealed major differences of spot intensities in both strains upon expression of<br />

the lipase operon. Proteins were grouped into those which were either down- or<br />

up- regulated in the lipase expression strain and those which were exclusively<br />

present either in the expression strain or in the control strain.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PV 06<br />

The RING-finger peroxins function as E3-ligases of the<br />

peroxisomal ubiquitination machinery<br />

F. El Magraoui 1 , H.W. Platta 1 , B.E. Bäumer 1 , D. Schlee 1 , J. Baumann 1 , W.<br />

Girzalsky 1 , R. Erdmann *1<br />

1 Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany<br />

The Peroxisomal Targeting Signal 1 (PTS1)-dependent peroxisomal matrix<br />

protein import is facilitated by the receptor protein Pex5p and can be divided<br />

into cargo recognition in the cytosol, membrane docking of the cargo-receptor<br />

complex, cargo release into the peroxisomal lumen and finally the recycling of<br />

the receptor back to the cytosol. The final step is controlled by the<br />

ubiquitination status of Pex5p. While polyubiquitinated Pex5p is degraded by<br />

the 26S proteasome, monoubiquitinated Pex5p is deubiquitinated and enters a<br />

new round of the receptor cycle. Recently, the E2-enzymes involved in Pex5p<br />

ubiquitination were identified as Ubc4p and Pex4p (Ubc10p), whereas the<br />

identity of the E3-ligases remained unknown. Here we report on the<br />

identification of the E3-ligases of the peroxisomal ubiquitination machinery. It<br />

is demonstrated that each of the three RING-peroxins Pex2p, Pex10p and<br />

Pex12p exhibit ubiquitin-protein isopeptide ligase activity. The three proteins<br />

differ in their specificity. Our results show that Pex2p mediates the Ubc4pdependent<br />

polyubiquitination whereas Pex12p facilitates the Pex4p-dependent<br />

monoubiquitination of Pex5p. The identification of the E3-enzymes completes<br />

the peroxisomal ubiquitination cascades acting on Pex5p.<br />

PV 07<br />

Characterization of the recombinant AAA-ATPases Pex1p<br />

and Pex6p.<br />

M.O. Debelyy 1 , D. Saffian 1 , S. Thoms 2 , V. Pawlak 1 , H.W. Platta 1 , R.<br />

Erdmann *1<br />

1<br />

Systembiochemie, Ruhr-Universität Bochum, Bochum, Germany<br />

2<br />

Department for Paediatrics and Neuropaediatrics, Georg-August-University of<br />

Göttingen, Göttingen, Germany<br />

The import of matrix proteins into the peroxisomal lumen is an ATP-dependent<br />

process. Cycling receptors recognize newly synthezised matrix proteins in the<br />

cytosol, ferry their cargo proteins to the peroxisomal membrane, release them<br />

into the lumen of the peroxisome and finally recycle back into the cytosol for<br />

further rounds of import. Recent studies suggest that this last step is ATPdependent<br />

and is carried out by the AAA-ATPases Pex1p and Pex6p, which are<br />

supposed to extract the receptors from the membrane. Thus, the AAA proteins<br />

Pex1p and Pex6p are motor proteins taking a prime role in peroxisome<br />

biogenesis. Until now, the stuides on these AAA peroxins has been hampered<br />

due to the lack of recombinantly purified proteins as they can not be<br />

overexpressed and purified from E. coli or yeasts. In or<strong>der</strong> to gain more<br />

detailed insight into the molecular mechanism of these ATPases, we have<br />

succeeded to express and purify Pex1p and Pex6p from Saccharomyces<br />

cerevisiae in insect cells using a baculvirus system. Immunoprecipitation<br />

studies of the recombinant proteins demonstrate that they form a heteromeric<br />

complex, which assembles in an ATP-dependent manner. We analyze the<br />

recombinant proteins by size exclusion chromatography and suggest that they<br />

can form high molecular weight complexes. These initial studies demonstrate<br />

that the recombinant proteins purified from insect cells represent a valuable tool<br />

to study the molecular architecture and function of the AAA peroxin complex.<br />

PV 08<br />

Towards definition of the interactome of Pseudomonas<br />

putida KT2440<br />

T. Dammeyer *1 , T. Chernikova 1 , S. Arias-Rivas 2 , T. Nechitaylo 1 , P.N.<br />

Golyshin 1 , K.N. Timmis 1,2<br />

1 Environmental Microbiology Laboratory, Helmholtz Centre for Infection<br />

Research, Braunschweig, Germany<br />

2 Institute of Microbiology, Technical University Braunschweig, Braunschweig,<br />

Germany<br />

189<br />

Proteins are the principal agents mediating most cellular functions. In general,<br />

they do so not as individual entities but rather as higher or<strong>der</strong> multi-protein<br />

complexes interacting in a variety of ways. Knowledge of these interactions is<br />

essential not only for un<strong>der</strong>standing the function of a cellular system, but also<br />

for optimization of its performance in biotechnological processes.<br />

Pseudomonas putida KT2440 is one of the best studied species of metabolically<br />

versatile and ubiquitous genus Pseudomonas and exhibits wide<br />

biotechnological potential due to its stress resistance, amenability for genetic<br />

manipulation and suitability as a heterologous expression host. In our study we<br />

aim to identify interaction partners of proteins that are either directly process<br />

relevant or of those that are regulated in response to process relevant stresses.

190<br />

We are currently establishing systems for genome-integrated, as well as broadhost-range<br />

plasmid based, affinity-tagging of bait open reading frames for copurification/precipitation<br />

of in vivo formed protein complexes. Such proteins<br />

can be identified by MS and and their binary interactions analyzed using a<br />

bacterial two-hybrid system. Knowledge of these interactions will help to<br />

uncover new metabolic interconnections and identify new points of intervention<br />

for the optimization of the cell factory.<br />

PW 01<br />

Investigating the role of hypothetical ABC transporters of<br />

Staphylococcus aureus in antimicrobial resistance<br />

A. Berscheid *1 , P. Sass 1 , G. Bierbaum 1<br />

1 Institute of Medical Microbiology, Immunology and Parasitology (IMMIP),<br />

University of Bonn, Bonn, Germany<br />

ATP-binding cassette transporters represent a large superfamily of proteins that<br />

can be found in all organisms, from bacteria to humans. Their main function is<br />

to pump a very diverse set of substances across biological membranes, while<br />

using the energy from ATP hydrolysis, however they can also be involved in<br />

nontransport cellular processes [1]. Since ABC transporters appear to support<br />

antimicrobial resistance by the mechanism of active drug efflux, we attempted<br />

to investigate the role of several ABC transporters of S. aureus, which either<br />

were upregulated upon antibiotic treatment or were differently expressed<br />

among strains with varying antibiotic susceptibility characteristics.<br />

Gene expression profiling of the susceptible strain S. aureus SG511 versus the<br />

more resistant VISA/MRSA strain SA137/93A revealed the divergent<br />

regulation of the hypothetical ABC transporters SA0420/SA0421 and<br />

SA2243/SA2242 [2], which were more highly expressed in strain SA137/93A.<br />

Furthermore, the hypothetical ABC transporter ATPase gene SA0192 was<br />

upregulated upon mersacidin treatment in several S. aureus strains [3].<br />

To analyze the putative function of these ABC transporters, their ATPase gene<br />

was deleted by allelic exchange mutagenesis in several S. aureus strains with<br />

different genomic backgrounds. Subsequently, the susceptibilities of the mutant<br />

strains to antimicrobial peptides and other antibiotics were tested. Here, our<br />

first observations revealed an increased susceptibility of the mutant strain S.<br />

aureus SA137/93G ΔSA2243 to nisin and vancomycin compared to the wild<br />

type strain. Further investigations on the deletion mutants of these ABC<br />

transporters may yield deeper insights into their roles in the lifestyle of S.<br />

aureus.<br />

[1] Davidson et al. (2008), Microbiol. Mol. Biol. Rev. 72(2): 317-364.<br />

[2] Sass and Bierbaum (2008), IJMM, accepted for publication.<br />

[3] Sass et al. (2008), BMC microbiology 8:186.<br />

PW 02<br />

Identification of the DASS Family dicarboxylate uptake<br />

system DccT of Corynebacterium glutamicum<br />

J.W. Youn *1 , E. Jolkver 2 , R. Krämer 2 , K. Marin 2 , V.F. Wendisch 1<br />

1 Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms<br />

University Muenster, Muenster, Germany<br />

2 Institute of Biochemistry, Cologne University, Cologne, Germany<br />

Many bacteria, e.g. Escherichia coli or Bacillus subtilis, can utilze C4carboxlyates,<br />

such as succinate, fumarate or L-malate, as carbon and energy<br />

sources. Different kinds of C4-carboxlyates transport systems, e.g. DctA, Dcu,<br />

MaeN or TtdT, have been intensively studied [2-3, 5], because of their<br />

importance for recognition of changed environmental conditions and transport<br />

of C4-carboxlyates [2].<br />

However, C. glutamicum cannot use tricarboxylic acid cycle intermediates such<br />

as succinate, fumarate or L-malate as sole carbon source. Citrate is the only<br />

described tricarboxylic acid cycle intermediate, which supports growth of C.<br />

glutamicum [4]. We isolated spontaneous mutants, which gained the ability to<br />

utilize succinate, fumarate and L-malate. DNA microarray analyses [6] showed<br />

an increased expression of cg0277, which subsequently was named dccT, in the<br />

mutants. Transcriptional fusion analysis revealed that a point mutation in the<br />

promoter region of dccT is responsible for higher expression of dccT. The<br />

overexpression of dccT was sufficient to enable C. glutamicum to grow on<br />

succinate, fumarate and L-malate as sole carbon sources. Biochemical analyses<br />

showed that DccT, which is a member of the divalent anion/Na + symporter<br />

family (DASS) [1], catalyzes the effective uptake of the dicarboxylates<br />

succinate, fumarate, L-malate, and likely also of oxaloacetate in a sodiumdependent<br />

manner.<br />

[1] Hall JA & Pajor AM (2007) J Bacteriol 189: 880-885.<br />

[2] Janausch IG, Zientz E, Tran QH, Kroger A & Unden G (2002) Biochim<br />

Biophys Acta 1553:39-56.<br />

[3] Kim OB & Unden G (2007) J Bacteriol189:1597-603.<br />

[4] Polen T, Schluesener D, Poetsch A, Bott M & Wendisch VF (2007) FEMS<br />

Microbiol Lett 273:109-119.<br />

[5] Tanaka K, Kobayashi K & Ogasawara N (2003) Microbiology 149:2317-<br />

2329.<br />

[6] Wendisch VF (2003) J Biotechnol 104:273-285.<br />

PW 03<br />

Function of CzcI in heavy metal resistance of Cupriavidus<br />

metallidurans CH34<br />

M. Herzberg *1 , J. Scherer 1 , C. Große 1 , D.H. Nies 1<br />

1 Inst. f. Biologie/Mikrobiologie, Universität Halle, Halle, Germany<br />

The β-proteobacterium Cupriavidus metallidurans CH34 harbors a great variety<br />

of heavy metal resistance determinants. The czcNICBADRSE-czcJ-czcP<br />

(cobalt, zinc, cadmium) determinant on plasmid pMOL30 is one of the most<br />

sophisticated and efficient heavy-metal resistance systems known. Its main<br />

product is the cation-proton-antiporter complex CzcCBA is essential for<br />

survival of C. metallidurans CH34 at high cobalt, zinc or cadmium<br />

concentrations. The czcICBA core determinant on plasmid pMOL30 arose by<br />

duplication of a similar determinant on chromosome 2 and the addition of<br />

further genes that were probably acquired by horizontal gene transfer. The<br />

czcICBA determinant has orthologs in related bacteria and may have existed in<br />

the Cupriavidus universal ancestor. CzcI seems to be a periplasmic metal<br />

binding protein.<br />

There is accumulating evidence that the CzcCBA protein complex transports<br />

the metals Co(II), Zn(II) and Cd(II) not from the cytoplasm but from the<br />

periplasm to the extracellular space. These cations must have been previously<br />

exported from the cytoplasm to the periplasm by inner membrane efflux<br />

systems like CzcD, DmeF (both proteins of the cation diffusion protein family,<br />

TC 2.A.4) or ZntA, CadA, PbrA, CzcP (P-type ATPases, TC 3.A.3). CzcI may<br />

play a role in storing periplasmic metal cations and delivering them to the<br />

CzcCBA efflux complex for export to the outside. The contribution of czcI to<br />

metal-resistance was investigated by using mutant strains with deletions in both<br />

czcI genes. These were characterized and complemented with czcCBA±czcI<br />

determinants from C. metallidurans and C. eutrophus strains H16 and JMP134.<br />

PW 04<br />

Analysis of the magnetosome directed iron transport in<br />

Magnetospirillum gryphiswaldense<br />

G. Poxleitner *1 , R. Uebe 1 , K. Junge 1 , D. Schüler 1<br />

1 Department Biologie I / Bereich Mikrobiologie, Ludwig-Maximilians-<br />

Universität, München, Germany<br />

The biomineralization of magnetite (Fe3O4) crystals is a complex mechanism,<br />

involving the uptake, accumulation and precipitation of large amounts of iron.<br />

Previous proteomic and genetic analysis of magnetospirilla revealed several<br />

candiate magnetosome-directed iron transporters: the CDF3 proteins<br />

MamB/MamM, which were deteced in the magnetosome-membrane, as well as<br />

MagA, which supposedly exhibited iron transport activity in vesicles generated<br />

from Escherichia coli.<br />

Isogenic deletion mutants of mamB or mamM showed a nonmagnetic<br />

phenotype and therefore indicate an essential role in magnetite<br />

biomineralization. Trans-complementation was succesful only for mamB,<br />

whereas in the mamM mutant the MamB protein encoded 6 kB downstream of<br />

mamM was no longer detectable.<br />

However, the expression of other proteins encoded further downstream of<br />

mamM within the mamAB operon was unaffected. This disproved the<br />

assumption that the deletion of the mamB gene might have caused polar effects.<br />

Further experiments including double complementation, tandemoverexpression<br />

of both proteins and crosslinking studies are in progress to<br />

reveal whether the stabilization of MamB expression requires the presence of<br />

MamM.<br />

MagA shows > 50% sequence similarity to the membrane domain of Kef-Clike<br />

K + -transporters. An insertion-duplication mutant of magA showed no<br />

significant phenotypic differences to the wildtyp. We are currently constructing<br />

an inframe deletion mutant of magA. Futhermore mCherry fusion proteins of<br />

MamB and MagA will be construced to exhibit their intracellular localisation.<br />

In conclusions, our preliminary results suggest that the magnetosome-directed<br />

iron transport is driven by MamB/MamM, whereas MagA seems to be not<br />

immediately involved in magnetosome formation.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PW 05<br />

Zinc uptake in Cupriavidus metallidurans CH34<br />

A. Kirsten *1 , A. Voigt 1 , J. Scherer 1 , D.H. Nies 1<br />

1 Biology/Molecular Microbiology, Martin Luther University, Halle, Germany<br />

Zinc is an essential cofactor in many enzymes. Cupriavidus metallidurans<br />

strain CH34 is a well-characterized metal-resistant bacterium known for its<br />

content of multiple efflux systems for transition metal cations, however,<br />

information concerning transition metal uptake is scarce. C. metallidurans lacks<br />

the highly efficient high affinity Zn(II)-uptake system ZnuABC, probably due<br />

to its adaption to environments containing high zinc concentrations. So, how<br />

does C. metallidurans take up zinc?<br />

C. metallidurans contains a member of the ZIP (Zrt/Irt-like, TC 2.A.5) protein<br />

family, an ortholog of ZupT from Escherichia coli. ZupTCm seems to transport<br />

Zn(II), Mn(II) and Co(II) ions. In C. metallidurans and in contrast to E. coli,<br />

zupT gene expression was induced by metal chelators like EDTA, TPEN or<br />

DTPA. Additionally, C. metallidurans contains the unusual high number of<br />

four members of the MIT (CorA metal ion transporter, TC 1.A.35) protein<br />

family, which usually transport many different transition metal cations in<br />

addition to the main substrate Mg(II). One of these, CorA1, was induced by<br />

magnesium starvation. Finally, transition metal cations like Zn(II) can enter the<br />

cell in the form of phosphate complexes, which are imported by the low affinity<br />

inorganic phosphate transporter PitA (TC 2.A.20.1.1). To determine the<br />

contribution of the four CorA-like proteins, ZupT and PitA to Zn(II) uptake in<br />

C. metallidurans single and multiple gene deletions were constructed using the<br />

Cre-Lox system. The resulting mutants were characterized in growth and metal<br />

uptake experiments.<br />

PW 06<br />

Time-resolved cw- and pulsed SDSL-EPR studies: Shedding<br />

light on structure and dynamics of a molecular switch<br />

S. Nicklisch *1 , I. Borovykh 2 , H.J. Steinhoff 2 , S. Morbach 1 , R. Kraemer 1<br />

1 Institute of Biochemistry, University of Cologne, Koeln, Germany<br />

2 Departement of Physics, University of Osnabrueck, Osnabrueck, Germany<br />

Upon hyperosmotic stress conditions, BetP, a secondary uptake system from<br />

Corynebacterium glutamicum is known to function as an autonomous transport<br />

unit, i.e. in the absence of any accessory protein. In doing so, it unifies the<br />

features of a transporter, a sensor for its stimulus (internal K + concentration)<br />

and an activity-regulator. Previous studies revealed that changes in the structure<br />

and/or orientation of the cytoplasmically exposed C-terminal domain of the<br />

carrier seem to be critically involved in stimulus sensing and/or signal<br />

transduction. Since the molecular mechanisms related to these processes are<br />

barely un<strong>der</strong>stood to date, we used site-directed spin labeling-electron<br />

paramagnetic resonance (SDSL-EPR) spectroscopy to probe the dynamics<br />

during BetP activation at strategically introduced cysteine residues within the<br />

C-domain. In addition, pulsed EPR techniques were applied to determine<br />

altered spin-spin distances in the non-active and active protein conformation.<br />

Focus was on the structure and structural changes of the C-terminal domain or<br />

adjacent protein and/or lipid domains within a reconstituted BetP trimer.<br />

Our results show that the C-domain of BetP un<strong>der</strong>goes structural changes upon<br />

a hyperosmotic-induced transporter activation referring to a shearing molecular<br />

switch. Thereby we elucidated a physiological response un<strong>der</strong> in vivo<br />

conditions on the molecular level.<br />

PW 07<br />

Function of the TolC like protein HgdD in heterocyst<br />

formation of Anabaena sp. PCC 7120<br />

P. Staron *1 , A. Hahn 2 , E. Schleiff 2 , I. Maldener 1<br />

1 Mikrobiologie / Organismische Interaktionen, EK Universität Tübingen,<br />

Tübingen, Germany<br />

2 Molekulare Zellbiologie <strong>der</strong> Pflanzen, JWG Universität Frankfurt, Frankfurt,<br />

Germany<br />

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 protects its<br />

nitrogenase from oxygen in differentiated cells called heterocysts. During<br />

morphological differentiation heterocysts form an extracellular glycolipid layer<br />

(HGL) as an O2 diffusion barrier. A mutant defective in alr2887, encoding a<br />

trimeric pore-forming outer membrane-barrel protein of the TolC family, is not<br />

able to grow on N2. While electron micrographs clearly showed the absence of<br />

the HGL layer, lipid analysis clarified that the mutant is not impaired in HGL<br />

synthesis. Since mutations in the ABC transporter DevBCA result in a very<br />

similar phenotype, Alr2887 could be the outer membrane component of an<br />

exporter for glycolipid moieties or enzymes involved in layer assembly. Like<br />

devBCA its expression depends on master regulators of heterocyst<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

differentiation. So, Alr2887 has been designated HgdD (heterocyst glycolipid<br />

deposition protein D). Consi<strong>der</strong>ing its importance for diazotrophic growth we<br />

aim to explore the exact role in HGL-formation. Excreted substrates will be<br />

revealed via comparative secretome analysis. Interaction partners will be<br />

identified in in vivo interaction studies and further analyzed in vitro. Up till now<br />

nothing is known about the formation of the HGL layer. With this approach we<br />

hope to learn about TolC´s function in Anabaena and cell wall formation in<br />

gram-negative bacteria in general.<br />

PW 09<br />

Characterisation of putative D-serine-Transporters in<br />

Staphylococcus saprophyticus<br />

L. Marlinghaus *1 , M. Korte 1 , S.G. Gatermann 1 , T. Sakinc 1<br />

1 Institut für Hygiene und Medizinische Mikrobiologie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

Staphylococcus saprophyticus is an important cause of urinary tract infections<br />

in young women. The amino acid D-serine occurs in relatively high<br />

concentrations in human urine and has a bacteriostatic or toxic effect on many<br />

non-uropathogenic microorganisms. The cytoplasmatic enzyme D-Serine-<br />

Deaminase DsdA, which is found in many uropathogens, enables S.<br />

saprophyticus to cleave D-serine into pyruvate and ammonia. This is probably a<br />

factor that makes it possible to colonize the urinary tract. In contrast to E. coli<br />

the dsd-operon does not encode a specific D-serine-transporter, but there are<br />

three genes in the genome of S. saprophyticus that encode for putative Dserine-transporters.<br />

It is therefore our approach to create single-, double- and<br />

triple-knock-out strains of these genes to characterise the D-serine-transport of<br />

S. saprophyticus via a radioactive labelled D-serine uptake assay. The results<br />

will presumably offer us more insights in to the physiology of virulence of S.<br />

saprophyticus.<br />

Because selective markers for S. saprophyticus are limited, we searched in<br />

clinical isolates for additional resistance determinants. We found a tetracycline<br />

resistant S. saprophyticus strain and isolated and sequenced a plasmid, which<br />

belongs to the pT181-family that is usually found in Staphylococcus aureus and<br />

S. epi<strong>der</strong>midis. It contains a gene encoding for a tetracycline effluxpump, which<br />

could be used as a selective marker for the generation of mutant-strains.<br />

PW 10<br />

Carboxylate transporters in Corynebacterium glutamicum.<br />

E. Jolkver *1 , J.W. Youn 2 , V.F. Wendisch 2 , R. Krämer 1 , K. Marin 1<br />

1 Institut für Biochemie, Universität zu Köln, Köln, Germany<br />

2 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

The apathogenic soil bacterium C. glutamicum is a widely distributed organism,<br />

which is able to grow on many different carbohydrates, alcohols, amino and<br />

carboxylic acids as single or combined sources of carbon and energy. Due to its<br />

ability to produce bulk amounts of amino and carboxylic acids it has gained<br />

high importance for the biotechnological production of L-glutamate and Llysine.<br />

Since the plasma membrane is impermeable for most substrates,<br />

transport processes are vital for the ability of both uptake and excretion of<br />

carboxylates. Several structurally different monocarboxylates are imported via<br />

MctC [1], whereas the import of dicarboxylates requires a point mutation in the<br />

promoter region of the DccT encoding gene [2]. DccT is a secondary active,<br />

Na + -dependent transporter of the DASS class with high similarity to the<br />

dicarboxylate transporter SdcS form S. aureus and the mammalian NaDC<br />

transporters. It mediates the uptake of succinate, fumarate, and malate at<br />

aerobic conditions and its specificity for oxaloacetate was demonstrated.<br />

During microaerobic incubation, C. glutamicum excretes lactate, succinate and<br />

acetate [3]. Deletion of the gene sucE encoding a putative transporter resulted<br />

in intracellular succinate accumulation, which points to a function of SucE as a<br />

putative succinate exporter. Moreover, pool sizes of lactate and acetate were<br />

affected, demonstrating the impact of a carefully balanced succinate pool.<br />

[1] Jolkver et al. (2008). J Bacteriol.<br />

[2] Youn et al. (2008). J Bacteriol.<br />

[3] Inui et al. (2004). J. Mol. Microbiol. Biotechnol.<br />

191

192<br />

PW 11<br />

ECF Transporters, A Novel Class Of Modular Vitamin-<br />

Uptake Systems In Prokaryotes<br />

O. Neubauer *1 , A. Alfandega 1 , P. Hebbeln 1 , D.J. Slotboom 2 , T. Eitinger 1<br />

1<br />

Institut für Mikrobiologie, Humboldt- Universität zu Berlin, Berlin, Germany<br />

2<br />

Department of Biochemistry, University of Groningen, Groningen,<br />

Netherlands<br />

ECF ("energy-coupling factor") transporters were recently identified, are<br />

abundant among prokaryotes and mediate the uptake of vitamins, transitionmetal<br />

ions and the substrates of salvage pathways [1, 2]. They are composed of<br />

highly diverse transmembrane substrate-capture proteins ("S"-components) that<br />

interact with a dedicated (groupI) or a shared (groupII) energizing module<br />

consisting of a conserved transmembrane protein ("T"-component) and pairs of<br />

ABC-ATPase domains ("A"-components). BioMNY is a groupI ECF-type<br />

transporter that catalyzes ATP-dependent high-affinity biotin uptake. In the<br />

absence of BioMN, the solitary S-unit BioY acts as a secondary active highcapacity<br />

biotin transporter [2]. As a prerequisite for biophysical analysis,<br />

protocols for overproduction and purification of BioY, BioMN (AT-module)<br />

and BioMNY (holotransporter) complexes were developed yielding the<br />

membrane proteins in milligram amounts. BioY was characterized by static<br />

light scattering as a monomer in detergent solution. The majority of groupII<br />

ECF transporters (> 460 cases) is distributed among Gram-positive bacteria<br />

including many human pathogens with restricted biosynthetic capacities. In<br />

these organisms up to 12 S-components are predicted to compete for the same<br />

AT-module. Shared use of the AT-module was shown experimentally for<br />

lactobacterial folate, pantothenate and thiamine transporters [1]. Questions of<br />

how the unrelated S-components specifically interact with the copies of a single<br />

AT-module to give transporters for defined vitamins are currently <strong>bei</strong>ng<br />

addressed.<br />

[1] Rodionov D.A. et al. (<strong>2009</strong>) J. Bacteriol. 191:42-51<br />

[2] Hebbeln P. et al. (2007) Proc. Natl. Acad. Sci. USA 104:2909-2914<br />

PW 12<br />

A secretin takes shape: Structural and functional analyses<br />

of a PilQ-comprising DNA translocator complex of Thermus<br />

thermophilus HB27<br />

J. Burkhardt *1 , B. Averhoff 1<br />

1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences,<br />

Goethe University Frankfurt/Main, Frankfurt/Main, Germany<br />

Thermus thermophilus HB27 is known for its extremely high competence for<br />

natural transformation and its ability to take up DNA from members of the<br />

archaea, bacteria and eukarya [1]. DNA binding and transport across the cell<br />

wall is mediated by a macromolecular transport machinery that involves 16<br />

distinct proteins such as pilin-like proteins, a secretin-like usher of the DNA<br />

translocator in the outer membrane as well as integral inner membrane proteins<br />

[2]. DNA-binding and uptake studies unravelled the distinct roles of individual<br />

proteins in the DNA translocation process. The secretin-like outer membrane<br />

protein PilQ was found to be essential for DNA binding on the cell surface [3].<br />

Purification of PilQ led to the identification of highly stable PilQ multimers<br />

resistant to 10% SDS and high temperatures of up to 100°C. Electron<br />

microscopical analyses of purified PilQ complexes revealed ring-like structures<br />

with a diameter of ~14 nm and a pore size of ~6-7 nm. These structures could<br />

serve as channels guiding the DNA translocator through the outer membrane.<br />

The function and the quaternary structure of the PilQ multimers will be<br />

discussed.<br />

[1] Schwarzenlan<strong>der</strong> C. and Averhoff B. (2006) FEBS J. 18: 4210-4218<br />

[2] Averhoff B. (<strong>2009</strong>) FEMS Microbiol. Rev., in press<br />

[3] Schwarzenlan<strong>der</strong> C., Haase W. and Averhoff B. (2008) Environ. Microbiol.,<br />

in press<br />

PW 13<br />

The K + transport system KtrAB of Vibrio alginolyticus:<br />

Transport properties, overproduction, purification of the<br />

complex, and reconstitution<br />

I. Hänelt *1 , M. vor <strong>der</strong> Brüggen 1 , M. Döbber 2 , D. Wunnick 2 , L. Sun<strong>der</strong>mann 1 , S.<br />

Löchte 1 , G. Schuurman-Wolters 3 , B. Poolman 3 , H.J. Steinhoff 2 , E.P. Bakker 1<br />

1 Mikrobiologie, Universität Osnabrück, Osnabrück, Germany<br />

2 Physik, Universität Osnabrück, Osnabrück, Germany<br />

3 Biochemistry, University of Groningen, Groningen, Netherlands<br />

In prokaryotes K + uptake is essential for vital processes, including turgor<br />

pressure regulation. Nakamura et al. described in 1998 KtrAB from V.<br />

alginolyticus as a new type of K + uptake system. Its activity depends on Na +<br />

ions. Ktr consists of 2 subunits: KtrB is the 50 kDa K + translocating subunit. It<br />

is a member of the superfamily of K + transporters (SKT), which have evolved<br />

from small K + channels of the KcsA/Kir-type by multiple gene duplications and<br />

gene fusions, forming covalently linked tetramers. The second subunit is KtrA,<br />

a 28 kDa membrane associated regulatory subunit. It confers velocity, cation<br />

coupling and K + selectivity to the KtrAB complex and is believed to regulate<br />

K + transport via a conformational switch induced by the binding of ATP to the<br />

protein.<br />

On my poster I will document the overproduction, isolation and reconstitution<br />

of an active KtrAB complex with an N-terminal His10-tag. With it I plan to do<br />

in vitro uptake experiments in proteoliposomes and examine the influence of<br />

different nucleotides and cations.<br />

The second aspect of the poster deals with the topology and the function of the<br />

long transmembrane M2C-helix from KtrB. A model based on EPR<br />

measurements of reconstituted single cystein mutants, on effects of mutants at<br />

single loci, and on PhoA/FLP fusion studies will be presented.<br />

PW 14<br />

Assembly and membrane interaction of TatA from<br />

Escherichia coli<br />

D. Mehner *1 , T. Brüser 1<br />

1 Institute of Biology / Microbiology, University of Halle-Wittenberg, Halle,<br />

Germany<br />

TatA is an essential component of the Tat protein translocation pathway of<br />

bacteria, archaea and plant plastids. Proteins that contain Tat-specific signal<br />

sequences bind to large TatBC complexes and TatA is believed to be directly<br />

involved in the following translocation step. TatA from Gram-positives and<br />

plant plastids is present in membrane associated and soluble populations. It is<br />

not known whether the soluble forms of TatA are functionally important,<br />

although a substrate targeting function has been postulated for TatA from<br />

Bacillus and Streptomyces species. Here we report an analysis of the<br />

localization and assembly of TatA from Escherichia coli. At wild type<br />

expression levels, the vast majority of TatA is membrane associated, al<strong>bei</strong>t a<br />

small population of TatA remains soluble. TatA shows a very strong selfinteraction<br />

that is partially SDS-resistant. A postulated functionally important<br />

amphipathic helix of TatA is important for the stability of the soluble part of<br />

this protein, indicating that it interacts with (or is part of) the soluble C-terminal<br />

domain. A systematic mutagenesis of the N-terminal putative trans-membranedomain<br />

of TatA revealed characteristics that are important for the membrane<br />

association of TatA.<br />

PW 15<br />

Mutational analysis of ligand-binding of the N-terminal<br />

PEX14 domain<br />

A. Neuhaus *1 , C. Neufeld 2 , F.V. Filipp 2 , B. Simon 2 , N. Schüller 3 , C. David 1 , R.<br />

Mirgalieva 1 , M. Willmanns 3 , M. Sattler 4 , W. Schliebs 1 , R. Erdmann 1<br />

1<br />

Inst. f. physiologische Chemie, Abt. Systembiochemie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

2<br />

EMBL Heidelberg, EMBL, Heidelberg, Germany<br />

3<br />

EMBL Hamburg, EMBL, Hamburg, Germany<br />

4<br />

Lehrstuhl f. biomolekulare NMR-Spektroskopie, TU München, München,<br />

Germany<br />

Protein import in peroxisomes depends on a complex and dynamic network of<br />

protein-protein interactions. PEX14 is a central component of the peroxisomal<br />

import machinery and binds the soluble receptors PEX5 and PEX19, which<br />

play important roles in the assembly of peroxisome matrix and membrane,<br />

respectively. The molecular recognition by PEX14 involves conserved aromatic<br />

side chains in the PEX5 WxxxF/Y motif and a similar motif in PEX19. NMR<br />

analysis revealed that PEX5 and PEX19 ligand helices bind competitively to<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

the same surface in PEX14(N) al<strong>bei</strong>t with opposite directionality (Neufeld,<br />

Filipp, Simon, Neuhaus, Schüller, David, Kooshapur, Madl, Erdmann,<br />

Schliebs, Wilmanns and Sattler, submitted for publication). By mutational<br />

analysis in vitroand in vivo, we show that PEX14 variants that selectively<br />

disrupt ligand binding are impaired in peroxisomal membrane localization.<br />

PW 16<br />

Farnesylation of Pex19p is essential for efficient<br />

peroxisomal membrane protein recognition<br />

R. Rucktäschel *1 , S. Thoms 1 , K. Alexandrov 2 , A. Halbach 1 , M. Pechlivanis 3 , J.<br />

Kuhlmann 3 , R. Volkmer-Engert 4 , H. Rottensteiner 1 , R. Erdmann 1<br />

1<br />

Systembiochemie, Ruhr-Universität, Bochum, Germany<br />

2<br />

Physikalische Biochemie, Max-Planck-Institut für molekulare Physiologie,<br />

Dortmund, Germany<br />

3<br />

Strukturelle Biologie, Max-Planck-Institut für molekulare Physiologie,<br />

Dortmund, Germany<br />

4<br />

Charité, Humboldt Universität, Berlin, Germany<br />

The conserved CaaX box protein Pex19p is required for peroxisome biogenesis.<br />

It binds to several peroxisomal membrane proteins (PMPs) and is needed for<br />

their targeting to peroxisomes. We show here that the complete pool of Pex19p<br />

is processed by farnesyl transferase in vivo. Characterisation of genomic<br />

mutations of PEX19 proved that farnesylation is essential for efficient<br />

peroxisome biogenesis. Yeast mutants defective in Pex19p farnesylation are<br />

characterised by a reduced steady-state level of several PMPs as well as a<br />

disturbed import of peroxisomal matrix proteins. In vitro and in vivo<br />

farnesylated Pex19p bound with strongly increased affinity to Pex19p binding<br />

sites in PMPs as demonstrated by two hybrid analysis, ligand blotting and<br />

fluorescence polarisation studies. This is likely due to a farnesylation-induced<br />

conformational change in Pex19p. Our results indicate that isoprenylation of<br />

Pex19p plays an important role in substrate protein recognition for the<br />

topogenesis of PMPs.<br />

PW 17<br />

The Phosphoinositol-3-Kinase Vps34p is required for<br />

Peroxisome Function and Degradation<br />

S. Grunau 1 , D. Lay 2 , S. Mindthoff *1 , W. Girzalsky 1 , W.W. Just 2 , R. Erdmann 1<br />

1<br />

Institut für Physiologische Chemie, Ruhr- Universität Bochum, Bochum,<br />

Germany<br />

2<br />

Biochemie- Zentrum <strong>der</strong> Universität Heidelberg, Universität Heidelberg,<br />

Heidelberg, Germany<br />

Many organelles in eukaryotic cells are exposed to a high variation concerning<br />

their protein composition and number. To adjust the required amount of<br />

organelles to the environmental conditions, both the biogenesis and degradation<br />

of theses organelles un<strong>der</strong>lie highly regulated control mechanisms. The specific<br />

degradation of peroxisomes is called pexophagy and occurs in two different<br />

ways, namely micropexophagy and macropexophagy. Pexophagy is inducible<br />

in S. cerevisiae with a shift from oleic acid- containing and therefore<br />

peroxisome inducing conditions to glucose- rich media. Some evidence exists<br />

that the membrane is marked for degradation by<br />

Phosphatidylinositolphosphates, phosphorylated <strong>der</strong>ivatives of the membrane<br />

phospholipid phosphatidylinositol (PtdIns).<br />

Here we identify Phosphatidylinositolphosphate synthesizing activity in<br />

purified peroxisomal membranes with radioactive labelled ATP and<br />

demonstrate by means of subcellular fractionation that the lipid kinase Vps34p,<br />

which is a vacuolar protein responsible for protein sorting, is associated with<br />

peroxisomes. Peroxisome biogenesis, analyzed with density gradient<br />

centrifugation and fluorescence microscopy, is not affected in vps34Δ cells.<br />

However, our work proved that Vps34p is essential for the regulated<br />

degradation of peroxisomes. As the steady state protein concentration of oleic<br />

acid inducible proteins is significantly reduced in vps34Δ cells, our data<br />

suggest a role for Vps34p in de-repression of glucose- repressed genes and<br />

induction of oleic acid- inducible genes.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PW 18<br />

The soluble PTS2-receptors of Saccharomyces cerevisiae are<br />

involved in the translocation of peroxisomal matrix proteins<br />

across the organellar membrane<br />

A. Hensel *1 , S. Grunau 1 , W. Girzalsky 1 , R. Erdmann 1<br />

1 Institut für Physiologische Chemie / Abt. Systembiochemie, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

The import of peroxisomal matrix proteins requires the presence of soluble<br />

receptor peroxins, which recognize newly synthesized peroxisomal matrix<br />

proteins in the cytosol and guide them to the peroxisomal import machinery.<br />

After cargo translocation and release through a so far unknown mechanism the<br />

receptors shuttle back to the cytosol.<br />

In Saccharomyces cerevisiae two different receptor-peroxins, the PTS1receptor<br />

Pex5p and the PTS2-receptor Pex7p are known. In contrast to Pex5p,<br />

Pex7p dependent cargo import needs the presence of the auxiliary proteins<br />

Pex18p and Pex21p.<br />

We analysed the PTS2-dependent import pathway to elucidate the events which<br />

occur after receptor docking to the peroxisomal membrane. The aim of this<br />

work addresses the question if Pex7p and Pex18p exhibit receptor function<br />

only, or if they have additional functions in the cargo translocation step across<br />

the peroxisomal membrane. Our results indicate that organellar associated<br />

Pex7p is resistant to proteinaseK treatment in wild-type strain. This finding<br />

indicates that Pex7p either enters the peroxisomal matrix together with the<br />

PTS2-cargo or is embedded in the membrane.<br />

In addition we show that the observed protease resistance of Pex7p is<br />

dependent on the presence of the coreceptors as well as on the presence of<br />

Fox3p. When either of them is deleted, Pex7p becomes accessible to<br />

proteinaseK.<br />

In contrast to Pex7p, Pex18p is not protected against proteinaseK degradation<br />

in wild-type cells, but becomes protected in strains where components of the<br />

importomer are missing.<br />

Finally we present a model which gives a hypothetical explanation for our<br />

observations.<br />

PW 19<br />

Crystal structure and biochemical characterization of the<br />

extracytoplasmatic binding protein GacH, involved in<br />

acarbose metabolism in Streptomyces glaucescens<br />

A. Licht *1 , A. Vahedi-Faridi 2 , S. Keller 3 , U.F. Wehmeier 4 , E. Schnei<strong>der</strong> 1<br />

1 Institut für Biologie, Humboldt-Universität zu Berlin, Berlin, Germany<br />

2 Institut für Chemie, Kristallographie, Freie Universität Berlin, Berlin,<br />

Germany<br />

3 Forschungsinstitut für Molekulare Pharmakologie (FMP), Campus Berlin-<br />

Buch, Berlin, Germany<br />

4 Fachbereich C, Chemische Mikrobiologie, Bergische Universität Wuppertal,<br />

Wuppertal, Germany<br />

193<br />

The secondary metabolite acarbose, produced by Actinoplanes sp. SE50/110<br />

and Streptomyces glaucescens GLA.O, is industrially used for the treatment of<br />

patients suffering from diabetes mellitus type II. The characteristic core<br />

structure of acarbose, the pseudo disaccharide acarviose, consists of a C7<br />

cyclitol bound via an imino bridge to 4-amino-4,6-dideoxyglucose and is<br />

therefore responsible for the inhibitory effect of α-glucosidases of competitors<br />

in the natural habitat. The acarbose structure is completed by a maltose residue.<br />

In addition, a "carbophor" activity of acarbose for the uptake of carbon sources<br />

was proposed: acarbose and related compounds might be used as acceptor<br />

molecules for glucose and oligosaccharides and thereby provide an additional<br />

carbon and energy source. The characterization of components of the identified<br />

acarbose gene cluster from Actinoplanes sp. SE50/110 (acb) and Streptomyces<br />

glaucescens GLA.O (gac) led to the model of an intra- and extracellular<br />

acarbose metabolism un<strong>der</strong> participation of ABC transport systems. ATP<br />

binding cassette (ABC) transporters couple substrate translocation across the<br />

cytoplasmic membrane to ATP hydrolysis.<br />

Binding studies with the extracellular substrate binding protein GacH via<br />

isothermal titration calorimetry and radiolabelled maltose as well as the high<br />

resolution structures of GacH in complex with various ligands identified the<br />

import system GacH-FG-(MsiK)2 as a high affinity maltose-/maltodextrin<br />

transporter. Maltose binding was strongly inhibited by maltodextrins and<br />

acarbose homologs but only weakly by acarbose. These data support the<br />

postulated "carbophor" activity as well as an acarbose recycling mechanism.

194<br />

PW 20<br />

The SURFE 2 R Technology as a tool for measuring<br />

electrophysiological transport activity of different classes of<br />

transporter<br />

I. Janausch *1 , P. Obrdlik 2 , R. Krause 3 , J. Englert 2 , K. Fendler 4 , D. Weitz 5 , H.<br />

Daniel 5 , B. Kelety 2<br />

1<br />

Molecular and Cell Biology, Iongate Biosciences GmbH, Frankfurt, Germany<br />

2<br />

Assay Development, Iongate Biosciences GmbH, Frankfurt, Germany<br />

3<br />

Development and Production, Iongate Biosciences GmbH, Frankfurt,<br />

Germany<br />

4<br />

Biophysical Chemistry, Max Planck Institute of Biophysics, Frankfurt,<br />

Germany<br />

5<br />

Lehrstuhl für Ernährungphysiologie, TU München, Freising, Germany<br />

The SURFE 2 R Technology permits direct electrophysiological analysis of<br />

transporters, ion channels and pumps. Since expression of bacterial transport<br />

proteins in oocytes or mammalian cells is difficult, electrical measurements<br />

were limited so far. This new method is based on a solid supported membrane<br />

and completely independent of radioactive or fluorescent probes. Different<br />

types of the devices are available in the range from table top units for academic<br />

research to multichannel workstations for higher-throughput pharmacological<br />

screening.<br />

Different types of membranes can be adsorbed to the sensor. The broad<br />

spectrum reaches from liposomes with reconstituted proteins to bacterial or<br />

mammalian membrane preparations. Besides the possibility for whole cell<br />

measurement exists. Therefore the method is also suitable for the determination<br />

of protein expression levels i.e. in clone identification. For the quality of<br />

pharmacological screening assays it is essential to find high expressing celllines.<br />

With the SURFE 2 R Technology we can analyze hundreds of clones per<br />

day without the need of membrane preparation or reconstitution of the transport<br />

protein.<br />

Here we present transport measurement data representing different microbial<br />

transporter classes. With the SURFE 2 R Technology we could analyze for<br />

example H + -driven (YdgR), Na + -driven (MelB), exchanger (NhaA) and lightdriven<br />

(ChR2, HR) proteins.<br />

PW 21<br />

Methionine uptake in Corynebacterium glutamicum by<br />

MetQNI and by MetPS, a novel methionine and alanine<br />

importer of the NSS neurotransmitter transporter family<br />

T. Mohrbach *1 , C. Trötschel 2 , M. Follmann 1 , J.A. Nettekoven 1 , L.R. Forrest 3 , R.<br />

Krämer 1 , K. Marin 1<br />

1 Institut für Biochemie, Universität zu Köln, Köln, Germany<br />

2 Plant Biochemistry, Ruhr-Universität Bochum, Bochum, Germany<br />

3 Independent Junior Research Group - Computational Structural Biology, Max<br />

Planck Institute of Biophysics, Frankfurt/Main, Germany<br />

The soil bacterium Corynebacterium glutamicum is a model organism in amino<br />

acid biotechnology. Here we present the identification of two different Lmethionine<br />

uptake systems including the first characterization of a bacterial<br />

secondary methionine carrier. The primary carrier MetQNI is a high affinity<br />

ABC-type transporter specific for L-methionine. Its expression is un<strong>der</strong> control<br />

of the transcription factor McbR, the global regulator of sulfur metabolism in C,<br />

glutamicum. Beside MetQNI a novel secondary methionine uptake system of<br />

the NSS (neurotransmitter sodium symporter) family was identified and named<br />

MetP. The MetP system is characterized by a lower affinity for methionine and<br />

uses Na + ions for energetic coupling. It is also the main alanine transporter in C.<br />

glutamicum and is expressed constitutively. These observations are consistent<br />

with models of methionine, alanine, and leucine bound to MetP, <strong>der</strong>ived from<br />

the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus.<br />

Complementation studies show that MetP consists of two components, a large<br />

subunit with 12 predicted transmembrane segments, and surprisingly, an<br />

additional subunit with one predicted transmembrane segment only. Thus, this<br />

new member of the NSS transporter family adds a novel feature to this class of<br />

carriers, namely the functional dependence on an additional small subunit.<br />

PW 22<br />

A simplified type IV secretion like system from Gram<br />

positive bacteria: Localization of the transport machinery<br />

in Enterococcus faecalis<br />

K. Arends *1 , A.M. Hanssen 2 , M.Y. Abajy 3 , E.K. Celic 3 , K. Schiwon 1 , W.<br />

Keller 3 , E. Grohmann 1<br />

1 Environmental Microbiology/Genetics, Technical University Berlin, Berlin,<br />

Germany<br />

2 Institute of Medical Biology, University of Tromsö, Tromsö, Norway<br />

3 IMB/Structural Biology, Karl-Franzens-University Graz, Graz, Austria<br />

The multiple antibiotic resistance plasmid pIP501 has an extremely broad-hostrange<br />

for conjugative transfer including a variety of Gram positive bacteria like<br />

Enterococcus faecalis and Staphylococcus aureus, multicellular Streptomyces<br />

and Gram negative Escherichia coli. The pIP501 transfer region consists of 15<br />

orfs and encodes 3 gene products with significant similarity to the type IV<br />

secretion system proteins (T4SS) VirB1, VirB4 and VirD4 from the<br />

Agrobacterium T-DNA transfer system responsible for virulence protein and T-<br />

DNA transport.<br />

A working model of the simplified T4SS (T4SLS) encoded on the pIP501<br />

plasmid was created by protein-protein interaction studies using the yeast-twohybrid<br />

system and in vitro pull down assays. The postulated DNA secretion<br />

complex is assembled in a manner reminiscent of a simplified T4SS.<br />

To localize the T4SLS transport machinery in vivo, E. faecalis JH2-2 (pIP501)<br />

cells were fractionated into the different cell compartments (cell wall, cell<br />

membrane, cytoplasm) and polyclonal antibodies specific for the pIP501<br />

transport proteins were used to detect the proteins by Western blotting. The<br />

Orf10 protein (VirD4 homolog) and Orf7 (VirB1 homolog) mainly localized to<br />

the cell wall fraction, whereas the Orf11 protein was found in all three<br />

compartments. The core proteins Orf6 and Orf9 localized to the cell envelope,<br />

they putatively stabilize the transfer channel. Orf5 was found to localize to the<br />

cell envelope in preliminary experiments. In a second approach we use<br />

microscopy techniques (confocal immuno-fluorescence and immuno-gold<br />

electron microscopy) to localize the Tra-proteins.<br />

PW 23<br />

ATPase activity, localisation and oligomeric state of ORF10,<br />

the VirD4 homologue of the pIP501-encoded type IV<br />

secretion system<br />

M.Y. Abajy *1 , M. Saleh 2 , A.M. Hanssen 3 , K. Arends 2 , W. Keller 1 , E.<br />

Grohmann 2<br />

1<br />

Institute of Mol. Biosciences/Structure Biology, Karl-Franzens-University<br />

Graz, Graz, Austria<br />

2<br />

Environmental Microbiology/Genetics, Technical University Berlin, Berlin,<br />

Germany<br />

3<br />

Institute of Medical Biology, University of Tromsø, Tromsø, Norway<br />

pIP501 is a multiple antibiotic resistance conjugative plasmid with an<br />

extremely broad host range, including all tested Gramm-positive bacteria, and<br />

Gramm-negative E. coli. The pIP501 transfer region encodes a simplified type<br />

IV secretion-like system. ORF10 encoded by pIP501 showed significant<br />

sequence similarity to the Agrobacterium type IV secretion system coupling<br />

protein VirD4. ORF10 is a putative VirD4-like coupling protein which could<br />

link the relaxosome consisting of ORF1 (the pIP501-encoded relaxase) bound<br />

to oriTpIP501 with the transport apparatus. In vivo yeast two-hybrid studies and in<br />

vitro pull-down assays showed that ORF10 interacts with ORF1. Investigations<br />

of the oligomeric state of ORF10 by glutaraldehyde cross-linking and size<br />

exclusion chromatography showed that ORF10 can form multimeric structures.<br />

The aggregation properties of ORF10 will be also analysed with negative<br />

staining and electron microscopy. ORF10 have been purified as 7xHis-fusion<br />

and showed ATP-binding/hydrolysis activity in vitro. The optimal conditions<br />

(buffer, divalent cations, pH and temperature), where ORF10 showed the<br />

highest activity, have been also determined. Two C-terminal deletion proteins<br />

of ORF10 (ORF10N442 and ORF10N475) have been achieved to improve its<br />

solubility and folding capacity and will be subjected to initial crystallization<br />

screens. The first 891 nucleotides of orf10, encoding the walker A & B<br />

motives, were cloned into the expression plasmid pQTEV, the corresponding<br />

protein have been purified and showed a lower activity as the full-length<br />

protein. Furthermore orf10 gene has been cloned into the E. coli / E. faecalis<br />

shuttle plasmid pMSP3535VA. Fractionation of E. faecalis cells expressing the<br />

7xHis-ORF10 showed a cell membrane localisation for ORF10. Immune-TEM<br />

of E. faecalis cells harbouring the pIP501 plasmid with gold-labled anti-ORF10<br />

antibodies showed also that ORF10 localized probably in the cell membrane.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PX 01<br />

Production and extraction of PHB in Iranian native<br />

methylotrophic microorganism<br />

F. Almasian *1 , N. Ghaemi 2 , H. Mahdavi 2 , M. Azin 3 , E. Zanjirian 1 , G.<br />

Mohammadzamani 1<br />

1<br />

Chemistry, Engineering Research Institute, Tehran, Iran<br />

2<br />

College of science, University of Tehran, Tehran, Iran<br />

3<br />

Biotechnology, Iranian research organization for science&technology,<br />

Tehran, Iran<br />

Poly-β-hydroxybutyrate (PHB) is an important biopolymer that accumulates by<br />

a wide variety of microorganisms as an intracellular storage source of carbon<br />

and energy. Although Polyhydroxybutyrate has many applications in medicine,<br />

agriculture, medical and food packaging however, the major problem<br />

associated with the industrial production of PHB is their high production cost.<br />

In this investigation methanol was used as a cheap substrate, for PHB<br />

production by Iranian native methylotrophic microorganism. For this purpose<br />

the strain was grown for production of PHB in mineral salt medium with<br />

methanol as carbon source. Various experiments were carried out at different<br />

pH, agitation, methanol concentrations and nitrogen concentration. PHB<br />

content was determined by gas chromatography. By these experiments,<br />

optimum PHB production conditions were determined for strain of<br />

methylotroph. The results showed optimum pH around 7, agitation 250 rpm,<br />

nitrogen concentration equal to 1 g.l–1 and methanol was found to be optimum<br />

at concentration equal to 17 g.l–1. In this conditions biomass dry weight was<br />

3.7 g.l–1 and PHB content was about 0.85 g.l–1. Then PHB, was extracted<br />

from lyophilized cells by chloroform: hypochlorite method, precipitated in 4<br />

voles methanol, and then dried in room temperature. molecular mass of purified<br />

polymer were estimated by gel-permeation chromatography. The results<br />

indicated that the molar mass of PHB extracted from Methylotrophic strain was<br />

about 6.2 .104g.mol-1.These experiments showed that the methylotrophic strain<br />

has potential for PHB production from methanol as a substrate.<br />

PX 02<br />

Crucial methionine residues responsible for reaction<br />

inactivation of oxidases: identification by MALDI-TOF/MS<br />

analysis and saturation mutagenesis<br />

S. Dorscheid *1 , F. Giffhorn 1<br />

1 Applied Microbiology, Saarland University, Saarbruecken, Germany<br />

It is a well-known phenomenon in biocatalysis that oxidases are affected by<br />

oxygen as it participates in the reaction, which stoichiometrically produces<br />

H2O2 while oxidizing a substrate. Although H2O2 can be removed by catalase,<br />

the transition of electrons from the substrate to O2 cannot be controlled, so that<br />

intermediate reactive oxygen species may affect the enzyme. We studied this<br />

phenomenon of reaction inactivation with pyranose 2-oxidase (P2Ox), an<br />

enzyme of biotechnological importance [1]. Here we report for the first time the<br />

identification of amino acid residues in P2Ox which had been oxidized during<br />

the reaction, and confine those whose oxidation might affect P2Ox activity [2].<br />

Following a bioconversion, P2Ox was proteolytically digested. Peptides were<br />

analysed using MALDI-TOF/MS for peptide mass mapping [3], and compared<br />

with those obtained by in silico digestions (ExPasy Proteomics Server). Thus<br />

we identified seven peptides each with a mass increase of 16 Da. As the<br />

peptides contained one methionine but no cysteine the sulfur moiety of the<br />

former had been oxidized [3]. We excluded six methionines from further<br />

consi<strong>der</strong>ation because of their peripherical locations and putative oxygen<br />

scavenger functions [4]. The remaining Met164, located at the active site, was<br />

replaced with glutamine by saturation mutagenesis. To demonstrate the<br />

improvement of the operative stability of P2OxM164Q, we performed<br />

bioconversions with repeated substrate addition. Thus we present a method to<br />

identify the most susceptible methionines and distinguish the more external<br />

ones from those buried inside the protein structure, the latter to be future targets<br />

for site-directed saturation mutagenesis.<br />

[1] Bastian et al. 2005. Appl. Microbiol. Biotechnol. 67: 654<br />

[2] Bannwarth et al. 2006. Biochemistry 45: 6587<br />

[3] Corless et al. 2003. Mass Spectrom. 17 : 1212<br />

[4] Levine et al. 1996. Proc. Natl. Acad. Sci. USA 93: 15036<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PX 03<br />

Feed stuff production by Iranian native pink pigmented<br />

facultative methlotropic microorganisms (PPFM)<br />

P. Jafari *1 , G. Mohammad Zamani 2 , F. Almasian 2 , B. Medi 2 , N. Ghaemi 2<br />

1 Biology, Azad Islamic University, Arak Branch, Tehran, Iran<br />

2 Chemistry, Engineering Research Institute, Tehran, Iran<br />

195<br />

Fishmeal is used for supplementation of animal feed, but it most frequently<br />

contaminated with mycotoxins. Limited availability of fishmeal and<br />

contamination of it in Iran, lead us to investigate a new nonconventional feed<br />

stuff. There is a huge potential for utilization of the methanol by<br />

methylotrophic microorganisms for production of food and feed. For the<br />

production of protein enriched feed stuff, up to 250 new obligate methylotrophs<br />

were isolated from soil. Various Shake flask cultivation experiments were<br />

carried out at different pH, temperature, agitation speed, and methanol<br />

concentration, for screening the isolate with the highest efficiency for<br />

production of biomass. Among the isolates, the PPFM strain P69, showed the<br />

highest biomass yield (6 g.l -1 ). This strictly aerobic, Gram-negative rod, motile<br />

bacteria was identified as a member of the genus Methylobacterium by 16S<br />

ribosomal DNA analysis.<br />

A statistical experimental design method was implemented to optimize<br />

experimental conditions of the purpose in 1 lit stirred tank fermenter. Agitation<br />

speed, aeration rate, initial methanol concentration, and pH were consi<strong>der</strong>ed as<br />

process parameters to be optimized. As the result of Taguchi analysis in this<br />

study, aeration rate and agitation speed were the most influencing parameters<br />

on biomass production. The highest biomass (~10 g.l -1 ) were obtained at<br />

aeration rate of 3 vvm, agitation speed of 800 rpm, pH 8, and 18 g.l -1 initial<br />

methanol concentration.<br />

An automatic two stage continuous culture system has been developed for feed<br />

stuff production. In this system, filtered substrate continuously supplied into a<br />

deep jet reactor of a 1 m3 capacity at a pre-determined rate (0.15 lit/h. After<br />

optimization with mathcad method, YX/S was 0.4 g.g-1 methanol (40 kg dried<br />

biomass per day). Empirical biomass formula was CH1.84O0.596N0.23 with<br />

Protein content up to 74%. Amino acids analysis of our product showed its<br />

nutritional value for animals as a replacement of fish meal<br />

PX 04<br />

Engineering of a glycerol utilization pathway for amino<br />

acid production by Corynebacterium glutamicum<br />

D. Rittmann 1 , S.N. Lindner *2 , V.F. Wendisch 2<br />

1 Institute of Biotechnology 1, Research Center Juelich, Juelich, Germany<br />

2 Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms<br />

University Muenster, Muenster, Germany<br />

Corynebacterium glutamicum is used for the annual production of >1,500,000<br />

tons of L glutamate and >750,000 tons of L lysine, but cannot utilize glycerol,<br />

the main by-product of biodiesel production (10% by weight). Here, C.<br />

glutamicum was engineered to grow on glycerol by heterologous expression of<br />

Escherichia coli glycerol utilization genes [1]. While expression of the E. coli<br />

genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase<br />

(glpD) was sufficient for growth on glycerol as sole carbon and energy source,<br />

additional expression of the aquaglyceroporin gene glpF from E. coli increased<br />

growth rate. Plasmid-borne expression of E. coli glpF, glpK and glpD enabled<br />

production of L-glutamate and of L-lysine from glycerol as sole carbon<br />

substrate as well as from glycerol-glucose blends [1]. The L-glutamate and Llysine<br />

yields on glycerol by the engineered strains were comparable to those of<br />

the parent strains on glucose [2, 3].With a growing biodiesel producing industry<br />

and increasing availability of glycerol, glycerol-based amino acid production<br />

may contribute to the biorefinery concept for bulk chemical production [4]).<br />

[1] Rittmann, D., S.N. Lindner, and V. F. Wendisch. 2008. Appl Environ<br />

Microbiol 74: 6216-6222.<br />

[2] Krings, E., K. Krumbach, B. Bathe, R. Kelle, V. F. Wendisch, H. Sahm, and<br />

L. Eggeling. 2006. J Bacteriol 188: 8054-61.<br />

[3] Radmacher, E., K. C. Stansen, G. S. Besra, L. J. Al<strong>der</strong>wick, W. N.<br />

Maughan, G. Hollweg, H. Sahm, V. F. Wendisch, and L. Eggeling. 2005.<br />

Microbiol 151: 1359-68.<br />

[4] Marx, A., V. F. Wendisch, R. Kelle, and S. Buchholz. 2006. In Biorefineries<br />

– Industrial Processes and Products. Status Quo and Future Directions (Kamm<br />

B., Gruber P.R., Kamm M., eds) WILEY-VCH, Weinheim, Germany: 201-216.

196<br />

PX 05<br />

Functional expression of porcine aminoacylase 1 in E. coli<br />

using a codon optimized synthetic gene and molecular<br />

chaperones<br />

R. Wardenga *1 , F.H. Hollmann 2 , O. Thum 3 , U. Bornscheuer 1<br />

1 Dept. of Biotechnology and Enzyme Catalysis, University of Greifswald,<br />

Greifswald, Germany<br />

2 Dept. of Biotechnology, Biocatalysis and Organic Chemistry, Delft University<br />

of Technology, Delft, Netherlands<br />

3 Evonik Goldschmidt GmbH, Essen, Germany<br />

Efficient recombinant expression of N-acyl-Laminoacylase 1 from pig kidney<br />

(pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized<br />

nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into<br />

vector pET-52(b) yielding an E. coli expressible pAcy1 gene. Formation of<br />

inclusion bodies was alleviated by co-expression of molecular chaperones<br />

resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger<br />

factor or GroEL–GroES, respectively. Facile purification was achieved via<br />

StrepTag affinity chromatography.<br />

Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter<br />

of cultivation broth. The protein was analyzed for structural and functional<br />

identity, and the performances of further described expression and purification<br />

systems for pAcy1 were compared.<br />

PX 06<br />

Regioselective hydroxylation of aromatic compounds by an<br />

extracellular fungal peroxygenase from Agrocybe aegerita<br />

M. Kinne *1 , M. Poraj-Kobielska 1 , E. Aranda 1 , R. Ullrich 1 , K. Scheibner 2 , K.E.<br />

Hammel 3 , M. Hofrichter 1<br />

1 Unit of Environmental Biotechnology, International Graduate School of<br />

Zittau (IHI), Zittau, Germany<br />

2 Unit of Biotechnology, Lausitz University of Applied Sciences, Senftenberg,<br />

Germany<br />

3 Institute for Microbial and Biochemical Technology, USDA Forest Products<br />

Laboratory, Madison, United States<br />

Selective hydroxylations of aromatic compounds are among the most<br />

challenging reactions in synthetic chemistry and have gained steadily<br />

increasing attention during recent years because hydroxylated aromatic<br />

precursors are used extensively in the chemical industry. For example, (R)-2-(4hydroxyphenoxy)propionic<br />

acid is an intermediate in the synthesis of<br />

enantiomerically pure aryloxyphenoxypropionic acid-type herbicides, in which<br />

the crop protection activity normally <strong>der</strong>ives from one enantiomer. (R)-2-(4hydroxyphenoxy)propionic<br />

acid is currently prepared from (R)-2phenoxypropionic<br />

with whole cells of the ascomycete Beauveria bassiana,<br />

which produces regioselective oxidases that catalyze this hydroxylation. We<br />

found that the extracellular heme-thiolate peroxygenase of Agrocybe aegerita<br />

catalyzed the H2O2-dependent hydroxylation of 2-phenoxypropionic acid to<br />

give the herbicide precursor 2-(4-hydroxyphenoxy)- propionic acid. The<br />

reaction proceeded regioselectively with an isomeric purity near 98%, yielding<br />

the desired R-isomer with an enantiomeric excess of 60%. 18 O-labeling<br />

experiments showed that the phenolic hydroxyl in 2-(4hydroxyphenoxy)propionic<br />

acid originated from H2O2, which establishes that<br />

the reaction is mechanistically a peroxygenation. In other work, we observed<br />

that the A. aegerita peroxygenase catalyzed the regioselective hydroxylation of<br />

the beta blocker propranolol to give 5-hydroxypropranolol, which is important<br />

because it is a human metabolite of propanolol. Our results raise the possibility<br />

that fungal peroxygenases may be useful for a variety of organic oxidations.<br />

PX 07<br />

Degradation of polycyclic musk fragrances by extracellular<br />

fungal oxidoreductases<br />

M. Poraj-Kobielska *1 , M. Kinne 2 , R. Ullrich 2 , K.E. Hammel 3 , G. Kayser 1 , M.<br />

Hofrichter 2<br />

1<br />

Unit of Environmental Process Engineering, International Graduate School of<br />

Zittau (IHI), Zittau, Germany<br />

2<br />

Unit of Environmental Biotechnology, International Graduate School of<br />

Zittau (IHI), Zittau, Germany<br />

3<br />

Institute for Microbial and Biochemical Technology, USDA Forest Products<br />

Laboratory, Madison, United States<br />

Musk-based perfumes have been used for many years in a variety of consumer<br />

products such as detergents, household cleaners, shampoos, and laundry<br />

detergents. In the mid-1990´s, questions arose, especially in Europe, about the<br />

safety of one class of these perfumes - the "polycyclic musk fragrances" (PMF).<br />

Attention focused on two particular chemicals - Tonalid (AHTN) and<br />

Galaxolide (HHCB). Both chemicals were subsequently listed by the European<br />

Commission as likely "persistent, bioaccumulative and toxic" (PBT)<br />

substances. For example, HHCB and AHTN were detected in sewage (6 µg<br />

HHCB/l, 4.4 µg AHTN/l) and in fish from sewage-contaminated water (159 mg<br />

HHCB/kg lipid, 58 mg AHTN/kg lipid) [1]. Current wastewater treatment<br />

techniques fail to remove these pollutants. We investigated the biodegradability<br />

of Tonalid by the extracellular heme-thiolate peroxygenases of Agrocybe<br />

aegerita (AaP) and Coprinellus radians (CrP), and by the chloroperoxidase<br />

(CPO) from Calariomyces fumago. The biocatalysts all converted Tonalid into<br />

various metabolites, probably via initial hydroxylations at various carbon<br />

positions. Incubation of Tonalid with CrP and H2O2 released four<br />

biotransformation metabolites with a maximum turnover of 24%. Similar<br />

results were observed for AaP (one reaction product, 15% turnover) and CPO<br />

(five reaction products, 14% turnover). Our results raise the possibility that<br />

fungal oxidoreductases may be useful for the oxidative degradation of PBT<br />

from wastewater.<br />

[1] Rimkus,G.G.Toxicol.Lett.1999,111,37-56.<br />

PX 08<br />

Production of glutaconate from glutamate in a recombinant<br />

Escherichia coli strain<br />

I. Djurdjevic *1 , J. Kim 1 , O. Zel<strong>der</strong> 2 , W. Buckel 1<br />

1<br />

Laboratorium für Mikrobiologie, FB Biologie, Philipps-Universität, Marburg,<br />

Germany<br />

2<br />

GVF/D - A030, BASF, Ludwigshafen, Germany<br />

Glutaconic acid together with a diamine can polymerize to a polyamide related<br />

to Nylon ® . The ideal material for biotechnological production of glutaconic<br />

acid is glutamic acid, which can be produced by sugar fermentation. The<br />

chemical deamination of α-amino acids to α,β-unsaturated acids is difficult. On<br />

the contrary, glutamate fermenting strict anaerobic bacteria can easily<br />

deaminate glutamate via α-ketoglutarate, (R)-2-hydroxyglutarate, and 2hydroxyglutaryl-CoA<br />

to (E)-glutaconyl-CoA that is decarboxylated to crotonyl-<br />

CoA [1]. In or<strong>der</strong> to convert Escherichia coli into a glutaconate producer we<br />

expressed six genes encoding 2-hydroxyglutarate dehydrogenase (HgdA),<br />

glutaconate CoA-transferase (GcdAB), and the activator (HgdC) from<br />

Acidaminococcus fermentans, as well as 2-hydroxyglutaryl-CoA dehydratase<br />

(HgdAB) from Clostridium symbiosum. The first enzyme of this new pathway,<br />

glutamate dehydrogenase, is already present in E. coli. The experimental data<br />

indicate that the recombinant E. coli strain indeed produced glutaconate in the<br />

medium though at a low level. The yield improves into the mM range by<br />

adding Fe 2+ and riboflavin, which form the prosthetic groups of the dehydratase<br />

and its activator [2]. The data further indicate that the concentration of<br />

glutaconate inside the cells is about 12-times higher than outside. Our aim is to<br />

use the cheaper glucose rather than glutamate as carbon source. This would<br />

yield in addition reduced ferredoxin and ATP that are required for the<br />

activation of the dehydratase. On the other hand, the use of glucose needs an<br />

acceptor for six electrons/mol, which cannot be oxygen due to the extreme<br />

oxygen sensitivity of HgdC.<br />

[1] Buckel W (2001) Appl Microbiol Biotechnol 57:263-273.<br />

[2] Hans M, Buckel W, Bill E (2000). Eur J Biochem 267:7082-93.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PX 10<br />

Biotechnological production of alkylquinolones<br />

H. Niewerth *1 , S. Fetzner 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

In Pseudomonas aeruginosa and many other pathogenic bacteria, production of<br />

virulence factors is controlled by quorum sensing (QS). One of the regulatory<br />

circuits of the QS system of P. aeruginosa is controlled by alkylquinolone<br />

(AQ) signalling molecules. Synthesis of HHQ (2-heptyl-4(1H)quinolone) via<br />

condensation of anthranilate and a β-keto fatty acid is directed by the pqsABCD<br />

genes; PqsH catalyzes hydroxylation of HHQ to form PQS (Pseudomonas<br />

quinolone signal, 2-heptyl-3-hydroxy-4(1H)quinolone)[1].<br />

For the biotechnological production of HHQ, the genes pqsABCD from P.<br />

aeruginosa PAO1 were expressed in P. putida KT2440, and the recombinant<br />

strain was cultivated with anthranilate and octanoate. RP-HPLC analysis<br />

indicated that about 14% of the added anthranilate was converted to HHQ; 30%<br />

and 70% of the produced HHQ were localized in the culture supernatant and<br />

the cell fraction, respectively. HHQ was extracted from cells with methanol,<br />

and isolated from culture supernatants with an adsorber resin. After<br />

concentrating the methanolic fractions, generation of a supersaturated solution<br />

by addition of water resulted in precipitation of HHQ. RP-HPLC analysis<br />

indicated that the precipitate contains small amounts of other alkylquinolones.<br />

A recombinant P. putida strain that synthesizes PqsH was used for the<br />

biotransformation of HHQ to PQS. Cells and supernatants were processed as<br />

described above, and significant amounts of PQS were detected in the cell<br />

fraction by RP-HPLC.<br />

The produced alkylquinolones will be useful for QS studies in P. aeruginosa<br />

and other bacteria, and for characterizing other biological activities of AQs.<br />

[1] Dubern JF, Diggle SP (2008) Mol. BioSyst. 4:882-888.<br />

PX 11<br />

Improved plasmids for heterologous protein production in<br />

Bacillus megaterium<br />

S. Stammen *1 , M. Gamer 1 , B.K. Müller 1 , J. Schwerk 1 , H. Schlums 1 , D. Jahn 1<br />

1 Institut für Mikrobiologie, Techische Universität Braunschweig,<br />

Braunschweig, Germany<br />

Bacillus megaterium is a Gram-positive soil bacterium which is known for its<br />

high protein secretion capability. A shuttle vector system for production and<br />

secretion of heterologous proteins in B. megaterium was developed. This<br />

plasmid based system employs the native homologous xylose-inducible<br />

promoter PxylA for target gene expression. Different optimisation strategies for<br />

the plasmid system were pursued and finally combined to increase the<br />

production of the protein of interest. To improve the transcriptional efficiency<br />

the -10 region of the promoter was changed into the predicted optimal<br />

sequence. The ribosome binding site (RBS) was changed to the consensus<br />

sequence for B. megaterium to further stimulate the protein production on the<br />

translational level. Finally, plasmids were designed which led to mRNA<br />

transcripts of the target gene with modified 5’ untranslated regions (5’UTR).<br />

The introduced modifications inside the 5’UTRs led to an increase in protein<br />

formation. For determination of heterologous protein production, the gene<br />

coding for green fluorescent protein (GFP) from jellyfish Aequorea victoria<br />

was cloned un<strong>der</strong> control of the xylose inducible PxylA of the novel plasmid<br />

constructs. Expression analyses were performed in B. megaterium to determine<br />

the amount of recombinantly produced GFP. After identification of promising<br />

new features for the expression vectors, these were combined in a novel<br />

generation of B. megaterium plasmids. These optimized production vectors for<br />

B. megaterium yielded up to 13 times more GFP compared to the previously<br />

described plasmids.<br />

PX 12<br />

Synthesis of cyanophycin with an altered composition by<br />

use of arginine-auxotrophic mutants of Saccharomyces<br />

cerevisiae<br />

A. Steinle *1 , A. Steinbüchel 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

Cyanophycin, a polyamide usually consisting of an aspartic acid backbone with<br />

arginine residues linked to each aspartate residue, represents an important<br />

precursor for various biotechnically employed substances. Especially for the<br />

synthesis of dipeptides, which are applied in various medical fields,<br />

cyanophycin plays an important role. In the present study mutants of<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Saccharomyces cerevisiae revealing defects in arginine biosynthesis were<br />

employed for synthesis of cyanophycin with an altered composition. For<br />

arginine biosynthesis five enzymes catalyze the conversion of glutamate to<br />

ornithine. L-ornithine is converted to citrulline by an ornithine<br />

carbamoyltransferase (ARG3), citrulline reacts with aspartate in an ATPdependent<br />

reaction catalyzed by argininosuccinate synthetase (ARG1) to Largininosuccinate.<br />

The latter is converted to L-arginine by argininosuccinate<br />

synthetase. The applied strains, referred to as strains Arg1 and Arg3, exhibit the<br />

respective deleted gene. All strains were transformed with the vector pYEX-<br />

BX::cphA6308, thus expressing cyanophycin synthetase (CphA) from the<br />

cyanobacterium Synechocystis sp. PCC 6308. This enzyme was chosen as it<br />

exhibits a wide substrate range in vitro (Aboulmagd et al. 2001). Cyanophycin<br />

isolated from the respective strains consisted of up to 7.3 mol% of citrulline<br />

(Arg1) and 5.5 mol% ornithine (Arg3) besides aspartate and arginine. Through<br />

variation of cultivation conditions the amount of incorporated citrulline could<br />

be increased to 20 mol% and the amount of ornithine to 8 mol%. The first<br />

achievement of in vivo synthesis of cyanophycin with constituents replacing<br />

arginine beside lysine largely expansions its field of application especially for<br />

synthesis of divergent dipeptides.<br />

PX 13<br />

Establishment of a novel anabolism-based addiction system<br />

with an artificially introduced mevalonate pathway for<br />

biotechnological processes<br />

J. Kroll *1 , A. Steinle 1 , R. Reichelt 2 , A. Steinbüchel 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

2 Institut für Medizinische Physik und Biophysik, Elektronenmikroskopie und<br />

Analytik, Westfälische Wilhelms-Universität Münster, Münster, Germany<br />

Plasmid stability in recombinant microorganisms is an important requirement<br />

for highly efficient plasmid-based production processes in biotechnology. To<br />

stably maintain plasmids, we developed an efficient and stringent novel class of<br />

anabolism-based addiction system. Outstanding for this system the cultivation<br />

is not addicted to a defined carbon source and can be used with complex media.<br />

This novel addiction system is based on two components: (i) an Escherichia<br />

coli HMS174(DE3) knock out mutant of the ispH gene coding for 4-hydroxy-3methylbut-2-enyl<br />

diphosphate reductase (EC 1.17.1.2) of the deoxyxylulose 5phosphate<br />

(DXP) pathway impairing synthesis of isopentenyl pyrophosphate<br />

(IPP) and (ii) a completely synthetic and episomal mevalonate (MVA) pathway<br />

as an alternative supplier of essential IPP. The latter is encoded by a plasmid<br />

which contains the genes for HMG-CoA reductases from Lactococcus lactis<br />

and Staphylococcus aureus plus HMG-CoA-synthase, MVA kinase, MVP<br />

kinase and MVPP decarboxylase from S. aureus. To demonstrate the<br />

functionality of this addiction system, a mutated cyanophycin synthetase gene<br />

(cphA6308C595S) was used. To determine plasmid stabilities, experiments un<strong>der</strong><br />

selective and non-selective conditions were carried out with the knock out<br />

mutant and two control strains, one harboring plasmid pCOLADuet 1::MVA1<br />

5::cphA6308, the other harboring a conventional expression plasmid pET-<br />

23a::cphA6308. The knock out mutant revealed a plasmid stability of 100%<br />

whereas the control strains exhibited plasmid stabilities of only 64% and 2%,<br />

respectively. Radiometric enzyme activity measurements for CphA revealed<br />

only 95% and 12.5% of the activity in the control strains harboring<br />

pCOLADuet-1::MVA1-5::cphA6308 or pET-23a::cphA6308, respectively, in<br />

comparison to the activity measured in the knock out mutant.<br />

PX 14<br />

Catalytic biofilms: A new concept for fine chemical<br />

production<br />

B. Halan *1 , K. Bühler 1 , A. Schmid 1<br />

1 Laboratory of Chemical Biotechnology, Department of Biochemical and<br />

Chemical Engineering, Technical University of Dortmund, Dortmund,<br />

Germany<br />

197<br />

Biofilms are ubiquitous surface associated microbial communities embedded in<br />

an extra cellular polymeric matrix which give the biofilm structure and<br />

strength. Bacterial cells in biofilms express phenotypic characteristics or<br />

adaptive responses to stress and are known for enhanced tolerance towards<br />

adverse environmental conditions, distinct from their planktonic counterparts<br />

[1] [2]. Bottlenecks in biocatalysis such as toxic substrates and/or products, and<br />

long term activity and stability of the biocatalyst may be overcome when<br />

applying biofilms for catalysis in organic chemistry [3]. In this regard, we<br />

introduce the concept and design of a microporous ceramic membrane based<br />

biofilm reactor and show its effectiveness in lab scale operation in terms of<br />

productivity and stability. Biotransformation experiments were carried out

198<br />

using Pseudomonas sp. strain VLB120ΔC as a biofilm biocatalyst for the<br />

production of enantiopure (S)-Styrene oxide from styrene. A microporous<br />

ceramic membrane was shown to be a suitable substratum as well as an<br />

efficient oxygen supply unit. A uniform and dense biofilm developed during 5<br />

days on this matrix without blocking the pores of the membrane. Due to this<br />

dual function of the ceramic membrane, the reactor configuration could be<br />

significantly simplified by eliminating additional packing materials and<br />

reducing the aqueous volume. In addition, an in situ substrate feeding and<br />

product recovery technique was implemented into this reactor. A maximum<br />

productivity of 28 g Laq -1 d -1 was achieved while the system is stable for more<br />

than 30 days. After this time, the experiment was actively terminated.<br />

In this presentation we will introduce this new and highly interesting reactor<br />

concept and characterize the development and activity of the used biocatalytic<br />

biofilm.<br />

[1] Costerton et al., 1987,Ann.Rev.Microbiol.1987.41:435-464<br />

[2] Stewart and Costerton 2001,Lancet. 358:135-138<br />

[3] Gross et al., 2007, Biotechnol Bioeng. 9999:1-12<br />

PX 15<br />

Maltose as booster for amino acid production with<br />

Corynebacterium glutamicum<br />

A. Henrich *1 , F. Krause 2 , B. Blombach 2 , R. Krämer 1 , B.J. Eikmanns 2 , G.M.<br />

Seibold 1<br />

1 Institute of Biochemistry, University of Cologne, 50674 Cologne, Germany<br />

2 Institute of Microbiology and Biotechnology, University of Ulm, 89069 Ulm,<br />

Germany<br />

For amino acid productivity of Corynebacterium glutamicum, the intracellular<br />

precursor supply is a factor of major importance. To increase the internal<br />

pyruvate concentration, the precursor for L-lysine and L-valine synthesis,<br />

pyruvate-dehydrogenase-complex-deficient (ΔaceE) C. glutamicum strains<br />

have been constructed [1]. These strains are dependent on acetate for growth<br />

and utilise glucose to produce amino acids. As in the presence of acetate the<br />

transcriptional regulator SugR represses the expression of ptsG, which encodes<br />

the glucose uptake system [2], ΔaceE strains do not produce amino acids during<br />

growth.<br />

We here show that the repression of ptsG during cultivation with acetate can be<br />

compensated by the addition of maltose. Growth experiments revealed that<br />

indeed the diminished utilisation of glucose in the presence of acetate is<br />

completely abolished when maltose is added to the culture broth, resulting in<br />

equal utilisation of all three carbon sources. We applied these results in the<br />

optimisation of L-valine production with C. glutamicum ΔaceE Δpqo<br />

(pJC4ilvBNCE) [3]: Indeed the addition of maltose to the fermentation broth<br />

caused the strain to produce L-valine already during growth phase.<br />

These results show that the rational combination of substrates increases the<br />

productivity of already existing C. glutamicum strains for amino acid synthesis<br />

by boosting the precursor supply during fermentation.<br />

[1] Blombach et al. (2007); Appl Environm Microbiol 73:2079-2084<br />

[2] Engels et al. (2007); J Bacteriol 189: 2955-2966<br />

[3] Blombach et al. (2008); Appl Microbiol Biotechnol 79: 471-479<br />

PX 16<br />

Expression of isomaltulose synthase (PalI) in Lactococcus<br />

lactis<br />

S. Scholtz *1 , K.J. Heller 1 , A. Geis 1<br />

1 Institute of Microbiology und Biotechnology, Max Rubner-Institut (Location<br />

Kiel), Kiel, Germany<br />

Isomaltulose synthase (PalI) from the Gram-negative bacterium<br />

Protaminobacter rubrum CBS 574.77 catalyzes conversion of sucrose to the<br />

major product isomaltulose and small amounts of other mono- and<br />

disaccharides. Thereby, an α-1,2 glycosidic bond is hydrolyzed and an α-1,6<br />

glycosidic bond is formed. Isomaltulose, commonly referred to as palatinose,<br />

has growing importance in industry, not only in the food sector but also in<br />

animal feed, medicines and cosmetics.<br />

For further studies we used gram positive Lactococcus lactis strains which have<br />

GRAS status. palI was cloned in strains Bu2-60 and MG1363. The nisin<br />

inducible commercial two component NICE expression system was used. The<br />

system consists of an expression vector called pNZ8037. Additionally, two<br />

different regulatory plasmids were used to mediate variable levels of<br />

expression. After removal of the DNA region encoding the Gram-negative<br />

signal sequence, the remaining palI gene was fused with the start codon of the<br />

nisA gene located directly downstream of the inducible promoter PnisA on<br />

pNZ8037. The palI gene was induced with different nisin concentrations to<br />

show dose-dependent expression.<br />

Enzymatic activity was detected by a rapid photometric test, the tetrazolium<br />

blue assay, which measures reducing sugars. More detailed information on<br />

activity of PalI and yields of isomaltulose after enzymatic conversion of<br />

sucrose were obtained by HPLC-RI, in which small amounts of the by-products<br />

glucose and fructose were seen as well.<br />

PX 17<br />

Enzymatic hydroxylation of dibenzofuran by fungal<br />

peroxygenases<br />

E. Aranda *1 , M. Kinne 2 , R. Ullrich 2 , M. Hofrichter 2 , G. Kayser 1<br />

1<br />

Unit of Environmental Process Engineering, International Graduate School<br />

Zittau, Zittau, Germany<br />

2<br />

Environmental Biotechnology, International Graduate School Zittau, Zittau,<br />

Germany<br />

This study describes ring-hydroxylations of dibenzofuran (DBF) at different<br />

positions by aromatic peroxygenases from the agaric mushrooms Agrocybe<br />

aegerita and Coprinellus radians. DBF is a heterocyclic model compound that<br />

had been used already in a number of degradation studies, since it is a persistent<br />

pollutant from diverse emission sources. Differences were observed in the<br />

extent of DBF conversion and the hydroxylation pattern of both peroxygenases.<br />

A. aegerita peroxygenase (AaP) converted almost 100% of DBF while C.<br />

radians peroxygenase (CrP) transformed only 40% un<strong>der</strong> the same conditions.<br />

Major metabolites formed were mono-hydroxylated DBF <strong>der</strong>ivatives.<br />

Moreover, a total of 11 metabolites – one tri-hydroxylated, eight dihydroxylated<br />

and two mono-hydroxylated products – were detected after the<br />

treatment of DBF with AaP in the presence of the radical scavenger ascorbic<br />

acid. These metabolites were tentatively identified by LC-MS analyses. In<br />

contrast to AaP, that was able to introduce up to three hydroxyl groups into the<br />

molecule, only di-hydroxylated and mono-hydroxylated DBF <strong>der</strong>ivatives were<br />

found in the reaction of CrP. In the absence of the radical scavenger, only<br />

smaller amounts of mono- and di-hydroxylated metabolites were detectable<br />

both in case of CrP and AaP (maybe part of the hydroxylation products was<br />

oxidatively polymerized). Based on these results, we propose that extracellular<br />

peroxygenases are involved in the oxidation of DBF and related aromatic<br />

heterocycles by certain fungi un<strong>der</strong> natural conditions. This type of reaction<br />

may be of general biotechnological and ecological interest, since selective<br />

oxygenations belong to the most difficult reactions in chemical synthesis and<br />

are known to initiate various microbial degradation pathways, respectively.<br />

PX 18<br />

Bioconversion of rye straw at elevated temperatures<br />

V. Bockemühl *1 , T. Ingram 2 , F. Hahn 1 , L. Popper 3 , I. Smirnova 2 , G. Brunner 2 ,<br />

G. Antranikian 1<br />

1<br />

Institute of Technical Microbiology, Hamburg University of Technology,<br />

Hamburg, Germany<br />

2<br />

Institute of Thermal Separation Processes, Hamburg University of<br />

Technology, Hamburg, Germany<br />

3<br />

Research and Development, SternEnzym, Ahrensburg, Germany<br />

Due to the shortage of fossil resources the bioconversion of biomass to high<br />

value products such as fine chemicals and biofuels has recently attracted the<br />

interest of scientists from academia and industry. The choice of the most<br />

suitable substrate and the process will be crucial for the success of the future<br />

biobased industry. In mainstream processes large quantities of chemicals, such<br />

as sulfuric acid, are added to hydrolyze the cellulosic material in or<strong>der</strong> to make<br />

it accessible for enzyme action. Here, we propose a novel process that allows<br />

the efficient conversion of rye straw to utilizable products without the use of<br />

chemicals. Liquid hot water treatment at elevated temperatures and a pressure<br />

of 50 bar was used to make the cellulosic material accessible for hydrolytic<br />

enzymes. The resulting liquid hydrolyzates as well as the solid residues were<br />

enzymatically converted to monomeric sugars (xylose/glucose) using heat<br />

stable enzymes from three different fungi (mesophilic and thermophilic). The<br />

enzymes were active between 50°C and 75°C at pH 4.0. These thermoactive<br />

enzyme systems contained endoglucanase, exoglucanase, β-glucosidase,<br />

endoxylanase and β-xylosidase activities. HPLC analysis showed that glucose<br />

and xylose are the major products formed and 98% of the initial xylan and 92%<br />

of the glucan was converted to monomeric sugars using a pretreatment<br />

temperature of 200°C. The described process using a fixed-bed reactor<br />

combines several advantages compared to other reactor types, namely<br />

significant energy savings since no biomass comminution is necessary, high<br />

solid-to-water-ratios and reduces by-product formation.<br />

We thank the Deutsche Bundesstiftung Umwelt (DBU) for financial support<br />

(AZ 13157-32).<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PX 19<br />

DNA-Transfer into Bacillus licheniformis by transconjugation<br />

and natural competence<br />

M. Rachinger *1 , A. Wollherr 1 , J. Bongaerts 2 , A. Ehrenreich 3 , R. Daniel 1 , W.<br />

Liebl 3 , H. Liesegang 1<br />

1 Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen,<br />

Germany<br />

2 Global R&D Laundry and Home Care, Henkel AG & Co. KGaA, Düsseldorf,<br />

Germany<br />

3 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising,<br />

Germany<br />

Bacillus licheniformis is an organism of great scientific and industrial interest<br />

[4,6]. The analysis of this important bacteria suffers from poor genetic<br />

accessibility. Therefore DNA transfer is a central problem for the preparation of<br />

genetic knock outs in B. licheniformis. Thus far there are only two published<br />

systems for the introduction of DNA into B. licheniformis, natural competence<br />

and the polyethylenglycol (PEG)-mediated transformation of protoplasts [1].<br />

Natural competence is described in many Bacillus species [2,5]. B.<br />

licheniformis 9945A, a close relative of B. licheniformis DSM13, exhibits a<br />

very efficient natural competence [3]. We have examined this strain to try to<br />

un<strong>der</strong>stand using comparative analysis why B. licheniformis DSM13 does not<br />

show a comparable natural competence.<br />

The PEG-transformation, as the most commonly used system, is a laborious<br />

procedure which suffers from poor reproducibility. Therefore we developed an<br />

alternative system based on trans-conjugative plasmid transfer.<br />

[1] Chang, and Cohen (1979) Molec. gen. Genet. 168, 111-115<br />

[2] Chen et al. (2007) Nat. Biotechnol. 25, 1007-1014<br />

[3] Gwinn, and Thorne (1963) J. Bacteriol. 87, 519-526<br />

[4] Rey et al. (2004) Genome Biol. 5, R77<br />

[5] Spizizen (1958) Proc. Natl. Acad. Sci.USA 8<br />

[6] Veith et al. (2004) J. Mol. Microbiol. Biotechnol. 7, 204–211<br />

PX 20<br />

Screening, isolation and characterisation of L-glucitoldehydrogenases<br />

for the production of D-sorbose<br />

S. Gauer *1 , F. Giffhorn 1 , G.W. Kohring 1<br />

1 Applied Microbiology, Saarland University, Saarbruecken, Germany<br />

The rare sugar D-Sorbose is interesting for use as a low calorie sweetener and<br />

building block for further syntheses of complex compounds. In the past, Dsorbose<br />

was produced chemically with a yield of about 70% and Huwig et al.<br />

[1] described a microbial conversion of L-glucitol to D-sorbose with a yield of<br />

95% using Pseudomonas spec. cells. However, this strain lost the activity<br />

during storage in the culture collection, but the N-terminal AA sequence<br />

revealed 85% identity to a putative ribitol-dehydrogenase from Bradyrhizobium<br />

japonicum USDA110. As the genome of this bacterium is sequenced<br />

completely, the isolation of the gene with PCR-techniques and therewith the<br />

expression of the ribitol-dehydrogenase in E. coli BL21 was easy to perform.<br />

An N-terminal His6-Tag was added to the protein for simple enrichment by<br />

affinity chromatography on Ni-sepharose giving a final specific activity of 0.9<br />

U/mg for the substrate L-glucitol. Other substrates of the enzyme are xylitol<br />

(2.4 U/mg), ribitol (2.9 U/mg), mannitol (10.3 U/mg) and D-glucitol (88<br />

U/mg), which would classify the enzyme as a D-glucitol dehydrogenase. In<br />

or<strong>der</strong> to isolate other L-glucitol-dehydrogenases, soil samples from different<br />

locations were screened for bacteria able to grow with L-glucitol as the sole<br />

carbon source. The crude extract from the isolate „K5“ exhibited a specific<br />

activity of 1.6 U/mg with L-glucitol, which is comparable to Pseudomonas<br />

spec. [2]. This enzyme is now subject to further biochemical characterization,<br />

cloning and sequencing.<br />

[1] Huwig et al., Carbohydrate Res. 281, 183-186 (1996);<br />

[2] Mayers-Küntzer et al., J. Biotechnol. 36, 157-164 (1994)<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PX 21<br />

Systematic expression analysis in continuous cultures of<br />

solventogenic and acidogenic Clostridium acetobutylicum<br />

cells<br />

C. Döring 1 , D. Krauß *2 , A. Ehrenreich 2<br />

1 Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen,<br />

Göttingen, Germany<br />

2 Lehrstuhl für Mikrobiologie, Technische Universität München, München,<br />

Germany<br />

Solvent producing Clostridia like Clostridium acetobutylicum are very<br />

interesting organisms for developing biorefinery technologies. Although the<br />

metabolic shift from the acidogenic to the solventogenic growth phase has been<br />

studied in consi<strong>der</strong>able detail in recent years, the regulation of the switch is still<br />

not well un<strong>der</strong>stood.<br />

Within the „SysMo“ collaborative project „COSMIC“ we perform<br />

transcriptional analysis to identify the key-regulatory elements of the shift and<br />

use the data as basis for mathematical modeling of the various interactions at<br />

cellular level.<br />

We prepared defined continuous culture experiments un<strong>der</strong> phosphate-limited<br />

conditions. The pH-dependent transition from acetogenesis to solventogenesis<br />

in the chemostat allows precise and reproducible acquisition of transcriptional<br />

data, a prerequisite for quantitative modelling.<br />

We compared the gene expression during the dynamic shift from the acidogenic<br />

to the solventogenic growth phase. When we focused on the ABE metabolism,<br />

the DNA microarray analysis pointed out that the acetoacetate decarboxylase<br />

(adc) and the sol-operon seem to be the main regulatory elements in this<br />

pathway.<br />

To investigate the consequences of decreasing hydrogenase activity on the<br />

expression of solventogenic genes, we constructed plasmids containing parts of<br />

antisense RNA for hydrogenase hydA. We verified transcription of antisense<br />

RNA by northern blot experiments for the detection of antisense RNA.<br />

Quantitative real-time RT-PCR confirmed the results of northern blot<br />

experiments. Therefore we measured the hydrogenase activity from all strains<br />

and analysed the solventogenic and acidogenic products in continuous culture<br />

as compared to the wildtyp.<br />

PX 22<br />

D-Glucitol Dehydrogenase as a model protein for the<br />

electroenzymatic production of enantiopure building blocks<br />

T. Klein *1 , P. Kornberger 1 , G. Janine 2 , H. Natter 2 , R. Hempelmann 2 , F.<br />

Giffhorn 1 , G.W. Kohring 1<br />

1 Applied Micorbiology, Saarland University, Saarbruecken, Germany<br />

2 Physical Chemistry, Saarland University, Saarbruecken, Germany<br />

D-Glucitol dehydrogenase or sorbitol dehydrogenase (SDH) was first isolated<br />

and characterized from the phototrophic bacterium Rhodobacter sphaeroides<br />

Si4 [1]. The SDH is a short chain alcohol dehydrogenase with a catalytically<br />

active homodimer consisting of 27 kDa subunits (256 amino acids) and the<br />

GXXXGXG cofactor binding motif. Cloning of the gene and heterologous<br />

overexpression of the SDH in E. coli resulted in a purified protein with a<br />

specific activity of 42.3 U/mg [3]. This enzyme preparation was used for<br />

crystallization and structure determination [2], which, in contrast to<br />

biochemical data, exhibited the possibility of a tetrameric organisation of the<br />

protein. Because the enzyme is well characterized and has no need for further<br />

cofactors like cations, it will be used in the 7thFP EU-project ERUDESP for the<br />

construction of electroenzymatic reactors, which are intended to use<br />

immobilized enzymes and cofactors on nanostructured electrodes for the<br />

production of enantiopure fine chemicals. A proof of concept has been<br />

published [4] with the enzyme attached via a mediator to the electrode. For<br />

direct binding of the SDH to the gold surface a His-tag was added to the gene,<br />

elongated by the addition of several Cys residues and expressed in E. coli<br />

BL21. Successful immobilization can be shown by SPR determination and<br />

activity of the enzyme by cyclovoltametry.<br />

[1] Schau<strong>der</strong> et al., Microbiol. 141, 1857-1863 (1995);<br />

[2] Philippsen et al., Acta Cryst. D 61, 347-379 (2005);<br />

[3] Stein et al., J. Bacteriol. 179, 6335-6340 (1997);<br />

[4] Gajdzik et al., J. solid state Electrochem. 11, 144-149 (2007)<br />

199

200<br />

PX 23<br />

Pseudomonas stutzeri DSM5190 T as a potential production<br />

strain for the compatible osmolyte hydroxyectoine<br />

B. Seip 1 , M. Stein 1 , E. Galinski 1 , M. Kurz *1<br />

1 Institut für Mikrobiologie und Biotechnologie, Rheinische Friedrich Wilhelms-<br />

Universität Bonn, Bonn, Germany<br />

Recently we reported the surprise discovery of hydroxyectoine (HOE)<br />

biosynthesis in the well known organism Pseudomonas stutzeri (strain<br />

DSM5190 T ). Here we present an evaluation of this strain with respect to its<br />

usability in large scale production.<br />

The actual industrial scale producer for HOE is Halomonas elongata. But in<br />

this organism HOE is only found un<strong>der</strong> special growth conditions and then in<br />

minor amounts alongside ectoine. Thus we have to use an arduous procedure<br />

for purification. P. stutzeri DSM5190 T in contrast produces nearly exclusively<br />

HOE and therefore has the potential to replace H. elongata in large scale<br />

biosynthesis. The organism can be grown in a lab scale fermenter at 5% salinity<br />

to reasonable cell densities within 2 days. Interestingly, like H. elongata the<br />

strain is milkable [Sauer and Galinski 1997], which greatly simplifies the<br />

recovery of the product. Although improvements are still necessary, with this<br />

we are positive to have found a new candidate for HOE production.<br />

PX 24<br />

Glycerol degradation by psychrophilic bacteria<br />

A. Lichtfuß *1 , G. Antranikian 1<br />

1<br />

Institute of Technical Microbiology, Hamburg University of Technology,<br />

Hamburg, Germany<br />

The growth of biodiesel consumption during the last years has led to an<br />

increased production of glycerol. Consequently, there is a need to find new<br />

applications for this product. Although glycerol already has versatile areas of<br />

application, the production still exceeds the need. Thus, when looking for new<br />

possible uses of glycerol, it is not only focused on chemical conversions, but<br />

also on microbial and enzymatic bioconversions.<br />

In this project, glycerol degradation by different psychrophilic bacteria was<br />

analyzed. The increasing interest in psychrophilic and psychrotolerant bacteria<br />

and their enzymes is due to the need for cold-active enzymes for various<br />

applications, such as food, detergent, chemical and pharmaceutical industries.<br />

Bacteria from two different habitats were chosen for this work: single strains<br />

isolated from samples taken in Spitsbergen and a bacterial consortium <strong>der</strong>ived<br />

from a sample taken from Mariana Trench in 10,000 m depth of the deep-sea<br />

bed. These bacteria were isolated at temperatures between 4°C and 15°C. The<br />

screening for glycerol degradation by the bacteria was performed in two<br />

consecutive steps. First, growth on medium containing glycerol as sole carbon<br />

source was analyzed. Subsequently, the decrease of glycerol in the medium was<br />

monitored by enzymatic determination and HPLC analysis. Overall, ten<br />

psychrophilic bacterial strains capable to degrade glycerol were identified.<br />

During aerobic growth at 15°C on 12.6 g/L glycerol, three of the strains<br />

completely depleted the substrate within 5 days.<br />

PX 25<br />

Genetic and biochemical studies on steroid degradation in<br />

Pseudomonas sp. strain Chol1<br />

A. Birkenmaier 1 , V. Suvekbala 1 , N. Jagmann 1 , B. Philipp *1<br />

1 Mikrobielle Ökologie, Universität Konstanz, Konstanz, Germany<br />

Bacterial transformation of natural steroids is of high relevance for producing<br />

pharmaceutical steroids, such as hormones or anti-inflammatory drugs. Despite<br />

its biotechnological importance, bacterial metabolism of steroid compounds is<br />

not well un<strong>der</strong>stood. Pseudomonas sp. strain Chol1 grows with the bile salt<br />

cholate, a surface-active steroid, as a source of carbon and energy. Strain Chol1<br />

degrades cholate by A-ring oxidation and β-oxidation of the acyl side chain to<br />

7,12-dihydroxy-androsta-1,4-diene-3,17-dione (DHADD). During β-oxidation,<br />

an acetyl- and a propionyl-residue are cleaved from the steroid skeleton.<br />

DHADD is converted to 3,7,12-trihydroxy-9,10-seco-1,3,5(10)-androstatriene-<br />

9,17-dione (THSATD), which is further degraded to CO2. To analyze reaction<br />

steps of the β-oxidation, transposon mutants were generated that had a block in<br />

the degradation of the acyl side chain, and accumulating degradation<br />

intermediates were identified by LC-MS/MS and NMR spectroscopy. Mutant<br />

R1 was interrupted in a gene encoding a putative acyl-CoA-dehydrogenase and<br />

accumulated the degradation intermediate 7α,12α-dihydroxy-3-oxopregna-1,4diene-20-carboxylate<br />

(DHOPDC). Mutant G12 was interrupted in a gene<br />

encoding a putative β-ketothiolase and accumulated degradation intermediates<br />

preceding DHOPDC. In cell extracts, oxidation of the A-ring, activation of<br />

cholate and DHOPDC with CoA and conversion of DHADD to THSATD<br />

could be shown. Enzymatic reactions for degradation of the acetyl- and the<br />

propionyl-residues were also detected. Based on these results, the degradation<br />

pathway from cholate to THSATD could be reconstructed in vitro. The<br />

deepened un<strong>der</strong>standing of metabolic pathways and the identification of novel<br />

genes for β-oxidation of steroid side chains might open new ways for metabolic<br />

engineering of bacteria used for biotransformation of natural steroids.<br />

PX 26<br />

Screening for molecules interfering with quorum sensing<br />

N. Weiland *1 , F. Symanowski 2 , N. Pinnow 1 , R. Schmitz-Streit 1<br />

1<br />

Institut für Allgemeine Mikrobiologie, CAU Kiel, Kiel, Germany<br />

2<br />

IFM-GEOMAR, Forschungsbereich 3: Marine Ökologie, CAU Kiel, Kiel,<br />

Germany<br />

Despite their apparent simplicity, bacteria can form complex associations with<br />

other organisms and act as "multicellular organisms" by the use of small signal<br />

molecules to communicate with one another and with their eukaryotic hosts<br />

(Bassler and Losick, 2006). This quorum sensing (QS) process enables a<br />

bacterial population to recognize and respond to their environment (Fuqua et<br />

al., 1996). The formation of biofilms for instance which are ubiquitous but also<br />

objectionable or even harmful is directly dependent on QS. In or<strong>der</strong> to prevent<br />

undesirable biofilms by interrupting bacterial communication, we aim to<br />

identify compounds which interfere with quorum sensing. As metagenomic<br />

libraries are rich sources for isolating novel bioactive compounds and genes,<br />

large insert libraries from the microbial consortia on Aurelia aurita tissues and<br />

a marine biofilm have been constructed and screened for quorum quenching<br />

compounds. Additionally, bacteria isolated from the surface of several marine<br />

eukaryotes were analyzed for quorum quenching activities.<br />

To screen for biomolecules interfering with intracellular or intercellular QS we<br />

constructed E. coli reporter strains. Using these reporter strains we identified<br />

several metagenomic clones and a few bacterial isolates from the surface of<br />

marine eukaryotes synthesizing biomolecules which interfere with quorum<br />

sensing. On or<strong>der</strong> to identify the quorum quenching compounds the respective<br />

metagenomic clones and isolates were further characterized using molecular<br />

tools.<br />

[1] Bassler B.L., Losick R. 2006. Cell, 125 (2): 237 - 246<br />

[2] Fuqua C. et al. 1996. Annual Review of Microbiology, 50: 727-751<br />

PX 27<br />

Synthetic polyester hydrolysis by a hydrolase from<br />

Thermobifida fusca KW3<br />

R. Wei *1 , T. Oeser 1 , C. Föllner 1 , W. Zimmermann 1<br />

1 Department of Microbiology and Bioprocess Technology, University of<br />

Leipzig, Leipzig, Germany<br />

Thermobifida fusca KW3 is a thermophilic actinomycete capable of degrading<br />

different synthetic polyesters such as poly (ethylene terephthalate) (PET) [1].<br />

An extracellular 28 kDa hydrolase (Tfu_0883) from T. fusca DSM 43793 has<br />

been recently identified. The enzyme consists of 261 amino acids and<br />

hydrolyzes PET and cutin, a plant polyester [2]. T. fusca produces a further<br />

hydrolase (Tfu_0882) with a very similar amino acid sequence and catalytic<br />

properties compared to Tfu_0883 [3].These enzymes can be applied for the<br />

surface modification of PET fibers in the textile industry [4]. Generation of a<br />

3D structural model of the polyester-degrading hydrolase Tfu_0882 from T.<br />

fusca KW3 by homology modeling suggested that its catalytic site is located in<br />

a strongly hydrophobic region of the enzyme enabling the hydrolysis of<br />

recalcitrant aromatic polyesters.<br />

[1] Alisch M, Feuerhack A, Müller, H, Mensak, B, Andreaus J, Zimmermann<br />

W (2004) Biocatalytic modification of polyethylene terephthalate fibres by<br />

esterases from actinomycete isolates. Biocatalysis and Biotransformation<br />

22:347-51<br />

[2] Kleeberg I, Welzel K, van den Heuvel J, Müller RJ, Deckwer WD (2005)<br />

Characterization of a new extracellular hydrolase from Thermobifida fusca<br />

degrading aliphatic-aromatic copolyesters. Biomacromolecules 6:262-70<br />

[3] Chen S, Tong X, Woodard RW, Du G, Wu J, Chen J (2008) Identification<br />

and characterization of bacterial cutinase. J Biol Chem 283(38):25854-62<br />

[4] Feuerhack A, Alisch-Mark M, Kisner A, Pezzin SH, Zimmermann W,<br />

Andreaus J (2008) Biocatalytic surface modification of knitted fabrics made of<br />

poly (ethylene terephthalate) with hydrolytic enzymes from Thermobifida fusca<br />

KW3b. Biocatalysis and Biotransformation 26(5): 357-64<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PX 28<br />

The Transportome of Corynebacterium glutamicum -<br />

Identification and characterization of transport proteins in<br />

a biotechnological workhorse<br />

K. Marin *1 , T. Radespiel 1 , E. Jolkver 1 , M. Follmann 1 , A. Wittmann 1 , M.<br />

Becker 1 , B. Boltres 1 , T. Mohrbach 1 , M. Ashfaq 1 , R. Krämer 1<br />

1 Institute of Biochemistry, University of Cologne, Cologne, Germany<br />

The description of cellular metabolic fluxes comprises the synthesis and<br />

degradation of intermediates as well as their import and export. Transport<br />

capacities are therefore determinants for the survival of bacteria in nature as<br />

well as for optimization of biotechnological productions. The apathogenic soil<br />

bacterium Corynebacterium glutamicum is one of the workhorses in<br />

biotechnology in particular for amino acid production. Furthermore the<br />

bacterium serves as a model organism for general aspects of biotechnological<br />

microbiology and systems biology.<br />

The analysis of the C. glutamicum genome revealed about 400 genes encoding<br />

putative transporters. They belong to more than 50 known transporter classes<br />

whereby ABC type and MFS transporter represent the dominant carrier<br />

families. Beyond this numerous unknown membrane proteins have to be<br />

consi<strong>der</strong>ed as putative transporters. Interestingly, less than 40 carriers were<br />

biochemically characterized. By a systematic approach we are aiming to create<br />

a knock out library for all transporter encoding genes. For the subsequent<br />

screening of this growing mutant collection we developed a MTP based<br />

cultivation system for C. glutamicum in or<strong>der</strong> to investigate numerous different<br />

growth conditions for the high number of different mutants in parallel. The<br />

application of this method revealed in combination with other cultivation<br />

methods candidates for new (putative) carriers for monocarboxylic acids, new<br />

amino acid import systems and ion channels. By classical biochemical analyses<br />

the transport activity was confirmed and kinetic parameters obtained as well as<br />

the substrate spectra. Finally the regulation of transport activities at the<br />

biochemical and gene expression level will be addressed.<br />

PX 29<br />

Production of industrially important chiral products using<br />

Gluconobacter oxydans enzymes<br />

P. Schweiger *1 , U. Deppenmeier 1<br />

1 Institut für Mikrobiologie & Biotechnologie, Universität Bonn, Bonn, Germany<br />

Many useful organic compounds, such as pharmaceuticals and food additives,<br />

with symmetric carbons and enantiomeric forms exist. It is common to have a<br />

single biologically active enatiomer, while the other not showing activity and<br />

sometimes having a harmful effect. In such cases chemically synthesized<br />

racemic mixtures cannot be used, especially for pharmaceuticals. Enzymes<br />

often attack their substrates regio- and stereoselectively making them naturally<br />

suited for the production of chiral intermediates in high enantiomeric excess.<br />

The Gluconobacter oxydans genome is known and contains 77 uncharacterized<br />

oxidoreductases. Investigation into their function via heterologous gene<br />

expression in E. coli has revealed many oxidoreductases that reduce α,βdiketones,<br />

α-ketoaldehydes and vinyl ketones and are capable of producing<br />

chiral intermediates that may find uses in industry (e.g. pharmaceutical, food<br />

additives and fragrance). Four enzymes reduced α,β-diketones, two of which<br />

did so stereospecifically producing 2-hydroxy ketones. Additionally, three<br />

enzymes reduced the olefinic bonds vinyl ketones. Two of these enzymes<br />

produced stereospecific products when the olefinic bond was substituted. These<br />

enzymatic reactions require expensive cofactors (e.g. NADPH), however<br />

increased yields and decreased production cost is achievable when cofactor is<br />

regenerated. Therefore, a coupled enzyme biotransformation scheme was<br />

developed that allows efficient cofactor regeneration and increased product<br />

yields.<br />

PX 30<br />

Deletion of membrane bound dehydrogenases in G. oxydans<br />

DSM 7145<br />

J. Voss 1 , A. Junker *2 , M. Kähler 3 , A. Ehrenreich 2 , W. Liebl 2<br />

1 Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen,<br />

Göttingen, Germany<br />

2 Lehrstuhl für Mikrobiologie, Technische Universität München, Freising,<br />

Germany<br />

3 Dr. Rieks GmbH, Uetersen, Germany<br />

Gluconobacter oxydans is unsurpassed in its ability to incompletely oxidize<br />

sugars, sugar alcohols and polyols in a regio- and stereoselective manner. The<br />

incomplete oxidation of substrates is catalyzed by membrane bound<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

dehydrogenases. Deletion of the major polyol dehydrogenase revealed that this<br />

enzyme has a broad substrate spectrum, ranging from C3-C6 polyols to 5-keto-<br />

D-gluconate, while the membrane bound D-glucose dehydrogenase seems only<br />

to be involved in the oxidation of D-glucose. Interestingly, the glucose<br />

dehydrogenase mutant was still able to grow on D-glucose maybe by increased<br />

flux through the central metabolic pathways.<br />

To increase the yield of biotechnologically important substances, such as Lsorbose<br />

or dihydroxyacetone, we have recently chromosomally replaced the<br />

glucose dehydrogenase by the major polyol dehydrogenase.<br />

Marker-less gene deletion in G. oxydans remains labor intensive. Therefore we<br />

develop a new procedure based on the use of upp (Gox0327) as a novel<br />

counter-selectable marker in the presence of the toxic pyrimidine analogue 5fluorouracil<br />

(5FU). The upp gene codes for a uracil phosphoribosyltransferase,<br />

that converts 5-fluorouracil into 5-fluoro-dUMP, a very potent inhibitor of<br />

thymidylate synthetase. Accordingly, while the wild type of G. oxydans 621H<br />

is sensitive to 5FU, an in frame upp deletion mutant is resistant. When upp was<br />

ectopically introduced into the deletion mutant, sensitivity to 5-FU can be<br />

restored. We use this as a strategy for counter-selection in a deletion strategy.<br />

PX 31<br />

Modified regulatory sequence is responsible for<br />

constitutive expression of galactitol-dehydrogenase in<br />

Rhodobacter sphaeroides D<br />

P. Kornberger 1 , C. Zimmer 2 , F. Giffhorn 1 , G.W. Kohring *1<br />

1<br />

Microbiology, Saarland University, Saarbruecken, Germany<br />

2<br />

Dept. of Biotechnology & Enzyme Catalysis, Ernst-Moritz-Arndt-University,<br />

Greifswald, Germany<br />

Rhodobacter sphaeroides D is a galactitol metabolizing gain of function mutant<br />

of the wild-type R. sphaeroides Si4 and was selected in a chemostat experiment<br />

based upon its ability to oxidise galactitol to L-tagatose. This reaction is<br />

performed by the enzyme galactitol dehydrogenase (GatDH), which is<br />

expressed constitutively in the mutant R. sphaeroides D only, although the gene<br />

is also present in the parent strain Si4 and an isoform in the type-strain R.<br />

sphaeroides 2.4.1 [1]. The GatDH gene has been identified and sequenced<br />

revealing that the 765 bp nucleotide sequence from strain D was identical to the<br />

gene from strain Si4, but different in 26 nucleotides to GatDH from strain 2.4.1.<br />

However, in contrast to both other strains, the GatDH gene from strain D<br />

showed a supplementary sequence located upstream of the 5’ end which<br />

contained two possible additional ribosomal binding sites and a promotor<br />

sequence at -12, similar to putative puf and puh promotor sequences of R.<br />

sphaeroides [2]. Un<strong>der</strong> the control of the pET promoter the genes from strains<br />

Si4 and 2.4.1 can also be expressed in an active form.<br />

GatDH is a secondary alcohol dehydrogenase with a broad substrate spectrum<br />

and a high enantio selectivity which let it appear as a very potent tool for the<br />

production of enantiopure building blocks in pharmaceutical and agrochemical<br />

syntheses. For application in electroenzymatic reactors the gene was modified<br />

with an N-terminal His(6)-tag coupled with additional cystein residues for<br />

immobilisation on the surface of gold electrodes and tested for activity with<br />

cyclovoltametry.<br />

[1] Schnei<strong>der</strong> KH et al, Microbiol. 141, 1865-1873 (1995);<br />

[2] Bauer CE, Chapter 58, in Blankenship et al (ed.) Anoxygenic<br />

Photosynthetic Bacteria, Kluwer Academic Publishers (1995)<br />

PX 32<br />

Heterologous production of mannosylglycerate in<br />

Halomonas elongata using both alternative biosynthetic<br />

pathways<br />

A. Meffert *1 , C. Lange 1 , A. Burdziak 1 , E.A. Galinski 1<br />

1 Institute of Microbiology & Biotechnology, Rheinische Friedrich-Wilhelms-<br />

Universität, Bonn, Germany<br />

201<br />

Halomonas elongata DSM 2581 T is used for the industrial production of the<br />

compatible solute ectoine according to the bacterial "milking technique" [1].<br />

Other compatible solutes from extremophilic microorganisms are less<br />

accessible, as for example mannosylglycerate (MG) of the thermophilic,<br />

mo<strong>der</strong>ately halophilic Rhodothermus marinus [2]. In response to fluctuations in<br />

both, growth temperature and salinity R. marinus produces this negatively<br />

charged osmolyte, which is widely distributed among (hyper)thermophilic<br />

microorganisms. Two alternative pathways for the synthesis of MG have been<br />

identified: a single-step pathway employing mannosylglycerate synthase (Mgs)<br />

and a two-step-pathway, which uses mannosylphosphoglycerate as an<br />

intermediate (Mpgs and the corresponding phosphatase). In addition,

202<br />

phosphomannose mutase and mannose-1-phosphate guanylyltransferase (helper<br />

enzymes) are needed to ensure supply of GDP-mannose [3].<br />

Using homologous recombination each of the alternative pathways for MG<br />

production of R. marinus (including helper genes) was placed un<strong>der</strong> the control<br />

of a gearbox/ο 70 promotor region (ectA-promotor), thus replacing the original<br />

ectoine gene cluster. The resulting strains (H. elongata KB10.1 and CLB1)<br />

were compared in their ability to synthesize this foreign compatible solute and<br />

to tolerate increased salinity and/or temperature. In addition, vector-encoded<br />

mannosylglycerate genes were co-expressed in or<strong>der</strong> to analyze the influence of<br />

enhanced enzyme levels on MG productivity. From this we were able to<br />

conclude that the salt-controlled two-step pathway is more efficient in H.<br />

elongata and that enhanced levels of synthesizing enzymes also improve<br />

results.<br />

* AM and CL contributed equally to this work.<br />

[1] Sauer T, Galinski EA (1998) Biotechnol Bioeng 57: 306-313<br />

[2] Alfredsson, GA. et al. (1988) J Gen Microbiol 134: 299-306<br />

[3] Burdziak, A. (2006) Dissertation, Universität Bonn<br />

PX 33<br />

Production of the rare compatible solute NAGGN in H.<br />

elongata and E. coli DH5α<br />

A. Korsten *1 , E.A. Galinski 1<br />

1 Institute of Microbiology & Biotechnology, Rheinische Friedrich-Wilhelms-<br />

Universität, Bonn, Germany<br />

The compatible solute NAGGN (N-acetyl-glutaminyl-glutamine-1-amide) has<br />

been reported from phototrophic bacteria like Thiocapsa halophila [1],<br />

rhizobia, e.g. Rhizobium meliloti [2] and some pseudomonads, as for example<br />

P. putida and P. aeruginosa [3]. Most of these organisms are characterized as<br />

mo<strong>der</strong>ately salt-tolerant soil or water bacteria. The solute NAGGN usually<br />

occurs in combination with other compatible solutes (e.g. trehalose, mannitol)<br />

and, therefore, at relatively low concentrations in the cells. The simultaneously<br />

synthesized co-solutes furthermore impair purification of the compatible solute<br />

NAGGN. In or<strong>der</strong> to facilitate production, the biosynthetic NAGGN genes of<br />

Pseudomonas putida KT2440 were integrated into the genome of Halomonas<br />

elongata by double homologous recombination, replacing the gene cluster of<br />

the natural compatible solute ectoine. As a consequence the NAGGN gene<br />

cluster is now un<strong>der</strong> control of the salt inducible promotor of the ectoine<br />

biosynthesis genes in the production strain H. elongata AKB. This strain<br />

facilitates purification of NAGGN due to the absence of any uncharged cosolutes.<br />

For reasons of comparison vector pK18-naggn, which was used for<br />

recombination steps in H. elongata, was also transferred into E. coli DH5α.<br />

Here too, heterologous expression of the NAGGN biosynthesis gene cluster<br />

enabled production of this rare, otherwise barely accessible compatible solute<br />

for further biotechnological investigations.<br />

[1] Galinski, E. A. (1992) Kompatible Solute aus Bakterien – Gewinnung,<br />

Anwendung, Struktur und Funktion. Habilitationsschrift. Universität Bonn<br />

[2] Smith, L.T.; Smith, G. M. (1989) An Osmoregulated Dipeptide in Stressed<br />

Rhizobium meliloti. Journal of Bacteriology 171: 4714-4717<br />

[3] Kets, P.W. et al. (1996) Mannitol, a Novel Bacterial Compatible Solute in<br />

Pseudomonas putida S12. Journal of Bacteriology 178: 6665–6670<br />

PX 34<br />

Salt-induced production of novel hydroxylated ectoine<br />

<strong>der</strong>ivatives by whole-cell biotransformation<br />

S. Vielgraf *1 , A. Meffert 1 , M. Stein 1 , E. Galinski 1<br />

1<br />

Institute of Microbiology & Biotechnology, Rheinische Friedrich-Wilhelms-<br />

Universität, Bonn, Germany<br />

Compatible solutes such as ectoines are accumulated by cells as a strategy to<br />

deal with different stress-situations in their environment, and in particular<br />

osmotic stress [1]. Un<strong>der</strong> certain environmental conditions (e.g. elevated<br />

temperature and decreased water activity) ectoine is hydroxylated<br />

stereospecifically by an Fe(II)/α-ketoglutarate-dependent dioxygenase [2;3].<br />

Ectoine hydroxylase (EctD) from the halophilic Halomonas elongata appears to<br />

accept an unusually broad spectrum of substrates, including synthetic ectoine<br />

<strong>der</strong>ivatives. For production of novel hydroxylated compounds for diverse<br />

biotechnological applications, we constructed a whole-cell biotransformation<br />

system in Escherichia coli DH5α, which comprises heterologously expressed<br />

hydroxylase (EctD), a functional citric acid cycle for the regeneration of the cofactor<br />

α-ketoglutarate and the salt-induced compatibles solute uptake systems<br />

proU/proP of the host for supply of educts. In or<strong>der</strong> to coordinate uptake of<br />

educts and conversion into hydroxylated products, the hydroxylase gene (ectD)<br />

was also placed un<strong>der</strong> the control of a salt-induced promoter, resulting in a salt-<br />

induced biotransformation system for hydroxylated ectoine <strong>der</strong>ivatives (E. coli<br />

DH5α VIBO).<br />

When the system was applied to homoectoine, an ectoine-<strong>der</strong>ivative with<br />

enlarged (7-membered) ring structure, cytoplasmic hydroxylation resulted in a<br />

continuous time-dependent accumulation of the product in the medium,<br />

suggesting permanent leakage from and impaired uptake back into the cells.<br />

This surprising observation can only be explained if one or both of the uptake<br />

systems (ProP/ProU) display a relatively higher affinity to the educt than to the<br />

product (the hydroxylated form of homoectoine).<br />

[1] Da Costa, et al. (1998) Adv Biochem Eng Biotechnol 61: 117-53.<br />

[2] Hausinger (2004) Crit Rev Biochem Mol Biol 39(1): 21-68.<br />

[3] Bursy et al. (2007) J Biol Chem 282: 31147-31155.<br />

PX 35<br />

A DmpA-homologous protein in Pseudomonas aeruginosa:<br />

A putative β-Peptidyl aminopeptidase<br />

V. Fuchs *1 , S. Wilhelm 1 , F. Rosenau 1 , K.E. Jäger 1<br />

1 Institute of Molecular Enzyme Technology, Heinrich-Heine-University<br />

Duesseldorf at Forschungszentrum Juelich, Juelich, Germany<br />

β-Peptidyl aminopeptidases are the first known enzymes that are able to<br />

hydrolyze a variety of short β-peptides. The substrate specificities of the<br />

peptidases vary greatly, but the enzymes have common structural properties.<br />

The general reaction of these enzymes is the N-terminal cleavage of β 3 -<br />

homoamino acids from oligopeptides, amides and esters. Recently, it was<br />

shown that these enzymes are also able to couple various β-amino acids to<br />

peptides.<br />

β-Peptides are extraordinarily resistant against degradation by many common<br />

peptidases and proteases and because of these properties, β-peptides are<br />

pharmaceutically interesting agents.<br />

All four known members were isolated from Proteobacteria. The β-Peptidyl<br />

aminopeptidase DmpA from Ochrobactrum anthropi was the first enzyme of<br />

this class to be purified, the other three enzymes originate from<br />

Sphingosinicella xenopeptidilytica, Sphingosinicella microcystinivorans and<br />

Pseudomonas sp. MCI3434.<br />

The putative fifth β-Peptidyl aminopeptidase which we found in Pseudomonas<br />

aeruginosa shows 45% identity to the protein sequence of O. anthropi. The<br />

corresponding gene was obtained by PCR and cloned in the expression vector<br />

pET22b(+). The overexpression was performed in Escherichia coli BL21(DE3)<br />

and the active enzyme was purified by anion-exchange and gelfiltration<br />

chromatography. The purified enzyme shows in a standard assay<br />

aminopeptidase activity.<br />

PX 36<br />

Optimizing a novel biofilm reactor for fine chemicals<br />

production<br />

R. Gross *1 , K. Bühler 1 , A. Schmid 1<br />

1 Lehrstuhl für Biotechnik, Fakultät Bio- und Chemieingenieurwesen,<br />

Technische Universität Dortmund, Dortmund, Germany<br />

Biofilms gain more and more attention as robust and highly productive<br />

biocatalysts for long-term applications in organic synthesis.<br />

We developed a novel two-phase tubular membrane biofilm reactor for fine<br />

chemicals production, specifically for the epoxidation of styrene to (S-)styrene<br />

oxide with an ee > 99.9% by the engineered strain Pseudomonas sp.<br />

VLB120ΔC [1]. In this study, we investigated structural features and reaction<br />

parameters of the catalytic biofilm system with the aim to maximize volumetric<br />

productivity.<br />

Two morphotypes of Pseudomonas sp. VLB120ΔC could be distinguished<br />

during biofilm cultivation composing a defined dynamic equilibrium and<br />

contributing with equal specific activity to the overall biofilm productivity.<br />

This indicates that the biofilm morphological structure was not limiting. As<br />

another possible limitation, mass transfer of product and substrate including<br />

oxygen has been investigated by varying flow rates and selection of different<br />

silicone membrane tube dimensions. We found that the volumetric productivity<br />

correlated with the specific surface area implying a significant contribution of<br />

optimal oxygen concentration to the biofilm performance.<br />

In this optimized system, product inhibition was observed at concentrations<br />

above 500 mM and 1.25 mM in organic phase and aqueous phase, respectively.<br />

The maximum volumetric productivity was improved to 87 g/(Laq day) for over<br />

50 days. Thus, this system can compete successfully with reported approaches<br />

based on suspended cells in continuous operation achieving productivities of 46<br />

g/(Laq day) for 2 days [2].<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

To conclude, this study contributes to a promising application for long-term<br />

sustainable production of fine chemicals by identifying and overcoming the<br />

limiting parameters of this novel catalytic biofilm system.<br />

[1] Gross, R., et al. Biotechnol Bioeng, 2007. 98(6): p. 1123-34.<br />

[2] Park, J.B., et al. Biotechnol Bioeng, 2007. 98(6): p. 1219-29.<br />

PX 37<br />

Heterologous expression of cyanophycin metabolism genes<br />

(cphA1 and cphB1) from Anabaena sp. PCC7120 in<br />

recombinant E. coli.<br />

Y. Abd El Karem *1 , R. Reichelt 2 , A. Steinbüchel 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhems-Universität Münster, münster, Germany<br />

2 Institut für Medizinische Physik und Biophysik, Westfälische Wilhems-<br />

Universität Münster, münster, Germany<br />

Several reports had studied the heterologous expression of cyanophycin<br />

synthetase gene (cphA) from cyanobacteria in recombinant E. coli. Our work<br />

aims at studying the complete metabolism of cyanophycin (CGP) in<br />

recombinant E. coli. Therefore, we have amplified and cloned the<br />

cyanophycinase gene (cphB1) alone or together with the cyanophycin<br />

synthetase (cphA1) as cluster (cph1 cluster) from the genomic sequence of the<br />

heterocyst-forming cyanobacterium Anabaena sp. PCC7120. The cph1 cluster<br />

was amplified with the own promoters by PCR, then cloned into the cloning<br />

vector pCR2.1-TOPO and transferred to E. coli S17-1. Firstly, CGP was<br />

accumulated inside the recombinant cells as small black granules with<br />

disruptive edges indicating the expression of cphA1. Subsequent degradation of<br />

CGP occurred by expression of cphB1 as demonstrated on SDS-PAGE. The<br />

average molecular weight of the partially degraded CGP was 3.5-18 kDa; the<br />

CGP content in the recombinant cells was 3.6% of cellular dry weight. The<br />

crude extracts of the recombinant cells showed degradation activity towards the<br />

main product of CGP degradation β-Asp-Arg dipeptide. We also succeeded in<br />

amplifing cphB1 gene from Anabaena with its own promoter and transferred it<br />

to E. coli S17-1 after cloning in pCR2.1-TOPO. The crude extracts of the<br />

recombinant exhibited degradation activity against CGP as shown on<br />

cyanophycin overlay agar plates.<br />

PX 38<br />

Cloning and overexpression of a novel aryl-malonic acid<br />

decarboxylase from Variovorax sp. HH01<br />

S. Horn *1 , M. Schilhabel 2 , C. Leggewie 3 , K.E. Jaeger 3 , H. Aygün 4 , W.R. Streit 1<br />

1 Microbiology & Biotechnology, University of Hamburg, Hamburg, Germany<br />

2 Institute for Clinical Molecular Biology, Univerity Hospital Schleswig-<br />

Holstein, Kiel, Germany<br />

3 Institute of Molecular Enzyme Technology, Heinrich Heine University<br />

Duesseldorf, Research Centre Juelich, Jülich, Germany<br />

4 BioSpring GmbH, Frankfurt am Main, Germany<br />

We have been investigating a novel and unique enzyme, arylmalonate<br />

decarboxylase (AMDase, E.C. 4.1.1.76, originating from Variovorax sp.<br />

HH01), which catalyses enantioselective decarboxylation of α-aryl-αmethylmalonates<br />

to give optically active α-arylpropionates of high<br />

enantiomeric excess in high yields. The strain harbouring this novel amdA gene<br />

was isolated from a soil sample with aryl malonic acid as the sole carbon and<br />

energy source. A 454 DNA sequence analysis of the isolate identified a gene<br />

cluster carrying the respective amdA gene together with three other genes<br />

possibly linked to the decarboxlyation of arylmalonate. Within the gene cluster<br />

a mandelate racemase (amdC) and a possible regulator (amdR) were identified.<br />

The respective amdA gene linked to the decarboxylation of arylmalonate<br />

encodes a protein with a Mw of 25 kDa. The enzyme has been cloned into an<br />

expression vector and is overexpressed in E.coli. HPLC analysis of the purified<br />

enzyme confirmed the degradation of phenyl malonic acid. Current work<br />

focuses on further biochemical characterization of the AmdA enzyme as well as<br />

a comprehensive look at the substrate spectrum.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PX 39<br />

Improving the thermostability of 1,5-Anhydro-D-fructose-<br />

Reductase by directed evolution<br />

M. Pitz *1 , F. Giffhorn 1<br />

1 Applied microbiology, Saarland University, Saarbuecken, Germany<br />

The 1,5-Anhydro-D-fructose-Reductase (AFR) from Sinorhizobium morelense<br />

catalyses the stereoselective reduction of 1,5-Anhydro-D-fructose (AF) to 1,5-<br />

Anhydro-D-mannitol as well as the conversion of a number of 2-keto aldoses<br />

(osones) to the corresponding manno-configurated aldoses. AFR is strictly<br />

NADPH-dependent, monomeric and has a molecular mass of 35,1 kDa [1]. As<br />

diverse osones can be readily prepared from gluco-configurated aldoses by<br />

regioselective oxidation with pyranose 2-oxidase (P2Ox) [2], rare<br />

pharmaceutical sugars become accessible by coupled redox conversions using<br />

P2Ox and AFR.<br />

In view of the potential of AFR in rare sugar synthesis, we had constructed<br />

NADH-accepting variants (AFR-A13G and AFR-A13G-S33D) by protein<br />

engineering, which were efficiently employed in bioconversions with cosubstrate<br />

regeneration [3].<br />

So far the thermostability of AFR was low and correlated with poor operative<br />

stability. Therefore, we performed directed evolution with AFR, targeting<br />

primarily at enzyme variants with enhanced thermostability. For this reason the<br />

gene encoding AFR-A13G of Sinorhizobium morelense S-30.7.5. was<br />

subjected to error-prone PCR and transformed in E. coli BL21(DE3) pLysS.<br />

Cells were grown in micro plates and lysis was induced by Triton X-100. An<br />

efficient screening protocol in micro scale was carried out, based on heating of<br />

the crude extrakt (10 min at 60°C) and subsequent activity assay by measuring<br />

the decrease of NADPH-absorbance (30°C, 340 nm). Then, AFR-clones with<br />

promising activities were cultivated in complex liquid cultures and reexamined.<br />

Currently 8 variants are studied in respect of heat stability, operative<br />

stability and acceptance of NADH. The detailed results will be presented.<br />

[1] Kühn et al. 2006. Appl. Environ. Microbiol. 72: 1248.<br />

[2] Bastian et al. 2005. Appl. Microbiol. Biotechnol. 67: 654.<br />

[3] Dambe et al. 2006. Biochemistry 45: 10030.<br />

PX 40<br />

Catabolism of the organic disulfide 4,4´-dithiodibutyric acid<br />

(DTDB) by Rhodococcus erythropolis strain MI2<br />

J.H. Wübbeler *1 , M. Wozniczka 2 , A. Steinbüchel 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität, Münster, Germany<br />

2 Management-Public Relations, DSMZ - Deutsche Sammlung von<br />

Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany<br />

DTDB is a putative precursor for the microbial synthesis of polythioester (PTE)<br />

[1] consisting of 4-mercaptobutyric acid (4MB) building blocks. Furthermore,<br />

DTDB is frequently applied in the construction of self-assembly monolayers<br />

formed on gold colloids or gold electrodes. Prior to metabolic engineering and<br />

the development of a process for production of novel PTE, like poly(4MB), it is<br />

essential to obtain bacteria able to utilize the respective PTE-precursors as<br />

carbon source to un<strong>der</strong>stand the catabolism [2, 3]. Since no microorganisms<br />

with the capability of utilizing DTDB as sole carbon source were known,<br />

enrichment cultures were accomplished. All characterised DTDB-utilizing<br />

strains were affiliated to the species Rhodococcus erythropolis. The<br />

chloramphenicol susceptible strain MI2 was suitable for transposon<br />

mutagenesis. Screening of 7.500 mutants yielded three mutants defective in<br />

DTDB-degradation. Extensive physiological studies revealed accumulation of<br />

interesting degradation intermediates in the cultures supernatant. Besides 4MB,<br />

the product of symmetrical cleavage of DTDB, an extraordinary compound<br />

identified as 4-oxo-4-sulfanylbutanoate was detected. Moreover, one of the<br />

mutants released remarkable high amounts of succinate into its supernatant, if<br />

cultivated with DTDB in addition to an utilizable carbon source like sodiumgluconate.<br />

Based on this knowledge, a putative degradation pathway for DTDB<br />

was proposed: DTDB is enzymatically cleaved into two molecules of 4MB,<br />

followed by an oxidation yielding 4-oxo-4-sulfanylbutanoic acid. A putative<br />

desulfhydrase catalyzes the sulfur abstraction, generating succinic acid and<br />

volatile hydrogen sulfide.<br />

[1] Lütke-Eversloh et al. (2001), Microbiology 147,11-19.<br />

[2] Bruland et al. (<strong>2009</strong>), In press doi:10.1074/jbc.M806762200<br />

[3] Wübbeler et al. (2008), Appl. Environ. Microbiol. 74,4028-4035.<br />

203

204<br />

PX 41<br />

StyA2B from Rhodococcus opacus 1CP, the first active<br />

representative of self-sufficient styrene monooxygenases<br />

D. Tischler *1 , D. Eulberg 2 , S. Lakner 2 , W.J.H. van Berkel 3 , S.R. Kaschabek 1 , M.<br />

Schlömann 1<br />

1 Environmental Microbiology, TU Bergakademie Freiberg, Freiberg, Germany<br />

2 Department of Microbiology, University Stuttgart, Stuttgart, Germany<br />

3 Laboratory of Biochemistry, Wageningen University, Wageningen,<br />

Netherlands<br />

Sequence analysis of a 9-kbp genomic fragment from the actinobacterium<br />

Rhodococcus opacus 1CP led to identification of an ORF encoding a novel<br />

fusion protein StyA2B with putative function in styrene metabolism. Whereas<br />

419 amino acids of the N-terminus of StyA2B are highly similar to the<br />

oxygenase subunit of two-component styrene monooxygenases (SMOs) from<br />

pseudomonads, the residual 154 amino acids of the C-terminus show significant<br />

homology to the smaller flavin-reducing subunit of these systems. Similar<br />

fusion proteins were recently found in the genomes of Nocardia farcinica<br />

IFM10152 and Arthrobacter aurescens TC1, however, cloning and functional<br />

expression of his10-StyA2B from strain 1CP revealed for the first time that the<br />

protein does in fact catalyze two functions. Highly stereoselective oxygenation<br />

of styrene and structurally related compounds to the corresponding oxides as<br />

well as a NADH-dependent reduction of flavin-containing cofactors were<br />

shown. Kinetic data of both activities are presented and compared to those<br />

available for two-component SMOs.<br />

The gene styA2B was found to be part of a gene cluster likely to be involved in<br />

styrene degradation. Most remarkably, an ORF (styA1) located directly next to<br />

styA2B was shown to encode an additional oxygenase subunit of a twocomponent<br />

SMO. The absence of single flavin-reductase component as well as<br />

the low oxygenating activity of StyA2B raises the question on a functional<br />

dependence between StyA1 and StyA2B.<br />

PX 42<br />

Global gene expression response of Escherichia coli to pcoumaric<br />

acid<br />

J. Schnei<strong>der</strong> *1 , V.F. Wendisch 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

Production of industrially relevant aromatic compounds from renewable<br />

resources could provide a competitive alternative to chemical synthesis routes.<br />

Escherichia coli is used to produce p-coumaric acid, which can be utilized as<br />

monomer or starting material for the production of novel polymers, flavours,<br />

pharmaceuticals, cosmetics and nutrition products [1]. Furthermore, p-coumaric<br />

acid is a precursor of several commercially interesting phenolic plant<br />

compounds, which also can be produced with recombinant E. coli. In the<br />

present study we extend our previous research [2] concerning the effect of<br />

aromatic compounds on the transcriptome of E. coli.<br />

Our goal was to characterize the specific transcriptome associated with the<br />

presence of p-coumaric acid and to identify potential targets for strain<br />

improvement. Therefore, we investigated the global gene expression changes of<br />

E. coli due to the presence of 3 mM p-coumaric acid in the growth medium. On<br />

the one hand, we found that the addition of p-coumaric acid caused the<br />

induction of genes coding for chemotaxis and flagellation, as well as of genes<br />

involved in the resistance against this compound. On the other hand, several<br />

genes belonging to the acid stress regulon were repressed.<br />

[1] Sariaslani F.S. (2007). Development of a Combined Biological and<br />

Chemical Process for Production of Industrial Aromatics from Renewable<br />

Resources. Annu. Rev. Microbiol., 61:51-69.<br />

[2] Polen T., Krämer M., Bongaerts J., Wubbolts M. & Wendisch V.F. (2005).<br />

The global gene expression response of Escherichia coli to L-phenylalanine. J.<br />

Biotechnol., 115:221-237.<br />

PX 43<br />

Pyruvate selection yields novel producer strains of<br />

Escherichia coli<br />

N. Trachtmann 1 , G. Sprenger *1<br />

1 Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany<br />

Microbial producer strains have usually been screened for production of<br />

valuable compounds. Here we present data on pyruvate-auxotroph Escherichia<br />

coli mutants which were subject to chemostat selection for endogenous<br />

pyruvate sources. First, a double deletion strain with lesions in both pyruvate<br />

kinase genes (pykA, pykF), and in the ppc gene (for TCA anaplerotic PEP<br />

carboxylase) was created. This mutant strain is auxotrophic and needs<br />

supplementation with exogenous pyruvate sources (e.g. D- or L-lactate,<br />

pyruvate, L-alanine) for growth on C-sources as glycerol, ribose or galactose.<br />

The gene pyc for pyruvate carboxylase (from Corynebacterium glutamicum)<br />

was inserted in this mutant strain´s chromosomal mal locus to improve<br />

pyruvate channelling into the TCA cycle. The mutant was then subject to<br />

parallel chemostat selections on glycerol with decreasing lactate amounts until<br />

growth without supplementation occurred. Strains obtained by this pyruvate<br />

selection apparently had opened up endogenous pathways to pyruvate which<br />

circumvented the pyk/ppc block. Among the various mutants thus selected we<br />

found those which use either the methylglyoxal bypass, or those which excrete<br />

2,3-dihydroxybenzoate, an intermediate of enterobactin synthesis. 2,3dihydroxybenzoate<br />

is <strong>der</strong>ived from the aromatic amino acid pathway<br />

intermediate chorismate by cleavage of the pyruvate side chain thus providing<br />

an endogenous pyruvate source which restored growth. The mutation is <strong>bei</strong>ng<br />

studied presently and results of its characterization will be presented<br />

PX 44<br />

Establishing an enzyme assay using 3-Sulfinopropionate (3-<br />

SP) and 3-sulfinopropionyl-CoA (3-SP-CoA) as substrates<br />

M. Schürmann *1 , N. Bruland 1 , J.H. Wübbeler 1 , A. Steinbüchel 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität, Münster, Germany<br />

3-SP was identified as intermediate in the catabolism of 3-mercaptopropionic<br />

acid in the bacterial strains Tetrathiobacter mimigardefordensis strain DPN7 T<br />

and Varivorax paradoxus strain TBAE6 [1, 2]. In both strains 3-SP is activated<br />

to 3-sulfinopropionyl-CoA (3-SP-CoA) by a CoA-ligase or a CoA-transferase,<br />

respectively. Afterwards, 3-SP-CoA is converted to propionyl-CoA catalyzed<br />

putatively in three steps thereby abstracting the sulfur as sulfite. 3-SP and 3-SP-<br />

CoA are not commercially available and the latter is hitherto unknown. To<br />

establish a reliable enzyme assay, both substrates are strictly necessary.<br />

Therefore, synthesis methods of either have to be established. 3-SP was<br />

synthesized and purified with a modified method according to Jolles-Bergeret<br />

[3]. It was subsequently analyzed by HPLC, GC/MS and NMR-spectroscopy.<br />

Synthesis of 3-SP-CoA starting from a cyclic anhydride of 3-SP, similar to the<br />

synthesis of other CoA-<strong>der</strong>ivatives was not possible. The cyclic anhydride of 3-<br />

SP is a very unstable compound and is converted to a polymer insoluble in<br />

methylenechloride [4]. Therefore, 3-chlorosulfinopropionylchloride as an<br />

activated form of 3-SP was synthesized starting from 3-mercaptopropionate.<br />

The subsequent reaction of this more stable compound with CoA should yield<br />

3-SP-CoA. To establish enzyme activity assays, the detection and<br />

quantification of 3-SP-CoA and other CoA <strong>der</strong>ivatives were necessary as well.<br />

This was achieved by HPLC applying a Nucleosil 100 column. In combination<br />

with a photo diode array different CoA-<strong>der</strong>ivatives could be identified<br />

according to their distinctive UV/VIS absorption.<br />

[1] Bruland et al. (<strong>2009</strong>) In press doi:10.1074/jbc.M8067622000<br />

[2] Wübbeler et al. (2008) Appl. Environ. Microbiol. 74,4028-4035.<br />

[3] Jolles-Bergeret, (1973), Eur. J. Biochem., 42,349-352.<br />

[4] Vasil’eva et al. (1984), Russian Chemical Bulletin, 33,570-575.<br />

PX 45<br />

Simple generation of site-directed point mutations in the<br />

Escherichia coli chromosome using Red ® /ET ®<br />

Recombination<br />

R. Heermann 1 , T. Zeppenfeld *2 , K. Jung 1<br />

1 Biologie I / Bereich Mikrobiologie, LMU München, München, Germany<br />

2 Commercial Center, Gene Bridges GmbH, Heidelberg, Germany<br />

Introducing point mutations into bacterial chromosomes is important for further<br />

progress in studies relying on functional genomics, systems- and synthetic<br />

biology, and for metabolic engineering. For many investigations, chromosomal<br />

systems are required rather than artificial plasmid based systems.<br />

Here we describe the introduction of a single point mutation into the<br />

Escherichia coli chromosome by site-directed mutagenesis without leaving any<br />

selection marker. We used Red ® /ET ® Recombination in combination with rpsL<br />

counter-selection to introduce a single point mutation into the E. coli MG1655<br />

genome, one of the widely used bacterial model strains in systems biology. The<br />

method we present is rapid and highly efficient. Since single-stranded synthetic<br />

oligonucleotides can be used for recombination, any chromosomal modification<br />

can be designed.<br />

Chromosomal modifications performed by rpsL counter-selection may also be<br />

used for other bacteria that contain an rpsL homologue, since Red ® /ET ®<br />

Recombination has been applied to several enteric bacteria before<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PX 46<br />

Investigation of tryptophane production from Basillus<br />

subtilis strain in the presence of indole and Iranian cane<br />

molass<br />

M. Dehghan Shasaltaneh *1 , J. Fooladi 1 , S.Z. Moosavi-Nejad 1<br />

1 Department of Biology; Faculty of Basic Sceince, Alzahra University, Tehran,<br />

Iran<br />

Chemical synthesis was the first methods applied to an industrial scale process<br />

for tryptophan production. By the end of the 80s, production of L-tryptophan<br />

was carried out through chemical, enzymatic and fermentation processes.<br />

Microbial fermentation strains allow the production of various amino acids<br />

from cheep and renewable carbon sources such as molasses, sucrose or glucose.<br />

Moreover, industrial production of L-tryptophan nowadays is mostly for feed<br />

and pharmaceutical purposes. L-tryptophan can be produced using varieties of<br />

microorganisms with tryptophan synthase activity. The aim of this study is to<br />

survey the production of L-tryptophan using Bacillus subtilis from Iranian<br />

molasses as C-source.<br />

In the present study, it has been optimized the condition of L-tryptophan<br />

production using a Bacillus subtilis ATCC 6633 strain with Iranian cane<br />

molass. Furthermore, thin layer chromatography (TLC) and high performance<br />

liquid chromatography (HPLC) were used for monitoring the amino acid<br />

tryptophan in culture medium. The maximum tryptophan production was<br />

determined as a function of molass concentration. The effect of indole, serine,<br />

pyridoxal phosphate (PLP) on tryptophan production was also investigated.<br />

Our results showed that the maximum biomass was obtained in the presence of<br />

1% molass concentration in culture medium. Indole caused tryptophan<br />

production and in the other experiment, adding serine in the presence of indole<br />

increased tryptophan production about 21%.<br />

The finding shows that the existence of pyridoxal phosphate had not significant<br />

effect on tryptophan production. Because it is reported that molass source is<br />

used for the fermentation process and moreover it contains L-serine as well as<br />

vitamins such as PLP, therefore we deduce that there is a consi<strong>der</strong>able amount<br />

of serine and enough amount of PLP in Iranian cane molass to produce<br />

tryptophan by Bacillus subtilis.<br />

PY 01<br />

Evaluation of 23SrRNA Point Mutation and rdxA Gene<br />

Deletion in Clarithromycin and Metronidazole Resistance<br />

of Helicobacter pylori Strains from the South West of Iran<br />

M. Kargar *1 , M. Baghernejad 1 , A. Doosti 2 , M. Homayoon 1<br />

1 Microbiology, Jahrom Azad University, Shiraz, Iran<br />

2 Biothecnology, Shahrekord Azad University, Shahrekord, Iran<br />

Background Helicobacter pylori rapidly acquires resistance to many classes of<br />

antibiotics after exposure to them. Resistance of Helicobacter pylori to eather<br />

clarithromycin or metronidazole has been associated with therapeutic failure<br />

and reduced eradication rates with multi - agent treatment regimens. The aim of<br />

this study is determination of point mutation 23SrRNA and rdxA gene deletion<br />

in H.pylori strains at south west of Iran.<br />

Material and Methods: In this cross sectional study, 263 patients with no<br />

history of antibiotic treatment evaluated. By using of biochemical test and<br />

amplification of gene ureC H.pylori strains identified. Antimicrobial<br />

susceptibility was performed according to the CLSI (Clinical and Laboratory<br />

Standards Institue) method. A2142G and A2143 mutations identified by PCR-<br />

RFLP method. Also specific primers were used for detection of A2142C<br />

mutation and inactivation of rdxA gene.<br />

Results: 84 strains (31.94%) confirmed by PCR. 19(22.62%) strains were<br />

resisted to clarithromycin and 49(58.33%) strains to metronidazole. Out of all<br />

clarithromycin resistant strains 68.42%, 15.79% and 10.53% had A2143G,<br />

A2142G, A2142C mutations. Also unknown mutation detected in one strain<br />

(5.33%). Deletion in rdxA gene observed in 2 metronidazole resistant strains.<br />

Conclusion: High clarithromycin resistance in these patients can indicate cross<br />

resistance between this antibiotic and other macrolids such as erythromycin.<br />

According to low frequency mutation in rdxA gene, analysis of mechanism<br />

contributed in metronidazole is suggested.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PY 02<br />

Monitoring of cell size, lipid droplet development and<br />

vitality in the oleaginous yeast Lipomyces lipofer using Flow<br />

Particle Image Analysis (FPIA) and Flowcytometry<br />

D. Raschke *1 , D. Knorr 1 , B. Soh Bejeng Ndikung 1<br />

1 Department of Food Biotechnology and Food Process Engineering, TU Berlin,<br />

Berlin, Germany<br />

Monitoring of the physiological state of cells is of utmost importance for the<br />

fermentation of microorganisms and cell cultures. The combination of the two<br />

methods, Flowcytometry and Flow Particle Image Analysis (FPIA), provides a<br />

powerful tool for measuring vitality, cell size and the development of<br />

intracellular storage compounds. In this approach the oleaginous yeast<br />

Lipomyces lipofer was used due to its ability of building visible intracellular<br />

lipid droplets.<br />

Using FPIA, both cell size and lipid droplet development of L. lipofer can be<br />

analyzed. FPIA visualizes the cells by using a strobe camera. Based on this data<br />

the cell size can be integrated using a mathematical grey-scale calculation.<br />

Flowcytometry can be used to determine the vitality of L. lipofer using a<br />

Propidium- Iodide/ Carboxy-Fluorescein-Diacetate double staining. The<br />

increase of intracellular lipid droplets can be measured using both the<br />

granularity, as well as the fluorescence after Nile Red staining.<br />

It could be shown that vitality was constantly high (above 85%) but slowly<br />

decreasing during growth in YED medium over 4 days. Based on the<br />

flowcytometry data it was demonstrated, that the lipid content increased during<br />

the abovementioned growth period. These findings were confirmed by FPIA<br />

data.<br />

PY 03<br />

Quantification of Yeast within 3 minutes using Methylene<br />

Blue Dye Reduction Test (MBRT)<br />

S. Nandy *1 , K.V. Venkatesh 1<br />

1<br />

Chemical Engineering Department, Indian Institute of Technology, Bombay,<br />

Mumbai, India<br />

To quantify CFU is continually challenged in all biological experiments.<br />

Among a number of different process to estimate CFU, what is the most<br />

efficient and inexpensive approach that the cell activity of any microorganism<br />

can be evaluated quickly? Despite the importance of the different approaches<br />

developed so far to differentiate between metabolic active or inactive cells<br />

suffers from major limitations like time consuming, inaccurate and expensive.<br />

Therefore, the situation needs to be further studied to develop an accurate,<br />

inexpensive and quick process to quantify CFU.<br />

An essential object of the present study is to provide an overview of one<br />

accurate method of detection of CFU within 3min based on wild-type Yeast,<br />

Saccharomyces cerevisiae. In addition, this study also detects the death phase<br />

in the ordinary optical density curve spectrophotometrically and rapidly with a<br />

highly accurate measurement result and also demonstrates the growth effect<br />

directly from MBRT slope. Here, we have developed and standardized one<br />

global techniques by monitoring the dye reduction rate at each time point to<br />

quantify CFU within very short time.<br />

This MBRT test correlates with CFU of all aerobic organisms up to a 1000 live<br />

cells as established by plating accurately within 3 minutes and further this assay<br />

has a wide application specially in food industry such as dairy, cheese, honey<br />

etc. The test developed is very simple, accurate, inexpensive and easy<br />

compared to available techniques.<br />

PY 04<br />

DNA repair pathways contribute to resistance against<br />

translational inhibitors in yeast<br />

S. Wemhoff *1 , R. Klassen 1 , F. Meinhardt 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität, Münster, Germany<br />

205<br />

The killer toxins PaT and zymocin produced by the yeasts Pichia acaciae and<br />

Kluyveromyces lactis display specific anticodon nuclease (ACNase) activities<br />

for tRNA Gln and tRNA Glu , respectively. However, tRNA depletion does not only<br />

impair translation but also affects genome integrity and concomitantly induces<br />

mutations. Accordingly, homologous recombination (HR), base excision repair<br />

(BER) and postreplication repair (PRR) contribute to resistance against both<br />

ACNases [1]. Here, we analysed DNA repair pathways with respect to their<br />

ability to confer resistance to other known translational inhibitors.<br />

Hypersensitivity of a BER mutant defective in two endonucleases (Apn1 and<br />

Apn2) acting on apurinic (AP) sites, revealed indeed a protective effect of BER

206<br />

to several translational inhibitors, including hygromycinB, erythromycin,<br />

geneticin and cycloheximide, whereas sensitivity to neomycin and<br />

paromomycin remained unaltered. A similar, but less pronounced effect was<br />

seen in a HR defective rad52 mutant. A rad18 mutant, defective in PRR,<br />

displayed articulate hypersensitivity to all compounds tested, including<br />

neomycin and paromomycin. Disruption of YKU80, which disables nonhomologous<br />

end joining, was previously shown to confer partial resistance to<br />

the yeast ACNase toxins, though it did not confer similar resistance to any of<br />

the above inhibitors, except for paromomycin. Translational inhibitors might in<br />

general affect DNA-integrity. However, since sensitivity profiles of mutants<br />

differ with respect to the drugs used, distinct DNA repair mechanisms appear to<br />

be differently involved. We are currently checking an increase of mutation<br />

frequencies in cells treated with ribosomal inhibitors.<br />

[1] Klassen, R., Wemhoff, S., Krause, J., and Meinhardt, F. (<strong>2009</strong>). Poster<br />

<strong>VAAM</strong> <strong>2009</strong><br />

PY 05<br />

Quantification of Yeast in 3 minutes using Methylene Blue<br />

Dye Reduction Test (MBRT).<br />

S. Nandy *1 , K. venkatesh 1<br />

1 Chemical engg Dept, Indian Institute of Technology, Bombay, mumbai, India<br />

To quantify CFU is continually challenged in all biological experiments.<br />

Among a number of different process to estimate CFU, what is the most<br />

efficient and inexpensive approach that the cell activity of any microorganism<br />

can be evaluated quickly? Despite the importance of the different approaches<br />

developed so far to differentiate between metabolic active or inactive cells<br />

suffers from major limitations like time consuming, inaccurate and expensive.<br />

Therefore, the situation needs to be further studied to develop an accurate,<br />

inexpensive and quick process to quantify CFU.<br />

An essential object of the present study is to provide an overview of one<br />

accurate method of detection of CFU within 3min based on wild-type Yeast,<br />

Saccharomyces cerevisiae. In addition, this study also detects the death phase in<br />

the ordinary optical density curve spectrophotometrically and rapidly with a<br />

highly accurate measurement result and also demonstrates the growth effect<br />

directly from MBRT slope. Here, we have developed and standardized one<br />

global techniques by monitoring the dye reduction rate at each time point to<br />

quantify CFU within very short time.<br />

This MBRT test correlates with CFU of all aerobic organisms up to a 1000 live<br />

cells as established by plating accurately within 3 minutes and further this assay<br />

has a wide application specially in food industry such as dairy, cheese, honey<br />

etc. The test developed is very simple, accurate, inexpensive and easy<br />

compared to available techniques.<br />

PZ 01<br />

Prevalence of intestinal parasites in a population in south of<br />

Tehran, Iran<br />

A. Rastegar Lari *1 , R. Alaghehbandan 2<br />

1<br />

Department of Microbiology, Iran University of Medical Sciences, Tehran,<br />

Iran<br />

2<br />

Research and Evaluation Department, Centre for Health Information, St.<br />

John’s, Canada<br />

The aim of this study was to describe epidemiologic characteristics of intestinal<br />

parasites in a population in south of Tehran, Iran. A retrospective crosssectional<br />

study of patients with suspicious intestinal parasitic infections referred<br />

to the Zakaria Razi Laboratory in Shahre-Ray, southern Tehran, Iran, was<br />

couducted from April 21, 2004 to October 20, 2005. All stool samples were<br />

examined and socio-demographic information were retrieved. Of 4,371 referred<br />

patients, 466 (239 males and 227 females) were laboratory diagnosed with<br />

intestinal parasites, with a period prevalence of 10.7%. Blastocystis hominis (B.<br />

hominis) and Giardia lamblia (G. lamblia) were the most frequent intestinal<br />

parasites. More than half of patients aged ≥ 18 years had a low level of<br />

educational attainment (e.g. illiterate, primary school, high school) (170/331,<br />

54.1%). Further, majority of patients were homemakers (42.3%, 140/331) or<br />

workers (28.1%, 93/331) employed in various business settings such as food<br />

industry and construction. Findings of this study showed that intestinal parasitic<br />

infections are still a major public health challenge in Iran that needs to be<br />

addressed. We believe that public education, improving sanitation conditions of<br />

un<strong>der</strong>developed areas/communities, community involvement, and supporting<br />

evidence-based practice/programs are the major keys to success in preventing<br />

the spread of intestinal parasitic infections in Iran.<br />

PZ 02<br />

Antibacterial activity of selected egyptian ethnomedicinal<br />

plants<br />

S. Selim *1 , S. El-Alfay 1 , M. Abdel Aziz 1 , H. Hamido 2 , M. Mashait 3 , M. Warrad 4<br />

1<br />

Botany Department, Microbiology section, Faculty of Sciences, Suez Canal<br />

University, Egypt, Ismailia, Egypt<br />

2<br />

Biology and Geology Department, Faculty of Education in Al-Arish, Suez<br />

Canal University, Egypt, Al-Arish, Egypt<br />

3<br />

Women Health Department, Faculty of Medicine, King Khaled University,<br />

Saudi Arabia, Abha, Saudi Arabia<br />

4<br />

Biology Department, Faculty of Sciences, King Khaled University, Saudi<br />

Arabia, Abha, Saudi Arabia<br />

Medicinal plants have recently received the attention of the antimicrobial<br />

activity of plants and their metabolites due to the challenge of growing<br />

incidences of drug-resistant pathogens. The aims of this study were to<br />

determine the antibacterial activities of plant extracts used as ethnomedicinal in<br />

Egypt. Investigations were carried out to assess the antibacterial efficiency of<br />

11 plant extracts (Ambrosia maritime, Artimisia cina, Curcuma longa,<br />

Cymbopogon proximus, Cyperus rotundus, Lepidium sativum, Myristica<br />

fragrans, Origanum majorana, Peganum harmala, Salvia officinalis and Senna<br />

alexandrina) used as ethnopharmacological among Egyptian native people<br />

against infections diseases. Crude methanol, ethanol, chloroform, hexane,<br />

acetone and aqueous extract of plants were tested for antibacterial activity in<br />

vitro against ten bacterial isolates, Bacillus cereus; Bacillus megaterium;<br />

Bacillus subtilis; Bordetella bronchisepta ATCC 4617; vancomycin resistance<br />

Enterococcus feacalis isolate (VRE); Escherichia coli; Pseudomonas<br />

aeruginosa; Salmonella enteritidis; Serratia marcescens and vancomycin<br />

resistance Staphylococcus aureus isolate (VRSA) using the disc diffusion<br />

method test. Discs were impregnated with 2 mg/ml of both extracts. Out of the<br />

crude extracts, the methanolic extract showed the highest activity than other<br />

extracts. P. harmala and S. officinalis exhibited highest antibacterial activity<br />

against gram positive and negative bacteria while the remaining medicinal<br />

plants extracts showed less activity. Results obtained herein, may suggest that<br />

the ethnomedicinal Egyptian plants possess antimicrobial activity and therefore,<br />

they can be used in biotechnological fields as natural preservative ingredients in<br />

food and/or pharmaceutical industry.<br />

PZ 03<br />

Effects of a transgenic amylopectin producing potato<br />

cultivar on carbon transformation processes in plant-soil<br />

systems<br />

S. Gschwendtner *1 , J. Esperschütz 1 , F. Buegger 1 , M. Reichmann 2 , J.C. Munch 1 ,<br />

M. Schloter 1<br />

1<br />

Institut für Bodenökologie, Helmholtz Zentrum München, Neuherberg,<br />

Germany<br />

2<br />

Institut für Pflanzenbau und Pflanzenzüchtung, Landesanstalt für<br />

Landwirtschaft, Freising, Germany<br />

From the two potato starch components amylose and amylopectin the second<br />

one is, due to its typical starch characteristics such as good adhesive properties,<br />

of greater interest for industry. To avoid the costly and environmentally<br />

damaging process of separating, genetic engineers developed a potato cultivar<br />

which contains only amylopectin by blocking the translation of starch synthase<br />

and thus amylose production through insertion of an artificial gene with<br />

antisense orientation to the starch synthase gene.<br />

The aim of this study is to investigate the influence of a transgenic amylopectin<br />

producing potato line (#1332) on carbon transformation processes in the plantsoil<br />

system with special focus on changes in rhizosphere microbial community<br />

patterns in comparison with the isogenic control (Walli ® ) and another<br />

conventional starch cultivar (Ponto ® ) in a greenhouse experiment over one<br />

vegetation period. To follow the carbon fluxes from plant to the rhizosphere the<br />

plants were exposed to 13 C-CO2 for two photoperiods in an airtight tent at<br />

different development stages.<br />

Rhizosphere microbial biomass (Cmic) was quantified via fumigationextraction;<br />

microbial rhizosphere community was characterized by<br />

phospholipid fatty acid (PLFA) analysis. To investigate the carbon distribution<br />

within plant itself and rhizosphere soil as well as the incorporation of plant<br />

<strong>der</strong>ived carbon into microbial rhizosphere community 13 C signatures in<br />

different plant parts, dissolved organic carbon (DOC), Cmic and PLFA profiles<br />

were determined. The presentation will give detailed results on the allocation<br />

pattern.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PZ 04<br />

Tolerability of N-chlorotaurine, a new endogenous<br />

antiseptic, in the bronchopulmonary system<br />

A. Pinna *1 , R. Geiger 2 , B. Treml 3 , L. Barnickel 4 , C. Walther 1 , S. Scholl-Bürgi 2 ,<br />

W. Gottardi 1 , R. Arnitz 1 , C. Sergi 4 , A. Löckinger 3 , M. Nagl 1<br />

1 Department of Hygiene and Medical Microbiology, Innsbruck Medical<br />

University, Innsbruck, Austria<br />

2 Pediatric University Hospital, Innsbruck Medical University, Innsbruck,<br />

Austria<br />

3 Anaesthesia University Hospital, Innsbruck Medical University, Innsbruck,<br />

Austria<br />

4 Department of Pathology, Innsbruck Medical University, Innsbruck, Austria<br />

Background: N-chlorotaurine, a long-lived oxidants produced by human<br />

leukocytes, can be applied in human medicine as an endogenous antiseptic. Its<br />

antimicrobial activity can be enhanced by ammonium chloride. In this study<br />

was observed its tolerability on the bronchopulminary system using a swine<br />

model.<br />

Methods: Anesthetized pigs inhaled test solutions of 1% NCT , 5% NCT, or 1%<br />

NCT plus 1% ammonium chloride, and 0.9% saline solution as a control,<br />

respectively. Lung function, blood oxygenation and circulation were monitored.<br />

After euthanasia at the end of the experiment lung samples for histology were<br />

removed.<br />

Results: Arterial pressure of oxygen (PaO2) decreased significantly in all<br />

animals. Compared to saline, only 1% NCT + 1% NH4Cl led to significantly<br />

lower PaO2 values at the endpoint. The increasing of Pulmonary Artery<br />

Pressure (PAP) was the same by every test solution. Histological investigations<br />

revealed no differences between the test and control groups and no<br />

ultrastructural changes of cells in transmission electron microscopy. The<br />

concentration of NCT tolerated in vitro by A549 lung epithelial cells was 0.25-<br />

0.5 mM.<br />

Conclusion: The endogenous antiseptic NCT was well tolerated at a<br />

concentration of 1% upon inhalation in the pig model. Addition of ammonium<br />

chloride in high concentration induces a statistically significant impact on blood<br />

oxygenation, which would require adjustment of dose.<br />

PZ 05<br />

Reduction of Salmonella spp. in two types of biological<br />

wastewater treatment systems<br />

T. Roggentin *1 , P. Roggentin 2 , S. Rönner-Holm 1 , S. Baumgarte 2 , N.C. Holm 1<br />

1 F & E, LimnoTec Abwasseranlagen GmbH, Hille, Germany<br />

2 Mikrobiologischer Verbraucherschutz, Institut für Hygiene und Umwelt,<br />

Hamburg, Germany<br />

For public health it is of interest to reduce the number of pathogens in effluents<br />

of wastewater treatment plants (WWTP) to a minimum.<br />

The municipal WWTP at Weißtal (NRW) uses two types of activated sludge<br />

processes, a conventional continuously operating activated sludge system and a<br />

sequential batch reactor (SBR). Both receive their wastewater from the same<br />

influent. These conditions allow a direct comparison of the reduction rates of<br />

salmonella for both systems.<br />

The Salmonella concentrations in influent samples varied in a wide range<br />

depending on seasonal and weather conditions. Both systems exhibit<br />

comparable salmonella reduction rates. The conventional system reduced its<br />

number in the range of 86 to 99.9 %, and SBR 88 to 99.9 %. These results have<br />

been confirmed with another SBR in Erfde (SH).<br />

For both systems we found a significant correlation between the salmonella<br />

concentration in the influent and its reduction rate. The higher the concentration<br />

of salmonella in the influent so much better was the reduction rate. From this<br />

point of view a separation of rain water from the wastewater stream seems to be<br />

advantageous.<br />

Furthermore, our results indicate that the main reduction mechanism within the<br />

treatment cycle of the SBR is the co-sedimentation of pathogens with activated<br />

sludge.<br />

The dominant salmonella serovars isolated from each sample were determined<br />

by bio- and serotyping. Interestingly, most of them represent rarely detected<br />

serovars and not those which are commonly known from registered diseases.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PZ 06<br />

In-vitro and in-vivo study of probiotic properties of<br />

Lactobacilli isolated from traditional fermented dairy<br />

products of Iran<br />

M. Tajabady Ebrahimi *1 , M. Heydari Nasr 2 , P. Jafari 3<br />

1 Biology, Azad Islamic University, Central Branch, Tehran, Iran<br />

2 Biology, Azad Islamic University, Parand Branch, Tehran, Iran<br />

3 Biology, Azad Islamic University, Arak Branch, Tehran, Iran<br />

The aim of this study was in-vitro and in-vivo evaluation of potential probiotic<br />

lactobacilli isolated from traditional fermented dairy products of Iran. Viability<br />

and survival of probiotic bacteria through passing stomach is an important<br />

parameter to reach intestine and providing therapeutic functions. A rapid<br />

screening method was used to isolate 56 acid tolerant lactobacillus spp from 30<br />

different samples. The isolates were characterized for survival rate at PBS (pH<br />

2.5) for 2h and then 25 resistant isolates (survival rate >107) were selected.<br />

Growth delay at the presence and absence of bile salts was determined. 18 acid<br />

and bile tolerant strains were identified by comparative 16S rDNA sequence<br />

analysis. These isolates were further evaluated for antagonistic activities against<br />

(E. coli, S. aureus, L. monocytogenes, Y. enterocolitica and L. inocua),<br />

adhesion and cholesterol assimilation. Competition, competitive exclusion and<br />

displacement of E. coli O157, L. monocytogenes and Salmonella spp. by<br />

Lactobacilli from adhesion on Caco-2 cell surfaces were studied. Lactobacilli<br />

were able to compete with, exclude and displace pathogenic bacteria, but the<br />

degree of inhibition of adhesion was strain dependent. Cholesterol assimilation<br />

in media, showed that all strains were able to assimilate cholesterol at varying<br />

levels ranging from 54 to 266 µg/ml.<br />

The effects of four most cholesterol assimilation strains, L. pentosus, L. brevis,<br />

L. plantarum and L. paraplantarum on blood lipids in rats were studied.<br />

Significant lowering of the serum concentrations of total cholesterol, lowdensity<br />

lipoprotein cholesterol, and triglycerides, in comparison with the<br />

control, were observed.<br />

These findings are important for development, selection and assessment of<br />

functions specific probiotics. After supplementary investigations and specific<br />

studies on their technological properties, these strains maybe suitable for used<br />

as probiotic starter or adjuvant cultures in dairy manufacture.<br />

PZ 07<br />

Screening and optimization of fed-batch production of<br />

Bacillus Isolates in the Broiler chicken<br />

P. Jafari *1 , F. Almasian 2 , G. Mohammad Zamani 2 , A. Askari 2 , M. Tajabadi<br />

Ebrahimi 3<br />

1 Biology, Azad Islamic University, Arak Branch, Tehran, Iran<br />

2 Chemistry, Engineering Research Institute, Tehran, Iran<br />

3 Biology, Azad Islamic University, Central Branch, Tehran, Iran<br />

207<br />

Spores from a number of different Bacillus spp. are currently <strong>bei</strong>ng used as<br />

poultry probiotics in maintaining animal health. Bacillus strains in the form of<br />

spores have been shown to reach the target intestine successfully.<br />

The isolation of 237 Bacillus spp. isolates from the gastrointestinal tract of<br />

poultry carried out by heat treatment of fecal material from broiler chickens.<br />

All of the isolates were characterized according to their morphological,<br />

physiological, and biochemical properties. All isolates sporulated efficiently in<br />

the laboratory. Bacterial spores were characterized for potential attributes (acid<br />

and bile tolerance, colonization and antimicrobial activity) that could account<br />

for their claimed probiotic properties. Our result showed that strain P75 have a<br />

very good probiotic properties and exhibit antimicrobial activity against a broad<br />

spectrum of bacteria, including E.coli, L.monocytogenes, S.aureus, S.pyogenes,<br />

S.typhi, S. paratyphi A and B, and V. cholerea. Importantly, this isolate was<br />

susceptible to most of the antibiotics tested, arguing that it would not act as<br />

donors for resistance determinants if introduced in the form of probiotic<br />

preparations.<br />

Optimized Batch fermentation of P75 with 20 g l-1 glucose in 1 lit fermenter<br />

resulted in 11 g.l-1 dry cell mass. A fed-batch fermentation process was<br />

developed in this research. A late-exponential phase was chosen as the best<br />

point for switching to a fed-batch process. Optimized fed-batch fermentation,<br />

feeding a medium of 100 g l-1 glucose gave the best results of 40.6 g l-1 dry<br />

cell mass which is much higher than the corresponding values in simple batch<br />

fermentation.<br />

Our results demonstrated that Bacillus sp. Strain 75 have a good probiotic<br />

potential and could inhibit most of the important poultry disease such as<br />

salmonella. In the other hand, high cell density culture of it is possible in<br />

fermenter. So after safety assessment, it could be used as a commercial product<br />

in poultry industry.

208<br />

PZ 08<br />

Horses with anaemia of unknown reason - a novel<br />

haemotrophic Mycoplasma species?<br />

S.M. Dieckmann *1 , M. Dieckmann 2 , M. Winkler 3 , M.M. Wittenbrink 1 , K.<br />

Hoelzle 1 , L.E. Hoelzle 1<br />

1 Institute of Veterinary Bacteriology, University of Zurich, Zurich, Switzerland<br />

2 independent horse surgery, Bommelsen, Bommelsen, Germany<br />

3 synlab.vet, Geesthacht, Geesthacht, Germany<br />

In Northern Germany horses with idiopathic anaemia were detected. They<br />

showed symptoms analogous to infections with haemotrophic mycoplasmas<br />

(HM) in other animals (e.g. reduced general condition, lethargic behaviour and<br />

reduced performance capacity), always with the following preliminary report:<br />

"it develops not very well in the last time; it is nerveless, loose condition and<br />

weight". HM infections are described for a various number of animals (e.g. pig,<br />

ruminants, dog and cat) as well as primates, but not yet for horses. First results<br />

substantiate the occurrence of uncultivable epicellular bacteria in the blood of<br />

these horses, e.g. resembling microscopic results to pigs infected with<br />

Mycoplasma suis. It was possible to obtain a 16S rDNA sequence with a<br />

homology of 94 % to known HM. There are urgent needs to elucidate the<br />

aetiology and importance of this novel infection, as it is the first description of<br />

a HM infection in horses. As most HM species are described as very host<br />

specific, this would be most likely the description of a novel haemotrophic<br />

Mycoplasma species. The importance of this infection, especially of the<br />

subclinical course, for the economic use of horses in sport and breeding is<br />

difficult to estimate, as no relevant data are available, and cannot be evaluated<br />

until the clinical manifestation and serial analyses are established. In case of a<br />

mild chronic course the owner has to count on a temporary reduced sporting<br />

achievement and in young horses on retardation of development. In fatal cases<br />

even death of horse may occur.<br />

PZ 09<br />

Differential Expression of nar-genes Encoding Respiratory<br />

Nitrate Reductase in Streptomyces coelicolor<br />

M. Fischer *1 , J. Al<strong>der</strong>son 2 , G. van Keulen 3 , G. Sawers 1<br />

1 Institut für Biologie/Mikrobiologie, Martin-Luther-Universität Halle-<br />

Wittenberg, Halle, Germany<br />

2<br />

Department Molecular Microbiology, John Innes Centre Norwich, Norwich,<br />

United Kingdom<br />

3<br />

Biological Sciences, University of Wales Swansea, Swansea, United Kingdom<br />

Streptomyces coelicolor is an obligate aerobic high-GC gram-positive<br />

actinobacterium. Despite only <strong>bei</strong>ng able to grow in the presence of oxygen, it<br />

has been shown that S. coelicolor can survive long periods without oxygen [1].<br />

The mechanisms un<strong>der</strong>lying anaerobic survival are not un<strong>der</strong>stood. The<br />

genome of S. coelicolor encodes a number of genes whose products are usually<br />

associated with anaerobic metabolism in facultative or obligate anaerobes,<br />

suggesting that some of these genes might be important for anaerobic survival.<br />

Amongst these genes are three operons (nar) encoding respiratory nitrate<br />

reductase. Using defined nar operon deletion mutants and promoter-exchange<br />

studies in combination with the development of an assay based on determining<br />

sensitivity/ resistance to the nitrate analogue chlorate, we demonstrate that the<br />

Nar enzymes are not functionally redundant, but rather are synthesised in<br />

different tissues or at different stages of growth. Nar1 enzyme is active in<br />

spores, while Nar2 synthesis is induced upon exposure of exponentially<br />

growing mycelium to anaerobic stress. Nar3 is most active during the stationary<br />

phase of growth. Although a triple nar knock-out mutant is unaffected in its<br />

ability to survive anaerobiosis, these results nevertheless suggest that the Nar<br />

enzymes have important roles during development and probably contribute to<br />

the general fitness of the organism.<br />

[1] van Keulen, G., J. Al<strong>der</strong>son, J. White, and R.G. Sawers (2007) The obligate<br />

aerobic actinomycete Streptomyces coelicolor A3(2) survives extended periods<br />

of anaerobic stress. Env. Microbiol. 9: 3143-3149.<br />

PZ 10<br />

Large linear plasmids in Micrococcus strains isolated from<br />

Argentinean high altitude wetlands.<br />

J.R. Dib *1 , M. Wagenknecht 2 , M.E. Farias 1 , F. Meinhardt 2<br />

1 Planta Piloto de Procesos Industriales Microbiológicos, Consejo Nacional de<br />

Investigaciones Cientifícas y Técnicas, Tucumán, Tucumán, Argentina<br />

2 Institut für Molekulare Mikrobiologie und Biotechnologie, Westfälische<br />

Wilhelms-Universität Münster, Münster, Germany<br />

High altitude wetlands are pristine and extreme environments in the northwest<br />

of Argentina. Three strains belonging to the genus Micrococcus, isolated from<br />

such environments, were found to harbor large linear plasmids (93-114 kb).<br />

Though for a number of Actinobacteria, including members of the genera<br />

Arthrobacter, Streptomyces, Mycobacterium and Rhodococcus, linear plasmids<br />

were found to exist, these are novel elements for Micrococcus spp. Three giant<br />

plasmids, pA1 (114 kb), pH5 (114 kb) and pV7 (93 kb) were separated and<br />

isolated by PFGE and electroelution, and were subsequently tested for<br />

sensitivity to exonuclease III and λ-exonuclease, which hydrolyze dsDNA in<br />

3´-5´ and 5´-3´ direction, respectively. Exonuclease III completely degraded the<br />

plasmids, whereas λ-exonuclease did not, indicating that the 5´ ends are<br />

protected, presumably by covalently attached terminal proteins. Uniform<br />

restriction patterns were obtained for pA1 and pH5; whereas pV7 was different.<br />

Consistently, by making use of a cloned pA1 fragment as the probe, Southern<br />

analysis revealed identical hybridization patterns for pA1 and pH5. Since<br />

Micrococcus strains harboring either pA1 or pH5 were isolated from different<br />

locations, these plasmids may play a role in the adaptation to extreme<br />

environmental conditions in Argentinean high altitude wetlands.<br />

Julián R. Dib thanks DAAD for financial support.<br />

PZ 11<br />

Denitrifying methanotrophs - their metabolism,<br />

biochemistry and ultrastructure<br />

M. Strous *1 , K. Ettwig 1 , M. Butler 1 , M. Kuypers 2 , F. Schreiber 2 , D. Lepaslier 3 ,<br />

E. Pelletier 3 , M. Jetten 1<br />

1<br />

Department of Microbiology, Radboud University Nijmegen, Nijmegen,<br />

Netherlands<br />

2<br />

Microbiology/Biosensing, Max Planck Institute for Marine Microbiology,<br />

Bremen, Germany<br />

3<br />

Genome Sequencing, Genopole, Paris, France<br />

Recently, a microbial consortium was found to couple anaerobic methane<br />

oxidation to denitrification [Raghoebarsing et al. 2006]. The consortium<br />

consisted of a bacterium representing a phylum without cultivated<br />

representatives and an archaeon distantly related to known<br />

methanogenic/methanotrophic archaea. Later, it appeared that the bacterium<br />

also oxidizes methane in the absence of the archaeon.<br />

Because this bacterium divides only once per month, it has not yet been isolated<br />

in pure culture. By metagenomics, transcriptomics, proteomics, biochemical<br />

analysis, 15N isotope labeling and electron microscopy of the enrichment<br />

culture, it was still possible to characterize the physiology of the novel<br />

denitrifying methanotroph.<br />

Surprisingly, we found a combination of anaerobic and aerobic pathways,<br />

which were both found to be expressed and functional.<br />

[1] Raghoebarsing AA, Pol A, van de Pas-Schoonen KT, Smol<strong>der</strong>s AJ, Ettwig<br />

KF, Rijpstra WI, Schouten S, Sinninghe-Damste JS, Op den Camp HJ, Jetten<br />

MS, Strous M (2006) A microbial consortium couples anaerobic methane<br />

oxidation to denitrification. Nature 440: 918-21.<br />

PZ 12<br />

Environmental studies on the distribution of the leptospires<br />

in surface waters of humid tropical regions<br />

H. Kaboosi *1 , M.R. Razavi 2 , A.S. Noohi 3<br />

1<br />

Islamic Azad University, Science and Research branch, Tehran, Iran, Islamic<br />

Azad University, Amol branch, Iran, Tehran, Iran<br />

2<br />

Pasteur Institute of Iran, Islamic Azad University, Science and Research<br />

branch, Tehran,Iran, Tehran, Iran<br />

3<br />

Islamic Azad University, Science and Research branch,Tehran,Iran, -, Tehran,<br />

Iran<br />

Objectives: Leptospirosis is consi<strong>der</strong>ed a reemerging disease that infects people<br />

who have contact with contaminated surface water. This disease is<br />

characterized by some researchers as the most common waterborne illness in<br />

the word, especially in humid tropical regions. In this research, environmental<br />

studies were conduced to distribution of the genus Leptospira in surface waters<br />

of humid tropical regions.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Methods: Environmental study was conduced in the Iranian Humid Tropical<br />

Provinces (IHTP). Within the IHTP, 12 bodies of surface waters were selected<br />

for sampling. Isolation of Leptospira was carried out according to the method<br />

of Wilson et al.<br />

Results: A total 35 samples were collected, and 11 samples (31.4%) were found<br />

to be positive. The temperature and pH of the surface water samples were<br />

within the range reported to be capable of supporting leptospiral viability in<br />

tropical areas.<br />

Conclusion: we attempted to examine the distribution of leptospires in surface<br />

waters within IHTP, by an enrichment culture method using a standard volume<br />

of sample material. Water samples were examinated for the presence of<br />

leptospires and 31% were positive. Since leptospirosis is water transmitted<br />

infection, all surface water is potential sources of infection; but this research<br />

has established that not all aquatic bodies of surface waters in humid tropical<br />

regions are equally of supporting a leptospiral population.<br />

PZ 13<br />

Isolation and characterisation of new Bdellovibrio<br />

bacteriovorus HD100 - mutants possessing a hostindependent<br />

lifecycle<br />

N. Roschanski *1 , U. Bergmann 2 , S. Klages 3 , M.W. Linscheid 2 , R. Reinhardt 3 , E.<br />

Strauch 1<br />

1 Department 4: Biological Safety, Fe<strong>der</strong>al Institute for Risk Assessment, Berlin,<br />

Germany<br />

2 Department of Chemistry, Humboldt-University to Berlin, Berlin, Germany<br />

3 Max Planck Institute for Molecular Genetics, Berlin, Germany<br />

Bdellovibrio bacteriovorus HD100 is a small, motile and predatory bacterium<br />

that belongs to the delta division of the Proteobacteria. A common phenotypic<br />

characteristic of its lifestyle is the growth and replication within the periplasm<br />

of a large variety of Gram-negative bacteria. Wild-type strains possess an<br />

obligate predatory lifecycle consisting of two developmental stages - a freeliving<br />

attack phase and an intracellular, filamentous growth phase.<br />

The nature of the obligate prey requirement for wild-type Bdellovibrios is still<br />

unclear. It is, however, remarkable that spontaneously occurring mutations<br />

were found that lead to prey-independent <strong>der</strong>ivatives which are able to grow on<br />

heat-killed prey bacteria or even in the absence of living prey.<br />

In 1992 T.W. Cotter & M.F. Thomashow detected, that host-independent<br />

mutants of the strain B. bacteriovorus 109J, which grow axenically on rich<br />

media, possess frame shift mutations in a small open reading frame within the<br />

hit locus. However, in another study was reported that sequence alterations<br />

within this region occurred only in three of seven mutants. Until now, no other<br />

genetic region, except the hit-locus, could be identified to be involved in the<br />

interaction between predator and prey.<br />

In our study we picked up these observations and isolated new saprophytic<br />

mutants in two different ways. Preliminary sequence analyses of the hit ORF<br />

(Bd0108) of 30 mutants indicate mutations within the coding sequence of the<br />

hit gene leading to frame shifts and thus affecting the primary structure of the<br />

putative polypeptide. These findings suggest that in all saprophytic mutants of<br />

strain HD100 the hit gene product is involved in the development of the prey<br />

independent phenotype.<br />

Recently we have started to analyse the complete genome sequence of mutant<br />

strains to un<strong>der</strong>stand more comprehensively how the changes within the<br />

Bdellovibrio-lifecycle takes place.<br />

PZ 14<br />

Development of a PNA-based RT-qPCR assay for detection<br />

of Legionella pneumophila<br />

J. Hilmes 1,2 , H.J. Kunte *1 , O. Seitz 2<br />

1 FG IV.1, Bundesanstalt für Materilaforschung und -prüfung (BAM), Berlin,<br />

Germany<br />

2 Department of Organic and Bioorganic Chemistry, Humboldt Universität zu<br />

Berlin, Berlin, Germany<br />

A selective and quantitative RT-PCR method is <strong>bei</strong>ng developed employing<br />

peptide nucleic acid (PNA) probes for the detection and quantification of living<br />

Legionella pneumophila cells. Living cells will be detected by isolation and<br />

cDNA-amplification of dotA mRNA. The dotA gene is involved in L.<br />

pneumophila virulence and is known to be a pathogenicity island. For<br />

quantitative detection of PCR-amplified dotA cDNA, we developed a set of<br />

specific PNA probes for L. pneumophila serogroup Sg-1, -6 and -12. It is<br />

planned to test these FIT-probes with other serogroups (Sg-4, -5, -6 and -10) of<br />

L. pneumophila and with other strains of Legionella such as L. longbeachae, L.<br />

erythra. The dotA-specific PNA probes contain the intercalator thiazole orange<br />

as a base substitute that serves as fluorescence dye. Upon hybridization with the<br />

target DNA, forced intercalation of thiazole orange (FIT) leads to an increase in<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

fluorescence. A unique feature of thiazole-containing PNA-probes is that<br />

emission of fluorescence light is highly sequence specific. [1], [2], [3]<br />

Therefore, detection of target DNA by FIT-probes depends not only on probe-<br />

DNA binding as known from Taqman probes but also on a perfect sequence<br />

match of probe and target. In addition, the specific binding characteristics of<br />

PNAs are less sensitive to changes in temperature and salt concentration<br />

compared to many other probes making a PNA-based PCR-assay less prone to<br />

false positive detection. To make the described PNA-based RT-qPCR of dotA<br />

mRNA a feasible method for routine detection of L. pneumophila, we are<br />

comparing our new fast and highly specific quantification method with the<br />

conventional culture methods.<br />

[1] O. Köhler, D. V. Jarikote, O. Seitz, ChemBioChem, 2005, 6, 66-77.<br />

[2] D. V. Jarikote, O. Köhler, E. Socher, O. Seitz, Eur. J. Org. Chem., 2005,<br />

3187.<br />

[3] E. Socher, O. Seitz, Methods Mol Biol.; 2008, 429, 187-197.<br />

PZ 15<br />

Distribution and activity of microorganisms in different<br />

zones of a silica sand capillary fringe<br />

D. Jost *1 , C. Gallert 1 , J. Winter 1<br />

1 Institut für Ingenieurbiologie und Biotechnologie des Abwassers (IBA),<br />

Karlsruhe - Institute of Technology (KIT), Karlsruhe, Germany<br />

The capillary fringe (CF) is highly important as a biological filter for water<br />

percolating through the unsaturated aquifer into the groundwater. Therefore the<br />

distribution and the activity of bacterial cells in a silica sand CF (355 - 710 µm<br />

grain size) were investigated. The experiments were performed in hele-shaw<br />

cells and Pseudomonas putida as a motile and aerobically growing<br />

microorganism. Besides, Corynebacterium glutamicum as an immotile and<br />

aerobic and Lactococcus lactis as an immotile and anaerobic bacterium were<br />

chosen. The bacterial cells were separately suspended in diluted medium and<br />

transported into the sand packing only by capillary forces. The tests revealed<br />

that after 3 days the majority of L. lactis cells were found in the lower, fully<br />

water saturated region, whereas most of P. putida cells were counted in the<br />

uppermost region of the CF. These results indicate that the different regions of<br />

the capillary fringe, providing anaerobic as well as aerobic conditions, define<br />

bacterial growth and distribution. By comparing the cell count and the activity<br />

of P. putida and C. glutamicum cells in the different CF regions, the P. putida<br />

cells showed a higher presence in the upper regions of the CF, especially in the<br />

transition zone. Thus, it can be assumed that the motility of bacteria also has an<br />

influence on their distribution. Concerning the enzyme activity of the<br />

microorganisms, a significant correlation (p < 0,001; Pearson r = 0,77) between<br />

the fluorescein diacetate hydrolysis and the cell count in the different CF<br />

regions was observed.<br />

PZ 16<br />

Important role of murE for the transfer of beta-lactam<br />

resistance from S. oralis to S. pneumoniae<br />

K. Todorova *1 , P. Maurer 1 , R. Hakenbeck 1<br />

1 Department of Microbiology, University of Kaiserslautern, Kaiserslautern,<br />

Germany<br />

209<br />

Beta-lactam resistance in Streptococcus pneumoniae, a major human pathogen,<br />

is essentially due to altered PBPs (Penicillin-binding proteins) with decreased<br />

affinity for these antibiotics. PBPs from penicillin-resistant isolates are encoded<br />

by mosaic genes with sequence blocks divergent from those of sensitive strains<br />

of up to 20% (Laible et al., 1991). Since the sequence structures of PBPs of<br />

resistant S. pneumoniae strains are homologous to PBPs of susceptible S. mitis<br />

and S. oralis strains, these species are suggested to be potential origins for<br />

resistant determinants (Reichmann et al., 1997).<br />

We have investigated the gene transfer from a high resistant S. oralis Uo5 to S.<br />

pneumoniae with chromosomal donor DNA. Selection with piperacillin,<br />

oxacillin and cefotaxime lead to the transfer of pbp2x, but further attempts to<br />

transfer pbp1a and/or pbp2b to these S. pneumoniae-mutants failed.<br />

DNA-microarray-analysis of a transformant, selected with piperacillin, which<br />

had a higher MIC, showed not only an altered pbp2x but also an altered murE<br />

(UDP-N-acetylmuramyl tripeptide synthetase). MurE catalyses the addition of<br />

the L-lysine residue to the UDP-linked muramyl dipeptide precursor during<br />

peptidoglycan biosynthesis. Using this strain as a recipient, transformants with<br />

alterations in pbp1a and pbp2b could be obtained.<br />

Further experiments demonstrated that there were no transformants with lowaffinity<br />

PBP1a or PBP2b in the absence of altered murE. These data suggest the<br />

important role of murE as a new resistance determinant.

210<br />

PZ 17<br />

Polyelectrolyte Nanoencapsulation of Bacteria through LbL<br />

Assembly<br />

S. Balkundi 1 , B. Franz *2 , C. Dahl 3 , Y. Lvov 1 , A. Prange 2<br />

1<br />

Institute for Micromanufacturing, Louisiana Tech University, Ruston, United<br />

States<br />

2<br />

Mikrobiologie und Lebensmittelhygiene, Hochschule Nie<strong>der</strong>rhein,<br />

Mönchengladbach, Germany<br />

3<br />

Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-<br />

Universität Bonn, Bonn, Germany<br />

The layer-by-layer (LbL) technique has been used to encapsulate the<br />

phototrophic purple sulfur bacterium Allochromatium vinosum and other<br />

bacteria of different genera and morphology with polyelectrolytes. Polycation /<br />

polyanion alternate adsorption of polyelectrolytes like<br />

poly(diallyldimethylammoniumchloride)/polystyrene sulfonate and poly-Llysine/chondroitin<br />

sulfate and others resulted in sequential alternation of<br />

charges and formation of organized shells of approx. 50 nm thickness. Polymer<br />

multilayer coating of living bacterial cells was confirmed by FE SEM (field<br />

emission scanning electron microscopy) and indirectly by Zeta potential<br />

readings measured during the encapsulation process.<br />

We could observe the formation of nanoshells for all bacteria un<strong>der</strong><br />

investigation indicating that encapsulation can be a general strategy for the<br />

production of microbial cells of controlled architecture and permeability.<br />

Bacterial cell division was not disturbed or inhibited. Furthermore the cells<br />

were metabolically active which was investigated in detail for growth on<br />

sulfide for A. vinosum.<br />

Our results indicate that encapsulation of bacteria might allow changing of cell<br />

colloidal stability, charge and other surface properties, to control bacterial<br />

metabolism and nutrition, to study bacterial adhesion to substrates (e.g.<br />

adhesion of A. vinosum to elemental sulfur) and might act as protective coating<br />

of bacteria.<br />

PZ 18<br />

Characterization of nitrifying organisms in Permafrost<br />

Soils of Lena Delta, Siberia<br />

T. San<strong>der</strong>s *1 , C. Fiencke 1 , E. Spieck 2 , E.M. Pfeiffer 1<br />

1 Institut für Bodenkunde, Universität Hamburg, Hamburg, Germany<br />

2 Biozentrum Klein Flottbek, Universität Hamburg, Hamburg, Germany<br />

Permafrost soils (Gelisols) cover about a quarter of the Earth´s land surface.<br />

Gelisols are in subsoils continuously frozen throughout the year and only the<br />

surface horizons, the so called active layers, thaw during the short vegetation<br />

period. The main characteristic of these soils is the extreme soil temperature<br />

regime which range from -30°C to +18°C. In this study one important part of<br />

the microbial N-cycle, the nitrification was investigated in typical wet arctic<br />

tundra sites of the Lena Delta, Northeast Siberia, Russia. During nitrification<br />

ammonia is oxidized in two steps via nitrite to nitrate. These steps are catalyzed<br />

by two groups of organisms the ammonia and nitrite oxidizers (AOB and<br />

NOB). Recently it was shown that not only Bacteria but also Archaea of the<br />

group of Crenarchaeota (AOA) are able to oxidize ammonia to nitrite [1].<br />

Different soils were analyzed: water saturated and organic rich soils of the<br />

polygonal tundra and dry sandy soils of the beach and inundation areas of the<br />

river Lena. In river inundation areas higher nitrification activities were found<br />

than in the peaty soils. In or<strong>der</strong> to differentiate Bacteria and Archaea ammonia<br />

oxidizing activity tests with streptomycin were performed.<br />

In enrichment cultures representatives of the genus Nitrosospira (AOB) and the<br />

genera Nitrospira and Nitrotoga [2] (NOB) were detected using DGGE and<br />

transmission electron microscopic (TEM) techniques. We could observe a shift<br />

of the nitrifying community depending on the incubation temperature,<br />

reflecting the presence of various ecological niches in this extreme ecosystem.<br />

[1] Könneke et. al. 2005, Nature 437(22): 543-546<br />

[2] Alawi et. al. 2007 ISME J 1:256 - 264<br />

PZ 19<br />

Isolation and characterization of two highly chromate<br />

resistant bacterial strains<br />

G. Sturm *1 , J. Jacobs 1 , J. Gescher 1<br />

1<br />

Institute for Biology II / Department of Microbiology, University Freiburg,<br />

Freiburg, Germany<br />

In the last decades chromium has become a wide spread pollutant in the<br />

environment. This is mainly due to anthropogenic factors, namely an often<br />

inadequate toxic waste management in leather tannery, dye-, car- and steel-<br />

industry. Consequently chromium has become the most important heavy metal<br />

pollutant in the European Union. Depending on its oxidation state chromium<br />

has varying levels of toxicity. Cr(VI) is the most toxic and biologically-mobile,<br />

whereas Cr(III) is a highly insoluble metal and therefore less toxic. Hence,<br />

microbial chromate-reduction is consi<strong>der</strong>ed to be a useful and economical<br />

possibility for bioremediation of contaminated field sites. We isolated and<br />

partly characterized two bacterial strains capable of tolerating high chromate<br />

concentrations. These strains – Leucobacter spec. and Aerococcus spec. - are<br />

resistant to chromate concentrations of up to 8 mM and 40 mM, respectively<br />

and show biphasic growth at higher chromate concentrations. Both strains show<br />

high chromate reductase activities in growth experiments. We could observe<br />

that Aerococcus spec. cells increased their cell diameters in correlation with<br />

ascending Cr(VI) concentrations. Leucobacter spec. cells instead showed an<br />

increased tendency to form cell-aggregates. These bacterial aggregates were<br />

examined for the distribution of living versus dead cells via fluorescence<br />

microscopy. More than 95% of all cells were shown to be viable. Dead cells<br />

were randomly distributed throughout the biofilm. We hypothesize that biofilm<br />

formation is a resistance strategy due to sorption of chromate to the matrix.<br />

PZ 20<br />

Challenge of filtration technique application for isolation of<br />

leptospires from surface water samples<br />

H. Kaboosi *1 , M.R. Razavi 2 , A.S. Noohi 3<br />

1<br />

Islamic Azad University, Science and Research branch, Tehran, Iran, Islamic<br />

Azad University, Amol branch, Amol, Iran, Tehran, Iran<br />

2<br />

Pasteur Institute of Iran, Tehran, Iran, _, Tehran, Iran<br />

3<br />

Islamic Azad University, Science and Research branch, Tehran, Iran, -,<br />

Tehran, Iran<br />

Objectives: Leptospirosis is a globally important zoonotic disease cause by<br />

filterable spirochetes from the genus Leptospira. Leptospirosis is transmitted to<br />

humans through environmental surface waters contaminated by the urine of<br />

domestic and wild mammals chronically colonized with Leptospira.The<br />

objective of this experimental work focused on challenge for application of<br />

membrane filter (with 0.45µm pore diameter) for isolation of Leptospira from<br />

surface water samples.<br />

Methods: Surface water samples were collected and passed through a sterile<br />

0.45µm pore size membrane filter. Then 1.0 milliliter of water samples was<br />

inoculated, in duplicate, into semisolid Ellinghausen – McCullough modified<br />

by Johnson – Harris (EMJH) medium. For control of this work, suspension of<br />

leptospires pure culture (1.5*10 6 leptospires per milliliter) was prepared and<br />

passed through 0.45µm pore diameter membrane filter and then enumerated<br />

with petroff-hausser bacterial counting chamber.<br />

Results: This research showed important aspect of using filtration method for<br />

isolation of Leptospira from surface water samples. Our results suggest for<br />

isolate nearly 100% of Leptospira from surface water samples, the optimal pore<br />

diameter should be less than 0.45µm.<br />

Conclusion: Although filtration method can be used to isolation leptospires<br />

from surface water samples, it is unclear whether this is a useful method for<br />

detection all of leptospires exist within surface water samples. However a large<br />

proportion of leptospires can be passed by membrane filter with a pore diameter<br />

commonly used to isolate leptospires from surface water samples (0.45µm pore<br />

size).<br />

PZ 21<br />

Soil microcosms for determination of growth by<br />

Steptomyces mirabilis P16B1 and superoxide dismutase<br />

production in heavy metal contaminated soil<br />

E. Schütze *1 , A. Schmidt 1 , M. Kästner 2 , E. Kothe 1<br />

1 Institute for Microbiology - Microbial Phytopathology, Friedrich Schiller<br />

University Jena, Jena, Germany<br />

2 Department of Bioremediation, Helmholtz Centre for Environmental Research<br />

- UFZ, Leipzig, Germany<br />

Streptomycetes are a dominat group of soil bacteria which belong to the group<br />

of Actinobacteria. They are known for their complex life cycle, including<br />

mycelial growth and spore production, as well as their production of secondary<br />

metabolites, among them a large number of antibiotics. In comparison to<br />

pristine soils, heavy metal contaminated soils show much higher numbers of<br />

Gram positive bacteria, with bacilli and streptomycetes dominating over Gram<br />

negative proteobateria or firmicutes. The extremely heavy metal resistant strain<br />

Streptomyces mirabilis P16B1, isolated from the former uranium mining site<br />

Wismut near Gera/Ronneburg, Thuringia, Germany, shows the ability to grow<br />

in highly contaminated soil from sample site K7 without the addition of any<br />

media ingredients even though there is a significantly higher content of a range<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

of heavy metals, including mobile and adsorbed fraction metals as determined<br />

by sequential extraction methods. Scanning electron microscopy was used to<br />

detect the mycelium of the strain on the surface and in the interior of the soil.<br />

First results of protein extraction from inoculated soil showed high superoxidedismutase<br />

(SOD) activity that provides a basis for further investigation of<br />

SODs from heavy metal resistant strains as well as their special protein<br />

expression clusters. Furthermore the influence of SOD on the pool of organic<br />

carbon within soil is investigated in microcosms.<br />

PZ 22<br />

Structure and function of a cofactor-independent<br />

dioxygenase belonging to the α/β-hydrolase fold family<br />

H. Janßen *1 , A. Albers 1 , R. Steiner 2 , S. Fetzner 1<br />

1 Institut für Molekulare Mikrobiologie und Biotechnlogie, WWU Münster,<br />

Münster, Germany<br />

2 Randall Division of Cell and Molecular Biophysics, King`s College London,<br />

London, United Kingdom<br />

The majority of oxygenases depend on a metal or organic cofactor for catalysis.<br />

In contrast, 2-methyl-3-hydroxy-4(1H)quinolone 2,4-dioxygenase (Hod), which<br />

catalyzes ring cleavage of its substrate to CO and N-acetylanthranilate, is a<br />

cofactor-independent enzyme evolutionarily related to α/β-hydrolases [1,2].<br />

The crystal structures of Hod [3] and of Hod-H251A complexed with organic<br />

substrate confirm the α/β-hydrolase fold and indicate structural conservation of<br />

residues (S101, D126, H251) which in α/β-hydrolases comprise the catalytic<br />

triad. Kinetic analyses of Hod proteins carrying individual amino acid<br />

replacements have been performed to assess the role of active-site residues in<br />

substrate activation and catalysis. Comparison of the pH dependence of kinetic<br />

parameters of Hod, Hod-H251A, and Hod-D126A confirms our previous<br />

proposal that H251 acts as a catalytically essential general base, forming a<br />

substrate anion [2], and suggests that D126 stabilizes the catalytic H251.<br />

Serine-101 is involved in substrate binding, and an adjacent His residue seems<br />

to be important for reactivity towards O2.<br />

Hod is active towards 2-alkyl-3-hydroxy-4(1H)quinolones with short alkyl<br />

chains. 2-Heptyl-3-hydroxy-4(1H)quinolone, also termed PQS (Pseudomonas<br />

quinolone signal), is a signalling molecule involved in the quorum sensing<br />

cascade of Pseudomonas aeruginosa. Since Hod catalyzes PQS cleavage, it is a<br />

useful tool to study PQS-dependent quorum sensing.<br />

[1] Fischer F, Künne S, Fetzner S (1999) J. Bacteriol. 181:5725-5733.<br />

[2] Frerichs-Deeken U, Ranguelova K, Kappl R, Hüttermann J, Fetzner S<br />

(2004) Biochemistry 43:14485-14499.<br />

[3] Steiner R, Frerichs-Deeken U, Fetzner S (2007) Acta Cryst. F63: 382-385.<br />

PZ 23<br />

Oxidation of inorganic sulfur compounds by heterotrophic<br />

Acidiphilium strains<br />

C. Janosch *1 , C. Thyssen 1 , W. Sand 1 , T. Rohwer<strong>der</strong> 1<br />

1 Biofilm Centre, University of Duisburg-Essen, Duisburg, Germany<br />

The alpha-proteobacterial genus Acidiphilium consists of chemoorganotrophic<br />

species, generally known as part of the acido-mesophilc microbial flora of<br />

leaching biotopes. One species, Acidiphilium acidophilum, is capable of<br />

chemolithotrophic growth on sulfur compound oxidation. However, also<br />

several strictly heterotrophic Acidiphilium strains show some potential for<br />

oxidation of inorganic sulfur species. We have now investigated the pathways<br />

for elemental sulfur and tetrathionate oxidation in two strains of Acidiphilium<br />

cryptum, DSM 2390 and JF-5. In both strains, the enzymatic oxidation<br />

activities are inducible by tetrathionate. In experiments with resting cells, strain<br />

JF-5 oxidized elemental sulfur and tetrathionate to sulfate without the formation<br />

of any detectable intermediates. In contrast, cells of strain DSM 2390 converted<br />

tetrathionate to sulfate via the main intermediates thiosulfate, sulfite and<br />

elemental sulfur. Biochemical and evolutionary aspects of this new feature<br />

among the heterotrophic Acidiphilium strains are discussed. In addition, this<br />

finding will possibly help to solve the long-standing questions about the<br />

biochemical nature of sulfur compound oxidation in mesophilic leaching<br />

bacteria.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PZ 24<br />

Attachment of leaching bacteria to mineral sulfides<br />

elucidated by a combination of AFM and EFM<br />

B. Florian *1 , N. Noël 1 , S. Bellenberg 1 , T. Rohwer<strong>der</strong> 1 , W. Sand 1<br />

1<br />

Aquatic Biotechnology, Biofilm Centre, University of Duisburg-Essen,<br />

Duisburg, Germany<br />

Bioleaching is the dissolution of metal sulfides, such as pyrite and chalcopyrite,<br />

by bacterial oxidation processes. Bacterial leaching is used for the winning of<br />

metals such as gold, copper, or zinc. In all cases, leaching bacteria form a<br />

biofilm on the mineral surface, which is their substrate. Consequently, the<br />

attachment to metal sulfides is critical for bioleaching performance. The aim of<br />

the study was to visualize the initial colonization of metal sulfides by pure and<br />

mixed cultures. Various strains of the genera Acidithiobacillus, Leptospirillum,<br />

Sulfobacillus and Acidiphilium were tested. Sessile and planktonic cells were<br />

visualized by fluorescence microscopy using DAPI-, Live/Dead-kit-, FISH-,<br />

lectin- and calcofluor- staining. Additionally, atomic force microscopy was<br />

used for the investigations on cell morphology, spatial arrangement of cells on<br />

metal sulfides and mineral surface topography. It was shown that the<br />

physiology and morphology of sessile cells were totally different as compared<br />

with planktonic ones. Interactions of different species resulted in increased<br />

production of extracellular polymeric substances (EPS) or caused non-attaching<br />

bacteria to be incorporated into a biofilm of adherent ones. Consequently<br />

biofilm formation was furthered. Based on these findings, bacterial attachment<br />

and, thus, metal extraction in industrial leaching processes may be optimized.<br />

Furthermore, strategies for the abatement of acid mine/rock drainage may be<br />

developed.<br />

PZ 25<br />

Prevention of microbially influenced corrosion by<br />

Extracellular Polymeric Substances<br />

A. Kuklinski *1 , M. Grooters 1 , A. Heyer 1 , W. Sand 1<br />

1<br />

Aquatic Biotechnology, Biofilm Centre, University of Duisburg-Essen,<br />

Duisburg, Germany<br />

Microbial influenced corrosion (MIC) of ferrous metals is a serious<br />

economical expense factor. Biofilms of sulfate-reducing bacteria (SRB) such<br />

as Desulfovibrio spp. are one of the main causes for MIC. As current<br />

conventional countermeasures are expensive, ineffective or environmentally<br />

harmful, there is a demand of new, environment-friendly concepts. The<br />

corrosion-mitigating effect of biofilms on metallic surfaces has been described<br />

recently. Their inhibiting effect is generally thought to be caused by oxygen<br />

depletion, the formation of passive layers or the adhesion prevention of<br />

detrimental microorganisms. Since some of the protective effects are ascribed<br />

directly or indirectly to the EPS, their application represents a promising new<br />

approach. However, extensive research is required before microbial EPS can be<br />

applied for corrosion inhibition in larger scale.<br />

Bacterial EPS of different origin and commercially available EPS such as<br />

Xanthan are used to coat alloyed and unalloyed steel. Their protective<br />

capabilities against MIC are assessed using a combination of atomic force and<br />

epifluorescence microscopy (AFM-EFM), fluorescence-in-situ hybridization<br />

(FISH) and weight-loss determinations. To elucidate the protective<br />

mechanisms, the chemical compositions of the applied EPS are analyzed.<br />

AFM images show the formation of dense EPS-layers, completely covering the<br />

surface structures of metal coupons. In MIC-simulations with Desulfovibrio<br />

vulgaris in Postgate medium C, the weight-loss of unalloyed steel was reduced<br />

by up to 74%. Additionally, EPS from SRB partially reduced the primary<br />

adhesion of D. vulgaris on alloyed steel. However, the stability of the EPScoatings<br />

in aqueous media has to be improved and the long-term stability has to<br />

be assessed.<br />

PZ 26<br />

Thio-Based Redox Regulation in Escherichia coli<br />

C. Lindemann *1 , L. Leichert 2 , N. Lupilova 2<br />

1 Medical Proteomic Center (MPC), AG: Redox Proteomics, Ruhr-Universität<br />

Bochum, Bochum, Germany<br />

211<br />

Reactive nitrogen species (RNS) and NO play an important role in the<br />

antimicrobial host defense. These RNS cause molecular and cellular damage,<br />

which eventually will lead to cell death. To survive the insult by RNS and NO<br />

from exogenous and possibly endogenous sources bacteria require protectionmechanisms<br />

to quickly detoxify those species. One of the main cellular targets<br />

of NO and RNS are presumably the cysteine-groups of proteins. Cysteine<br />

modifications can potentially modulate the activity of proteins. However, so far<br />

little is known about the target proteins of RNS and the nature of the<br />

modifications the proteins un<strong>der</strong>go upon RNS-stress treatment. We plan to<br />

study the effects of RNS and NO on proteins in Escherichia coli. We will be

212<br />

using a novel mass spectrometry based method, which allows us to determine<br />

the amount of thiol-modifided proteins un<strong>der</strong> RNS or NO stress conditions in<br />

vitro and in vivo. Proteins identified as RNS-sensitive un<strong>der</strong> aerobic and<br />

anaerobic conditions will be characterized biochemically to elucidate their<br />

possible functions in the cellular response to NO-stress. Our investigation will<br />

be focused on the in vivo role of those proteins and the characterisation of the<br />

thiol-modification of their RNS-sensitive thiol groups. We will test if RNS<br />

sensitive proteins differ in their sensitivity towards different RNS-species,<br />

which would imply that distinct reactive nitrogen species could affect<br />

independent physiological processes in bacteria. Ultimately, we hope this study<br />

will expand our un<strong>der</strong>standing of protection mechanisms against NO-damage<br />

and shed light on the connection between NO-stress and cellular stress.<br />

PZ 27<br />

Regulation and limiting steps of C1 metabolism in<br />

Methylobacterium extorquens AM1<br />

H. Pondelikova - Smejkalova *1 , T.J. Erb 1 , G. Fuchs 1<br />

1 Mikrobiologie, Fakultät Biologie, Albert-Ludwigs Universität, Freiburg,<br />

Germany<br />

Methylobacterium extorquens AM1 is an aerobic α-Proteobacterium type II<br />

methylotroph that can grow on reduced one-carbon compounds such as<br />

methanol as sole source of carbon and energy, which makes this organism an<br />

interesting target for biotechnological applications.<br />

The assimilation of C1 compounds proceeds in this facultative methylotroph<br />

via the serine cycle, which has been well established by the group of R.<br />

Quayle [1] . This cycle starts with glyoxylate, and 1 formaldehyde and 1 CO2 are<br />

assimilated to form 1 acetyl-CoA. Since this bacterium does not have a<br />

glyoxylate cycle for the assimilation of acetyl-CoA, another pathway must<br />

exist.<br />

Here we show that Methylobacterium uses the ethylmalonyl-CoA pathway, a<br />

novel acetate assimilation pathway that has been described originally in<br />

Rhodobacter sphaeroides [2,3] . All enzymatic activities of the ethylmalonyl-CoA<br />

pathway were detected in cells grown on methanol. The reactions of the<br />

ethylmalonyl-CoA pathway were integrated into the scheme of methanol<br />

assimilation in Methylobacterium in which methanol and CO2 contribute nearly<br />

equally to cell carbon.<br />

Moreover, all enzymes of the central carbon metabolism were studied un<strong>der</strong><br />

different growth conditions (C1, C2 and C4 compounds) to detect rate-limiting<br />

steps for cell growth and regulation of the individual steps.<br />

[1] Reviewed in: Anthony, C. (1982). The biochemistry of methylotrophs.<br />

[2] Erb, TJ, Berg, IA, Brecht, V, Müller, M, Fuchs, G, Alber, BE (2007),<br />

PNAS.<br />

[3] Erb, TJ, Retey, J, Fuchs, G, Alber, BE (2008), JBC.<br />

PZ 28<br />

Identification of bacteria isolated from a pharmaceutical<br />

environment<br />

G. Rieser *1 , S. von Brehmer 2 , G. Schuffenhauer 2 , M. Wenning 1<br />

1 Mikrobielle Ökologie, TU München, Freising, Germany<br />

2 Qualitätskontrolle Mikrobiologie, IDT Biologika, Dessau-Rosslau, Germany<br />

In the pharmaceutical industry, production lines for sterile products are located<br />

in clean rooms to prevent contamination. Four different categories of clean<br />

rooms have been defined and a continuous monitoring of contamination levels<br />

is required. Due to legal requirements, such an environmental monitoring must<br />

include the determination of the number of cfu as well as the identification of<br />

the microbes detected.<br />

One commonly used method for identification of microorganisms in the<br />

pharmaceutical industry is the API system (BioMérieux). In this work we used<br />

Fourier-transform infrared (FT-IR) spectroscopy to identify bacteria isolated<br />

within an environmental monitoring process and compared the reliability of<br />

identifications obtained by FT-IR spectroscopy with the commercial API<br />

system. Since the main microbiological contaminants in these environments are<br />

spore forming bacilli and gram-positive cocci, the test was restricted to these<br />

two groups. API 50CHB and API 20E for bacilli and ID 32STAPH were the<br />

systems used from BioMérieux. 16S rDNA sequencing for bacilli and rpoB<br />

gene sequencing for staphylococci served as reference methods for unequivocal<br />

identification. 149 randomly picked isolates from a current monitoring were<br />

tested (94 gram positive cocci and 55 bacilli). Results show that FT-IR<br />

spectroscopy obtained a significantly higher rate of correctly identified strains<br />

at the species level in comparison to the API system and support the suitability<br />

of FT-IR spectroscopy as an alternative identification method for isolates from<br />

these environments. Furthermore, including new strains, e. g. from this study,<br />

in the FT-IR databases will adapt them to the microflora of pharmaceutical<br />

clean rooms and will further improve identification results significantly.<br />

PZ 29<br />

The Escherichia coli peptidyl-prolyl isomerase PpiD - the<br />

periplasmic trigger factor for newly-translocated proteins?<br />

Y. Matern 1 , B. Barion 2 , S. Behrens-Kneip *3<br />

1 P26: Nosocomial Infections of the El<strong>der</strong>ly, Robert-Koch-Institut, Berlin,<br />

Germany<br />

2 Institut für Mikrobiologie und Genetik, Abt. Molekulare Genetik und<br />

Präparative Molekularbiologie, Georg-August-Universität Göttingen,<br />

Göttingen, Germany<br />

At least three periplasmic folding factors are known to assist in the periplasmic<br />

transit and folding of the β-barrel proteins destined for insertion into the outer<br />

membrane of Escherichia coli: the chaperone and peptidyl-prolyl isomerase<br />

(PPIase) SurA, the small general chaperone Skp, and the chaperone and<br />

protease DegP. Biochemical and genetic evidence suggests that among these,<br />

SurA plays a pivotal role in the maturation of this class of outer membrane<br />

proteins (OMPs). The periplasmic PPIase PpiD, which is anchored to the inner<br />

membrane, has been reported to be capable of complementing the in vivo<br />

function of SurA [1]. However, whereas SurA primarily acts as a chaperone<br />

[2], the surA complementing function of PpiD was shown to depend on its<br />

PPIase activity [1]. We have further investigated the role of PpiD in protein<br />

folding. We show that an influence of PpiD on OMP maturation is clearly<br />

observable only in cells that lack both, the SurA and Skp chaperones. Increased<br />

production of PpiD rescues surA skp cells from lethality, reduces their strongly<br />

elevated sigma E and Cpx envelope stress responses, and partially restores OMP<br />

maturation. We also show that a PpiD protein lacking its PPIase-domain<br />

functions in vivo and exhibits chaperone activity in vitro. Our data support a<br />

model in which PpiD primarily acts as a general chaperone for newlytranslocated<br />

envelope proteins. In particular, we propose that PpiD performs a<br />

chaperone function at the periplasmic side of the inner membrane similar to that<br />

of the cytosolic chaperone trigger factor at the ribosome.<br />

[1] Behrens et al. (2001) EMBO J. 20, 285-294.<br />

[2] Dartigalongue and Raina (1998) EMBO J. 14, 3968-3980.<br />

PZ 30<br />

Structure-function analysis of the periplasmic chaperone<br />

SurA of Escherichia coli<br />

Y. Matern *1 , D. Marsh 2 , S. Behrens-Kneip 1<br />

1 P26:, Robert-Koch-Institut, Berlin, Germany<br />

2 Abt. Spektroskopie, Max-Planck-Institut für biophysikalische Chemie,<br />

Göttingen, Germany<br />

The periplasmic chaperone SurA, which also exhibits peptidyl-prolyl isomerase<br />

(PPIase) activity, plays a key role in the biogenesis of the integral β-barrel outer<br />

membrane proteins (OMPs) of Escherichia coli. SurA preferentially interacts<br />

with non-native OMP folding intermediates by specifically recognizing peptide<br />

sequences that are characteristic of this class of proteins [1,2]. A peptide<br />

binding site in SurA that appears to be responsible for its substrate specificity<br />

has recently been located by crystal structure determination of complexes<br />

between model peptides and fragments of SurA [3]. Unexpectedly, binding of<br />

one of the peptides also induced the dimerization of SurA. We have asked<br />

whether the in vitro peptide-SurA and SurA-SurA interactions reflect in vivo<br />

activities of SurA. By using a ToxR-based E. coli two-hybrid system [4] and<br />

site-directed mutagenesis we show that SurA indeed dimerizes in vivo and that<br />

dimer formation involves some of the peptide-SurA and SurA-SurA contacts<br />

observed in the crystal structure as well as additional contacts yet to be<br />

identified. However, our current data also suggest that dimerization of SurA is<br />

no prerequisite for its function in OMP maturation. Furthermore, while the<br />

above studies affirm the proposed peptide binding site in SurA as a site for in<br />

vivo substrate binding, the analysis of its interaction with other known OMP<strong>der</strong>ived<br />

SurA binding peptides [2] by electron paramagnetic resonance (EPR)<br />

spectroscopy suggests that additional sites for selective peptide binding may<br />

exist in SurA. Further studies are currently un<strong>der</strong>taken in or<strong>der</strong> to substantiate<br />

these preliminary results.<br />

[1] Bitto and McKay (2003) J. Biol. Chem. 278: 49316-39322.<br />

[2] Hennecke et al. (2005) J. Biol. Chem. 280: 23540-23548.<br />

[3] Xu et al. (2007) J. Mol. Biol. 373: 367-381.<br />

[4] Hennecke et al. (2005) Protein Eng. Des. Sel. 18:477-86.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PZ 31<br />

Production of biogenic amines by wine-related<br />

microorganisms and their detection by RP-HPLC in<br />

German Wines<br />

E. Kaschak *1 , P. Sebastian 1 , P. Pfeiffer 1 , H. König 1<br />

1<br />

Institut für Mikrobiologie und Weinforschung, Johannes Gutenberg<br />

Universität, Mainz, Germany<br />

Biogenic amines may occur in different food products such as fish, seafood,<br />

cheese and wine. About nine biogenic amines have been found in wine.<br />

Ingestion of biogenic amines can cause different pseudo-allergic reactions like<br />

an increase in systolic blood pressure, migraine, urticaria, rhinitis and in few<br />

cases an anaphylactic shock has been observed [1]. Furthermore the<br />

consumption of biogenic amines in wine is problematic since ethanol and<br />

acetaldehyde inhibit the activity of mono- and diamine oxidases, which<br />

normally degrade biogenic amines.<br />

In addition the EU will probably regulate the upper limits of biogenic amines in<br />

wine in the near future. This was the reason for our present study about the<br />

production and distribution of biogenic amines in German wines. Biogenic<br />

amines producing bacterial strains were identified and the conditions for<br />

production were analysed. The detection method was improved involving solid<br />

phase extraction before <strong>der</strong>ivatization of the amines with ortho-Phtalaldehyd<br />

(OPA), reversed-phase HPLC with gradient elution and fluorimetric detection<br />

[2]. This study included more than 60 German wines from the 13 most<br />

important wine growing regions.<br />

[1] Askar et al. (1996). Biogene Amine in <strong>der</strong> Ernährung. Springer Verlag.<br />

[2] Pfeiffer, P. und Orben, C. (1996). Deutsche Lebensmittel-Rundschau 92,<br />

39-42.<br />

PZ 32<br />

Acetone production by Escherichia coli via a new metabolic<br />

pathway<br />

A. May 1 , S. Piehl *1 , R.J. Fischer 1 , H. Bahl 1<br />

1 Institut für Biowissenschaften/Mikrobiologie, Universität Rostock, Rostock,<br />

Germany<br />

In the classic ABE fermentation by Clostridium acetobutylicum a mixture of<br />

solvents is produced (acetone, butanol, ethanol). The heterologous expression<br />

of the corresponding genes in an industrial production strain such as E. coli is<br />

one possibility to yield acetone as the only product. Here we present a new<br />

metabolic pathway for an acetate independent acetone formation in E. coli. The<br />

production is based on plasmid-mediated expression of thiolase A (ThlA) and<br />

acetoacetate decarboxylase (Adc) from Clostridium acetobutylicum in<br />

combination with thioesterase II (TEII) from Bacillus subtilis or YbgC from<br />

Haemophilus influenzae. TEII and YbgC showed thioesterase activity in vitro<br />

with acetoacetyl-CoA as substrate. The corresponding genes teII or ybgc were<br />

cloned together with thlA and adc from C. acetobutylicum as an operon un<strong>der</strong><br />

control of the lac or clostridial thl promoters. Among several strains,<br />

production of acetone up to 66 mM (3.8 g/l) could be demonstrated in strain E.<br />

coli HB101.<br />

PZ 33<br />

Biochemical characterization of a highly active new type of<br />

intracellular PHB depolymerase activity mobilizing and<br />

converting PHB quantitatively to (R)-3-HB within 20 min<br />

D. Pfeiffer *1 , D. Jendrossek 1<br />

1 Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany<br />

Bacteria significantly differ in the speed of intracellular mobilization of<br />

accumulated PHA. Mineral salts medium-grown cells of Ralstonia eutropha, for<br />

example, need about one week of incubation in carbon source free mineral salts<br />

medium to mobilize most of previously accumulated PHB. PHB mobilization<br />

times up to almost 2 years (in distilled water) have been described for a<br />

Legionella strain [1]. Rapid mobilization of accumulated PHB within 20 h has<br />

been determined for Hydrogenophaga pseudoflava in carbon source free<br />

mineral salts medium [2]. In Azohydromonas lata rapid mobilization and<br />

secretion of high amounts of 3-hydroxybutyrate (3HB) occurred at pH 3 - 4 [3].<br />

In conclusion, bacteria highly differ in their velocity of intracellular PHB<br />

mobilization. In this contribution we investigated the PHB mobilization system<br />

of A. lata. Evidence for the presence of two different PHB granule-attached<br />

PHB depolymerases were found. One PHB depolymerase activity was active at<br />

alkaline pH (i-PHB8 depolymerase) and shared characteristics of previously<br />

described PHB depolymerases. The other activity was most active at pH 5 (i-<br />

PHB5 depolymerase) and consi<strong>der</strong>ably differed from all known PHB<br />

depolymerases.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

213<br />

[1] James, B. W., Mauchline, W. S., Dennis, P. J., Keevil, C. W., and Wait, R.<br />

(1999) Appl Environ Microbiol 65(2), 822-827<br />

[2] Yoon, S. C., and Choi, M. H. (1999) J Biol Chem 274(53), 37800-37808<br />

[3] Lee, S. Y., Lee, Y., and Wang, F. (1999) Biotechnol Bioeng 65(3), 363-368<br />

PZ 34<br />

Formation of Volatile Metal(loid) Species by Intestinal<br />

Microorganisms<br />

R. Diaz-Bone *1 , T. VAN DE WIELE 2<br />

1<br />

Institute of Environmental Analytical Chemistry, University of Duisburg-<br />

Essen, Essen, Germany<br />

2<br />

Laboratory of Microbial Ecology and Technology, University of Ghent, Gent,<br />

Belgium<br />

Methylation and hydrogenation of metal(loid)s by microorganisms are<br />

widespread and well-known processes in the environment, by which mobility<br />

and in most cases toxicity are significantly enhanced in comparison to<br />

inorganic species. Though the human gut contains a highly diverse and active<br />

microbiocenosis, little is known about the occurrence and importance of this<br />

process in the human intestine. Recent in vivo studies demonstrated that<br />

ingested inorganic bismuth is metabolized to volatile and neurotoxic trimethyl<br />

bismuth in the human gut. In this study, we compared the capability of<br />

intestinal microorganisms to volatilize different metal(oid)s (Ge, As, Sn, Sb,<br />

Te, Hg, Pb and Bi) as well as the nonmetal selenium. Due to both ethical and<br />

experimental consi<strong>der</strong>ations, an in vitro gastrointestinal model, the Simulator of<br />

the Human Intestinal Ecosystem (SHIME), was used.<br />

These experiments clearly showed that intestinal microorganisms are capable to<br />

volatilize As, Se, Te, Sb and Bi from inorganic salts. In dependence on the<br />

element concentration and the part of the large intestine simulated, different<br />

species were detected. Most surprising was the detection of highly toxic arsine<br />

(AsH3), which was the most important volatile arsenic compounds found in the<br />

majority of the experiments. In addition to methylated species of Se, Te, Sb and<br />

Bi, arsenic and selenium species containing both methyl and thiomethyl (-<br />

SCH3) groups were identified using simultaneous elemental (ICP-MS) and<br />

molecular detection (EI-MS) hyphenated to gas chromatography.<br />

These results suggest that the intestinal microbiota can significantly increase<br />

the mobility and toxicity of orally ingested metal(loid)s. Further studies are<br />

necessary to investigate the extent of this process as well as the availability of<br />

metal(loid)s from different sources for microbial transformations.<br />

PZ 35<br />

Formation of Volatile Metal(loid) Species by Intestinal<br />

Microorganisms<br />

R. Diaz-Bone *1 , T. VAN DE WIELE 2<br />

1<br />

Institute of Environmental Analytical Chemistry, University of Duisburg-<br />

Essen, Essen, Germany<br />

2<br />

Laboratory of Microbial Ecology and Technology, University of Ghent, Gent,<br />

Belgium<br />

Methylation and hydrogenation of metal(loid)s by microorganisms are<br />

widespread and well-known processes in the environment, by which mobility<br />

and in most cases toxicity are significantly enhanced in comparison to<br />

inorganic species. Though the human gut contains a highly diverse and active<br />

microbiocenosis, little is known about the occurrence and importance of this<br />

process in the human intestine. Recent in vivo studies demonstrated that<br />

ingested inorganic bismuth is metabolized to volatile and neurotoxic trimethyl<br />

bismuth in the human gut. In this study, we compared the capability of<br />

intestinal microorganisms to volatilize different metal(oid)s (Ge, As, Sn, Sb,<br />

Te, Hg, Pb and Bi) as well as the nonmetal selenium. Due to both ethical and<br />

experimental consi<strong>der</strong>ations, an in vitro gastrointestinal model, the Simulator of<br />

the Human Intestinal Ecosystem (SHIME), was used.<br />

These experiments clearly showed that intestinal microorganisms are capable to<br />

volatilize As, Se, Te, Sb and Bi from inorganic salts. In dependence on the<br />

element concentration and the part of the large intestine simulated, different<br />

species were detected. Most surprising was the detection of highly toxic arsine<br />

(AsH3), which was the most important volatile arsenic compounds found in the<br />

majority of the experiments. In addition to methylated species of Se, Te, Sb and<br />

Bi, arsenic and selenium species containing both methyl and thiomethyl (-<br />

SCH3) groups were identified using simultaneous elemental (ICP-MS) and<br />

molecular detection (EI-MS) hyphenated to gas chromatography.<br />

These results suggest that the intestinal microbiota can significantly increase<br />

the mobility and toxicity of orally ingested metal(loid)s. Further studies are<br />

necessary to investigate the extent of this process as well as the availability of<br />

metal(loid)s from different sources for microbial transformations.

214<br />

PZ 36<br />

Structural and mutational analysis of a salicylate 1,2dioxygenase<br />

activity from Pseudaminobacter<br />

salicylatoxidans<br />

L. Steimer *1 , S. Bürger 1 , I. Matera 2 , M. Ferraroni 2 , F. Briganti 2 , A. Stolz 1<br />

1 Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany<br />

2 Department of Chemistry, University of Florence, Florence, Italy<br />

The bacterium Pseudaminobacter salicylatoxidans salicylatoxidans produces a<br />

ring-fission dioxygenase which cleaves gentisate and several<br />

monohydroxylated aromatic compounds, such as salicylate, substituted<br />

salicylates, and 1-hydroxy-2-naphthoate. The encoding gene is homologous to<br />

previously described gentisate 1,2-dioxygenases and 1-hydroxy-2-naphthoate<br />

dioxygenases from other bacterial sources. The structure of the enzyme has<br />

been solved and it was demonstrated that the enzyme belongs to the cupin<br />

superfamily. The holoenzyme consists of 4 identical subunits. Each subunit<br />

contains one catalytically active Fe(II)-ion which is complexed by 3 histidine<br />

residues. We are currently attempting to analyse the molecular basis for the<br />

unique ability of this dioxygenase to oxidatively cleave a wide range of<br />

monohydroxylated aromatics. Therefore, from the crystal structure several<br />

amino acid residues were identified that are presumably involved in substrate<br />

binding. These amino acid residues were changed by site-directed mutagenesis.<br />

The enzyme variants were analysed for the conversion of gentisate, salicylate,<br />

5-methylsalicylate, 5-fluorosalicylate, and 1-hydroxy-2-naphthoate and the<br />

fundamental kinetic parameters determined. These experiments revealed that<br />

only a single amino acid exchange was necessary to convert the enzyme into<br />

one which specifically cleaves 1-hydroxy-2-naphthoate and demonstrating only<br />

residual activities with the other substrates. In addition, a different mutant was<br />

identified, which converted gentisate nearly with the same rate as the wildtype<br />

enzyme, but almost completely lost its activity for the other substrates tested.<br />

We are currently using the information obtained from these and other mutants<br />

in or<strong>der</strong> to identify the structural characteristics which allow the enzyme to<br />

convert monohydroxylated aromatics.<br />

PZ 37<br />

Microbial community characterisation of the CO2 reservoir<br />

in saline aquifer in Ketzin, Germany<br />

D. Morozova *1 , M. Wandrey 2 , A. Vieth 3 , H. Würdemann 2<br />

1 GeoForschungsZentrum Potsdam, Geoengineering, Potsdam, Germany<br />

2 Geoengineering, GeoForschungsZentrum Potsdam, Potsdam, Germany<br />

3 GeoForschungsZentrum Potsdam, Organic Geochemistry, Potsdam, Germany<br />

The investigations on the possibilities to reduce the CO2 emissions are<br />

receiving a great attention as driven by the global warming effects of the CO2<br />

gas, accumulated in the atmosphere. Within the CO2SINK project, in the<br />

frames of the international „Greenhouse-gas Removal Apprenticeship and<br />

Student Program“ (GRASP), sponsored by the European Commission, we<br />

investigate the effects of un<strong>der</strong>ground CO2 storage by injecting into saline<br />

aquifer near Ketzin, Germany. The main emphasis of the microbial monitoring<br />

is put onto locating, identifying and analysing the composition and activity of<br />

the microbial community, unravelling the origin and fate of dissolved organic<br />

matter, and characterising microbial life in extreme habitats and its influence on<br />

mineralization as well as their impact on the technical effectiveness of the CO2<br />

storage technique.<br />

The first results of the fluid samples analyses revealed high diversity of the<br />

saline aquifer inhabitants. The deep biosphere community was dominated by<br />

the haloalkaliphilic fermentative bacteria and extremophilic organisms,<br />

coinciding with reduced conditions, high salinity and pressure. Beside<br />

halophilic bacteria the sulphate reducing bacteria were found, known to be<br />

involved in corrosion processes. The reactions between the microorganisms and<br />

the minerals of both the reservoir rock and the cap rock may cause major<br />

changes in the structure and chemical composition of the rock formations,<br />

corrosion at the casing and the casing cement around the well. Analyses of<br />

microbial community composition and its changes provide information about<br />

the efficiency and reliability of the long-term CO2 storage technique.<br />

PZ 38<br />

Detection of hepatitis b virus by loop mediated Isothermal<br />

amplification(LAMP)<br />

E. Moslemi *1 , M.H. Shahhosseiny 2 , K. Parivar 1 , T. Nejad sattari 1<br />

1<br />

Science and research branch, Cellular and molecular department, Azad<br />

University, Tehran, Iran<br />

2<br />

Shahryar unit/Quds branch, Microbiology department, Azad University,<br />

Tehran, Iran<br />

Background/objectives: Hepatitis b virus (HBV) belongs to hepadnaviridea<br />

family which is one of the main factors of hepatocellular carcinoma and liver<br />

diseases.<br />

The HBV detection methods (serological and molecular) have their own<br />

limitations, so the using ability of them in all diagnosis centers is not possible.<br />

In this study it has been tried to apply new novel LAMP technique on serum<br />

samples by using specific primer designed for HBs region.<br />

The advantage of this method is that the reaction can be performed by using a<br />

simple heater and there is no need to developed thermal cycler.<br />

Material and methods: in this study we use HBsAg positive serum samples.<br />

DNA was extracted by DNP kit and then PCR reaction was optimized by using<br />

specific primers. All Elisa positive samples were studied by PCR.<br />

6 specific primers were designed for LAMP technique and then the LAMP<br />

reaction was set up on the samples.<br />

At the end of the reaction SYBR Green was used for identifying negative and<br />

positive products.<br />

Result: 21.24% (18)of Elisa positive samples had negative result in both PCR<br />

and LAMP techniques.17.7%(15) of PCR negative samples were positive<br />

result in LAMP tests. By using PCR we can determine 40 copies/ml but by<br />

novel LAMP technique we can detect 10 copies/ml.<br />

Conclusion: in compression the use of 3 Elisa, PCR and LAMP technique on<br />

samples with definite virus particles the LAMP technique had more specificity<br />

and sensivity.<br />

PZ 39<br />

Cellular damage and storage stability of Lactobacillus<br />

rhamnosus GG dried in selected disaccharides un<strong>der</strong><br />

vacuum.<br />

E.O. Sunny-Roberts *1 , D. Knorr 1<br />

1 Department of Food Biotechnology and Food Process Engineering, Berlin<br />

University of Technology, Berlin, Germany<br />

The survival of cell concentrates of Lactobacillus rhamnosus GG (LGG) in<br />

selected dissacharides after drying un<strong>der</strong> vacuum was evaluated. Drying is a<br />

useful technique for preserving foods, agricultural products and<br />

pharmaceuticals. However, biological materials can be irreversibly damaged<br />

during this treatment resulting in substantial loss of viability and activity. In<br />

this paper, disaccharide systems (trehalose, sucrose and lactose) were used as<br />

protectants for L. rhamnosus GG. Among the solutes examined, trehalose<br />

improved the recovery of viable cells after drying as well as during storage at<br />

4°C and 25°C. Stored vacuum-dried trehalose systems were found as viscous<br />

syrups which indicated that they were not glassy. Damage of cell membrane<br />

and reduction in colony sizes occurred as a result of dehydration inactivation.<br />

Comparison of the conventional techniques with flow cytometric viability<br />

assessment after drying revealed the occurrence of certain cell population<br />

which were stressed and lost their ability to grow on agar. The presence of such<br />

non-culturable bacteria with a high degree of membrane intactness in food<br />

might be critical as they may be active in excreting toxic or food spoilage<br />

metabolites. The protecting ability of trehalose on bacterial cells was only<br />

against lysozyme and pepsin actions. These results could have some relevance,<br />

especially in un<strong>der</strong>developed countries, for the production of functional<br />

confections.<br />

PZ 40<br />

Identification of genes essential for magnetotaxis within the<br />

genome of Magnetospirillum gryphiswaldense<br />

S. Ullrich *1 , C. Jogler 1 , E. Katzmann 1 , D. Schüler 1<br />

1 Department Biologie I Bereich Mikrobiologie, Ludwig-Maximilians-<br />

Universität, München, Germany<br />

Most genes required for magnetosome synthesis in Magnetospirillum<br />

gryphiswaldense are clustered within several operons of the 130 kb conserved<br />

genomic „magnetosome island“ (MAI), which however contains substantial<br />

numbers of pseudogenes, genes of unknown function, and transposases. To<br />

elucidate the subset of MAI genes essential for magnetotaxis, we combined the<br />

genotyping of spontaneous non-magnetic mutants and a systematic deletion<br />

analysis of large regions within the MAI and outside the MAI that were<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

identified by comparative genome analysis as putative candidates for<br />

magnetosome genes.<br />

All analysed spontaneous non-magnetic mutants had single or multiple<br />

deletions within several magnetosome operons including the mamAB and the<br />

mms6 cluster, indicating the essential role of these genes in magnetosome<br />

formation. For the construction of large deletions both Cre-lox excision and a<br />

RecA-mediated double crossover strategy was employed. Consistently with the<br />

analysis of spontaneous mutants, a deletion of the mamAB operon resulted in a<br />

non-magnetic phenotype. Preliminary results show that the mms6 operon is<br />

involved in magnetite biomineralization. Deletion of the region between the<br />

mamAB and the mamXY operon in the non-magnetic spontaneous mutant MSR-<br />

1B indicates that the MAI is not required for growth of MSR-1. We also<br />

demonstrated by insertion-duplication mutagenesis that certain targets outside<br />

the MAI are not essential for growth but important for magnetosome formation.<br />

We conclude that the Cre-lox excision system is a powerful method for genome<br />

engineering in MSR-1, and that a large fraction of MAI genes is necessary for<br />

magnetotaxis, but not required for growth.<br />

PZ 41<br />

Rumen fungi as direct-fed microbial: an uncovered<br />

approach for animal productivity<br />

A.K. Puniya *1 , S.S. Dagar 1<br />

1 Dairy Microbiology Division, National Dairy Research Institute, Karnal, India<br />

Escalating demand for improved production efficiency of domesticated animals<br />

has resulted in the development of different approaches for rumen microbial<br />

manipulation. These approaches mainly comprised of dietary modulations of<br />

ruminants and/ or live microbial supplementation. The microbial feed additives<br />

are natural growth enhancer and termed as direct-fed microbial (DFM) that<br />

include viable cultures of yeasts, moulds or bacteria. Their mode of action<br />

encompasses secretion of antimicrobial substances, growth promoters,<br />

bioactive compounds and fiber degrading enzymes besides, enhancing host<br />

immune response. Conventionally, yeasts and aerobic fungi were prominently<br />

used as DFM for ruminants. But, recently use of anaerobic fungi as DFM is<br />

emphasized because of its ability to produce wide array of fibrolytic enzymes in<br />

the rumen. Degradation of lignified plant cell-wall is an important feature of<br />

rumen fungi because rhizoids of rumen fungi penetrate plant tissues much<br />

better than bacteria and protozoa. These fungi have also been found to produce<br />

conjugated linoleic acid that has many health promoting attributes to the<br />

consumers. Our research group have isolated fungi from rumen/ faecal samples<br />

of different domestic and wild, ruminants and non-ruminants. These fungi have<br />

been found to possess significant in vitro hydrolytic enzyme activities viz.<br />

cellulase, filter paperase, cellobiase and xylanase. The oral administration of<br />

fibrolytic fungi was also reported to increase growth rate, rumen fermentation<br />

and nutrient digestibility in cattle/ buffalo calves and also enhanced milk<br />

production non-significantly in buffaloes. Hence, in this paper it has been<br />

reviewed that the fiber-degrading ruminal fungi are prevalent in domestic as<br />

well as wild ruminants and non-ruminant herbivores, and their fibrolytic<br />

potential could be exploited through their use as live animal feed additives for<br />

domesticated ruminants for improved animal nutrition and/ or enhanced milk<br />

production.<br />

PZ 42<br />

Membrane fluidity guides bacterial surface motility<br />

C. Holz *1 , D. Opitz 1 , J. Mehlich 2 , B.J. Ravoo 2 , B. Maier 1<br />

1<br />

Institut für Allgemeine Zoologie und Genetik, Westfälische Willhelms-<br />

Universität Münster, Münster, Germany<br />

2<br />

Institut für Organische Chemie, Westfälische Wilhelms-Universität Münster,<br />

Münster, Germany<br />

Bacterial surface motility enables bacteria to form microcolonies, colonise<br />

human host cells and abiotic surfaces, and is often required for biofilm<br />

formation. Twitching motility is powered by polymeric cell appendages called<br />

type IV pili. They act as grappling hooks that support motility by a cycle of<br />

pilus elongation, surface adhesion and retraction. It is very poorly un<strong>der</strong>stood<br />

how bacteria control the velocity and direction of twitching.<br />

We investigated twitching motility of the human pathogen Neisseria<br />

gonorrhoeae on different surfaces including glass-supported membranes to<br />

mimic cell surfaces. We found that bacteria twitch with a velocity of ~1µm/sec<br />

and that movement is persistent on a time scale of around 8sec. Velocity and<br />

persistence increased with decreasing fluidity of solid supported membranes.<br />

On micropatterned surfaces, bacterial movement was confined to the least fluid<br />

regions, i.e. we found that motility was guided by surface fluidity. Our<br />

experiments reveal an unprecedented physical mechanism for controlling the<br />

direction of twitching motility and we hypothesize that this mechanism is<br />

involved in formation of microcolonies during infection.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PZ 43<br />

Changes of the proteome of Halomonas elongata in response<br />

to osmotic stress analysed by LC-ESI-MS/MS<br />

S. Faßben<strong>der</strong> *1 , D. Burdziak 2 , G. Lentzen 3 , I. Bagyan 3 , F. Siedler 4 , B. Scheffer 4 ,<br />

F. Pfeiffer 4 , D. Oesterhelt 4 , H.P. Klenk 5 , S. Schuster 6 , H.J. Kunte 1<br />

1 FG IV.1, Bundesanstalt für Materialforschung und –prüfung (BAM), Berlin,<br />

Germany<br />

2 Life Science Group, BioRad, München, Germany<br />

3 Forschung und Entwicklung, bitop, Witten, Germany<br />

4 Abt. Membranbiochemie, Max-Planck-Institut für Biochemie, Martinsried,<br />

Germany<br />

5 Mikrobiologie, Deutsche Sammlung von Mikrooorganismen und Zellkulturen<br />

(DSMZ), Braunschweig, Germany<br />

6 Department of Biochemistry and Molecular Biology, Pennsylvania State<br />

University, University Park, United States<br />

Halomonas elongata, a halophilic γ-proteobacterium, is used by industry for the<br />

production of the compatible solute ectoine. Ectoine can function as a cell<br />

stabilizer and is therefore a much sought-after compound in cosmetic industry<br />

and in medical applications e.g. <strong>der</strong>matology. To design more efficient<br />

producer strains for ectoines, we determined the sequence of the H. elongata<br />

genome using the pyrosequencing technique and annotated the genome<br />

automatically and manually. The genome consists of 4.062.437 bp and is<br />

predicted to encode 2711 putative soluble proteins and 762 potential proteins<br />

carrying transmembrane domains. In or<strong>der</strong> to find proteins that are involved in<br />

osmoregulation, the soluble proteom and membrane proteom of H. elongata<br />

were analyzed by 1D-SDS-PAGE followed by LC-MS/MS. Comparing the<br />

membrane proteom of cells grown at different salinities revealed that certain<br />

proteins of the flagellar motor were present only in cells from high salt medium<br />

indicating that the motility of the cells changes with the salt concentration.<br />

Inoculated on swarming agar, cells of H. elongata were non-motile on low salt<br />

agar (0.1 M NaCl), while cells at higher salinities were motile. In cells from<br />

high salt medium a putative thioredoxin-like protein was detectable, which is<br />

encoded by an ORF annotated as yhgI. Deletion of yhgI created a mutant that is<br />

unable to tolerate more than 1.7 M NaCl. The wild-type salt tolerance in the<br />

mutant could be restored by expressing yhgI from a plasmid. Interestingly, yhgI<br />

from Escherichia coli could complement the yhgI-deletion in H. elongata as<br />

well. These results showed that proteom analysis is a promising tool in<br />

identifying new osmoregulatory mechanisms in halophilic bacteria.<br />

PZ 44<br />

Analysis of [FeFe]-Hydrogenase H-Cluster biosynthesis<br />

I. Husemann *1 , A. Silakov 2 , W. Lubitz 2 , T. Happe 1<br />

1 Plant Biochemistry, Ruhr-University Bochum, Bochum, Germany<br />

2 Bioinorganic Chemistry, Max-Planck Institute, Mülheim a.d. Ruhr, Germany<br />

215<br />

The active site of [FeFe] hydrogenase, the so called H-cluster consists of a<br />

[4Fe4S] cluster connected to a [2Fe2S] subcluster. The irons of the subcluster<br />

are coordinated by CO and CN ligands and a CO bridge, a dithiolate bridge<br />

connects the sulphur atoms [1]. Two radical S-adenosylmethionine proteins<br />

(HydE, HydG) and a GTPase (HydF) are required for the assembly of an active<br />

H-cluster. Homologues of these maturation factors are common in all<br />

organisms containing [FeFe]-hydrogenase [2]. To get active HydE, HydF and<br />

HydG, the proteins have to be expressed in concert [3]. At current state the role<br />

of the proteins in the maturation process is not completely un<strong>der</strong>stood.<br />

Clostridium acetobutylicum naturally provides all three maturation factors, so<br />

we choose it as an overexpression system to overexpress active HydE, HydF<br />

and HydG. The proteins can be isolated in high amounts via StrepTagII-affinity<br />

purification. To analyse the single steps of the maturation process we<br />

established an in vitro maturation assay, in which we can recover the activity of<br />

inactive hydrogenase apoproteins of different species up to 100% compared to<br />

its native activity. Furthermore we did first biochemical and biophysical<br />

characterisations of the maturation factors and the apoprotein to get an idea<br />

what happens in the maturation process.<br />

[1] J. W. Peters, W. N. Lanzilotta, B. J. Lemon, L. C. Seefeldt, Science 1998,<br />

282, 1853.<br />

[2] M. C. Posewitz, P. W. King, S. L. Smolinski, L. P. Zhang, M. Seibert, M. L.<br />

Ghirardi, Journal of Biological Chemistry 2004, 279, 25711.<br />

[3] S. E. McGlynn, S. S. Ruebush, A. Naumov, L. E. Nagy, A. Dubini, P. W.<br />

King, J. B. Bro<strong>der</strong>ick, M. C. Posewitz, J. W. Peters, Journal of Biological<br />

Inorganic Chemistry 2007, 12, 443.

216<br />

PZ 45<br />

Pseudomonas aeruginosa PAO1 preferentially grows as<br />

aggregates in liquid batch culture and disperses upon<br />

starvation<br />

D. Schleheck *1 , N. Barraud 2 , J. Klebensberger 2 , J. Webb 3 , D. McDougald 2 , S.<br />

Rice 2 , S. Kjelleberg 2<br />

1 Microbial Ecology, University of Konstanz, Konstanz, Germany<br />

2 Centre for Marine Bio-Innovation, University of New South Wales, Sydney,<br />

Australia<br />

3 School of Biological Sciences, University of Southampton, Southampton,<br />

United Kingdom<br />

In natural and artificial environments, bacteria predominantly grow in biofilms,<br />

and often disperse from biofilms as freely suspended single-cells. In the present<br />

study, the formation and dispersal of planktonic cellular aggregates, or<br />

‘suspended biofilms’, by Pseudomonas aeruginosa in liquid cultures was<br />

closely examined. Cultures were monitored by detailed growth curves, and<br />

plankton samples were analyzed by particle-size scanning by laser-diffraction<br />

(LDA) in the range 0.5 - 00 µm diameter, and by microscopy of aggregates<br />

collected by sedimentation. Interestingly, LDA indicated that up to 90% of the<br />

total planktonic biomass consisted of cellular aggregates in the size range 10 –<br />

400 µm diameter during the growth phase, as opposed to individual cells.<br />

However, upon carbon or nitrogen starvation, or during oxygen limitation, the<br />

planktonic aggregates dispersed into single cells, resulting in an increase in<br />

optical density (OD) independent of cellular growth. During growth, planktonic<br />

aggregates contained viable cells and extracellular DNA (eDNA), and<br />

starvation resulted in loss of viable cells, an increase in dead cells and eDNA,<br />

and release of metabolites and superinfective bacteriophage into the culture<br />

supernatant. Furthermore, carbon starvation induced a marked decrease in<br />

intracellular concentration of the second messenger cyclic di-GMP. Thus, what<br />

traditionally have been described as planktonic, individual cell cultures, are in<br />

fact composed of suspended biofilms, and such suspended biofilms have<br />

behaviours and responses (e.g. dispersal) similar to surface-associated biofilms.<br />

In addition, we suggest that this planktonic biofilm model system can provide<br />

the basis for a detailed analysis of the synchronized biofilm life cycle of P.<br />

aeruginosa.<br />

PZ 46<br />

Pseudomonas aeruginosa porphobilinogen synthase in<br />

complex with the antibiotic alaremycin<br />

C. Schulz *1 , I. Heinemann 1 , M. Jahn 1 , D. Jahn 1<br />

1 Institut für Mikrobiologie, TU Braunschweig, Braunschweig, Germany<br />

Tetrapyrroles participate in fundamental biological processes such as<br />

photosynthesis and respiration. The structural core of these molecules implies a<br />

highly conserved biosynthetic pathway which comprises seven enzymatic<br />

reactions starting from the common precursor molecule 5-aminolevulinic acid<br />

(ALA) to protoheme. The well characterized porphobilinogen synthase (PBGS)<br />

catalyzes the asymmetric condensation of two molecules of ALA to form<br />

porphobilinogen. The structure of Pseudomonas aeruginosa PBGS in complex<br />

with the isolated natural antibiotic alaremycin from Streptomyces sp. A012304<br />

has been solved lately. Kinetic parameters un<strong>der</strong>score the function of<br />

alaremycin as an inhibitor of tetrapyrrole biosynthesis. Now the PBGS from<br />

Streptomyces sp. A012304 has been isolated and characterized in reference to<br />

own produced antibiotic alaremycin. Current crystallisation studies of the<br />

Streptomyces sp. A012304 PBGS with its inhibitor will provide further insights<br />

into the molecular strategy of Streptomyces sp. to protect itself from the<br />

detrimental effect of the antibiotic alaremycin<br />

PZ 47<br />

Characterization of the major outer membrane porins from<br />

Providencia stuartii<br />

Q.T. Tran *1 , M. Kozhinjampara 1 , A. Regli 2 , M. Ullrich 1 , J.M. Pagès 2 , M.<br />

Winterhalter 1 , H. Weingart 1<br />

1 School of Engineering and Science, Jacobs University Bremen, Bremen,<br />

Germany<br />

2 Laboratoire UMR-MD1, Facultés de Médecine et de Pharmacie, Université de<br />

la Méditerranée, Marseille, France<br />

Providencia are pathogenic bacterial agents involved in hospital-acquired<br />

infections. They appear among the most frequently isolated species in clinic<br />

and cause urinary tract infections with severe symptoms e.g. kidney stone.<br />

Providencia stuartii is one of the most antibiotic-resistant species in the family<br />

of Enterobacteriaceae. Various clinical isolates of P. stuartii demonstrate a<br />

multidrug resistance phenotype with a very high level of resistance to<br />

carbapenems and cephalosporins up to the last generation such as cefepime and<br />

cefpirome. These antibiotics are known to use porins as major influx pathway<br />

into the cells.<br />

Currently, two porins (PROSTU_01774 and PROSTU_03464) were identified<br />

in P. stuartii ATCC 25827. They shared about 50% amino acid sequence<br />

identity with OmpF and OmpC from E. coli and 74% identity with each other.<br />

Little is known about these porins as well as their roles in Providencia. Overexpression<br />

and purification of the two porins in P. stuartii ATCC 29914 was<br />

carried out for electrophysiological characterization using planar lipid bilayer<br />

technique. The translocation of several selected antibiotic molecules through<br />

these proteins was studied. Moreover, we aim to create knockout mutants in<br />

or<strong>der</strong> to study the functionality of the porins and to un<strong>der</strong>stand more about the<br />

role of these porins in drug resistance of Providencia stuartii.<br />

PZ 48<br />

Novel quorum quenching clones from Rhizobium sp.<br />

NGR234<br />

D. Krysciak *1 , J. Riethausen 1 , M. Quitschau 2 , S. Grond 3 , W. Streit 1<br />

1 Microbiology and Biotechnology, University of Hamburg, Hamburg, Germany<br />

2 Institute for Organic Chemistry, University of Goettingen, Göttingen, Germany<br />

Quenching microbial quorum sensing represents a useful, novel strategy to<br />

control microbial infections. Of consi<strong>der</strong>able interest are enzymes capable of<br />

hydrolyzing N-acyl-L-homoserine lactones (NAHLs) and thus their ability to<br />

block undesirable NAHL-mediated quorum sensing phenotypes e.g. biofilm<br />

formation. A genomic library of Rhizobium sp. NGR234 was screend for the<br />

presence of cosmids conferring N-acyl homoserine lactone degradation ability.<br />

By using the biosensor strain Agrobacterium tumefaciens NTL4 with a traIlacZ<br />

gene fusion we identified at least six loci that interfere with bacterial<br />

quorum sensing. In this study we report the isolation and biochemical<br />

characterisation of two open reading frames (designated as 1612 and 2545)<br />

involved in the degradation of N-(3-oxooctanoyl)-L-homoserine lactone (3oxo-C8-HSL).<br />

Sequence analysis of ORF 1612 revealed no significant<br />

similarities to other known lactonases, but shares a "HxHxDH" zinc-binding<br />

motif that is conserved in several groups of metallohydrolases. Exogenous<br />

addition of crude extracts of E. coli expressing 1612 as well as purified protein<br />

to Pseudomonas aeruginosa PAO1 cultures reproducibily inhibited motility and<br />

biofilm formation. Sequence analysis of gene 2545 revealed weak similarities<br />

to a dienelactone hydrolase family. Crude extracts of protein 2545 exogenous<br />

added to Pseudomonas cultures resulted in inhibited motility. Overexpression<br />

and purification of protein 2545 are ongoing. Additionally purified 1612<br />

protein was shown to degrade 3-oxo-C8-HSL resulting in a reduced production<br />

of pyocyanine in P. aeruoginosa PAO1 and violaceine in the reporter strain<br />

Chromobacterium violaceum CV026. Current work focuses on the detailed<br />

analysis of the cleaving mechanisms with high-performance liquid<br />

chromatography-mass spectrometry.<br />

PZ 49<br />

Development of techniques for genetic manipulation of<br />

members of the Roseobacter clade<br />

T. Piekarski *1 , P. Tielen 1 , D. Jahn 1<br />

1 Institute of Microbiology, Technische Universität Braunschweig,<br />

Braunschweig, Germany<br />

Bacteria of the Roseobacter clade belong to the family Rhodobacteraceae of α-<br />

Proteobacteria. They colonize diverse marine habitats and show a high<br />

physiological diversity.<br />

Since no methods for the genetic manipulation of the Roseobacter group are<br />

described, we developed a suitable system for their genetic engineering.<br />

We tested six different species belonging to the Roseobacter clade.<br />

To determine possible selectable markers, we tested these strains for their<br />

susceptibility to different antibiotics and correlated our results with their<br />

genetic repertoire.<br />

Different methods of DNA transfer as conjugation, electroporation and<br />

transformation of chemo-competent cells were tested to identify an appropriate<br />

technique of gene transfer. Plasmid transfer is hin<strong>der</strong>ed due to incompatibility<br />

reactions of natural plasmids present in many strains of the Roseobacter group.<br />

We analysed different common broad-host range vectors for transfer efficiency<br />

and plasmid stability in the Roseobacter cells. Moreover, promoter recognition<br />

was demonstrated by fluorescent flavinmononucleotide-based fluorescent<br />

protein (FbFP) expression driven by a kanamycin resistance gene promoter.<br />

Furthermore, we constructed anr and dnr knock-out mutants by homologous<br />

recombination with the plasmid pEX18Ap to characterize the regulation of<br />

anaerobic growth of the species Dinoroseobacter shibae.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PZ 50<br />

Miniaturized calorimetry – a new technology for the<br />

monitoring of biofilm activity<br />

F. Buchholz *1 , A. Wolf 2 , J. Lerchner 2 , H. Harms 1 , T. Neu 3 , T. Maskow 1<br />

1<br />

Environmental Microbiology, Helmholtz-Centre for Environmental Research -<br />

UFZ, Leipzig, Germany<br />

2<br />

Institute for Physical Chemistry, Technische Universität Bergakademie<br />

Freiberg, Freiberg, Germany<br />

3<br />

River Ecology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig,<br />

Germany<br />

In nature bacteria predominantly occur as sessile communities called biofilms.<br />

They are involved in a variety of beneficial or detrimental reactions and cost<br />

e.g. billions of dollars yearly by equipment damage, product contamination,<br />

energy losses and medical infections. For a better un<strong>der</strong>standing of biofilm<br />

processes as for the development of countermeasures monitoring techniques are<br />

required. Following the microbial activity via the metabolic heat by<br />

calorimetric measurements offers a possibility which works non-invasively and<br />

non-destructively, is easily combinable with other methods and provides realtime-information.<br />

Even though calorimetry was applied successfully for the<br />

investigation of planctonic microorganisms, only few studies concerning the<br />

investigation of surface-associated cells are known. Biofilm examinations<br />

usually require a flow-mode-system, but due to high costs and low throughput<br />

capacities conventional flow-through-calorimetry is rarely used. However,<br />

biofilm poisoning or detachment could be detected calorimetrically within a<br />

few minutes. The presented chip-calorimetric method provides a new approach<br />

for the investigation of biofilms. It offers a flow-through mode as well as highthroughput<br />

capacities due to exchangeable flow-cells, which allow separation<br />

of cultivation and measurement.<br />

In the experiments different biofilms of Pseudomonas putida PAW340 cells<br />

were calorimetrically investigated. The results indicate that biofilm activity can<br />

be described accurately with this technique. Furthermore the potential of chipcalorimetry<br />

to monitor and investigate antibiotic action against microorganisms<br />

growing in a biofilm was examined. Surface adhered P. putida PAW340 cells<br />

were exposed to different antibiotic treatments. The real-time observation of the<br />

processes qualifies this technique as a monitoring tool, but additionally<br />

physiological information can be <strong>der</strong>ived.<br />

PZ 51<br />

Effects of micro elements on aspartase activity and its<br />

relationships to other Amidohydrolases<br />

I.S. Okonkwo *1<br />

1 Industrial Physics, Enugu State University of Technology, Enugu, Nigeria<br />

The enzyme-aspartate- is involved in nitrogen mineralization in soils. It was not<br />

until recently that the activity of this enzyme was detected in soils, and a<br />

method was developed for its assay. The method was used in studies of the<br />

effects of the salts of 18 trace elements on the activity of aspartase in four<br />

fieldmoist soils and their air-dried counterparts. At 7 mol g-1 soil, all the trace<br />

elements inhibited aspartase activity in the soils. With most of the elements,<br />

greater inhibition was found in air-dried than in field-moist soils. Among the<br />

trace elements studied, Ag + and Hg 2+ were the most effective inhibitors of<br />

aspartase activity; >78% when added at 7 mol g-1 soil. The least inhibition<br />

(22%) was with Ni + added to the field-moist Inland soil and the greatest (98%)<br />

was with Ag + in the air-dried Plain soil. Aspartase activity was significantly<br />

correlated with the contents of organic C (r=0.78***, P

218<br />

PZ 55<br />

Formation of discrete Fe- and Al-rich precipitates during<br />

microbial sulfate reduction at low pH conditions (pH 3-5)<br />

J. Meier *1 , D. Fortin 2<br />

1<br />

Department of Lake Research, Helmholtz Centre for Environmental Research -<br />

UFZ, Magdeburg, Germany<br />

2<br />

Department of Earth Sciences, University of Ottawa, Ottawa, Canada<br />

Low pH environments are generally characterized by high concentrations of<br />

dissolved metal cations (e.g. Al, Fe). Due to alkalinity generation and hydrogen<br />

sulfide production, dissimilatory sulfate reduction may lead to the formation of<br />

secondary minerals, such as aluminum hydroxides and iron sulfides. In this<br />

study, mineral formation and the effect of elevated concentrations of dissolved<br />

Al and Fe(II) onto bacterial growth and activity at low pH were investigated<br />

using an acidotolerant, autotrophic enrichment culture dominated by species of<br />

the genus Thermodesulfobium. Transmission electron microscopy revealed the<br />

formation of compact aggregates of cells and amorphous Al-rich precipitates,<br />

whereas iron sulfides precipitated as crystalline nanoparticles indicating two<br />

completely different mechanisms of metal removal.<br />

PZ 56<br />

Heterologous Expression of Bacterial Carotenoid<br />

Biosynthesis Genes in Escherichia coli.<br />

Plasmid versus Chromosomal integration<br />

C. Albermann *1 , S. Ghosh 1 , N. Trachtmann 1 , G. Sprenger 1<br />

1 Institut für Mikrobiologie, Universität Stuttgart, Stuttgart, Germany<br />

The introduction of new metabolic activities into a host organism is an<br />

important assignment of metabolic engineering. Multi-copy plasmids may be<br />

best choice for the cloning and expression of recombinant genes, in particular<br />

for the maximum overproduction of one desired protein. However, the strong<br />

gene expression could be unfavorable for an increased productivity in<br />

metabolic engineering applications. To overcome this problem, heterologous<br />

genes can be cloned and expressed e.g. on low-copy plasmids. But the general<br />

disadvantage of recombinant plasmids is their possible structural instability and<br />

the need of selection markers to maintain the vectors in the host cell. In this<br />

presentation we describe a fast and efficient method for the integration of<br />

heterologous expression cassettes into a specific locus on the E. coli<br />

chromosome using the λ-Red recombinase. [1] The biosynthesis genes needed<br />

for the formation of different C40-carotenoids in E. coli were integrated into<br />

redundant genes in the E. coli chromosome. As preferred locus we used genes<br />

responsible for the degradation of rare sugars, like lacZYA, fucIK, rhaBAD,<br />

rbsDK, and others. By this way the integration into these loci can be monitored<br />

easily using a screening approach based on differential medium (MacConkeyagar<br />

plates containing the corresponding sugar). The heterologous biosynthesis<br />

of phytoene, lycopene, β-carotene, as well as of xanthophyll carotenoids will be<br />

presented. The heterologous expression of plasmid-coded genes and<br />

chromosomal-integrated genes will be compared and their disadvantage and<br />

advantage will be discussed.<br />

[1] K.A. Datsenko and B.L. Wanner, 2000, PNAS, 97, 6640-6645.<br />

PZ 57<br />

Survival of different Deinococcus radiodurans strains after<br />

exposure to mono- and polychromatic UV radiation and<br />

dessiccation<br />

A. Bauermeister * 1 , R. Moeller 1 , G. Reitz 1 , P. Rettberg 1 ,<br />

1 Radiation Biology Division, German Aerospace Center (DLR), Cologne,<br />

Germany<br />

The aim of this work was to help elucidate the mechanisms of resistance of<br />

Deinococcus radiodurans to mono- and polychromatic UV radiation and<br />

desiccation. For this purpose, different DNA repair-deficient mutant strains of<br />

D. radiodurans were irradiated with UVC (254 nm), UVA/B (280-400 nm) and<br />

UVA (315-400 nm) radiation as well as subjected to air-drying, drying in a<br />

desiccator and in vacuum. Strain ΔrecA was very sensitive to desiccation and<br />

UVC radiation compared to the wild-type, but showed no loss of resistance<br />

after irradiation with UVA/B and UVA. This emphasizes the importance of<br />

homologous recombination for the repair of DNA doube strand breaks (DSB)<br />

induced by UVC radiation and desiccation. The wild-type like resistance of<br />

ΔrecA to UVA and UVA/B radiation as well as the sensitivity of ΔpprA to<br />

these treatments led to the assumption that protection from oxidative stress<br />

plays a greater role in resistance to UVA and UVB than the repair of DNA<br />

DSB (induced by reactive oxygen species), and even might help avoid their<br />

formation. IrrE possesses a regulatory function in inducing different DNA<br />

repair or damage protection genes after stress treatment, including recA and<br />

pprA. The strain ΔirrE therefore exhibited higher sensitivity to desiccation and<br />

UV radiation of all qualities. When dried cells of the wild-type strain were<br />

irradiated with mono- and polychromatic UV radiation, they showed higher<br />

leves of resistance than when irradiated in suspension. This could be related to<br />

the induction of a protection system, especially of oxidative stress protection,<br />

during slow air-drying, which subsequently protects the cells from the oxidative<br />

stress produced by radiation as well.<br />

PZ 58<br />

Frequency of gastrointestinal parasites in dogs and cats<br />

referred in the city of Belgrade and they role in the<br />

epidemiology of the parasitic zoonoses<br />

S. Katić- Radivojević *1 , D. Ćulafić 2 , M. Kerkez 3<br />

1<br />

Department of Parasitology, Faculty of Veterinary Medicine, Belgrade, Serbia<br />

and Montenegro<br />

2<br />

Institute of Digestive Diseases, Clinical Center of Serbia, Belgrade, Serbia and<br />

Montenegro<br />

3<br />

Institute of Pulmonary Diseases, Clinical Center of Serbia, Belgrade, Serbia<br />

and Montenegro<br />

Fecal samples from 1755 dogs and 327 cats were examined for the presence of<br />

helminths and protozoan forms. From the total samples, 486 (27.7%) dogs and<br />

103 (31.5%) cats presented at least one parasite. The main genus of parasite in<br />

dogs were Ancylostoma(12.7%), Giardia (8.5%),Cystoisospora (4.4%),<br />

Toxocara (2.6%), and Cryptosporidium (2.2 %). The occurrence of<br />

Ancylostoma was associated to male dogs, ol<strong>der</strong> than one year, while Giardia,<br />

Cryptosporidium, Cystoisospora and Toxocara were associated to dogs<br />

younger than one year (P ≤ 0.05). Infection with all detected species were<br />

significantly higher (P≤ 0.05) in military working (100%) and stray dogs<br />

(93.3%) versus household pets (50.8%). Among cats, the most frequent<br />

parasites were Cryptosporidium (11.3%), Giardia (8.3%), Cystoisospora<br />

(8.3%), Toxocara (6.1%), and Ancylostoma (2.1%). Cryptosporidium and<br />

Cystoisospora were more prevalent in cats younger than one year (P ≤ 0.05).<br />

The high prevalence of zoonotic parasites registered in the dogs and cats<br />

population from a highly urban area in south-eastern Europe indicated a<br />

potential risk to human health.<br />

HDID 01<br />

The Viable but not Culturable Paradigm<br />

R. Colwell *1<br />

1 University of Maryland and Johns Hopkins University Bloomberg School of<br />

Public Health, Collage Park and Baltimore, United States<br />

A well-studied, long-term survival mechanism employed by Gram-positive<br />

bacteria is formation of endospores. For Gram-negative bacteria, the<br />

assumption has been that a survival state does not exist. However, a dormancy<br />

state has been described for Gram-negative bacteria and designated as the<br />

viable but nonculturable strategy of nonspore-forming cells. A variety of<br />

environmental factors are involved in induction of the viable but non-culturable<br />

(VBNC) state and Vibrio cholerae provides a useful paradigm for the VBNC<br />

phenomenon. It is now accepted that plate counts cannot be relied upon to<br />

enumerate or detect VBNC cells. Therefore, direct methods employing<br />

fluorescent staining, molecular genetic probes, and other molecular methods<br />

have proven both useful and reliable in detecting and enumerating both<br />

culturable and non-culturable cells. A predictive model for cholera, a bacterium<br />

that employs the VBNC strategy, has been developed and, based on ground<br />

truth data gathered using molecular methods, combining them with data<br />

obtained by remote sensing, employing satellites, an improved un<strong>der</strong>standing of<br />

this disease has been achieved. It is clear that microbiology in the twenty-first<br />

century is not only driven by new tools, but also by new paradigms.<br />

HDID 02<br />

Physiological aspects of the viable but non-culturable<br />

response<br />

D. McDougald *1 , Z. Moore 2 , S. Kjelleberg 1<br />

1<br />

Centre for Marine Bio-Innovation, University of New South Wales, Sydney,<br />

Australia<br />

2<br />

Ecowise Environmental, University of New South Wales, Sydney, Australia<br />

The identification of the viable but non-culturable response more than 25 years<br />

ago prompted an intense decade of debate as to the relevance of this state in<br />

both the environment and in health related settings. However, extensive studies<br />

using a variety of different techniques strongly support that these cells are truly<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

viable. Stress adapted cells show delay in entry into the VBNC state, indicating<br />

that there is a close relationship between stress and nonculturability. DNA and<br />

RNA hybridisation studies show that there are two phases of VBNC formation<br />

with the first involving loss of culturability with maintenance of cellular<br />

integrity and intact RNA and DNA (and thus possibly viability), and the second<br />

typified by a gradual degradation of nucleic acids, the products of which partly<br />

remain inside the cells and partly diffuse into the extracellular space. Results<br />

show that there is a small population of cells that can be resuscitated within the<br />

VBNC population but that percentage of the population diminishes over time.<br />

The potential for disease caused by undetected non-culturable cells is still an<br />

issue that warrants consi<strong>der</strong>ation.<br />

HDID 03<br />

Indicators to pursuit living deads<br />

U. Obst *1<br />

1<br />

Institute for Functional Interfaces (IFG), Forschungszentrum Karlsruhe,<br />

Karlsruhe, Germany<br />

Thirty years after the detection of viable but not culturable bacteria by Colwell<br />

and coworkers culturability is no longer the only and ultimate indicator for<br />

viability and infectivity. A growing number of culture-independent procedures<br />

provide better insights into cells balancing between life and death. While death<br />

of unicellular organisms is defined as the irreversible loss of cell integrity<br />

(lysis) or irreversible damage of the genome, dormancy, change of cell<br />

structures, or other reactions against a hostile environment are consequences of<br />

a bacterial stress response and not necessarily indicate cell death. Therefore,<br />

growth of non-culturable cells after a transfer to better life conditions does not<br />

mean resuscitation, but rather a re-activation. The detection of dormant bacteria<br />

in the environment is still difficult and requires novel tools for detecting stress<br />

indicators. The detection of stress responses and structural changes is especially<br />

relevant during and after disinfection, or other biocidal measures. A wellknown<br />

example is the up-regulation of PBP 5 in Enterococcus faecalis as<br />

shown by Del Mar Lleo and co-workers which can be used to identify<br />

beginning dormancy e.g. in oligotrophic and cold environments. This upregulation,<br />

however, is limited to only a few species, and thus is not suitable as<br />

a general stress parameter. For the identification of all-purpose stressparameters<br />

we propose 1. quantitative DNA analysis during dormancy and<br />

reactivation; 2. analysis of the expression of central stress response genes<br />

including rpoS, sigma factors but also others; and 3. determination of structural<br />

changes in cell morphology. Appropriate parameters and methods will be<br />

discussed.<br />

HDID 04<br />

Non-growing cells and maintenance metabolism in dense<br />

cultures of E. coli in membrane bioreactors<br />

U. Szewzyk *1 , C. Keil 1 , S. Müller 1<br />

1 Environmental Microbiology Group, Technische Universität Berlin, Berlin,<br />

Germany<br />

An environmental isolate of E. coli was cultivated in a continuously fed<br />

membrane bioreactor (MBR) with complete biomass retention. The cells<br />

reached very high densities after several days of cultivation and after that time<br />

did not further increase in density. Examination of the biomass revealed, that<br />

the stable cell density was achieved by reducing cell division to almost zero. In<br />

reverse it was concluded, that the biomass not only stopped growing but also<br />

had very little loss due to cell lysis. The long term starvation of the cells in the<br />

MBR is due to competition of the cells for limiting substrates.<br />

The biomass was sampled during the total time of continuous cultivation (up to<br />

3 month) and examined for activity and viability parameters. The data indicate<br />

a division of the population in several (at least three) subpopulations. Several<br />

models for longtime survival un<strong>der</strong> non-growth permitting conditions and<br />

relation to culturabilty will be presented.<br />

HDID 05<br />

A physiological approach to determine the survival of subseafloor<br />

prokaryotes un<strong>der</strong> energy deprivation.<br />

F. Mathes *1 , H. Sass 1 , R.J. Parkes 1<br />

1<br />

School of Earth and Ocean Sciences, Cardiff University, Cardiff, United<br />

Kingdom<br />

The deep biosphere is potentially the largest prokaryotic habitat on Earth [1]<br />

and active microbial communities have been detected in very old and deep<br />

sediments [2]. With ongoing ageing of sediment layers easily degradable<br />

organic matter is depleted by microorganisms resulting in the accumulation of<br />

recalcitrant kerogen. Hence, microorganisms face starvation and ultimately<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

death. As deep subsurface organisms must have adapted to such conditions<br />

their response to nutrient limitation may differ from that of their near surface<br />

relatives in terms of maintenance of viability, ability to use very low substrate<br />

concentrations and resuscitation after starvation. However, starvation-survival<br />

experiments have been mostly performed on pelagic microorganisms.<br />

Therefore, we compare the response of near surface and subsurface<br />

Photobacterium strains from the North Sea and Mediterranean sapropels [3] to<br />

energy deprivation.<br />

Our results showed consi<strong>der</strong>able resistance of near surface Photobacterium<br />

strains towards starvation. For strain SAMA2 no significant decline in total cell<br />

numbers occurred over a 1 Year starvation period. This was accompanied by a<br />

high proportion of "live" (~85%) and FISH-detectable cells (~70%).<br />

Culturability decreased over time but interestingly was higher un<strong>der</strong> aerobic<br />

than anaerobic conditions. Surprisingly strains from the sapropels were more<br />

sensitive towards starvation in terms of total cell counts whereas "live" and<br />

FISH-detectable cells were similar to their near surface relatives. The<br />

concentration of volatile fatty acids in the medium was reduced during<br />

incubation despite that, to our knowledge, Photobacterium strains are not able<br />

to utilise these anaerobically for dissimilatory processes.<br />

[1] Whitman et al. 1998 Proc. Natl. Acad. Sci. USA 95: 6578-6583.<br />

[2] Roussel et al. 2008 Science 320: 1046<br />

[3] Süß et al. 2008 Microb. Ecol. 55: 371-383<br />

HDID 06<br />

The case for safe disinfection of drinking water with<br />

chlorine : breaking the nucleic acids and repair systems !<br />

E. Le Guen *1 , M.H. Phe 1 , M. Hajj Chehade 1 , C. Merlin 2 , M. Dossot 1 , J.C.<br />

Block 1<br />

1 LCPME, UMR 7564 CNRS - Nancy-University, Villers-lès-Nancy, France<br />

2 LCPME, UMR 7564 CNRS - Nancy University, Vandoeuvre-lès-Nancy, France<br />

Chlorine (Cl2) is the most widely used bactericidal agent to disinfect drinking<br />

water. It is consi<strong>der</strong>ed as a strong oxidant affecting many cell functions. One<br />

problem in water disinfection is linked to the control of its efficiency, which<br />

requires mandatory methods such as culturing bacteria on standard nutritive<br />

agar media. These methods are time-consuming and un<strong>der</strong>estimate the number<br />

of viable bacteria, especially after Cl2 application. Recently, we have<br />

developed a method enabling rapid detection of nucleic acid damage by<br />

combining Sybr-II fluorochrome staining and flow cytometry. This new method<br />

allows the observation of intracellular nucleic acid alterations upon Cl2<br />

treatment. However, it doesn´t give any indication on either the damage<br />

extension or reversibility given that bacteria have repair systems.<br />

In this work, we hypothesized that successful disinfection can only occurred<br />

when the applied Cl2 concentration leads to severe intracellular nucleic damage<br />

as well as strong alteration of cellular machineries, including the DNA repair<br />

system. We investigated Cl2 effects on the SOS system of Salmonella<br />

typhimurium, and compared it to the loss of membrane permeability to<br />

propidium iodide, DNA integrity assessed by Sybr-II staining, and bacterial<br />

cultivability on TGA agar medium.<br />

This study shows that Cl2 has pleiotropic effects on bacteria at the different<br />

cellular organisation. First, Cl2 reacted at the cellular surface increasing the<br />

membrane permeability. Second, Cl2 diffused inside the cell and damaged<br />

nucleic acid. Third, Cl2 induced the SOS system. These new results support our<br />

hypothesis that efficient and safe disinfection (low risk of bacterial repair and<br />

regrowth) should be definitively achieved when a dramatic reduction in the<br />

fluorescence of DNA/RNA fluorochromes is observed. Rapid measurement of<br />

such nucleic acid alteration by fluorochrome based staining (within 1 hour) can<br />

be a new alternative method to control disinfection effectiveness.<br />

HDID 07<br />

Community analysis and taxonomic identification of<br />

drinking water bacteria with respect to live/dead status<br />

L. Kahlisch *1 , K. Henne 1 , J. Draheim 1 , L. Groebe 1 , I. Brettar 1 , M. Höfle 1<br />

1 Dept. Vaccinology & Applied Microbiology, HZI-Helmholtz Center for<br />

Infection Research, Braunschweig, Germany<br />

219<br />

Though the water treatment process aims at eliminating or killing the bacteria,<br />

the drinking water still shows a diverse microflora, partially due to re-growth<br />

after the treatment process. Molecular methods such as 16S rRNA based<br />

fingerprints and sequencing can provide an insight into the taxonomic<br />

composition of the drinking water microflora (Eichler et. al. 2006). However,<br />

the physiological state of the bacteria is a critical question and still remains to<br />

be assessed. It is of great health relevance for drinking water, especially with<br />

respective to pathogenic bacteria, to distinguish live and dead bacteria at the

220<br />

taxonomic level, i.e. to estimate the live and dead fraction of each bacterial<br />

species in drinking water.<br />

To distinguish live and dead bacteria at the taxonomic level, we combined three<br />

methods i) a staining procedure indicating membrane-injured cells (for the used<br />

BacLight Kit, Promega, the latter are usually consi<strong>der</strong>ed as dead cells), ii) a<br />

Fluorescence Activated Cell Sorting (FACS) of the injured and intact bacteria,<br />

and iii) molecular analyses of DNA/RNA extracted from the sorted bacteria<br />

enabling analysis of the species level.<br />

The analysis of drinking water samples revealed that a consi<strong>der</strong>able fraction of<br />

the bacteria is membrane-injured. 16S rRNA based fingerprint analysis of the<br />

bacterial DNA and RNA showed that many bacterial species have live and dead<br />

fractions, while a few species were on either side, i.e. completely injured, or not<br />

injured at all. The relevance of the non-injured species for the drinking water<br />

will be discussed.<br />

HDID 08<br />

Use of propidium monoazide for live-dead distinction<br />

A. Nocker *1<br />

1<br />

Quality of Life, Netherlands Organisation for Applied Scientific Research<br />

(TNO), Zeist, Netherlands<br />

Sample treatment with propidium monoazide (PMA) prior genomic DNA<br />

extraction and subsequent PCR analysis is increasingly <strong>bei</strong>ng used to<br />

preferentially detect cells with intact cell membranes. PMA is believed to<br />

selectively enter cells with compromised cell membranes (consi<strong>der</strong>ed dead)<br />

whereas it is excluded from intact cells. Once inside the dead cells, PMA<br />

intercalates into the DNA and can be covalently cross-linked to it upon<br />

exposure to bright visible light. The resulting modification of the DNA strongly<br />

interferes with its PCR amplification and thus leads to exclusion of dead cells<br />

from the analysis. This presentation addresses selected applications of the<br />

technology, current limitations, and possible future research directions.<br />

HDID 09<br />

Transcriptional activity around bacterial cell death reveals<br />

molecular biomarkers for cell viability<br />

R. Kort *1 , B.J. Keijser 1 , M.P.M. Caspers 1 , F.H. Schuren 1 , R. Montijn 1<br />

1<br />

Netherlands Organisation for Applied Scientific Research (TNO), Zeist,<br />

Netherlands<br />

Background: In bacteriology, the ability to grow in selective media and to form<br />

colonies on nutrient agar plates is routinely used as a retrospective criterion for<br />

the detection of living bacteria. However, the utilization of indicators for<br />

bacterial viability -such as the presence of specific transcripts or membrane<br />

integrity- would overcome bias introduced by cultivation and reduces the time<br />

span of analysis from initiation to read out. Therefore, we investigated the<br />

correlation between transcriptional activity, membrane integrity and<br />

cultivation-based viability in the Gram-positive model bacterium Bacillus<br />

subtilis.<br />

Results: We present microbiological, cytological and molecular analyses of the<br />

physiological response to lethal heat stress un<strong>der</strong> accurately defined conditions<br />

through systematic sampling of bacteria from a single culture exposed to<br />

gradually increasing temperatures. We identified a coherent transcriptional<br />

program including known heat shock responses as well as the rapid expression<br />

of a small number of sporulation and competence genes, the latter only known<br />

to be active in the stationary growth phase.<br />

Conclusions: The observed coordinated gene expression continued even after<br />

cell death, in other words after all bacteria permanently lost their ability to<br />

reproduce. Transcription of a limited number of genes correlated with cell<br />

viability un<strong>der</strong> the applied killing regime. The transcripts of the expressed<br />

genes in living bacteria - but silent in dead - include those of essential genes<br />

encoding chaperones of the protein folding machinery and can serve as<br />

molecular markers for bacterial cell viability.<br />

HDID 10<br />

Definition of death- relevance for public health and risk<br />

regulation.<br />

M. Exner *1<br />

1 Institue for Hygiene and Public Health, University of Bonn, Bonn, Germany<br />

The Aim of Public Health and risk regulation concerning food, water for human<br />

consumption, medical devices, pharmaceuticals, cosmetics, technical processes,<br />

disinfection and sterilization processes is to assess the microbial safety of<br />

media, processes or products and to verify that there is no risk which can harm<br />

health of the public and/ or individuals. In addition it is essential to possess<br />

methods by which acute risks can be verified and be brought un<strong>der</strong> control.<br />

Instruments and methods which were used for public health purposes to verify<br />

the microbial safety for the microbial evaluation of water, soil and air were<br />

developed and described for the first time by R. Koch 1883. Even if he taken<br />

into account spores as an alternative form of life together with the vegetative<br />

form most of the methods for the verification used in public health were<br />

methods of culturability of vegetative cells. Until today these methods are even<br />

used for more than 100 years with tremendous success. Therefore these<br />

methods which were also taken as evidence for the death of microorganisms<br />

seemed to be sufficient longtime for public health purposes.<br />

With new methods characterizing the status of cryptobiosis of microorganisms<br />

and verifying persisters, VBNC, starving cells, dormant cells, injured cells by<br />

molecular methods of measuring respiratory activity, Protein syntheses,<br />

enzymatic activity and membrane integrity and Trojan horses for<br />

microorganisms like amoebae the consequences for risk regulation and public<br />

health may be consi<strong>der</strong>able. The applicability of these methods for risk<br />

regulation and public health has to be validated and will be discussed in the<br />

presentation.<br />

In either case we are in the beginning of a transition period for microbial<br />

methods for public health purposes.<br />

HDID P 01<br />

Living and non-living bacteria in groundwater and<br />

drinking water<br />

G. Preuß *1 , E. Ziemann 1 , N. Zullei-Seibert 1<br />

1 Institut für Wasserforschung GmbH, Zum Kellerbach 46, Schwerte, Germany<br />

Several fluorescence staining methods were proofed to determine bacteria in<br />

water samples collected from a groundwater catchment area used for drinking<br />

water production. The investigation aimed at comparing the ability of<br />

microscopic methods for the assessment of hygienic drinking water quality.<br />

The samples were treated with DNA-binding dyes (DAPI, SYBR Green II,<br />

SYTO 62) to estimate total bacteria counts and with PI, that penetrates the<br />

membranes of non-living cells only. Additionally cFDA was used for viability<br />

analysis after incubation at 30°C. Colony forming units (cfu/ml) were estimated<br />

using cultivating methods according to the German drinking water regulation.<br />

In surface water dyes with DAPI, SYBR Green II and SYTO 62 showed<br />

comparable results between 1,6*10 6 and 2,8*10 6 cells/ml. In groundwater and<br />

drinking water cell counts were between 3,9*10 4 and 8,4*10 4 cells/ml. In these<br />

samples the highest results were found with SYBR Green II, detecting 43%<br />

more cells than the assay with DAPI.<br />

Using SYTO 62 or SYBR Green II gave the same results as the sum of cFDAactive<br />

cells and dead cells detected with PI. The rate of active cells was<br />

between 7,8% in surface water and 1,4% in drinking water. Only 0,02% of<br />

these cells could be detected on nutrient plates.<br />

This results confirm the actual knowledge about viable but not cultivable<br />

bacteria (VBNC) in nutrient-poor water. In addition of the standard culture<br />

methods fluorescence microscopic techniques can be used as sensitive<br />

parameters to assess the efficiency of water treatment processes.<br />

HDID P 02<br />

Comparison between PMA-PCR and DNase-PCR methods<br />

for the discrimination of live and dead bacteria<br />

J. Varela Villarreal *1 , T. Schwartz 1 , U. Obst 1<br />

1 Institut für Technische Chemie-Wasser- und Geotechnologie,<br />

Forschungszentrum Karlsruhe GmbH, Karlsruhe, Germany<br />

Viable but non-cultivable bacteria are a serious issue in public health. Viability<br />

of bacteria is still overall determined by its ability to grow and produce<br />

colonies, despite it is well-known that this ability is not enough to demonstrate<br />

if a bacteria is alive due to the fact that it can be in a VBNC state. A reliable<br />

method for the detection of viable bacteria is a great challenge for water and<br />

food monitoring.<br />

Nowadays DNA-based methods are used for the detection and characterization<br />

of bacteria. One of the mayor disadvantages of these techniques is that they can<br />

not distinguish between DNA of live and dead cells. In view of the fact that<br />

propidium monoazide (PMA) has the property of intercalating free DNA and<br />

suppressing its subsequent amplification, this substance has been recently used<br />

in or<strong>der</strong> to differentiate the different physiological states of bacteria: viable<br />

cells with intact cell membrane and dead cells with harmed cell membrane.<br />

Following a similar idea already named in 2000 by Nogva et al., DNases could<br />

also be used for the live-dead differentiation, since it can destroy free DNA<br />

before the extraction and amplification of DNA that belong to living cells.<br />

These methods have been applied in this work to see if small amounts of living<br />

pathogens can be detected in drinking water and to see if bacterial population<br />

analysis can be done in drinking water samples. The results and the comparison<br />

between these viability tests and standard culture methods are presented here.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

HDID P 03<br />

Effects of electrical Polarization on bacterial biofilm<br />

formation and bacterial activity<br />

A. Rumpf *1 , C. Weidlich 2 , K.M. Mangold 2 , G. Schaule 1 , H.C. Flemming 1,2<br />

1 Applied Microbiology, IWW Water Centre, Mülheim/Ruhr, Germany<br />

2 Karl-Winnacker-Institut, DECHEMA e.V., Frankfurt/Main, Germany<br />

3 Universität Duisburg-Essen, Biofilm Centre, Duisburg, Germany<br />

The aim of this study was to influence electrostatic interactions during biofilm<br />

development by means of electrical polarization to investigate an inhibitory<br />

effect on biofilm growth and bacterial activity. For that purpose different<br />

polarization routines focusing on pulsed potential on surfaces such as Indiumtinn-oxide<br />

(ITO) were applied resulting in varying surface properties but no<br />

chemical reactions in the electrolyte.<br />

Primary adhesion of bacteria could not be avoided by constant or pulsed<br />

polarization. But biofilm growth was reduced significantly. When pulsed<br />

polarization (+/- 600 mV) was applied in a routine of 60 s, biofilm growth of<br />

drinking water bacteria was significantly inhibited. While the non polarized<br />

control assay reached a thickness of approximately 100 µm, the polarized<br />

biofilm remained a monolayer with evenly distributed bacteria over a period of<br />

one week.<br />

In or<strong>der</strong> to investigate a physiological effect of electrical polarization, PCR-<br />

DGGE analysis of drinking water biofilms after one week of polarization (+/-<br />

600 mV) and control was performed. The PCR-DGGE-analysis showed<br />

differences in the pattern between polarized and control biofilms indicating an<br />

influence on population diversity. When biofilms (polarized/control) were<br />

allowed to regenerate for 24 h after polarization, the population diversity<br />

changed again. These results confirm an influence of pulsed potential on<br />

drinking water biofilm population diversity. ATP measurements showed a<br />

decreased intracellular ATP content in polarized bacteria compared to the<br />

control assay indicating stressed bacteria. Treatment with pipemidic acid<br />

revealed a loss in cell elongation in the polarized bacteria indicating an<br />

inhibitive effect on growth. These results indicate that the microbial adhesion<br />

process is not affected by pulsed potential but that polarization in the applied<br />

routine strongly affects the viability of adhering cells, effectively suppressing<br />

further growth.<br />

The financial support by the Ar<strong>bei</strong>tsgemeinschaft industrieller<br />

Forschungsvereinigungen AiF is gratefully acknowledged (No. 174 ZN/2). We<br />

kindly acknowledge the Phd scholarship of Deutsche Bundesstiftung Umwelt<br />

(DBU) for A. Rumpf.<br />

HDID P 04<br />

Survey and analysis of anticacinogenic role of some<br />

vitamins and enzymes used in cosmetics and hygienic<br />

creams in natural and combined form by<br />

Salmonella/microsome assay and identification them by<br />

TLC and column chromatography<br />

M. Hatefi *1<br />

1<br />

Microbiology, Islamic Azad University branch oloom va tahghighat, Tehran,<br />

Iran<br />

This study is a precise analysis that defines antimutagenicity and<br />

anticacinogenesis effects of nonenzymatic antioxidants such as vitamins A and<br />

E, some water soluble vitamins such as vitamin B and C.<br />

In addition, antimutagenicity effects of some enzymes that used recently in<br />

cosmetics and hygienic industries in the world and the three additive materials<br />

to cosmetic products were investigated as well. Among these tested substances,<br />

vitamins A and E presented the best antimutgenicity results and among natural<br />

substances, propolis showed interesting result either.<br />

Antimutagenicity assay has been performed according to the protocol<br />

developed by Professor B. Ames and his colleagues by means of two mutant<br />

strains of Salmonella typhymurium TA100 and TA97 against two mutagenic<br />

substances named azide sodium and potassium permanganate in the presence<br />

and the absence of microsomal homogenate of mouse liver (S9). Based upon<br />

their theory nearly 80% of mutagenic substances were carcinogenic too.<br />

Finding results such as the number of revertants in tester strain TA100 and<br />

TA97 and the percentage of mutation inhibition against two mutagenic<br />

substances in absence or presence of (S9) were entered in SPSS software<br />

program and processed statistically. The significant differences ( ) between the<br />

means of revertants per plate of the sample in relation to the mutagens were<br />

calculated using Tuckey Honest Significant Difference (HSD) test for unequal<br />

sample sizes and some other statistical tests such as Levene, Anova and Welch.<br />

The final criterion used to interpret the results of significant increase<br />

(enhancement effect) or decrease (inhibition effect) in the number of salmonella<br />

revertant showed a reversion rate >50% or

222<br />

and a neighboring eutrophic lake. Therefore, we expect a strong effect of lake<br />

trophic status on microbial communities involved in the transformation of<br />

nitrogen, a key parameter of lake trophy. Vertical patterns of microbial<br />

community composition will be analyzed by Denaturing Gradient Gel<br />

Electrophoresis and Fluorescence in situ Hybridization.<br />

Total cell counts using SYBR Green II staining revealed higher microbial<br />

abundances in the eutrophic compared to the oligotrophic lake. HPLC-based<br />

techniques [1] and SYBR Green II staining indicated depth-dependent changes<br />

of viral abundances as well. Noteworthy, an important impact of viruses on<br />

freshwater bacterial population dynamics was suggested recently [2].<br />

[1] Rathmann, C., Stolle, P., Auling, G. (2008): Microbial Ecology Revised due<br />

to New Methods for Quantification of Bacteriophages, RAISEBIO-HIGRADE-<br />

Summerschool Leipzig, Germany 22 - 25/09/2008<br />

[2] Filippini, M., Buesing, N. & Gessner, M. O. (2008): Temporal dynamics of<br />

freshwater bacterio- and virioplankton along a littoral-pelagic gradient.<br />

Freshwater Biology 53: 1114-1125.<br />

HDID P 08<br />

How to quantify living sulfate reducing bacteria in sediment<br />

samples?<br />

K. Röske *1 , J. Schirrmeister 2 , I. Röske 2<br />

1<br />

Institut für Mikrobiologie, Sächsische Akademie <strong>der</strong> Wissenschaften zu<br />

Leipzig, Leipzig, Germany<br />

2<br />

Institut für Mikrobiologie, Technische Universität Dresden, Dresden, Germany<br />

Molecular methods, based on isolated DNA are commonly applied to<br />

investigate the microbial diversity in a wide range of different habitats. A<br />

disadvantage of these methods, that has to be accepted so far, is that not only<br />

DNA <strong>der</strong>iving from living bacteria is amplified; also free DNA and DNA from<br />

dead organisms will be amplified during PCR. Propidium monoazide (PMA)<br />

can not penetrate intact cells. But, if the cell membrane is damaged it can<br />

penetrate the cell and binds covalently on the DNA. This prevents an isolation<br />

of this DNA, and therefore, a subsequent amplification of bacteria without an<br />

intact cell membrane. The aim the study was to make a first effort to quantify<br />

living sulfate reducing bacteria in different sediment samples from a drinking<br />

water reservoir. The samples were treated with PMA according to Nocker et al.<br />

(2007). To analyze the reduction of isolated DNA after PMA treatment, DNA<br />

was isolated from identical samples after or without PMA treatment and<br />

measured with the Quant-it PicoGreen-System. Afterwards, sulfate reducing<br />

bacteria (SRB) were quantified by real time PCR using primers for the<br />

amplification of parts of the gene for dissimilatory sulfite reductase (dsrAB). In<br />

the majority of the samples, the DNA concentration was lower in the PMA<br />

treated samples compared to the untreated controls. This difference was often<br />

greater in samples from lower sediment depth (8 to 10 cm). The results of the<br />

real time PCR showed that in part the reduction of the DNA concentration was<br />

probably due to an elimination of DNA from dead SRB. No trend to a wi<strong>der</strong><br />

difference in the quantity of SRB in PMA treated and untreated samples in a<br />

special sediment horizon was observed. More investigations need to be done to<br />

analyze the potential of PMA for the differentiation of living and dead SBR in<br />

sediment samples.<br />

HDID P 09<br />

Involvement of PHB metabolism in Legionella virulence<br />

and culturability<br />

P. Auraß *1 , A. Flieger 1<br />

1 FG11 Bakterielle Infektionen, Robert Koch-Institut, Wernigerode, Germany<br />

Legionella pneumophila, the causative agent of a fatal pneumonia, is an<br />

intracellular parasite of eukaryotic cells. In the environment, it colonizes and<br />

replicates in amoebae. If inhaled by humans, bacteria infect alveolar cells in a<br />

way that is mechanistically similar to the amoeba infection. In addition to the<br />

Legionella Dot/Icm Type IVB protein secretion system and its effectors,<br />

several other compounds are essential for host-cell colonization and<br />

exploitation. In search for novel Legionella virulence factors, we employed a<br />

new screening technique allowing fast screening of several thousand clones of a<br />

transposon mutagenized Legionella library. Thereby we isolated a clone<br />

harbouring a resistance cassette within the bdhA/patD operon. By analyzing the<br />

locus, we recognized its role in Legionella virulence and persistence. The<br />

operon encodes two enzymes, one with homology to the poly-βhydroxybutyrate<br />

(PHB) dehydrogenase BdhA, which could therefore be an<br />

enzyme of PHB degradation. The other is PatD, a patatin-like phospholipase<br />

(PLP) family protein. Investigating the bdhA/patD- mutant by means of<br />

enzymatic assays, microscopy, intracellular replication assays, as well as<br />

fourier transform infrared spectroscopy (FTIR) unfolds a role of bdhA/patD in<br />

virulence and survival. The mutant was unable to replicate intracellulary,<br />

contained higher PHB amounts than wildtype bacteria, possessed less<br />

phospholipase activity and showed increased culturability. The study for the<br />

first time shows that Legionella PHB metabolism is involved in virulence and<br />

culturability, and therefore offers a new target of inhibition and avoidance of<br />

Legionella reservoires.<br />

HDID P 10<br />

Survival of simualted space conditions hy haloarchaea.<br />

H. Stan-Lotter *1 , M. Dornmayr-Pfaffenhuemer 1 , S. Fendrihan 2 , A. Holzinger 1 ,<br />

T.K. Polacsek 1 , M. Grösbacher 1<br />

1<br />

Molecular Biology, University of Salzburg, Salzburg, Austria<br />

2<br />

President, Romanian Bioresource Centre and Advanced Research Association,<br />

Bucharest, Romania<br />

The search for extraterrestrial life has been declared as a goal for the 21th<br />

century by several space agencies. Potential candidates are microorganisms on<br />

or in the surface of moons and planets, such as Mars. Numerous space probes<br />

have been sent to Mars, some of which were not sterilized and crashed onto the<br />

planet. Therefore Mars is possibly already contaminated with terrestrial<br />

microorganisms. The question if there is viable or non-viable microbial life will<br />

be explored by in situ robotic detection.<br />

We are focussing on the application of fluorescent probes for the detection of<br />

viability of haloarchaeal strains, following exposure to simulated space<br />

conditions (e.g. desiccation, irradiation with UV). Using the LIVE/DEAD<br />

BacLight TM kit, it was estimated that the D37 (dose of 37% survival) for<br />

Halococcus dombrowskii DSM14522 and Halobacterium salinarum NRC-1<br />

ATCC700922 was about 400 kJ/m 2 , when cells were embedded in halite and<br />

about 1 kJ/m 2 , when cells were in liquid cultures. The data from fluorescent<br />

staining indicated a slightly higher cellular activity than was <strong>der</strong>ived from the<br />

determination of CFUs. A second method for assessment of viability is the<br />

BacLight TM Bacterial Membrane Potential kit. The fluorescent membrane<br />

potential indicator dye DiOC2(3) gave strong signals with Hcc. dombrowskii<br />

and the control organism E. coli; as expected, the membrane potential was<br />

diminished by the uncoupler CCCP, resulting in change of color. Reaction<br />

times were generally longer with Hcc. dombrowskii than with E. coli.<br />

The advantages of staining with fluorescent dyes are rapid results on membrane<br />

intactness and membrane potential, but more data are needed for a better<br />

correlation to cellular viability.<br />

HDID P 11<br />

Fluorescence in situ hybridisation (FISH) as alternative<br />

method for the detection of viable coliform bacteria in<br />

drinking water<br />

M. Hügler *1 , K. Böckle 1 , I. Eberhagen 1 , C. Beimfohr 2 , K. Thelen 2 , B. Hambsch 1<br />

1 Mikrobiologie, DVGW-Technologiezentrum Wasser, Karlsruhe, Germany<br />

2 Mikrobiologie, Vermicon AG, München, Germany<br />

The monitoring of microbiological contaminants in water supplies requires fast<br />

and sensitive methods for the specific detection of indicator organisms or<br />

pathogens. The standard cultivation methods are too time-consuming to match<br />

the requirements of mo<strong>der</strong>n water safety management, i.e. coming close to<br />

online technology. The FISH technology has been proven to represent a<br />

sensitive molecular method for the specific detection of microorganisms.<br />

Within the TECHNEAU project – an integrated project funded by the European<br />

Commission (contract number 018320) – it was investigated if FISH can be<br />

used to directly detect and quantify E. coli and coliform bacteria in drinking<br />

water samples. Two approaches were used: Either the direct detection of single<br />

E.coli and coliform bacteria cells on the filter membrane or an approach<br />

including an incubation step on a nutrient agar plate for a few hours before the<br />

subsequent staining of micro-colonies. Both approaches were optimized for the<br />

analysis of spiked water samples. For the validation of the protocol, the effects<br />

of heat and chlorine disinfection were tested in water samples spiked with pure<br />

cultures. The results obtained by the FISH technique were compared to results<br />

obtained by culture-based methods and by total cell counts.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

HDID P 12<br />

Application of broad band UV light overcomes drawbacks<br />

of conventional monochromatic UV disinfection<br />

J. Süß *1 , U. Obst 1 , T. Schwartz 1<br />

1 Institute for Technical Chemistry, Water Technology and Geotechnology<br />

Division, Microbiology of natural and technical surfaces Department,<br />

Forschungszentrum Karlsruhe, Eggenstein-Leopoldshafen, Germany<br />

Microbes possess different mechanisms to restore DNA lesions caused by UV<br />

light (254nm) and might overcome disinfection barriers. This problem could be<br />

solved by the use of excimer lamps which emit light over a broa<strong>der</strong> range in the<br />

UVC area. In this study we compared different UV sources with special regard<br />

to sustainability. As UV injured bacteria are not cultivable but alive a molecular<br />

biology concept was applied. It is based on the assumption that UV-induced<br />

DNA alterations inhibit PCR. Six reference strains and wastewater effluent<br />

samples were irradiated using microwave driven Hg- and excimer lamps (XeCl,<br />

XeBr). Cultivation (CFU) and molecular biology analyses (qPCR, DGGE) were<br />

combined for the estimation of inactivation and regeneration of the bacteria<br />

over a time period of at least 24 hours. Both, monochromatic and broadband<br />

UV rays caused strong DNA injuries in the reference microbes. DNA repair<br />

was detected in almost all bacteria after conventional UV disinfection but was<br />

strongly reduced when excimer lamps were used. In case of the most effective<br />

UV source (XeBr) post-irradiation activity as well as the expression of repair<br />

and stress genes were analysed in more detail. Wastewater bacteria exhibited a<br />

higher resistance against UV rays and therefore an increased regeneration<br />

potential. Subsequent population analyses demonstrated an UV source<br />

depended re-growth of wastewater bacteria, which became obvious after 24<br />

hours. Our study indicates that also UV-robust species can be effectively<br />

inactivated by broad band UV light. In wastewater treatment technologies the<br />

use of UV-based techniques should be reconsi<strong>der</strong>ed with regard to prolonged<br />

disinfection efficiencies.<br />

HDID P 13<br />

Antimicrobial efficacy testing of a novel silver-based<br />

nanocomposite additive<br />

S. Egger 1 , E. Kuhn 1 , R. Lehmann 1 , M. Height 2 , M. Loessner 1 , M. Schuppler *1<br />

1 Institute of Food Science and Nutrition, ETH Zurich, Zurich, Switzerland<br />

2 High Performance Additives, HeiQ Materials, Bad Zurzach, Switzerland<br />

Nanostructured materials offer great potential for bringing sophisticated<br />

properties into numerous applications such as textiles, plastics and coatings. A<br />

novel silver nanocomposite pow<strong>der</strong> material that may be readily incorporated in<br />

various materials was investigated for its antimicrobial efficacy on a selection<br />

of different bacteria and fungi. The minimal inhibitory concentrations (MICs)<br />

and bactericidal concentrations (MBCs) were determined by exposing the<br />

microorganisms to varying concentrations of the silver nanocomposite pow<strong>der</strong>.<br />

In addition, MICs and MBCs were also determined for silver nitrate and silver<br />

zeolite in or<strong>der</strong> to compare the antimicrobial activity of the silver<br />

nanocomposite pow<strong>der</strong> to an available standard. The antimicrobial functionality<br />

of polystyrene samples functionalized with the novel silver nanocomposite<br />

pow<strong>der</strong> was also tested. Furthermore, the polystyrene material containing the<br />

antimicrobial nanocomposite additive was investigated for its ability to prevent<br />

biofilm formation. Although the determined minimal inhibitory concentration<br />

varied for the different microorganisms tested, the results from this study<br />

demonstrate the strong antimicrobial activity of the novel silver nanocomposite<br />

pow<strong>der</strong>. The polystyrene samples functionalized with the silver nanocomposite<br />

pow<strong>der</strong> revealed a strong antimicrobial activity, and effectively inhibited<br />

biofilm formation during the entire test period of 6 weeks.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

HDID P 14<br />

Test for viability of acidophilic sulfur oxidizing bacteria<br />

J. Huergo *1 , S. Bellenberg 2 , C.F. Leon Morales 3 , T. Rohwer<strong>der</strong> 2 , W. Sand 2 , E.<br />

Donati 1<br />

1<br />

CINEFI - CONICET, Universidad Nacional de La Plata, La Plata, Argentina<br />

2<br />

Aquatische Biotechnologie - Biofilm Centre, Universität Duisburg-Essen,<br />

Duisburg, Germany<br />

3<br />

Aquatische Mikrobiologie - Biofilm Centre, Universität Duisburg-Essen,<br />

Duisburg, Germany<br />

Acidophilic microorganisms capable of inorganic iron and/or sulfur compounds<br />

oxidation have not only an emergent biotechnological use but also an important<br />

environmental impact. In the latter case, those microorganisms are involved in<br />

the formation and control of acid mine drainage. These acidic, heavy metalcontaminated<br />

waters tend to pollute rivers and groundwater. On the other hand,<br />

acidophilic microorganisms can be employed for heavy metal recovery from<br />

sulfidic ores. For un<strong>der</strong>standing their role in both processes, it is very important<br />

to discriminate between metabolically active and inactive bacterial cells. There<br />

are some fluorometric assays for determining bacterial cell viability, e.g.,<br />

propidium iodide is a nucleic acid dye which is reported to stain only cells with<br />

compromised membranes. These test systems, although widely used for<br />

neutrophilic bacteria, are still controversial when applied to acidophilic<br />

bacteria. The aim of this work was to determine if there is a relation between<br />

the metabolic activity of acidophilic sulfur oxidizing microorganisms and the<br />

permeability of their plasma membranes to propidium iodide. Obligately<br />

chemolithotrophic bacteria were grown on sulfur compounds as inorganic<br />

electron donors. Bacterial viability was assessed using various methods<br />

including MPN and specific fluorescence dyes.<br />

HDID P 15<br />

Occurrence of viable but not cultivable Escherichia coli in a<br />

drinking water distribution system<br />

L. Mezule 1 , S. Larsson 2 , T. Juhna *2<br />

1 Department of Water Engineering and Technology, Riga Technical University,<br />

Riga, Latvia<br />

223<br />

Escherichia coli is widely used as an indicator of fecal contamination of<br />

drinking water. E.coli concentration is routinely determined with culture based<br />

methods, even though in oligothrophic environment (e.g.drinking water) they<br />

may enter viable but not cultivable (VBNC) state. The aim of this study was to<br />

estimate the occurrence of VBNC E. coli in a water supply system in which<br />

culturable E.coli is not usually found.<br />

The sampling of water and biofilm (on pipes surfaces) from a water supply<br />

system was carried out over the period of more than one year. The system was<br />

supplied with chlorinated drinking water which was produced from chemically<br />

coagulated surface water and groundwater (Riga, Latvia). The biofilm samples<br />

were collected both with plug flow and completely mixed reactors during 2–3<br />

week expose to drinking water. The water samples prior analyses were<br />

concentrated with tangential ultrafiltration method. All samples were analysed<br />

by cultivation on nutrient-rich selective culture media and by direct viable<br />

count (cells which elongate in nonselective media after treatment with nalidixic<br />

acid) combined with fluorescent in situ hybridization (DVC-FISH method).<br />

From 12 biofilm samples collected from 6 sampling sites at different times no<br />

cultivable E. coli was detected, however, all of the samples were FISH positive<br />

for E. coli and more than 50 % of the samples contained DVC positive E. coli.<br />

Results showed that initially (1-2 weeks) VNBC E.coli is harboring the surfaces<br />

of the pipes (up to 500 cells/cm2) and then they are slowly released to water<br />

phase. In all biofilm samples positive hyperbolic correlation between biomass<br />

concentration and number of FISH-DVC E.coli was observed.<br />

We conclude that VNBC is important mode of E.coli „living“ in drinking<br />

water networks and their fate is dependent on interaction between surfaces and<br />

water phase. The study contributes to un<strong>der</strong>standing of ecology of E.coli in<br />

drinking water and provision on public health safety.

224 AUTOREN<br />

Abajy, M.Y. PP 38<br />

PW 23<br />

PW 22<br />

Abd El Karem, Y. PX 37<br />

Abdel Aziz, M. PZ 02<br />

PL 01<br />

Abdillahi Ibrahim, R. PP 08<br />

Abdul Rahim, R. PB 04<br />

Abdullah, N. PB 04<br />

Abed, R.M.M. PE 08<br />

Abraham, W.R. PN 50<br />

Abu Laban, N. PA 37<br />

Ackermann, M. HDID P 07<br />

PO 30<br />

Ackermann, S. PN 14<br />

Adamek, M. PO 01<br />

Adrian, L. PS 54<br />

Akob, D. PN 59<br />

PN 70<br />

Aktas, M. PJ 02<br />

Alaghehbandan, R. PZ 01<br />

Alawi, M. PN 29<br />

PO 23<br />

Alber, B.E. KN 03<br />

Albermann, C. PZ 56<br />

KT 06<br />

Albers, A. PZ 22<br />

Albers, S. KS 05<br />

Albers, S.V. PB 10<br />

PB 15<br />

PB 11<br />

KB 04<br />

Alborn, D. PM 14<br />

Albrecht, A. KL 03<br />

Albrecht, D. PJ 14<br />

Albrecht, R. KI 03<br />

Albrecht, T. PP 31<br />

Aldahash, A.A. PN 01<br />

Al<strong>der</strong>son, J. PZ 09<br />

Alexandrov, K. PW 16<br />

Alfandega, A. PW 11<br />

Al-Halabi, L. PZ 53<br />

Alisch, R. FGD 06<br />

Al-Karablieh, N. PJ 22<br />

Allers, T. PB 25<br />

Allgaier, S. KD 01<br />

Almasian, F. PX 01<br />

PZ 07<br />

PX 03<br />

Alyamani, E.J. PN 05<br />

Amann, R. PO 25<br />

KJ 06<br />

FGE 04<br />

PO 48<br />

PG 06<br />

PG 07<br />

PO 09<br />

Amon, J. PS 59<br />

Anacker, S. KC 02<br />

Andries, K. PP 08<br />

FGF 04<br />

Anetzberger, C. PS 03<br />

Angel, R. PO 02<br />

Angelov, A. PN 57<br />

PG 04<br />

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Ankele, E. KC 03<br />

Anna, B. PO 37<br />

Anneser, B. PN 34<br />

Antelmann, H. PP 39<br />

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PN 47<br />

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KT 05<br />

PX 24<br />

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PX 17<br />

PX 06<br />

Archer, D. H 13<br />

Ardalan, R. PP 10<br />

Arends, K. KH 06<br />

PW 22<br />

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Arnold, M. PR 01<br />

Ashfaq, M. PX 28<br />

Askari, A. PZ 07<br />

Astashov-Frauenhoffer, M. PN 81<br />

Ataka, K. PQ 04<br />

Auchter, M. PS 20<br />

Auling, G. PC 01<br />

HDID P 07<br />

PO 30<br />

Auraß, P. HDID P 09<br />

Averhoff, B. KH 04<br />

PW 12<br />

Avrahami, S. PN 55<br />

Axmann, I. KM 01<br />

Aygün, H. PX 38<br />

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Bachmann, A. PR 19<br />

PR 20<br />

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Baghernezhad, M. PP 10<br />

Bagyan, I. PZ 43<br />

Bahl, H. PV 01<br />

PS 28<br />

PZ 32<br />

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KA 02<br />

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Bakker, E.P. PW 13<br />

Balck, A. PZ 53<br />

Bald, D. FGF 04<br />

PP 08<br />

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Bandow, J. PP 13<br />

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Barion, B. PZ 29<br />

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Barkowits, K. PS 41<br />

Barnickel, L. PZ 04<br />

Barraud, N. PZ 45<br />

Barroso, V. FGF 01<br />

Barthel, S. PS 28<br />

Basché, T. FGG 05<br />

Basner, A. KT 05<br />

Bathe, F. PH 11<br />

Bathe, S. PO 01<br />

PN 13<br />

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Baum, C. PP 15<br />

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PN 34<br />

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KN 05<br />

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Behrens-Kneip, S. PZ 30<br />

PZ 29<br />

Beimfohr, C. HDID P 11<br />

Belden, W.J. H 07<br />

Bellack, A. KB 02<br />

Bellenberg, S. PZ 24<br />

HDID P 14<br />

Benndorf, D. PN 69<br />

PN 84<br />

Berendonk, T. PN 56<br />

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Berg, I. PB 13<br />

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Bierbaum, G. PC 05<br />

PC 06<br />

PW 01<br />

PS 48<br />

PP 28<br />

Bihlmaier, K. KS 02<br />

Bijtenhoorn, P. KU 06<br />

Bill, E. PS 25<br />

Bimet, F. PK 01<br />

Birkenmaier, A. PX 25<br />

Birrer, C. PH 02<br />

Bischoff, J. PN 19<br />

Bischoff, S. PN 59<br />

Bischofs, I. KR 02<br />

Biswas, R. PP 31<br />

Bizet, C. PK 01<br />

Blank, L.M. KT 02<br />

FGD 03<br />

Blaser, M. PA 25<br />

PA 26<br />

Blättel, V. PI 01<br />

Bleichrodt, F. PS 12<br />

Bleiziffer, I. PP 19<br />

Blissenbach, B. PS 33<br />

Block, J.C. HDID 06<br />

Bloemendal, S. KE 03<br />

Blombach, B. PX 15<br />

Blumberg, M. PN 54<br />

Blümel, M. PO 34<br />

Bobeth, C. KE 07<br />

Bock, R. KF 06<br />

KC 07<br />

Böckeler, K. PF 03<br />

Bockemühl, V. PX 18<br />

Böckle, K. HDID P 11<br />

Boerngen, K. KS 04<br />

Boes, N. KO 01<br />

Boetius, A. PO 25<br />

Böhm, J. PH 04<br />

Böhme, K. KQ 03<br />

PU 08<br />

Bohne, A. PE 04<br />

Boiangiu, C.D. PA 13<br />

Boland, W. KD 05<br />

Boland Nazar, R. PP 03<br />

Bölker, M. KE 06<br />

Boll, M. PA 40<br />

PA 12<br />

PA 19<br />

Bollin, R.P. KM 04<br />

Boltres, B. PX 28<br />

Bondarev, V. PR 20<br />

PR 19<br />

Bongaerts, J. PX 19<br />

PR 17<br />

Bonnefoy, V. KU 04<br />

Bormann, J. PH 13<br />

Börner, T. KF 01<br />

Bornscheuer, U. PX 05<br />

Borovskaya, A. PN 85<br />

Borovykh, I. PW 06<br />

Borriss, R. PI 05<br />

PP 45<br />

Bös, N. PS 49<br />

Bosch, J. PA 34<br />

Bosecker, K. PO 37<br />

Both, L. PR 26<br />

Bott, M. PQ 01<br />

PM 23<br />

KO 06<br />

KN 04<br />

Böttger, R. PN 35<br />

Boursillon, D. HDID P 05<br />

Bowien, B. PR 10<br />

Braendle, M. PM 04<br />

Brakhage, A. KP 06<br />

PJ 15<br />

PP 18<br />

KR 05<br />

PH 15<br />

Bramadathan, K.N. PP 06<br />

Bramkamp, M. PM 13<br />

KI 04<br />

PM 07<br />

PM 02<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Brandes, I. PO 17<br />

Braumann, I. PG 01<br />

PH 26<br />

Braus, G.H. PH 16<br />

Braus-Stromeyer, S.A. PH 16<br />

Brefort, T. FGD 01<br />

Brehm, S. PS 10<br />

Breidenbach, B. PO 44<br />

Breitling, R. KI 07<br />

Brenneis, M. PU 01<br />

Bretschnei<strong>der</strong>, U. PD 02<br />

PD 01<br />

Brettar, I. HDID 07<br />

Breuker, A. PN 46<br />

PO 37<br />

Breves, R. PN 28<br />

Briganti, F. PZ 36<br />

Bringer, S. PQ 01<br />

Brochier-Armanet, C. FGC 01<br />

Brock, M. FGF 05<br />

KN 02<br />

Brocker, M. PM 23<br />

Bröcker, M. PQ 06<br />

Broekmans, M. KF 04<br />

Brötz-Oesterhelt, H. FGA 02<br />

Brown, D.W. FGD 02<br />

Brucher, B. PD 03<br />

PD 02<br />

Brul, S. FGD 07<br />

Bruland, N. PX 44<br />

Brumfeld, V. H 11<br />

Brune, A. PN 38<br />

PN 62<br />

PO 10<br />

Brüning, S. PP 15<br />

Brunner, G. PX 18<br />

Brüser, T. KS 06<br />

PR 23<br />

PW 14<br />

Brust, D. KD 02<br />

Brzonkalik, K. PH 27<br />

Brzuszkiewicz, E. PG 02<br />

Buchholz, F. PZ 50<br />

Buck, S. PH 02<br />

Buck, U. PO 09<br />

Buckel, W. PX 08<br />

PA 05<br />

PA 13<br />

Bücking, C. KA 04<br />

Budinova, R. PR 10<br />

Buegger, F. PZ 03<br />

Buffing, M. PR 27<br />

Bühler, B. KT 02<br />

Bühler, K. PX 36<br />

PX 14<br />

Burchhardt, G. PS 61<br />

Burdziak, A. PX 32<br />

Burdziak, D. PZ 43<br />

Bürger, S. PZ 36<br />

Burghardt, T. KI 06<br />

KJ 04<br />

Burian, M. KG 02<br />

Burkhardt, E.M. PN 59<br />

Burkhardt, J. PW 12<br />

Burkovski, A. PS 59<br />

PS 58<br />

KO 06<br />

Burmester, A. KE 04<br />

PH 22<br />

Bürstel, I. PQ 10<br />

Busch, A. PQ 11<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

PQ 09<br />

Busse, S. PU 13<br />

Bussey, K. PN 28<br />

Butler, M. PZ 11<br />

Buttermann, D. PH 18<br />

Büttgenbach, S. PZ 53<br />

Büttner, D. PP 12<br />

Cabezas, A. PO 44<br />

Cao, X.H. PG 06<br />

Carius, A.B. KM 04<br />

Casero, D. H 08<br />

Caspers, M.P.M. HDID 09<br />

Castruita, M. H 08<br />

Casutt, M.S. KN 01<br />

Caucci, S. PN 56<br />

Celic, E.K. PW 22<br />

PP 38<br />

Chacinska, A. H 10<br />

Charuvi, D. H 11<br />

Chatzinotas, A. PN 76<br />

PN 67<br />

PN 56<br />

PN 71<br />

Chavarría, M. PS 11<br />

Chen, H. PO 31<br />

Chen, X. PI 05<br />

Cherdchim, B. FGD 05<br />

Chernikova, T. PV 08<br />

Chhatwal, G.S. FGF 01<br />

PP 07<br />

PP 06<br />

PP 04<br />

Chisholm, S.W. PU 06<br />

Chow, R.K.K. PH 33<br />

Chuartzman, S.G. H 11<br />

Circolone, F. KH 05<br />

Cizmowski, C. KS 03<br />

Claessen, D. FGA 05<br />

Claus, H. PI 01<br />

Clausen, M. KG 03<br />

Clemons, Jr., W.M. KH 03<br />

Cokus, S. H 08<br />

Colwell, R. HDID 01<br />

Commichau, F.M. FGG 03<br />

Conrad, R. PO 28<br />

PO 02<br />

PA 26<br />

PA 25<br />

Conrads, G. PP 42<br />

Cramer, A. PS 20<br />

Cramer, P. KB 03<br />

Crnovcic, I. PT 11<br />

Ćulafić, D. PZ 58<br />

Cypionka, H. PR 18<br />

PN 23<br />

PN 31<br />

PN 17<br />

PB 26<br />

KB 05<br />

PN 49<br />

Dagar, S.S. PZ 41<br />

Dahl, C. PR 09<br />

PZ 17<br />

PR 06<br />

Daims, H. PB 14<br />

Dalle, F. KL 06<br />

Dam, R. PM 24<br />

Dambeck, M. PB 23<br />

Dammeyer, T. PV 08<br />

Daniel, H. PW 20<br />

Daniel, R. PN 57<br />

PG 02<br />

PO 41<br />

PO 47<br />

PX 19<br />

PJ 14<br />

PR 30<br />

Darban, D. PP 02<br />

Darfeuille, F. PU 11<br />

David, C. PW 15<br />

De Groot, P. FGD 07<br />

de Koning, L. FGD 07<br />

De Koster, C. FGD 07<br />

de Lorenzo, V. PS 11<br />

de Maria, L. PC 02<br />

de Vos, W.M. PA 41<br />

Debelyy, M.O. PV 07<br />

Deckert, J. PU 12<br />

Defeu Soufo, H.J. KI 02<br />

Deghmane, A.E. PP 30<br />

Dehghan Shasaltaneh, M. PX 46<br />

Dekker, H. FGD 07<br />

Dempwolff, F. FGF 03<br />

PM 19<br />

Depkat-Jakob, P.S. PJ 03<br />

Depke, M. PJ 21<br />

Depmeier, W. PB 03<br />

Deppenmeier, U. PX 29<br />

PA 24<br />

Dersch, P. PU 08<br />

PP 26<br />

PH 34<br />

PP 25<br />

KQ 03<br />

Desch, A. KH 04<br />

Deutscher, J. PP 30<br />

Deutzmann, J. PN 32<br />

Devreese, B. PN 69<br />

Diaz-Bone, R. KB 01<br />

PZ 34<br />

PZ 35<br />

PA 07<br />

Dib, J.R. PZ 10<br />

Dieckmann, M. PZ 08<br />

Dieckmann, S.M. PZ 08<br />

Die<strong>der</strong>ich, A. KC 02<br />

Die<strong>der</strong>ichs, K. PD 04<br />

Diekert, G. PA 08<br />

PA 04<br />

PA 20<br />

Dienst, D. KM 01<br />

Diethmaier, C. PS 01<br />

Dietrich, K. KK 01<br />

Dikfidan, A. PA 18<br />

Dillard, J. KL 04<br />

Dimitrova, A. PR 10<br />

Dinkla, K. FGF 01<br />

Dischinger, J. PC 06<br />

PC 05<br />

Dittmann, E. PM 25<br />

KF 02<br />

PT 01<br />

Djurdjevic, I. PX 08<br />

Do Huu, N. PH 03<br />

Döbber, M. PW 13<br />

Dobler, N. PR 06<br />

Döhlemann, G. KR 01<br />

Donati, E. HDID P 14<br />

Dönhöfer, A. FGG 04<br />

Donovan, C. PM 13<br />

AUTOREN 225<br />

Doosti, A. PY 01<br />

Döring, C. PV 01<br />

PX 21<br />

Döring, K. PM 26<br />

Dornmayr-Pfaffenhuemer, M.HDID<br />

P 10<br />

Dörr, T. PP 23<br />

Dorscheid, S. PX 02<br />

Dossot, M. HDID 06<br />

Dougan, D.A. KI 03<br />

Downie, J.A. PM 11<br />

Dräger, G. PC 01<br />

Draheim, J. HDID 07<br />

Drake, H. PN 22<br />

PN 36<br />

PJ 03<br />

KG 05<br />

Drath, M. KC 01<br />

Drees, S. KS 05<br />

Dreier, A. PN 82<br />

Drepper, F. PQ 08<br />

Drepper, T. KH 05<br />

Drews, M. PO 38<br />

Driessen, A. PB 11<br />

KS 05<br />

Dübel, S. PZ 54<br />

PZ 53<br />

Dubilier, N. PN 16<br />

Dumke, I. PO 13<br />

Dunlap, J.C. PH 01<br />

H 07<br />

Dünnwald, P. PS 26<br />

Dürre, P. PS 21<br />

PS 10<br />

PJ 09<br />

PV 01<br />

Dutow, P. PM 26<br />

Dwidjosiswojo, Z. HDID P 06<br />

Eberhagen, I. HDID P 11<br />

Eberl, L. H 06<br />

Ebert, B. KT 02<br />

Edwards, A. PM 11<br />

Egert, M. PN 28<br />

Egger, S. HDID P 13<br />

Eggert, T. KH 05<br />

Ehlers, C. PU 09<br />

PB 22<br />

Ehrenhofer-Murray, A. PB 19<br />

Ehrenreich, A. PX 19<br />

PR 17<br />

PX 30<br />

PR 30<br />

PX 21<br />

PV 01<br />

Eikmanns, B. PM 23<br />

PX 15<br />

PR 11<br />

PS 20<br />

PP 14<br />

Einsle, O. KA 06<br />

Eisenacher, M. KU 07<br />

Eisenbarth, K. PO 45<br />

Eisenreich, W. H 01<br />

PR 13<br />

Eitinger, T. PW 11<br />

El Magraoui, F. PV 06<br />

El-Alfay, S. PL 01<br />

PZ 02<br />

Elend, C. KT 05<br />

Elleuche, S. KE 02

226 AUTOREN<br />

KU 01<br />

Ellrott, A. PJ 24<br />

El-Safey, E.M. PN 01<br />

Elsner, M. PD 06<br />

Engelen, B. PN 31<br />

PN 17<br />

PN 23<br />

PN 49<br />

Engelhardt, H. PL 03<br />

Engelmann, S. PP 39<br />

Engh, I. KH 02<br />

KE 03<br />

Englert, J. PW 20<br />

Enseleit, M. PN 78<br />

Entian, K.D. FGH 02<br />

Entian, M. PS 50<br />

Erb, T.J. KN 03<br />

PZ 27<br />

Erdmann, R. PP 32<br />

PV 07<br />

PV 06<br />

PW 18<br />

PW 15<br />

PW 16<br />

KS 03<br />

PW 17<br />

KU 07<br />

Erker, W. FGG 05<br />

Ermler, U. PA 21<br />

PQ 04<br />

PA 23<br />

Ernst, S. PO 42<br />

Esche, J. PM 14<br />

Eschenhagen, M. PN 65<br />

PO 36<br />

PN 35<br />

Esperschütz, J. PZ 03<br />

Espinosa, J. KC 01<br />

Essen, L.O. PM 17<br />

Esser, D. PB 18<br />

Ettwig, K. PZ 11<br />

Etzel, K. PB 03<br />

Etzkorn, M. PS 26<br />

Eulberg, D. PX 41<br />

Euringer, K. PN 60<br />

Euzeby, J. FGE 04<br />

Evers, S. PR 17<br />

Evguenieva-Hackenberg, E. KQ 06<br />

Ewers, C. PP 35<br />

PP 11<br />

KL 05<br />

Exner, M. HDID 10<br />

Eylert, E. H 01<br />

Fairhead, M. KT 03<br />

Faisal, I. PO 49<br />

Faivre, N. PN 62<br />

Fälker, S. PP 43<br />

Fallschissel, K. PO 45<br />

PP 27<br />

PO 46<br />

Farias, M.E. PZ 10<br />

Faßben<strong>der</strong>, S. PZ 43<br />

Fechter, I. PH 32<br />

Fedtke, I. PP 37<br />

Fegeler, W. PJ 16<br />

Feger, S. PC 06<br />

Fehrmann, C. PJ 16<br />

Felbeck, H. PJ 14<br />

Feldbrügge, M. KD 04<br />

Fendler, K. PW 20<br />

Fendrihan, S. HDID P 10<br />

Ferraroni, M. PZ 36<br />

Ferrero, F. PO 08<br />

Fetzer, I. PN 71<br />

Fetzner, S. PX 10<br />

PZ 22<br />

Fichtel, J. PN 31<br />

Fichtner, N. PN 35<br />

Fiencke, C. PZ 18<br />

Filipp, F.V. PW 15<br />

Finster, K. PN 70<br />

Fischer, F. KR 06<br />

Fischer, M. PZ 09<br />

Fischer, R.J. PR 04<br />

PS 29<br />

PV 01<br />

PS 28<br />

PZ 32<br />

PR 14<br />

Fischer, R. PH 11<br />

KH 01<br />

Fischer, S. PD 04<br />

PB 25<br />

Fischer, U. PC 04<br />

Fleck, C. KN 02<br />

Fleischer, R. KO 03<br />

Flemming, H.C. HDID P 06<br />

HDID P 03<br />

Flieger, A. HDID P 09<br />

Flores, E. H 09<br />

Florian, B. PZ 24<br />

Follmann, M. PX 28<br />

PR 22<br />

PW 21<br />

Föllner, C. PX 27<br />

Fooladi, J. PX 46<br />

Forchhammer, K. KC 01<br />

PS 02<br />

PF 01<br />

PE 02<br />

Forrest, L.R. PW 21<br />

Fortin, D. PZ 55<br />

Fösel, B.U. PN 30<br />

Fraas, S. PA 21<br />

Fraatz, M. FGD 03<br />

Frank, C. PR 02<br />

Frank, U. PQ 06<br />

Frankenberg, S. PN 62<br />

Frankenberg-Dinkel, N. PS 41<br />

PS 50<br />

PS 53<br />

PQ 11<br />

Franz, B. PZ 17<br />

Fränzel, B. PS 23<br />

Franzki, B. FGD 01<br />

Frasch, H.J. PC 07<br />

Freese, H.M. PN 09<br />

Freese, S. PH 21<br />

Frenzel, P. PO 11<br />

PN 18<br />

PN 06<br />

Frerichs, J. PN 39<br />

PN 42<br />

Frick, I.M. PP 04<br />

FGF 01<br />

Frick, V.O. PJ 08<br />

Fried, L. PS 19<br />

PS 13<br />

Friedl, T. PN 78<br />

Friedrich, B. PQ 10<br />

H 12<br />

PZ 52<br />

KN 05<br />

Friedrich, C. PR 12<br />

PR 16<br />

Friedrich, L. KG 04<br />

Friedrich, M. PN 54<br />

Friedrich, M.W. PO 29<br />

PO 44<br />

PN 48<br />

PO 07<br />

Friedrich, T. FGG 04<br />

PS 08<br />

Friedrichs, C. PP 07<br />

PP 06<br />

Frielingsdorf, S. PZ 52<br />

Frimmel, F.H. PN 13<br />

Fritzsche, A. PA 34<br />

Frixel, S. PV 04<br />

Fröde, D. PZ 53<br />

Frühwirth, S. PS 39<br />

Fuchs, B. PR 02<br />

KK 01<br />

KJ 06<br />

Fuchs, G. PR 13<br />

PR 21<br />

PB 08<br />

PB 13<br />

PZ 27<br />

PB 12<br />

KN 03<br />

PR 28<br />

Fuchs, J. PG 05<br />

Fuchs, V. PX 35<br />

Fuhrer, T. PB 23<br />

Funes, S. KI 01<br />

Funk, A. KP 06<br />

Funken, H. PV 05<br />

Futschik, M. PU 06<br />

Gabriel, G. PS 42<br />

Galinski, E. PX 33<br />

PX 32<br />

PX 23<br />

PX 34<br />

Gallert, C. PZ 15<br />

Gallinger, C. PS 35<br />

Gamer, M. PX 11<br />

Ganzert, L. PO 12<br />

García-González, E. PP 45<br />

Gardebrecht, A. PJ 14<br />

Gärdes, A. PJ 20<br />

PJ 19<br />

Gärtner, A. PO 43<br />

Gasser, I. PO 05<br />

Gatermann, S. KL 03<br />

PP 33<br />

PP 21<br />

PW 09<br />

Gaubig, L. KQ 05<br />

Gauer, S. PX 20<br />

Gaupp, R. PP 31<br />

Gayer, S. PS 07<br />

Gehrke, A. PJ 15<br />

Gehrke, F. KN 06<br />

Geiger, R. PZ 04<br />

Geis, A. PX 16<br />

Geisel, A.C. PO 26<br />

Geisler, L. PN 25<br />

Geissinger, O. PO 10<br />

Gemeinholzer, B. KU 03<br />

Gemperli, A.C. PQ 02<br />

Genersch, E. PP 45<br />

Georg, J. PU 07<br />

KM 01<br />

Georgi, T. KO 06<br />

Gerber, S.A. H 07<br />

Gerhards, D. FGH 05<br />

Gerischer, U. PS 12<br />

Gescher, J. KI 02<br />

KA 04<br />

PZ 19<br />

PA 09<br />

PN 14<br />

Gesell Salazar, M. PM 24<br />

Gesing, S. PH 14<br />

Geyer, W. PN 71<br />

Ghaemi, N. PX 03<br />

PX 01<br />

Ghanegaonkar, S. KT 06<br />

Ghareeb, H. PJ 04<br />

FGD 01<br />

Ghosh, S. PZ 56<br />

Giebler, J. PN 76<br />

Giesbert, S. PH 18<br />

Giffhorn, F. PX 22<br />

PX 02<br />

PX 31<br />

PX 20<br />

PX 39<br />

Girzalsky, W. PV 06<br />

PW 18<br />

PW 17<br />

KS 03<br />

Gittel, A. PN 83<br />

Glaeser, J. KQ 02<br />

PN 75<br />

PS 09<br />

PS 22<br />

PN 77<br />

Glaeser, S. PN 75<br />

PN 77<br />

Glanz, S. KC 05<br />

PE 06<br />

Glaubitz, S. PN 52<br />

Gleinser, M. PJ 05<br />

Glöckner, F.O. KK 01<br />

FGE 04<br />

PN 08<br />

KK 01<br />

PG 06<br />

FGE 01<br />

Glöer, J. PS 38<br />

Gniese, C. PN 39<br />

Gödeke, J. PN 41<br />

Goebel, W. H 01<br />

Goecke, F. PJ 07<br />

PN 40<br />

Goerke, C. PP 05<br />

KG 02<br />

Göhring, N. PP 37<br />

Goldberg, A. H 02<br />

Goldet, G. KA 05<br />

Golyshin, P.N. PV 08<br />

Gomaa, M. PC 01<br />

Gomolla, D. PQ 03<br />

Göpel, Y. PU 03<br />

Görke, B. PU 03<br />

PS 36<br />

Gottardi, W. PZ 04<br />

Göttfert, M. KG 04<br />

Göttlicher, J. PN 14<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Gottschalk, G. PG 02<br />

PR 30<br />

Götz, F. PP 31<br />

PP 19<br />

PP 22<br />

Götz, P. PV 01<br />

Govorun, V. PN 85<br />

Grammel, H. PV 02<br />

KM 04<br />

PA 16<br />

Grantcharova, N. KJ 02<br />

Grasse, N. KM 02<br />

Graue, J. PN 23<br />

Graumann, P. KI 02<br />

FGF 03<br />

PM 20<br />

PM 19<br />

PF 02<br />

Grein, F. PR 09<br />

Gribaldo, S. FGC 02<br />

Griebler, C. PN 64<br />

PN 34<br />

PN 63<br />

Grie<strong>der</strong>, K. KT 03<br />

Griffin, B.M. PA 03<br />

Grimm, F. PR 06<br />

Grimpo, J. PR 15<br />

Groebe, L. HDID 07<br />

Groebel, K. PJ 10<br />

Grohmann, E. KH 06<br />

PP 38<br />

PP 16<br />

PW 22<br />

PW 23<br />

Gronau, K. PR 01<br />

Grond, S. KU 06<br />

PZ 48<br />

Grooters, M. PZ 25<br />

Grösbacher, M. HDID P 10<br />

Gross, R. PX 36<br />

Grossart, H.P. PN 77<br />

PN 75<br />

PN 87<br />

Große, C. PW 03<br />

Grossmann, M. FGH 05<br />

Grosz, M. PN 77<br />

Grote, J. PN 26<br />

PN 43<br />

Grunau, S. PW 17<br />

PW 18<br />

Grundmann, O. PA 22<br />

Gschwendtner, S. PZ 03<br />

Gu, J.D. PH 33<br />

Gudiseva, H.V. KN 06<br />

Guenther, S. PP 35<br />

Guezguez, J. FGA 04<br />

Guggenberger, C. PS 13<br />

Guljamow, A. PM 25<br />

Gunka, K. KU 05<br />

Günther, S. PN 65<br />

Gunzer, M. PJ 15<br />

Gust, G. PN 87<br />

Gutjahr, M. PJ 21<br />

Haag, C. KD 04<br />

Haagsma, A.C. PP 08<br />

FGF 04<br />

Haas, D. H 03<br />

Haase, S. PP 22<br />

Häberer, K. FGF 03<br />

Hackermüller, J. PU 11<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Hädrich, A. PN 53<br />

Haesler, F. PN 03<br />

Hagemann, A. PB 20<br />

Hagemann, M. KF 02<br />

KC 02<br />

Hahn, A. PW 07<br />

Hahn, F. PX 18<br />

Hahnke, R. PN 15<br />

Hahnke, S. PR 18<br />

Hai, B. PN 03<br />

Hajj Chehade, M. HDID 06<br />

Hakenbeck, R. PS 14<br />

PZ 16<br />

Halacheva, V. PR 10<br />

Halan, B. PX 14<br />

Halbach, A. PW 16<br />

Halbedel, S. KI 07<br />

Hall, M. PH 25<br />

Hallam, S.J. PG 05<br />

Hallmann, C. PN 78<br />

Hamann, A. KD 02<br />

PH 10<br />

Hambruch, E. KS 03<br />

Hambsch, B. HDID P 11<br />

Hamido, H. PL 01<br />

PZ 02<br />

Hammel, K.E. PX 07<br />

PX 06<br />

Hammer, E. PJ 21<br />

Hamoen, L.W. KI 07<br />

Haneburger, I. PS 18<br />

Hänelt, I. PW 13<br />

Hanke, T. PQ 01<br />

Hannig, M. PJ 08<br />

Hansen, S. PP 41<br />

Hanssen, A.M. PW 22<br />

PW 23<br />

Hantke, K. PJ 11<br />

Hanzel, J. PN 68<br />

Happe, T. PE 07<br />

KC 04<br />

PQ 07<br />

PA 17<br />

PZ 44<br />

KF 05<br />

KF 03<br />

PA 43<br />

Harald, H. PB 01<br />

Har<strong>der</strong>, J. KK 04<br />

PR 02<br />

PA 18<br />

PA 21<br />

PN 15<br />

Harms, A. PS 41<br />

Harms, H. PN 71<br />

PN 56<br />

PN 67<br />

PZ 50<br />

PN 68<br />

PN 76<br />

Härtner, T. PT 07<br />

Hartung, T. PJ 08<br />

Hasenberg, M. PJ 15<br />

Hashemi Aghdam, Y. PP 01<br />

Hassan, M.A. PB 04<br />

Hasselt, K. PS 58<br />

Haszprunar, G. KU 03<br />

Hatefi, M. HDID P 04<br />

Hatzenpichler, R. PB 14<br />

PB 16<br />

Haus, S. PV 01<br />

Hauser, E. PS 42<br />

Hauser-Gerspach, I. PN 81<br />

Haußmann, U. PG 03<br />

KR 06<br />

Havemann, J. PH 24<br />

Hebbeln, P. PW 11<br />

Heck, B. KL 03<br />

Hecker, M. PJ 21<br />

PR 01<br />

PG 06<br />

PS 52<br />

PJ 14<br />

KN 05<br />

PP 39<br />

Hedrich, S. PN 33<br />

Heeg, K. PN 58<br />

Heermann, R. KO 02<br />

PS 36<br />

PS 42<br />

PX 45<br />

PS 07<br />

Hegemann, P. KU 02<br />

Heichlinger, A. PM 09<br />

Hei<strong>der</strong>, J. PA 44<br />

PA 11<br />

Heidrich, G. PR 03<br />

Heidrich, N. KQ 04<br />

Height, M. HDID P 13<br />

Heilmann, C. PP 19<br />

PJ 16<br />

Heimel, K. KR 01<br />

Heimerl, T. KJ 04<br />

Hein, K. KO 04<br />

Heindl, H. PO 43<br />

Heinekamp, T. PP 18<br />

Heinemann, I. PZ 46<br />

Heinemann, K. PP 34<br />

Heinrich, J. KO 04<br />

Heinrich, S. PP 17<br />

Heintz, D. PA 19<br />

Heipieper, H.J. PR 27<br />

Held, C. PR 30<br />

Helena, M. PN 50<br />

Heller, C. PN 82<br />

Heller, E.M. KL 04<br />

Heller, J. KD 03<br />

Heller, K.J. PX 16<br />

Hempel, D.C. PH 28<br />

Hempel, K. PG 06<br />

Hempelmann, R. PX 22<br />

Hemschemeier, A. KC 04<br />

PE 07<br />

PQ 07<br />

PA 17<br />

PA 43<br />

Hendrischk, A.K. PS 39<br />

Hengge, R. PS 51<br />

PU 13<br />

Henne, K. HDID 07<br />

Hennecke, H. H 04<br />

Hennig, S. PP 32<br />

Henrich, A. PX 15<br />

PR 26<br />

Henrichfreise, B. PM 14<br />

Hense, B.A. KR 04<br />

Hensel, A. PW 18<br />

Hensel, G. KD 06<br />

Hensel, M. KG 01<br />

Hensel, R. PA 07<br />

KB 01<br />

PB 20<br />

AUTOREN 227<br />

PB 24<br />

Hentschel, U. PN 73<br />

PJ 25<br />

Heppe, M. PO 31<br />

Herbst, K. PP 26<br />

Herlemann, D.P.R. PO 10<br />

Hernandez-Alvarez, B. PD 05<br />

Herndl, G.J. PN 43<br />

Heroven, A.K. PU 08<br />

KQ 03<br />

Herrmann, J. KI 01<br />

KS 02<br />

Herrmann, M. HDID P 07<br />

PO 30<br />

PN 53<br />

PN 55<br />

PJ 08<br />

Hertweck, C. KP 06<br />

KR 05<br />

PT 01<br />

FGA 03<br />

FGA 03<br />

Hertwig, F. PS 52<br />

Herzberg, M. PW 03<br />

Herzog, B. PB 06<br />

Hess, W. PU 07<br />

PU 06<br />

KM 01<br />

Hettinger, S. PH 11<br />

Heusipp, G. FGF 02<br />

PP 43<br />

Heydari Nasr, M. PZ 06<br />

Heyer, A. PZ 25<br />

Heyer, R. PU 01<br />

Hiergeist, A. PS 34<br />

Hiery, E. KO 06<br />

Hieter, P. PG 05<br />

Higgs, P. PF 04<br />

Higuera Sobrino, J.J. PA 43<br />

Hilberg, M. PA 30<br />

Hildebrandt, P. PJ 21<br />

Hildenbrand, C. PM 08<br />

Hill, C. PP 14<br />

Hillen, W. PS 55<br />

Hillmann, A. PS 36<br />

Hillmann, F. KA 02<br />

PS 29<br />

Hilmes, J. PZ 14<br />

Hinton, J. PU 14<br />

PU 05<br />

Hippauf, M. PP 36<br />

Hippler, M. PQ 09<br />

KF 05<br />

PQ 08<br />

Hiromoto, T. PQ 04<br />

Hirschmann, I. KF 05<br />

Hitkova, I. KO 02<br />

Ho, A. PO 11<br />

Hoelzle, K. PZ 08<br />

PJ 10<br />

Hoelzle, L.E. PZ 08<br />

PJ 10<br />

Hoff, B. KP 02<br />

PT 03<br />

PT 02<br />

PH 29<br />

PH 04<br />

PT 08<br />

Hoffmann, C. PL 03<br />

Hoffmann, M. PN 84<br />

Hoffmann, S. PU 11

228 AUTOREN<br />

Höfle, M. HDID 07<br />

Hofmann, E. PQ 11<br />

Hofrichter, M. PX 07<br />

PX 06<br />

KT 04<br />

PH 30<br />

PH 03<br />

KE 07<br />

PX 17<br />

PH 09<br />

Hohloch, M. HDID P 05<br />

Höhne, M. FGB 03<br />

Holert, J. PR 18<br />

Hollmann, F.H. PX 05<br />

Holm, N.C. PZ 05<br />

Holz, C. PZ 42<br />

Holzinger, A. HDID P 10<br />

Homayoon, M. PP 10<br />

PY 01<br />

Homeier, T. KL 05<br />

PP 11<br />

Homonnay, Z.G. PN 88<br />

Hoppert, M. PN 82<br />

PM 26<br />

PN 78<br />

Horn, M. PN 36<br />

PN 22<br />

Horn, M.A. KG 05<br />

PJ 03<br />

Horn, M. KK 03<br />

Horn, S. PX 38<br />

Horn, U. KR 05<br />

Hornung, C. KU 06<br />

Hortschansky, P. PH 15<br />

Horz, H.P. PP 42<br />

Hoth, N. PN 39<br />

Hubberten, H.W. PO 12<br />

Hube, B. KL 06<br />

Huber, B. KB 01<br />

PA 07<br />

Huber, H. PB 02<br />

PB 03<br />

KI 06<br />

KB 06<br />

PN 04<br />

KJ 04<br />

Huber, T. KN 01<br />

Hübner, S. PS 01<br />

Huddar, M. KT 07<br />

Huergo, J. HDID P 14<br />

Hug, J. KR 02<br />

Hugenholtz, P. PO 10<br />

Hügler, M. HDID P 11<br />

PN 16<br />

KB 05<br />

Hultgren, S. H 05<br />

KJ 03<br />

Hummel, P. PQ 10<br />

Humpf, H.U. FGD 02<br />

Hung, C. KJ 03<br />

Hunke, S. KO 03<br />

PP 09<br />

Hurwitz, R. PU 12<br />

Husemann, I. PZ 44<br />

Hüser, A. PR 22<br />

Hust, M. PZ 54<br />

PZ 53<br />

Hütz, A. KJ 07<br />

Idoine, A. KF 06<br />

Ikeda-Ohtsubo, W. PN 62<br />

PO 10<br />

Ikeno, S. PB 04<br />

Ilbert, M. KN 06<br />

Iliev, D. KC 06<br />

Ilina, E. PN 85<br />

Ilmberger, N. PI 02<br />

Imhoff, J.F. PO 38<br />

PO 34<br />

PJ 07<br />

PO 33<br />

PN 16<br />

PN 40<br />

PO 43<br />

Ingram, T. PX 18<br />

Ingvorsen, K. PN 83<br />

Isaeva, A. PN 85<br />

Isenhardt, S. PR 24<br />

Ishida, K. PT 01<br />

Ismail, W. PR 13<br />

Itzek, A. PP 06<br />

PP 07<br />

Jäckel, U. PP 36<br />

PO 46<br />

PO 50<br />

PO 42<br />

PO 45<br />

Jacobs, J. PE 07<br />

KC 04<br />

Jacobs, J. PZ 19<br />

Jaeger, K.E. PX 38<br />

PL 04<br />

Jaekel, U. PA 33<br />

PA 38<br />

Jafari, P. PZ 07<br />

PX 03<br />

PZ 06<br />

Jäger, A. PS 39<br />

Jäger, D. PU 09<br />

Jäger, K.E. PX 35<br />

PV 05<br />

PR 24<br />

Jäger, S. PP 24<br />

Jagmann, N. PX 25<br />

Jahn, D. PZ 49<br />

PP 34<br />

PZ 46<br />

PQ 06<br />

PX 11<br />

Jahn, M. PZ 46<br />

Jahn, S. PS 16<br />

Jain, S. KL 04<br />

Jakob, U. KN 06<br />

Jakobs, D. PN 50<br />

Janausch, I. PW 20<br />

Janine, G. PX 22<br />

Janosch, C. PZ 23<br />

Jansen, A. PC 02<br />

Janssen, D. FGB 01<br />

Janßen, H. PZ 22<br />

Janssen, H. PR 14<br />

PV 01<br />

Jansson, M. PH 17<br />

Janus, D. PH 04<br />

PT 02<br />

Jayamani, E. PA 13<br />

Jehmlich, N. PN 44<br />

KJ 05<br />

Jellen-Ritter, A. PU 01<br />

Jendrossek, D. PZ 33<br />

Jensen, G.J. KH 03<br />

Jessberger, N. PS 58<br />

PS 59<br />

Jetten, M. PZ 11<br />

Jogler, C. PZ 40<br />

PG 07<br />

PO 48<br />

Johannes, J. PA 35<br />

PA 39<br />

John, P. PN 47<br />

Johnsen, U. PB 23<br />

Jolkver, E. PW 02<br />

PX 28<br />

PW 10<br />

Jordan, E. PZ 54<br />

Joseph, B. H 01<br />

Jost, D. PZ 15<br />

Jost, G. PN 26<br />

PN 52<br />

Jost, G. PN 43<br />

Josten, M. PC 05<br />

Juhna, T. HDID P 15<br />

Jung, H. PS 08<br />

Jung, K. FGG 04<br />

KO 02<br />

PS 18<br />

PS 19<br />

PS 36<br />

PS 42<br />

PS 37<br />

PX 45<br />

PS 07<br />

PS 13<br />

PS 03<br />

Junge, K. PW 04<br />

PM 18<br />

Jungfer, C. PN 21<br />

Junglas, B. KJ 04<br />

Junker, A. PX 30<br />

Juretzek, T. PJ 13<br />

Jürgens, K. PN 26<br />

PN 52<br />

Jürgens, K. PN 43<br />

Jurk, K. PJ 16<br />

Just, W.W. PW 17<br />

Kaase, M. KL 03<br />

Kaboosi, H. PZ 12<br />

PZ 20<br />

Kahl, B.C. PP 15<br />

Kähler, M. PX 30<br />

Kahlisch, L. HDID 07<br />

Kahmann, R. FGD 01<br />

FGF 06<br />

PJ 04<br />

KR 01<br />

Kai, M. KP 03<br />

Kaim, G. KM 06<br />

Kajahn, I. PJ 07<br />

Kalinka, J. PJ 21<br />

Kalinowski, J. PR 22<br />

Kamerewerd, J. PH 17<br />

Kamke, J. PJ 12<br />

Kammann, C. PN 72<br />

PN 74<br />

Kammler, L. PR 09<br />

Kämper, J. KR 01<br />

Kämpfer, P. PO 20<br />

PO 46<br />

PP 36<br />

PP 27<br />

PO 45<br />

Kaplan, A. H 11<br />

Käppel, E. PJ 19<br />

Kappelmeyer, U. PR 27<br />

Karami, N. FGE 03<br />

Karcher, D. KC 07<br />

Kargar, M. PP 10<br />

PY 01<br />

Karolewiez, A. PN 51<br />

Karpowicz, S. H 08<br />

Kaschabek, S.R. PX 41<br />

Kaschak, E. PZ 31<br />

Kaschner, M. PL 04<br />

Kassahun, A. PN 39<br />

Kaster, A.K. KM 05<br />

Kästner, M. PZ 21<br />

PH 07<br />

PN 60<br />

Katic Radivojevic, S PZ 58<br />

Katzmann, E. PZ 40<br />

PM 15<br />

Kaufenstein, M. PF 02<br />

Kaufmann, C. PN 70<br />

Kaulfersch, M. PN 67<br />

Kavalchuk, K. PS 31<br />

Kaya, M. PN 29<br />

Kayser, G. PX 07<br />

PX 17<br />

KT 04<br />

Kazunori, N. PB 04<br />

Kehr, J.C. KF 02<br />

Kehrel, B. PJ 16<br />

Keijser, B.J. HDID 09<br />

Keil, C. HDID 04<br />

Kelety, B. PW 20<br />

Keller, S. PW 19<br />

Keller, U. PT 11<br />

PH 24<br />

Keller, W. PP 38<br />

PW 23<br />

PW 22<br />

Kellermann, C. PN 63<br />

Kembou, F. PS 30<br />

Kemler, M. KK 07<br />

Kemper, M. PH 02<br />

Kempf, C. PH 11<br />

Kempken, F. PG 01<br />

PH 26<br />

KD 01<br />

Kempkes, R. PR 26<br />

Kengen, S.W.M. PA 41<br />

Kerkez, M. PZ 58<br />

Kern, M. KA 03<br />

PA 29<br />

PA 27<br />

Kerzenmacher, S. KA 04<br />

Kettenbach, A.N. H 07<br />

Keuter, S. PO 23<br />

Khanna, M. PJ 01<br />

Khiyami, M.A. PN 05<br />

Khodakaramian, G. PO 03<br />

Khodaverdi, V. PS 46<br />

Khosravani, A. PP 40<br />

Kiekebusch, D. PM 17<br />

Kierul, K. PI 05<br />

Kiesel, B. PN 56<br />

PN 67<br />

Kim, J. PX 08<br />

Kinashi, H. PO 03<br />

Kinne, M. PX 07<br />

PX 17<br />

PX 06<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Kirchberg, J. PP 12<br />

Kirchen, S. PM 04<br />

Kirchhoff, H. PQ 09<br />

PQ 08<br />

Kirsten, A. PW 05<br />

Kist, M. FGF 03<br />

Kistler, C. PS 08<br />

Kjelleberg, S. PZ 45<br />

HDID 02<br />

Klaffl, S. PR 11<br />

Klages, S. PZ 13<br />

Klähn, S. KC 02<br />

Klassen, R. PY 04<br />

FGH 04<br />

Klebensberger, J. PZ 45<br />

Klein, B. PA 16<br />

Klein, K. PL 04<br />

Klein, S. PP 24<br />

Klein, T. PX 22<br />

Kleindienst, S. PN 23<br />

PO 25<br />

Kleine, B. PP 33<br />

KL 03<br />

Klenk, H.P. PZ 43<br />

KU 03<br />

Kletzin, A. PB 21<br />

Klimova, M. PN 29<br />

Kling, A. PB 11<br />

Klingl, A. PB 03<br />

Klinner, U. KE 06<br />

PH 21<br />

PH 20<br />

Klis, F. FGD 07<br />

Klix, V. PH 01<br />

Klockow, C. PN 08<br />

Klug, G. KQ 02<br />

PS 22<br />

PS 09<br />

KO 05<br />

PS 39<br />

Klug, K. PO 46<br />

Kluge, M. PH 30<br />

KT 04<br />

Kluge, S. PN 84<br />

Klüsener, S. PJ 02<br />

Kniemeyer, O. PJ 15<br />

PP 18<br />

KR 05<br />

Knittel, K. KK 01<br />

PO 25<br />

KJ 06<br />

PO 09<br />

Knopf, B. PJ 06<br />

Knorr, D. PR 07<br />

PZ 39<br />

PY 02<br />

Knorr, J. PR 24<br />

Knörzer, P. KF 03<br />

Knura, T. PB 24<br />

Koch, J. PS 40<br />

Koch, K. KK 02<br />

Köcher, S. PR 25<br />

Kock, D. PO 13<br />

PO 14<br />

Kock, J. PB 22<br />

Kockelkorn, D. PB 08<br />

PB 12<br />

Koenig, F. KM 03<br />

Koerdt, A. PM 16<br />

PB 10<br />

Kohler, T. KL 02<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Köhler, T. PN 62<br />

Kohlmann, Y. KN 05<br />

Kohls, K. PE 08<br />

Kohring, G.W. PX 20<br />

PX 31<br />

PX 22<br />

Kokoschka, S. PN 82<br />

Kolb, M. PS 56<br />

PS 55<br />

Kollath-Leiß, K. KD 01<br />

Kölzer, S. PA 30<br />

König, H. PI 01<br />

PZ 31<br />

PJ 06<br />

PO 24<br />

König, J. KD 04<br />

König, S. PJ 09<br />

Könneke, M. PN 49<br />

PB 26<br />

KB 05<br />

Kopka, B. PL 04<br />

Kopke, K. PT 03<br />

Köpke, M. PR 30<br />

Korehi, H. PN 46<br />

Kornberger, P. PX 31<br />

PX 22<br />

Korsten, A. PX 33<br />

Kort, R. HDID 09<br />

Korte, M. PP 21<br />

PW 09<br />

Kortmann, J.F. PU 02<br />

Kostka, J. PN 70<br />

PN 59<br />

Kostrzewa, M. PN 85<br />

Kothe, E. PZ 21<br />

Kotzerke, A. PN 07<br />

Koul, A. FGF 04<br />

PP 08<br />

Kouril, T. PB 18<br />

Kozhinjampara, M. PZ 47<br />

Kraemer, R. PS 40<br />

KS 04<br />

PW 06<br />

Kraft, B. PN 49<br />

Krakat, N. PN 27<br />

Kralik, S.M. KI 03<br />

Krämer, R. PX 15<br />

PX 28<br />

PE 05<br />

PW 02<br />

PR 22<br />

PW 10<br />

PW 21<br />

Krätzer, C. PA 24<br />

Krause, D. PV 01<br />

Krause, F. PX 15<br />

Krause, J. FGH 04<br />

Krause, R. PW 20<br />

Krause, S. PN 18<br />

Krauß, D. PX 21<br />

Krauss, J. PA 32<br />

Krauß, N. KO 03<br />

Krauss, U. PL 04<br />

Kravietz, D. KC 04<br />

Krawietz, D. KF 05<br />

PA 43<br />

Kraxenberger, T. PS 19<br />

PS 13<br />

Krebs, P. PN 56<br />

Krehenbrink, M. PM 11<br />

Kreher, S. PA 20<br />

Kremling, A. PS 07<br />

Kretzschmar, U. PS 46<br />

Kristensen, H.H. PC 02<br />

Krohne, G. PJ 25<br />

Kroll, J. PX 13<br />

Kroneck, P. KA 06<br />

PA 21<br />

PA 23<br />

Kropat, J. H 08<br />

Kroutil, W. FGB 02<br />

Krüger, M. PN 42<br />

PN 54<br />

PO 29<br />

PN 39<br />

PO 13<br />

PO 32<br />

PN 17<br />

Krukenberg, V. PN 82<br />

Krull, R. PP 34<br />

PH 28<br />

Kruse, M. PO 19<br />

Kruse, T. PC 02<br />

Krysciak, D. PZ 48<br />

Kube, M. PO 48<br />

PG 07<br />

Kück, U. KP 02<br />

PH 04<br />

PH 17<br />

PE 06<br />

PH 29<br />

KC 05<br />

PT 08<br />

KE 03<br />

PH 05<br />

PT 02<br />

PT 03<br />

KH 02<br />

Kueper, U. PB 01<br />

PN 04<br />

Kües, U. FGD 05<br />

PH 23<br />

Kuhlgert, S. PQ 08<br />

Kuhlmann, J. PW 16<br />

Kuhn, E. HDID P 13<br />

Kuhn, R. PN 69<br />

Kühn, A. PJ 21<br />

Kühner, D. PP 17<br />

Kuklinski, A. PZ 25<br />

Kumar, H. PP 16<br />

Kung, J.W. PA 12<br />

Kunin, V. PO 10<br />

Kunte, H.J. PZ 14<br />

PR 03<br />

PZ 43<br />

Kuntze, K. PA 40<br />

Küper, U. KI 06<br />

Kurnia, F. PO 49<br />

Kürnsteiner, H. KP 02<br />

Kurz, M. PX 23<br />

Küsel, K. PN 59<br />

PN 55<br />

PN 70<br />

PN 53<br />

Kusian, B. PR 10<br />

Kutscher, B. PP 13<br />

Kuttler, C. KR 04<br />

Kuypers, M. PZ 11<br />

PA 27<br />

Kwon, J.H. KF 04<br />

AUTOREN 229<br />

Labes, A. PN 40<br />

PO 43<br />

PJ 07<br />

Labrenz, M. PN 26<br />

PN 52<br />

PN 43<br />

Laera, G. PN 69<br />

Lakner, S. PX 41<br />

Lambertz, C. PE 07<br />

Lammirato, C. PH 07<br />

Lang, C. PM 18<br />

Lang, E. PO 17<br />

PO 16<br />

Lang, G. PJ 07<br />

Lange, C. PX 32<br />

PB 17<br />

Lange, D. PA 28<br />

Lange, M. PB 01<br />

Langenbach, K. PN 60<br />

Langenhorst, F. PN 70<br />

Langhammer, P. PR 02<br />

Langklotz, S. PM 06<br />

Laros, J.J. H 07<br />

Larribe, M. PP 30<br />

Larsson, S. HDID P 15<br />

Lassak, J. PS 44<br />

Latif, A. PO 49<br />

Latus, A. PM 09<br />

Lay, D. PW 17<br />

Layer, G. PA 14<br />

PA 06<br />

Le Guen, E. HDID 06<br />

Lebedeva, E. PB 14<br />

Lechner, U. PN 61<br />

PS 54<br />

Leclerque, A. PK 03<br />

Lee, B. PF 04<br />

Legewie, S. KM 01<br />

Leggewie, C. PX 38<br />

Lehmann, C. PS 61<br />

Lehmann, D. PR 04<br />

Lehmann, R. HDID P 13<br />

Lehner, J. PF 01<br />

Lehnik-Habrink, M. FGG 03<br />

PS 15<br />

Leichert, L. PZ 26<br />

KN 06<br />

Leis, A. PL 03<br />

Leischner, K. KM 02<br />

Lentzen, G. PR 03<br />

PZ 43<br />

Lenz, O. PQ 10<br />

Leon Morales, C.F. HDID P 14<br />

Lepaslier, D. PZ 11<br />

Lerchner, J. PZ 50<br />

Lessing, F. PJ 15<br />

Lestari, R. PO 49<br />

Letarov, A. PN 85<br />

Lewis, K. PP 23<br />

PP 41<br />

Liaimer, A. PT 01<br />

Liao, Y.F. FGG 05<br />

Licht, A. PW 19<br />

Lichtfuß, A. PX 24<br />

Lickfeld, M. PH 02<br />

PH 19<br />

Liebl, S. PG 04<br />

Liebl, W. PX 30<br />

PX 19<br />

PR 17<br />

PR 30

230 AUTOREN<br />

PG 04<br />

PN 57<br />

PM 26<br />

Liebner, S. KK 04<br />

PN 19<br />

Liers, C. PH 09<br />

PH 03<br />

KE 07<br />

Liesack, W. PN 74<br />

PN 72<br />

Liesegang, H. PG 04<br />

PO 47<br />

PJ 14<br />

PX 19<br />

PR 30<br />

Lill, H. FGF 04<br />

PP 08<br />

Lill, R. FGH 01<br />

Lin, P.J. PH 28<br />

Lindell, D. PU 06<br />

Lindemann, C. PZ 26<br />

Lindenstrauß, U. KS 06<br />

Lindner, S.N. PX 04<br />

Linge, H. PP 04<br />

FGF 01<br />

Linne, U. KI 02<br />

FGF 06<br />

Linnerbauer, S. KO 02<br />

Linscheid, M.W. PZ 13<br />

Lippert, M.L. PA 44<br />

Lipps, G. PB 15<br />

Lipski, A. PO 19<br />

PO 12<br />

PB 05<br />

Lissner, U. PM 24<br />

Litzinger, S. PD 04<br />

Liu, A. KR 02<br />

Liu, S.J. KR 06<br />

PG 03<br />

Liu, Y. PN 36<br />

Löchte, S. PW 13<br />

Löckinger, A. PZ 04<br />

Lod<strong>der</strong>s, N. PO 20<br />

PP 27<br />

Loessner, M. HDID P 13<br />

Logemenn, J. PN 31<br />

Longen, S. KS 02<br />

Löper, D. PT 08<br />

Lorenzo, C. PP 24<br />

Lorenzo Fajardo, J.C. PS 41<br />

Loros, J.J. PH 01<br />

Löwe, J. PM 17<br />

Lu, Y. KU 02<br />

Lübben, M. KS 05<br />

Lubitz, W. PZ 44<br />

Lucchini, S. PU 05<br />

PU 14<br />

Luce, K. PH 12<br />

Lück, C. PJ 13<br />

Lüddeke, F. PA 18<br />

Lü<strong>der</strong>s, T. PN 52<br />

Ludwig, W. PO 27<br />

PO 26<br />

FGE 04<br />

KK 01<br />

KK 01<br />

Luecker, S. KK 03<br />

Lue<strong>der</strong>s, T. PN 64<br />

Lüer, C. PQ 03<br />

Lüke, C. PN 06<br />

Lünenschloß, A. PS 17<br />

Luo, Z.H. PH 33<br />

FGD 04<br />

Lupilova, N. PZ 26<br />

Lüttmann, D. PS 36<br />

Lvov, Y. PZ 17<br />

M Tóth, E. PN 88<br />

Maassen, N. KE 06<br />

Ma<strong>der</strong>, D. PP 37<br />

Madhusudan, S. PS 31<br />

Maerker, C. PS 32<br />

Mager, A. PA 10<br />

Mahdavi, H. PX 01<br />

Maier, B. KG 03<br />

PZ 42<br />

Maier, T. PN 85<br />

PK 01<br />

Maier, U.G. PO 10<br />

Majcherczyk, A. FGD 05<br />

Majzlan, J. PN 14<br />

Makarewicz, O. PS 60<br />

Maldener, I. PF 01<br />

PW 07<br />

Malow, M. PO 08<br />

Man<strong>der</strong>s, E. FGD 07<br />

Mangold, K.M. HDID P 03<br />

Mann, M.S. KA 02<br />

Marchfel<strong>der</strong>, A. PU 01<br />

PB 25<br />

Márialigeti, K. PN 88<br />

Marin, K. PE 05<br />

PW 02<br />

PR 22<br />

PX 28<br />

PW 10<br />

PW 21<br />

Markert, S. PJ 14<br />

Marlinghaus, L. PP 21<br />

PW 09<br />

Marrero-Coto, J. PB 19<br />

Marsh, D. PZ 30<br />

Martens-Habbena, W. PO 39<br />

Martin, E. PO 50<br />

PO 42<br />

Martinez, P. PR 27<br />

Mascaraque, V. PR 13<br />

Masepohl, B. PS 05<br />

PS 06<br />

Mashait, M. PL 01<br />

PZ 02<br />

Maskow, T. PZ 50<br />

Matera, I. PZ 36<br />

Matern, Y. PZ 29<br />

PZ 30<br />

Mathes, F. HDID 05<br />

Matsubara, K. PA 36<br />

Matura, A. PI 03<br />

Maurer, P. PZ 16<br />

Maurischat, S. PJ 17<br />

May, A. PZ 32<br />

May, F. PN 42<br />

Mayer, B. PS 42<br />

Mayer, C. PR 05<br />

PD 04<br />

PN 37<br />

Mayer, F. PN 04<br />

Mayer, M. KJ 07<br />

PN 30<br />

Mayser, P. PT 10<br />

Mazé, A. PP 30<br />

McDougald, D. PZ 45<br />

HDID 02<br />

McDowall, A. KH 03<br />

McInerney, J. FGC 03<br />

Meckenstock, R. PN 60<br />

PA 39<br />

PA 34<br />

PA 35<br />

PA 37<br />

Medger, A. PV 03<br />

Medi, B. PX 03<br />

Meffert, A. PX 32<br />

PX 34<br />

Mehlich, J. PZ 42<br />

Mehlig, L. PN 65<br />

PO 36<br />

Mehner, D. PW 14<br />

Meier, J. PZ 55<br />

Meinhardt, F. FGH 04<br />

PZ 10<br />

PY 04<br />

Meisinger, C. H 10<br />

Melis, A. PQ 07<br />

Mengel, C. FGD 01<br />

Menzel, K.D. KD 05<br />

Merchant, S. H 08<br />

Mercker, M. KD 01<br />

Merlin, C. HDID 06<br />

Mertins, S. H 01<br />

Mesecke, N. KS 02<br />

Metz, S. KO 05<br />

Metzger, R. PJ 18<br />

Meyer, A.H. PD 06<br />

Meyer, B. FGH 02<br />

PS 47<br />

Meyer, C. PB 02<br />

PB 01<br />

KJ 04<br />

Meyer, E. FGD 01<br />

PJ 04<br />

Meyer, H.E. KU 07<br />

Meyer, T.F. PP 09<br />

Meyer, V. PH 32<br />

Meyerdierks, A. KJ 06<br />

Meyer-Klaucke, W. PQ 04<br />

Mezule, L. HDID P 15<br />

Michaela, B. PN 60<br />

Michaelis, W. PN 54<br />

Michalzik, M. PZ 53<br />

Michel, K.P. KC 01<br />

Michels, I. KU 07<br />

Michie, K. PM 17<br />

Mientus, M. PN 57<br />

Mika, F. PU 13<br />

Mikkat, S. KF 02<br />

Milbredt, D. PT 05<br />

Millat, T. PV 01<br />

Miltner, A. PH 07<br />

Mindthoff, S. PW 17<br />

Mirgalieva, R. PW 15<br />

Mirzakhan, L. PO 40<br />

Mirzakhan, Z. PO 40<br />

PI 04<br />

Mirzanamadi, F. PI 04<br />

Mishra, A. PE 06<br />

Mitschke, J. PU 07<br />

PU 06<br />

Mittag, M. KC 06<br />

Mobaiyen, H. PP 01<br />

Moeker, N. KS 04<br />

Moell, A. PM 05<br />

Moeller, R. PR 29<br />

PZ 57<br />

mohabati mobarez, A. PM 01<br />

Mohammad Zamani, G. PZ 07<br />

PX 03<br />

Mohammadzamani, G. PX 01<br />

Mohrbach, T. PX 28<br />

PW 21<br />

Moissl-Eichinger, C. KK 05<br />

Mojtabavi, M. PP 03<br />

Möker, N. PS 43<br />

Moldt, J. PS 39<br />

Mölleken, K. PM 14<br />

Möller, I. PN 42<br />

Molzahn, L. PH 02<br />

Mondschein, A. PI 03<br />

Monk, I.R. PP 14<br />

Montijn, R. HDID 09<br />

Moore, E.R.B. FGE 03<br />

Moore, Z. HDID 02<br />

Moosavian, M. PP 02<br />

Moosavi-Nejad, S.Z. PX 46<br />

Morbach, S. PS 40<br />

PS 43<br />

PW 06<br />

KS 04<br />

Morbitzer, D. PM 08<br />

Mori, M. PB 04<br />

Moritz, F. PH 09<br />

PH 03<br />

Moritz, M.M. HDID P 06<br />

Moritz, P. PB 24<br />

Morozova, D. PZ 37<br />

Moser, J. PP 24<br />

PQ 06<br />

Moslemi, E. PZ 38<br />

Mueller, V. PN 04<br />

Muhl, D. PS 58<br />

Muller, Y. PS 58<br />

Müller, A. PS 05<br />

KH 03<br />

Müller, B.K. PX 11<br />

Müller, C. PR 27<br />

Müller, D. PB 02<br />

Müller, H. PO 05<br />

Müller, J. KR 04<br />

PA 06<br />

Müller, M. PS 14<br />

Müller, N. PA 02<br />

Müller, S.I. PJ 11<br />

Müller, S. HDID 04<br />

PN 65<br />

Müller, V. PS 47<br />

PR 25<br />

PA 42<br />

Müller, V.S. PP 09<br />

Mullineaux, C. KM 03<br />

Müllner, M. PS 25<br />

Munch, J.C. PN 24<br />

PN 03<br />

PZ 03<br />

Musat, F. PA 33<br />

PA 38<br />

Musiol, E.M. PT 07<br />

Muth, G. PM 09<br />

FGA 04<br />

PM 10<br />

Mutter, A. PB 03<br />

Mutzel, R. PF 03<br />

Mygind, P.H. PC 02<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Nachtigall, J. PP 45<br />

Nacke, H. PO 41<br />

Nagel, K. PJ 07<br />

Nagel, M. PP 28<br />

Nägele, V. PN 48<br />

PO 07<br />

Nagl, M. PZ 04<br />

Nahaie, M.R. PP 01<br />

Nähter, A. PN 48<br />

Nait-Abdallah, J. PP 30<br />

Nandy, S. PY 03<br />

PY 05<br />

Nannapaneni, P. PS 52<br />

Narberhaus, F. PM 06<br />

KQ 05<br />

PU 02<br />

PJ 02<br />

Näther, A. PO 07<br />

PR 10<br />

Natter, H. PX 22<br />

Naumann, B. PQ 09<br />

Navarro-González, M. FGD 05<br />

Nechitaylo, T. PV 08<br />

Nejad sattari, T. PZ 38<br />

Nentwich, M. PS 57<br />

Nettekoven, J.A. PW 21<br />

Neu, T. PZ 50<br />

Neubauer, O. PW 11<br />

Neubauer, P. FGB 04<br />

Neubauer, S. PS 60<br />

Neufeld, C. PW 15<br />

Neuhaus, A. PW 15<br />

Neumann, A. PH 27<br />

Neumann, C. PJ 16<br />

PP 15<br />

Neumann, L. PO 04<br />

PN 12<br />

Neumann, S. PP 33<br />

Neupert, J. KC 07<br />

Neve, S. PC 02<br />

Nevo, R. H 11<br />

Nghi, D.H. PH 09<br />

Ngugi K., D. PN 38<br />

Nickelsen, J. PE 04<br />

KC 03<br />

Nicklisch, S. PW 06<br />

Niebler, M. PG 07<br />

PO 48<br />

Nie<strong>der</strong>weis, M. PL 03<br />

Nieland, S. FGD 06<br />

KP 04<br />

Nielsen, A.K. PC 02<br />

Nies, D.H. PW 03<br />

PW 05<br />

Niess, J.H. KG 06<br />

Niewerth, H. PX 10<br />

Nijenhuis, I. PR 27<br />

Nimtz, M. PP 24<br />

Nitsche-Schmitz, D.P. PP 04<br />

PP 06<br />

PP 07<br />

FGF 01<br />

Niyogi, K.K. PQ 09<br />

Noack, S. PV 01<br />

Nocker, A. HDID 08<br />

Noël, N. PZ 24<br />

Nold, N. PS 21<br />

Noll, M. PO 08<br />

PO 15<br />

PN 50<br />

Nolting, N. PH 08<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Noohi, A.S. PZ 12<br />

PZ 20<br />

Novak, J. PE 05<br />

Nowaczyk, M. KM 02<br />

Nowrousian, M. PH 01<br />

KH 02<br />

PH 17<br />

PH 14<br />

Nuss, A.M. PS 22<br />

Obrdlik, P. PW 20<br />

Obst, U. PM 04<br />

HDID P 12<br />

HDID P 02<br />

PN 51<br />

PN 21<br />

HDID 03<br />

Ochrombel, I. PR 22<br />

OConnell, S. PH 25<br />

Oelgeschläger, E. PA 31<br />

Oeljeklaus, S. KU 07<br />

Oeser, T. PX 27<br />

Oesterhelt, D. PZ 43<br />

Off, S. PN 29<br />

Ohad, I. H 11<br />

Okonkwo, I.S. PT 09<br />

PZ 51<br />

Okoye, O. PT 09<br />

Oldiges, M. PA 16<br />

Opitz, D. PZ 42<br />

KG 03<br />

Oppermann, B. PN 54<br />

Orcutt, B. PO 25<br />

Ortel, I. PH 24<br />

Osadnik, H. PR 23<br />

Osiewacz, H.D. KD 02<br />

KE 01<br />

PH 12<br />

PH 10<br />

Ostendorf, E. PQ 09<br />

Ott, M. KI 01<br />

PS 42<br />

Ott, V. PS 40<br />

Otto, A. KN 05<br />

Otzen, C. FGF 05<br />

Overmann, J. PN 48<br />

KJ 07<br />

KK 06<br />

PQ 05<br />

PO 31<br />

PO 07<br />

PN 30<br />

Öztürk, B. PN 08<br />

Pachulec, E. KL 04<br />

Pag, U. PC 02<br />

Pagès, J.M. PZ 47<br />

Palese, L.L. PN 69<br />

Palinska, K. PE 08<br />

Palmer, K. PN 22<br />

Panakova, M. KE 06<br />

Pané-Farré, J. PJ 21<br />

Pang, K.L. PH 33<br />

Panhorst, M. PS 04<br />

Pape, M. PA 17<br />

Papenfort, K. PU 05<br />

PU 14<br />

Parey, K. KM 05<br />

PA 23<br />

Parivar, K. PZ 38<br />

Parkes, R.J. HDID 05<br />

Parthasarathy, A. PA 05<br />

Passoth, V. PH 20<br />

PH 21<br />

Pasztor, L. PP 22<br />

Patallo, E.P. PT 04<br />

PT 05<br />

Paukner, A. PS 30<br />

Paulick, A. PM 16<br />

Pawlak, V. PV 07<br />

Pawlik, J. PJ 25<br />

Pechlivanis, M. PW 16<br />

Pechter, K. FGG 02<br />

Pecoraro, V. PM 08<br />

Peers, G. PQ 09<br />

Pellegrini, M. H 08<br />

Pelletier, E. PZ 11<br />

Peplies, J. KK 01<br />

FGE 04<br />

Pereira, I.A.C. PR 09<br />

Perera, J. PR 13<br />

Pérez-de-Mora, A. PO 18<br />

Perner, M. PN 11<br />

Persicke, M. PR 22<br />

Pesavento, C. PS 51<br />

Peschel, A. PP 37<br />

KL 01<br />

KL 02<br />

Peters, G. PP 20<br />

PP 19<br />

Petersen, J. PN 11<br />

Petersen, N. PO 48<br />

PG 07<br />

Petri, A. PB 12<br />

Petroutsos, D. KF 05<br />

Petzold, M. PN 65<br />

Peuser, V. PS 09<br />

Peykov, S. PR 10<br />

Pfanner, N. H 10<br />

Pfannes, K. PN 60<br />

Pfeiffer, D. PZ 33<br />

Pfeiffer, E.M. PZ 18<br />

Pfeiffer, F. PZ 43<br />

Pfeiffer, P. PZ 31<br />

Pflüger-Grau, K. PS 11<br />

Pförtner, H. PS 15<br />

PM 26<br />

Phe, M.H. HDID 06<br />

Philipp, B. PX 25<br />

Philipps, G. KC 04<br />

PA 43<br />

Piechulla, B. KP 03<br />

Piehl, S. PZ 32<br />

Piekarski, T. PP 24<br />

PZ 49<br />

Pierik, A.J. PA 08<br />

Pietack, N. PS 01<br />

PR 01<br />

Pilak, O. PQ 04<br />

Pilloni, G. PN 64<br />

Pimpirev, C. PO 12<br />

Pinkner, J. KJ 03<br />

Pinna, A. PZ 04<br />

Pinnow, N. PX 26<br />

Pinske, C. PA 15<br />

Piotrowski, M. PT 08<br />

Pirch, T. PS 42<br />

PS 03<br />

Pisa, K. PN 04<br />

Pitz, M. PX 39<br />

Plagens, A. PB 20<br />

Platta, H.W. PV 07<br />

AUTOREN 231<br />

PV 06<br />

KS 03<br />

Plitzko, J. PM 15<br />

PL 03<br />

Podkaminski, D. PU 05<br />

Poetsch, A. PR 08<br />

PS 17<br />

KR 06<br />

PR 22<br />

PG 03<br />

PS 23<br />

Pöggeler, S. KE 02<br />

PH 06<br />

PH 04<br />

PH 17<br />

PH 08<br />

PH 01<br />

KU 01<br />

PH 29<br />

Pohl, M. PL 04<br />

Pohlmann, A. KN 05<br />

Polacsek, T.K. HDID P 10<br />

Pollice, A. PN 69<br />

Pöllinger, C. PM 14<br />

Pollok, K. PN 70<br />

Poncet, S. PP 30<br />

Pondelikova - Smejkalova, H. PZ 27<br />

Poolman, B. PW 13<br />

Popiol, M. PN 86<br />

Popp, F. KA 04<br />

Popper, L. PX 18<br />

Poraj-Kobielska, M. PX 06<br />

PX 07<br />

Porta, M.A. PJ 13<br />

Pottkämper, J. PI 02<br />

Potzkei, J. KH 05<br />

Poxleitner, G. PW 04<br />

Prange, A. PZ 17<br />

Pratscher, J. PO 28<br />

Praveen Kumar, S. PS 52<br />

Preising, J. KG 06<br />

Preissner, K.T. PJ 08<br />

Prestele, M. KI 01<br />

Preuß, G. HDID P 01<br />

PN 10<br />

Pritzkow, W. PN 50<br />

Probian, C. PN 15<br />

Proctor, R.A. PP 20<br />

Pross, E. PS 27<br />

Prüße, E. KK 01<br />

Pruteanu, M. PU 13<br />

Puls, M. KH 05<br />

Puniya, A.K. PZ 41<br />

Pust, J. PO 30<br />

HDID P 07<br />

Qi, S.W. PG 03<br />

Quast, C. KK 01<br />

Quentmeier, A. PR 16<br />

PR 12<br />

Quitschau, M. PZ 48<br />

KU 06<br />

Rabenau, A. PP 13<br />

Rabus, R. PA 28<br />

PA 27<br />

Rachel, R. KI 06<br />

PB 02<br />

PB 07<br />

KB 02<br />

PB 03<br />

PB 11

232 AUTOREN<br />

KB 06<br />

PB 01<br />

PN 04<br />

KJ 04<br />

Rachinger, M. PX 19<br />

Rademacher, C. PS 06<br />

Radespiel, T. PX 28<br />

Rafiei, A. PA 26<br />

Raine, A. KQ 04<br />

Raja Venkatesh, G. PS 30<br />

Rajkumari, R. PP 06<br />

Ramos-Vera, H. PB 13<br />

PB 12<br />

Rangrez, A. PP 16<br />

Rapp, E. PN 69<br />

PN 84<br />

Rappl, K. PO 27<br />

PO 26<br />

Rasch, G. KC 01<br />

Rasche, D. PP 27<br />

Raschke, D. PY 02<br />

Rastegar Lari, A. PZ 01<br />

Rath, F. PH 32<br />

Rather, L. PR 13<br />

Rathmann, C. HDID P 07<br />

PO 30<br />

Rattei, T. KK 03<br />

Rau, J.E. PC 04<br />

Raulf, F. PA 35<br />

Rav-Hon, O. H 11<br />

Ravn, B. PC 02<br />

Ravoo, B.J. PZ 42<br />

Raza, W. PM 22<br />

Razavi, M.R. PZ 20<br />

PZ 12<br />

Regli, A. PZ 47<br />

Rehling, P. KS 01<br />

Rehm, N. KO 06<br />

Reich, Z. H 11<br />

Reichelt, R. PX 37<br />

PX 13<br />

Reichenbach, B. PU 03<br />

Reichl, U. PN 25<br />

PN 69<br />

PN 84<br />

Reichmann, M. PZ 03<br />

Reignier, J. PU 11<br />

Reihlen, P. PS 43<br />

Reimold, C. KI 02<br />

Reinartz, B. KU 07<br />

Reinhardt, R. PZ 13<br />

PG 07<br />

PO 48<br />

Reinhart, F. PS 25<br />

Reinhold, A. PA 04<br />

Reinhold, B. PS 14<br />

Reißmann, S. FGF 01<br />

PP 07<br />

PP 06<br />

PP 04<br />

Reitner, J. PN 82<br />

Reitz, G. PZ 57<br />

KB 06<br />

PR 29<br />

Rengstl, B. KC 03<br />

Resch, A. PP 22<br />

Rettberg, P. KB 06<br />

PR 29<br />

PZ 57<br />

Reuss, M. PV 01<br />

Reuther, J. PR 15<br />

PS 57<br />

Rexroth, S. PQ 03<br />

KF 04<br />

KM 03<br />

Rice, S. PZ 45<br />

Richnow, H.H. PA 40<br />

KJ 05<br />

PN 44<br />

Richter, M. FGE 04<br />

Richter, S. PN 63<br />

Rickert, E. PB 09<br />

Riebe, O. KA 02<br />

PS 29<br />

Riedel, C.U. PP 14<br />

PJ 05<br />

KG 06<br />

Riedel, T. PN 64<br />

Riedele, C. PN 25<br />

Rie<strong>der</strong>, R. PU 12<br />

Riemer, J. KS 02<br />

Rieser, G. PZ 28<br />

Riethausen, J. PZ 48<br />

Riethmueller, V. HDID P 05<br />

Ringelberg, C. PH 01<br />

Rittmann, D. PX 04<br />

Rodepeter, S. KL 03<br />

Rodloff, A.C. PP 07<br />

PP 06<br />

Roe<strong>der</strong>, R. PN 58<br />

Roggentin, P. PZ 05<br />

Roggentin, T. PZ 05<br />

Rögner, M. KM 02<br />

KF 04<br />

KM 03<br />

PR 08<br />

PQ 03<br />

PG 03<br />

Rohde, M. FGF 01<br />

Rohwer<strong>der</strong>, T. PZ 23<br />

PZ 24<br />

KU 04<br />

HDID P 14<br />

Romann, E. PN 30<br />

Römling, U. KJ 02<br />

Rönner-Holm, S. PZ 05<br />

Roschanski, N. PZ 13<br />

Rosenau, F. PP 29<br />

PV 05<br />

PX 35<br />

PR 24<br />

Rosenstiel, P. PJ 18<br />

Röske, I. PN 35<br />

HDID P 08<br />

PO 36<br />

PN 65<br />

Röske, K. PO 36<br />

HDID P 08<br />

Rosselló-Móra, R. FGE 04<br />

Roth, A. PH 34<br />

Roth, K. PH 22<br />

Roth, T. FGA 04<br />

PM 10<br />

Rothe, F.M. FGG 03<br />

Rother, D. PR 16<br />

Rother, M. PU 04<br />

PA 31<br />

Rottensteiner, H. PW 16<br />

Rücker, O.L. PQ 05<br />

Rückert, C. PR 22<br />

Rucktäschel, R. PW 16<br />

Rudat, J. PD 01<br />

PD 03<br />

PD 02<br />

Rudigier, Y. PH 22<br />

Rudolf, C. PA 16<br />

Ruedrich, J. PN 78<br />

Ruehl, J. KT 02<br />

Rühle, T. PQ 07<br />

Ruiz, J. PS 37<br />

Rullkötter, J. PN 31<br />

Rumpf, A. HDID P 03<br />

Rupprecht, J. KF 06<br />

Rychlik, N. PB 14<br />

PB 16<br />

Saeger, M. KM 04<br />

Saffian, D. PV 07<br />

Sahl, H.G. PC 05<br />

FGA 01<br />

PC 02<br />

PM 14<br />

Sahm, H. PQ 01<br />

Sahm, K. PN 45<br />

PN 47<br />

Said, N. PU 12<br />

PU 14<br />

Sakai, K. PB 04<br />

Sakinc, T. PW 09<br />

PP 21<br />

PP 33<br />

Saleh, M. PW 23<br />

Salgado-Pabón, W. KL 04<br />

Salman, V. PO 22<br />

Sand, W. PZ 24<br />

PZ 25<br />

KU 04<br />

PZ 23<br />

HDID P 14<br />

San<strong>der</strong>, G. PP 20<br />

San<strong>der</strong>, J. KM 02<br />

San<strong>der</strong>s, T. PZ 18<br />

Sanganas, O. KA 05<br />

Sass, H. HDID 05<br />

Sass, P. PW 01<br />

Sass, V. PC 02<br />

Sastalla, I. PP 04<br />

Satke, K. PN 27<br />

Sattler, C. PU 04<br />

Sattler, M. PW 15<br />

Sauer, M. PP 34<br />

Sauer, U. PS 11<br />

PB 23<br />

Saum, R. PS 47<br />

Saum, S. PR 25<br />

Savitsky, S. PN 45<br />

Sawers, G. PZ 09<br />

KA 01<br />

PP 12<br />

PA 15<br />

Say, R.F. PR 28<br />

Schäberle, T. PC 07<br />

Schachschal, S. PI 03<br />

Schacht, M. PE 08<br />

Schachtschabel, D. KD 05<br />

Schäfer, B. PH 21<br />

FGH 03<br />

Schäfer, J. PP 27<br />

PP 36<br />

Schäfer, K. KO 04<br />

Schäfers, C. PH 05<br />

Schäkermann, M. PM 06<br />

Schär, J. H 01<br />

Scharf, D.H. PH 15<br />

Schaule, G. HDID P 03<br />

Schauss, K. PN 03<br />

PN 07<br />

Schäwe, R. PN 71<br />

Scheel, M. PS 29<br />

Scheer, M. PS 41<br />

KO 01<br />

Scheerer, P. KO 03<br />

Scheffer, B. PZ 43<br />

Scheibe, A. PN 55<br />

Scheibner, K. PH 30<br />

PX 06<br />

Scheibner, O. KE 04<br />

Scherer, J. PW 03<br />

PW 05<br />

Scherer, P. PO 04<br />

PN 12<br />

PN 27<br />

Scherlach, K. KP 06<br />

KR 05<br />

Scheuffele, S. PU 04<br />

Schiel, B. PS 21<br />

PS 10<br />

Schier, N. PH 11<br />

Schierack, P. PP 35<br />

Schilhabel, A. PA 08<br />

Schilhabel, M. PX 38<br />

Schillhabel, M. PB 16<br />

Schilling, J. PP 43<br />

FGF 02<br />

Schimek, C. PH 22<br />

KE 04<br />

Schink, B. KA 06<br />

PN 09<br />

PA 02<br />

PA 03<br />

PN 32<br />

Schipper, C. KU 06<br />

Schippers, A. PO 13<br />

PN 46<br />

PO 32<br />

PO 14<br />

PO 37<br />

Schirawski, J. FGF 06<br />

FGD 01<br />

PJ 04<br />

PT 10<br />

Schirrmann, T. PZ 54<br />

Schirrmeister, J. HDID P 08<br />

Schiwon, K. PW 22<br />

KH 06<br />

Schlag, M. PP 31<br />

Schlebusch, M. PE 02<br />

Schlee, D. PV 06<br />

Schlegel, K. PS 47<br />

Schleheck, D. PZ 45<br />

Schlei<strong>der</strong>, J. PR 05<br />

Schleifer, K.H. PO 27<br />

PO 26<br />

FGE 04<br />

Schleiff, E. PW 07<br />

Schlicht, M. KD 05<br />

Schlicker, C. PS 02<br />

Schliebs, W. PW 15<br />

KS 03<br />

PP 32<br />

KU 07<br />

Schlink, F. PE 01<br />

Schlömann, M. PN 33<br />

PX 41<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

PO 21<br />

Schlömer, S. PO 13<br />

Schloter, M. PN 24<br />

PZ 03<br />

PN 07<br />

PB 05<br />

PO 18<br />

PN 03<br />

Schluesener, D. KR 06<br />

Schlums, H. PX 11<br />

Schlüsener, D. PS 17<br />

Schmalenberger, A. PO 15<br />

Schmaljohann, R. PO 38<br />

Schmalz, G. PB 03<br />

Schmeinck, A. KE 05<br />

Schmid, A. FGD 03<br />

PX 14<br />

KT 02<br />

PX 36<br />

Schmid, F. PO 06<br />

Schmidt, A. PZ 21<br />

Schmidt, F. PN 44<br />

PM 24<br />

KJ 05<br />

Schmidt, H. PN 02<br />

Schmidt, I. PN 28<br />

Schmidt, M. PN 61<br />

Schmidt, S. PA 42<br />

Schmitt, K. PE 06<br />

Schmitt, S. PJ 12<br />

Schmitz, H.P. PH 19<br />

PH 02<br />

Schmitz-Streit, R. PX 26<br />

PB 22<br />

PJ 18<br />

PS 38<br />

PU 09<br />

Schneemann, I. PJ 07<br />

Schnei<strong>der</strong>, E. PW 19<br />

Schnei<strong>der</strong>, J. PX 42<br />

Schnei<strong>der</strong>, T. FGA 01<br />

PM 14<br />

PC 02<br />

Schnell, S. FGH 05<br />

Schnetz, K. PS 31<br />

PS 33<br />

PS 30<br />

Schnitzlein, K. KT 07<br />

Schobert, M. PS 49<br />

PS 41<br />

KO 01<br />

Scholl-Bürgi, S. PZ 04<br />

Scholtz, S. PX 16<br />

Scholz, I. PU 07<br />

Schönheit, P. PB 23<br />

Schopf, S. PB 07<br />

Schott, J. PA 03<br />

Schottkowski, M. KC 03<br />

Schramm, A. PN 83<br />

PF 04<br />

Schreiber, F. PZ 11<br />

PA 27<br />

Schreiber, K. KO 01<br />

Schreiber, L. KJ 06<br />

Schroeckh, V. KP 06<br />

Schroe<strong>der</strong>, K. PJ 16<br />

Schröter, G. KS 05<br />

Schubert, B. PS 41<br />

Schubert, K. KJ 07<br />

Schubert, T. PZ 52<br />

PA 04<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Schuenemann, V.J. KI 03<br />

Schuffenhauer, G. PZ 28<br />

Schühle, K. PA 11<br />

Schuldes, J. PN 57<br />

PG 02<br />

Schüler, D. PZ 40<br />

PW 04<br />

KK 06<br />

PM 18<br />

PS 24<br />

PG 07<br />

PM 15<br />

PO 48<br />

Schüller, N. PW 15<br />

Schulte, L. PU 05<br />

Schulz, A. PR 07<br />

Schulz, C. PZ 46<br />

Schulz, F. PN 80<br />

Schulz, S. PN 24<br />

PO 18<br />

Schulz-Vogt, H. PO 22<br />

Schulz-Vogt, H.N. PR 20<br />

PR 19<br />

Schumacher, J. PP 44<br />

Schumacher, M. PP 17<br />

Schumann, P. PK 01<br />

Schuppler, M. HDID P 13<br />

Schuren, F.H. HDID 09<br />

Schürmann, J. KP 06<br />

Schürmann, M. PX 44<br />

Schuster, N. PB 14<br />

Schuster, S. PZ 43<br />

Schütz, B. PA 09<br />

Schütze, E. PZ 21<br />

Schütze, I. PN 37<br />

Schuurman-Wolters, G. PW 13<br />

Schwaiger, A. PM 02<br />

Schwartz, D. PC 03<br />

Schwartz, T. PM 04<br />

HDID P 12<br />

HDID P 02<br />

PN 21<br />

PN 51<br />

Schwarz, C. PE 04<br />

Schwarz, J. PS 22<br />

Schwarz, K. PA 41<br />

Schwarz, P. PS 20<br />

Schwarz, R. H 11<br />

Schwarz, W.H. PA 32<br />

Schwarzer, M. PR 17<br />

Schwe<strong>der</strong>, T. PG 06<br />

PJ 14<br />

Schweiger, P. PX 29<br />

Schwerk, J. PX 11<br />

Schwibbert, K. PR 03<br />

Sczodrok, S.D. PU 02<br />

Sebastian, P. PZ 31<br />

Seebah, S. PJ 20<br />

Seggewiß, J. PP 20<br />

Segmüller, N. KD 03<br />

Seibold, G.M. PX 15<br />

PR 26<br />

Seifert, J. PO 21<br />

PN 33<br />

PN 39<br />

Seifert, R. PO 29<br />

PN 54<br />

PN 11<br />

Seiler, S. KE 03<br />

Seip, B. PX 23<br />

Seitz, H. PR 03<br />

Seitz, O. PZ 14<br />

Seitz, S. KC 06<br />

Selesi, D. PA 37<br />

PA 39<br />

Selim, S. PZ 02<br />

PL 01<br />

Selimovic, D. PJ 08<br />

Selmer, T. PA 13<br />

Selvin, J. KP 05<br />

Selzer, M. PU 04<br />

Serci, S. PP 29<br />

Sergi, C. PZ 04<br />

Serio, A.W. FGG 02<br />

Sevvana, M. PS 58<br />

Seyfarth, K. PB 21<br />

Shahhosseiny, M.H. PZ 38<br />

Shao, L. KC 03<br />

Sharma, C. PU 06<br />

PU 09<br />

KQ 02<br />

PU 07<br />

PU 11<br />

Shelest, E. PP 18<br />

Shen, Q. PM 22<br />

Shima, S. PQ 04<br />

Shimoni, E. H 11<br />

Shirai, Y. PB 04<br />

Shouche, Y. PP 16<br />

Shrestha, P.M. PN 74<br />

PN 72<br />

Shutinoski, B. FGF 02<br />

Sibbald, M.J.J. PP 39<br />

Sichau, K. PQ 05<br />

Siebers, B. PB 19<br />

PA 36<br />

KR 03<br />

PB 18<br />

Siedler, F. PZ 43<br />

KI 06<br />

Siegert, M. PO 32<br />

PN 54<br />

Siegl, A. PN 73<br />

Siemens, H. PO 31<br />

Sietmann, R. PG 06<br />

Sievert, S. PB 01<br />

PJ 14<br />

PN 16<br />

Silakov, A. PZ 44<br />

Simon, B. PW 15<br />

Simon, E. KC 02<br />

Simon, J. PA 27<br />

KA 03<br />

PA 10<br />

PA 29<br />

Simon, L. PM 20<br />

Simon, M. PO 47<br />

Sitte, J. PN 70<br />

Sittka, A. PU 11<br />

Sizova, I. KU 02<br />

Skarstad, K. KI 05<br />

Skovhus, T. PN 83<br />

Slotboom, D.J. PW 11<br />

Smirnova, I. PX 18<br />

Smith, D.M. PA 05<br />

Sobke, A.C.S. PJ 08<br />

Soboh, B. KA 01<br />

Soh Bejeng Ndikung, B. PY 02<br />

Sommer, E. PM 03<br />

Sonenshein, A.L. FGG 02<br />

Soppa, J. PM 08<br />

PU 01<br />

AUTOREN 233<br />

PB 25<br />

PB 23<br />

PB 17<br />

Sørensen, K. PN 83<br />

Sorger-Herrmann, U. PS 04<br />

Sorgo, A. FGD 07<br />

Sosinska, G. FGD 07<br />

Sourjik, V. PM 03<br />

Spaete, K. PS 40<br />

Spall, S.K. KI 03<br />

Specht, M. FGF 03<br />

Speer, F. PH 21<br />

Speth, V. FGF 03<br />

Spieck, E. PN 29<br />

PO 19<br />

PO 23<br />

PZ 18<br />

PB 14<br />

PB 16<br />

Spielvogel, A. PH 32<br />

Sprenger, G. PZ 56<br />

KT 06<br />

PX 43<br />

Spröer, C. PK 01<br />

Srivastava, V. PJ 01<br />

Stadler, P. PU 11<br />

Stagge, S. PV 04<br />

Stagni, M.S. PQ 04<br />

Stahl, D.A. PO 39<br />

Stahl, U. PH 32<br />

Stahlhut, G. PR 10<br />

Stahmann, K.P. FGD 06<br />

KT 07<br />

KP 04<br />

Stammen, S. PX 11<br />

Standfest, S. KB 05<br />

PB 26<br />

Standfest, T. PS 21<br />

Stanley, W. KS 03<br />

Stan-Lotter, H. HDID P 10<br />

Staron, P. PW 07<br />

Stary, E. PS 56<br />

Staufenberger, T. PO 43<br />

Steen, A. PS 49<br />

KO 01<br />

Stefan, F. PH 20<br />

Steffen, W. PQ 02<br />

Steglich, C. PU 07<br />

PU 06<br />

Stegmann, E. PC 07<br />

Stehle, T. PP 31<br />

Steil, L. PS 52<br />

PM 24<br />

PJ 21<br />

Steimer, L. PZ 36<br />

Stein, A. KM 06<br />

Stein, M. PX 34<br />

PX 23<br />

Steinbrenner, C. PN 35<br />

Steinbüchel, A. PX 12<br />

PX 37<br />

PX 13<br />

PX 40<br />

PX 44<br />

Steiner, R. PZ 22<br />

Steinhoff, H.J. PW 13<br />

PW 06<br />

Steinle, A. PX 12<br />

PX 13<br />

Steinsiek, S. PV 04<br />

Steinwand, M. PZ 54

234 AUTOREN<br />

Stephan, C. KU 07<br />

Sterr, Y. PN 02<br />

Steuber, J. KM 06<br />

PQ 02<br />

KN 01<br />

Stief, P. PO 48<br />

Stieglmeier, M. KK 05<br />

Stirnberg, M. PE 05<br />

Stock, T. PU 04<br />

Stockdreher, Y. PR 06<br />

Stoepel, J. PG 05<br />

Stoll, R. H 01<br />

Stolle, P. HDID P 07<br />

PO 30<br />

PC 01<br />

Stolz, A. PZ 36<br />

Stolz, T. PP 25<br />

Storbeck, S. PA 14<br />

PA 06<br />

PP 24<br />

Strahler, J.R. KN 06<br />

Stratmann, T. PS 33<br />

Straub, J. PU 01<br />

Straube, E. PJ 08<br />

Strauch, E. PZ 13<br />

Streit, W. KU 06<br />

PB 16<br />

PI 02<br />

PZ 48<br />

PN 11<br />

KT 05<br />

PX 38<br />

Striebeck, P. KM 02<br />

Stripp, S. KA 05<br />

Strous, M. PZ 11<br />

Struck, J.M. PN 16<br />

Stück, A. PS 08<br />

Studenik, S. PA 08<br />

PA 20<br />

Stührmann, T. KK 04<br />

Stülke, J. PS 15<br />

KU 05<br />

FGG 03<br />

PS 01<br />

PR 01<br />

Stupperich, E. PS 34<br />

PS 35<br />

Sturm, G. PZ 19<br />

Su, J. PS 02<br />

Suess, B. PU 10<br />

Sulaiman, A. PB 04<br />

SulfoSYS consortium. KR 03<br />

Süling, J. PO 38<br />

PO 34<br />

Sultana, M. PO 21<br />

Sun<strong>der</strong>mann, L. PW 13<br />

Sunny-Roberts, E.O. PZ 39<br />

Süß, J. HDID P 12<br />

Süssmuth, R. PP 45<br />

Suvekbala, V. PX 25<br />

Svensson, L. FGE 03<br />

Syldatk, C. PD 02<br />

PH 27<br />

PD 01<br />

PD 03<br />

Symanowski, F. PX 26<br />

Szabados, F. KL 03<br />

Szewzyk, U. PN 58<br />

HDID 04<br />

PN 86<br />

Tabatabaei, M. PB 04<br />

Taha, M.K. PP 30<br />

Tajabadi Ebrahimi, M. PZ 07<br />

PZ 06<br />

talebi bezmin abadi, A. PM 01<br />

Tandeau de Marsac, N. PM 25<br />

KF 02<br />

Tank, M. PO 33<br />

Tarne, P. PN 58<br />

Taubert, M. KJ 05<br />

PN 44<br />

Taupp, M. PG 05<br />

Tausendschön, M. PR 25<br />

Tavanaii Sani, A. PP 03<br />

Taviani, M. PN 82<br />

Taylor, M. KK 03<br />

PJ 12<br />

Tedin, K. PJ 17<br />

Teeling, H. PN 08<br />

FGE 01<br />

Teichert, B. PO 32<br />

Temme, N. PH 31<br />

ten Brink, F. KA 06<br />

ter Haseborg, E. PN 13<br />

Terashima, M. KF 05<br />

Tetsch, L. FGG 04<br />

Teufel, R. PR 13<br />

Teutsch<strong>bei</strong>n, J. PP 18<br />

Thanbichler, M. PM 17<br />

PM 05<br />

Thauer, R.K. KM 05<br />

PQ 04<br />

Theilmann, W. PM 26<br />

Thelen, K. HDID P 11<br />

Thewes, S. PF 03<br />

Thiel, V. PJ 07<br />

PO 33<br />

Thiele-Bruhn, S. PN 07<br />

Thiemer, B. PP 12<br />

Tholen, S. KU 05<br />

Thomas, F. PA 07<br />

KB 01<br />

Thomm, M. PB 03<br />

KB 03<br />

Thoms, S. PW 16<br />

PV 07<br />

Thomsen, J. PU 09<br />

Thöny-Meyer, L. KT 03<br />

Thormann, K. KJ 01<br />

PB 10<br />

PS 44<br />

PM 16<br />

PN 41<br />

PJ 02<br />

Thorsten, B. PO 47<br />

Thronicker, O. PN 86<br />

Thullner, M. PN 68<br />

Thum, O. PX 05<br />

Thürmer, A. PJ 14<br />

Thyssen, C. PZ 23<br />

Tichy, E. PS 56<br />

Tielen, P. PZ 49<br />

PP 34<br />

Tietze, M. PE 06<br />

Timmis, K.N. PV 08<br />

Tindall, B.J. PM 12<br />

PP 24<br />

Tischler, D. PX 41<br />

Tjaden, B. PU 01<br />

PB 20<br />

PB 24<br />

Todorova, K. PZ 16<br />

Toetzke, F. PN 26<br />

Tönniges, A. PN 71<br />

Török, G. PN 88<br />

Totsche, K.U. PA 34<br />

Trachtmann, N. PZ 56<br />

PX 43<br />

Tran, Q.T. PZ 47<br />

Tran-van, T. PH 16<br />

Trautwein, K. PA 28<br />

Treml, B. PZ 04<br />

Treude, T. PO 25<br />

Triller, W. PN 35<br />

Trötschel, C. KR 06<br />

PR 08<br />

PS 23<br />

PS 17<br />

PR 22<br />

PW 21<br />

Truscott, K.N. KI 03<br />

Tudzynski, B. KE 05<br />

PP 44<br />

FGD 02<br />

Tudzynski, P. PH 18<br />

PH 13<br />

KD 03<br />

PH 31<br />

KP 01<br />

Türck, M. PS 48<br />

Uebe, R. PS 24<br />

PW 04<br />

Ulbricht, K. PN 79<br />

Uliczka, F. PP 25<br />

Ullmann, J. PD 05<br />

Ullmann, K. PA 19<br />

Ullrich, M. PJ 19<br />

PM 23<br />

PJ 20<br />

PZ 47<br />

PJ 24<br />

Ullrich, M.S. PJ 22<br />

Ullrich, R. PX 06<br />

PH 03<br />

KT 04<br />

KE 07<br />

PX 17<br />

PH 09<br />

PX 07<br />

PH 30<br />

Ullrich, S. PZ 40<br />

Ultee, A. PO 24<br />

Unbehauen, M. PN 12<br />

Unden, G. FGG 05<br />

PS 26<br />

PS 25<br />

Unger, C. PJ 13<br />

Unosson, C. FGE 03<br />

Urich, T. PB 21<br />

Urlaub, H. PU 12<br />

Ussery, D.W. FGE 02<br />

Vahedi-Faridi, A. PW 19<br />

Valdebenito, M. PJ 11<br />

Valdez, M. PM 26<br />

Valevich, M. KL 04<br />

Valla, S. PM 24<br />

Vamanu, E. PA 01<br />

van Baarle, S. KI 04<br />

van Berkel, W.J.H. PX 41<br />

VAN DE WIELE, T. PZ 35<br />

PZ 34<br />

van den Berg, M. PG 01<br />

PH 26<br />

Van <strong>der</strong> Does, C. KL 04<br />

van <strong>der</strong> Laan, M. H 10<br />

van <strong>der</strong> Oost, J. PA 41<br />

van Dijl, J.M. PP 39<br />

van Hijum, S. PS 52<br />

van Keulen, G. PZ 09<br />

van Pée, K.H. PI 03<br />

PT 05<br />

PT 04<br />

Varela Villarreal, J. HDID P 02<br />

Vater, J. PP 45<br />

Veith, A. PB 21<br />

Venkatesh, K.V. PY 03<br />

PY 05<br />

Vera, M. KU 04<br />

Vergauwen, K. PP 08<br />

Vianna, M. PP 42<br />

Vielgraf, S. PX 34<br />

Vieth, A. PZ 37<br />

Vincon, V. FGD 01<br />

Virus, S. PQ 06<br />

Vitt, S. PA 42<br />

Vödisch, M. KR 05<br />

Vogel, A. PN 02<br />

Vogel, J. PU 12<br />

KQ 02<br />

PU 06<br />

PU 05<br />

PU 07<br />

PU 11<br />

PU 09<br />

PU 14<br />

KQ 01<br />

Vogelmann, J. FGA 04<br />

PM 10<br />

Voget, S. PO 47<br />

Vogler, S. PO 21<br />

Vogt, C. PN 44<br />

KJ 05<br />

PA 40<br />

Vogt, S. PQ 04<br />

Vogts, T. PH 21<br />

Voigt, A. PW 05<br />

Voigt, B. PG 06<br />

PI 05<br />

Völker, U. PS 52<br />

PJ 21<br />

PM 24<br />

Volkmer-Engert, R. PW 16<br />

Völlmecke, C. KS 05<br />

Vollmeister, E. KD 04<br />

Vollmers, J. PO 47<br />

Vollstedt, C. PI 02<br />

Volz, J. PA 29<br />

von Bergen, M. PN 44<br />

KJ 05<br />

von Brehmer, S. PZ 28<br />

von Eiff, C. PP 20<br />

von Wallbrunn, C. FGH 05<br />

vor <strong>der</strong> Brüggen, M. PW 13<br />

Vorburger, T. KM 06<br />

Voss, B. PU 07<br />

Voss, J. PX 30<br />

Voytsekh, O. KC 06<br />

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PH 33<br />

Vulic, M. PP 23<br />

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PP 41<br />

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Wägele, J.W. KU 03<br />

Wagenknecht, M. PZ 10<br />

Wagner, A. PS 54<br />

Wagner, D. PB 05<br />

PN 19<br />

PO 12<br />

KK 02<br />

PB 09<br />

KK 04<br />

KE 05<br />

Wagner, G.H. KQ 04<br />

Wagner, K. FGF 02<br />

PP 43<br />

Wagner, M. PB 14<br />

KK 03<br />

PB 16<br />

PB 15<br />

Wagner, N. PC 03<br />

Wahl, R. KR 01<br />

Waidmann, M. PP 14<br />

Waidner, B. FGF 03<br />

Waldhuber, A. PS 08<br />

Waldmann, J. PN 08<br />

FGE 01<br />

Waldminghaus, T. KI 05<br />

Waldvogel, E. PR 15<br />

Walker, A.K. KN 06<br />

Walther, C. PZ 04<br />

Walther, J. PA 14<br />

PA 06<br />

PP 24<br />

Walther, P. PP 14<br />

Waltimo, T. PN 81<br />

Wandrey, M. PZ 37<br />

Wang, F. PE 04<br />

Wang, Y. PA 39<br />

Wanner, G. KK 06<br />

PG 07<br />

PO 48<br />

Wardenga, R. PX 05<br />

Warkentin, E. PQ 04<br />

PA 21<br />

Warrad, M. PL 01<br />

PZ 02<br />

Warscheid, B. KU 07<br />

Wartenberg, D. PP 18<br />

Waschewski, N. KF 04<br />

Waschkowitz, T. PG 02<br />

Wassenaar, T.M. FGE 02<br />

Wasserburger, N. KJ 04<br />

Wassmann, M. PR 29<br />

Wätzlich, D. PQ 06<br />

Webb, J. PZ 45<br />

Weber, A. PS 42<br />

Weber, M. FGE 01<br />

Weber, T. PS 27<br />

PT 07<br />

Webner, K. PA 22<br />

Webster, N. KK 03<br />

Wecker, P. PJ 24<br />

Wehmeier, U.F. PW 19<br />

Wei, L. PO 48<br />

PG 07<br />

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PJ 22<br />

PZ 47<br />

PJ 24<br />

Weiss, A. PN 02<br />

Weissenmayer, B. PF 03<br />

Weitbrecht, K. PO 29<br />

PN 54<br />

Weitz, D. PW 20<br />

Weitzel, B. PO 38<br />

Weiz, A.R. PT 01<br />

Welling, S. PM 20<br />

Wellner, S.A. PO 20<br />

Welsink, T. PU 14<br />

Welte, W. PD 04<br />

Wemhoff, S. FGH 04<br />

PY 04<br />

Wendisch, V.F. PW 02<br />

PS 04<br />

KT 01<br />

PX 04<br />

PW 10<br />

PX 42<br />

Wenning, M. PZ 28<br />

Wenter, R. KK 06<br />

Wenzel, M. KG 04<br />

Werner, I. PA 22<br />

Wesche, A. PS 49<br />

Wessel, M. PJ 02<br />

Westermann, M. PA 04<br />

Wetzel, J. KE 04<br />

PH 22<br />

Wetzel, S. PI 03<br />

Whalan, S. KK 03<br />

Wichmann, R. PR 12<br />

Wick, L. PN 68<br />

PN 76<br />

Wick, S. PM 17<br />

Widdel, F. PA 27<br />

PA 22<br />

PA 33<br />

Wiedemann, I. PM 14<br />

PC 02<br />

Wiedemann, N. H 10<br />

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PP 11<br />

PP 35<br />

KL 05<br />

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PJ 07<br />

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PR 24<br />

PX 35<br />

PP 29<br />

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PW 15<br />

Wimmer, R. PC 02<br />

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Winkler, C. PA 29<br />

Winkler, M. PZ 08<br />

KF 03<br />

Winkler, R. KR 05<br />

PP 18<br />

Winter, J. PO 01<br />

PZ 15<br />

PN 13<br />

Winter, T. PP 39<br />

Winterberg, B. FGF 06<br />

Winterhalter, M. PZ 47<br />

Wirth, K. PI 01<br />

Wirth, R. KI 06<br />

PB 07<br />

KB 02<br />

PB 02<br />

PB 06<br />

KJ 04<br />

Wirtz, C. PP 05<br />

Wischer, D. PB 26<br />

KB 05<br />

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PZ 08<br />

Wittmann, A. PX 28<br />

Woestemeyer, J. KE 04<br />

Wohlleben, W. PS 27<br />

PC 07<br />

PM 09<br />

PS 57<br />

PR 15<br />

PM 10<br />

PT 07<br />

Wöhnert, J. FGH 02<br />

Wolf, A. PZ 50<br />

PN 80<br />

PN 79<br />

Wolf, D. KR 02<br />

Wolf, J. PP 32<br />

Wolff, D. PR 08<br />

Wolke, S. PJ 15<br />

Wolkenhauer, O. PV 01<br />

Wollherr, A. PX 19<br />

Wolters, D. PG 03<br />

KR 06<br />

PS 23<br />

Wolz, C. KG 02<br />

PP 05<br />

Worm, K. PN 61<br />

Wöstemeyer, J. PH 22<br />

Wozniczka, M. PX 40<br />

Wray, V. PP 24<br />

Wrede, C. PN 82<br />

Wright, A.D.G. PB 04<br />

Wright, T. PN 46<br />

Wu, H. PM 22<br />

Wu, Y.R. PH 33<br />

FGD 04<br />

Wübbeler, J.H. PX 40<br />

PX 44<br />

Wunnick, D. PW 13<br />

Würdemann, H. PZ 37<br />

Wüst, P.K. KG 05<br />

Yaghobi, R. PP 10<br />

Yarza, P. FGE 04<br />

Youn, J.W. PW 02<br />

PW 10<br />

Yu, W. PP 31<br />

Yurkov, A. KK 07<br />

AUTOREN 235<br />

Zadra, I. KP 02<br />

Zafari, D. PK 02<br />

Zahiri, A. KR 01<br />

Zakaria, M.R. PB 04<br />

Zanjirian, E. PX 01<br />

Zaparty, M. PB 18<br />

KR 03<br />

Zapf, K. PP 34<br />

Zaprasis, A. PN 36<br />

Zarzycki, J. PR 21<br />

Zechlau, M. PR 14<br />

PS 28<br />

Zedelius, J. PA 38<br />

PA 27<br />

Zegber, I. PQ 10<br />

Zehner, S. KG 04<br />

Zeides, S. PM 26<br />

Zeiger, L. PV 02<br />

Zekert, N. KH 01<br />

Zel<strong>der</strong>, O. PX 08<br />

Zeller, M.E. KB 03<br />

Zeppenfeld, T. PX 45<br />

Zeth, K. PD 05<br />

KI 03<br />

Zeyer, J. KK 04<br />

Zhou, X. KO 03<br />

Zhurina, D. PM 23<br />

Ziebandt, A.K. PP 22<br />

Ziegelmüller, K. PN 31<br />

Ziegler, C. PS 40<br />

Ziegler, S. PN 14<br />

Ziegler, U. PJ 10<br />

Ziemann, E. PN 10<br />

HDID P 01<br />

Ziemert, N. PT 01<br />

Zigann, K. PS 07<br />

Zilliges, Y. KF 02<br />

Zimmer, B. PS 36<br />

Zimmer, C. PX 31<br />

Zimmermann, A.C. PF 02<br />

Zimmermann, M. KE 06<br />

Zimmermann, W. PX 27<br />

Zintel, S. PH 10<br />

Zitzmann, N. PN 81<br />

Zolghadr, B. PB 11<br />

Zoll, S. PP 31<br />

Zoltner, M. KS 05<br />

Zorin, B. KU 02<br />

Zorll, I. PN 47<br />

Zorn, H. FGD 03<br />

Zubair, A. PH 25<br />

Zullei-Seibert, N. HDID P 01<br />

PN 10<br />

Zumsteg, A. PN 26<br />

Zuther, K. PT 10<br />

Zverlov, V.V. PA 32

236 PERSONALIA AUS DER MIKROBIOLOGIE 2008<br />

Personalia aus <strong>der</strong> Mikrobiologie 2008:<br />

Habilitationen:<br />

Dirk Wagner habilitierte sich am<br />

3. Oktober 2007 an <strong>der</strong> Universität<br />

Potsdam (Microbial perspectives<br />

of the methane cycle in<br />

permafrost ecosystems in the<br />

eastern Siberian Arctic: implications<br />

for the global methane budget).<br />

Günter Fritz habilitierte sich am<br />

7. Februar 2008 an <strong>der</strong> Universität<br />

Konstanz (The Role of Metal<br />

Ions in Signalling and Catalysis).<br />

Thorsten Brinkhoff habilitierte<br />

sich am 13. Februar 2008 an <strong>der</strong><br />

Universität Oldenburg (Diversität<br />

und Physiologie mariner heterotropher<br />

Bakterien).<br />

Knut Ohlsen habilitierte sich am<br />

16. April 2008 an <strong>der</strong> Universität<br />

Würzburg (Untersuchungen <strong>zur</strong><br />

Pathophysiologie von Staphylococcus<br />

aureus).<br />

Andreas Tauch habilitierte sich<br />

am 29. April 2008 an <strong>der</strong> Universität<br />

Bielefeld (Genom- und Postgenomforschung<br />

an industriell<br />

und medizinisch relevanten Corynebakterien).<br />

Bodo Philipp habilitierte sich am<br />

9. Juli 2008 an <strong>der</strong> Universität<br />

Konstanz (Bacterial biodegradation:<br />

metabolic pathways and cellcell<br />

interactions).<br />

Oliver Kurzai habilitierte sich am<br />

9. Juli 2008 an <strong>der</strong> Universität<br />

Würzburg (Vom Kommensalen<br />

zum Krankeheitsererger – mikrobielle<br />

und immunologische Voraussetzungen<br />

für Virulenzvariation).<br />

Matthias Labrenz habilitierte<br />

sich am 9. Oktober 2008 an <strong>der</strong><br />

Universität Rostock (Microbes<br />

and microbial lifestyles within<br />

unusal environments).<br />

Rainer Cramm habilitierte sich<br />

an <strong>der</strong> Humboldt-Universität zu<br />

Berlin (Bakterielle NO-Stressproteine:<br />

Untersuchungen <strong>zur</strong> Molekularbiologie,<br />

Biochemie und<br />

physiologische Funktion).<br />

Gabriele Pradel habilitierte sich<br />

am 12. November 2008 an <strong>der</strong><br />

Universität Würzburg (Proteine<br />

<strong>der</strong> Transmissionsstadien des<br />

Malariaerregers Plasmodium sp.<br />

und ihre Bedeutung für Interventionen).<br />

Charles Franz habilitierte sich am<br />

26. November 2008 an <strong>der</strong> Universität<br />

Karlsruhe. (Molecular biological<br />

investigations into the<br />

diversity, functionality and safety<br />

of lactic acid bacteria, in particular<br />

the enterococci).<br />

Rufe:<br />

Michael Bott von <strong>der</strong> Universität<br />

Düsseldorf übernahm am 15.<br />

November 2007 die W3-Professor<br />

für Biotechnologie 1 an <strong>der</strong><br />

Universität Düsseldorf sowie die<br />

Leitung des Instituts für Biotechnologie<br />

1 am Forschungszentrum<br />

Jülich.<br />

Heide Schulz-Vogt vom Institut<br />

für Mikrobiologie <strong>der</strong> Universität<br />

Hannover übernahm am 1.<br />

November 2007 die W2-Professur<br />

für Mikrobiologie am Max-<br />

Planck-Institut für Marine Mikrobiologie,<br />

Bremen.<br />

Wolfgang Liebl von <strong>der</strong> Universität<br />

Göttingen übernahm am 1.<br />

Februar 2008 die W3-Professur<br />

für Mikrobiologie an <strong>der</strong> Technischen<br />

Universität München.<br />

Thomas Rudel vom Max-Planck-<br />

Institut für Infektionsbiologie Berlin<br />

übernahm am 21. Februar<br />

2008 die W3-Professur für Mikrobiologie<br />

an <strong>der</strong> Universität Würzburg.<br />

Thorsten Selmer von <strong>der</strong> Universität<br />

Marburg übernahm am 1.<br />

März 2008 die W2-Professur für<br />

Enzymtechnologie an <strong>der</strong> Fachhochschule<br />

Aachen, Standort<br />

Jülich.<br />

Jörg Hacker von <strong>der</strong> Universität<br />

Würzburg übernahm am 1. März<br />

2008 die Präsidentschaft des<br />

Robert-Koch-Instituts in Berlin.<br />

Annegret Wilde von <strong>der</strong> HU Berlin<br />

übernahm am 1. April 2008 die<br />

W2-Professur am Institut für<br />

Mikrobiologie und Molekularbiologie<br />

<strong>der</strong> Universität Gießen.<br />

Johann Hei<strong>der</strong> von <strong>der</strong> TU Darmstadt<br />

übernahm am 1. April 2008<br />

eine W3-Professur für Mikrobiologie<br />

an <strong>der</strong> Universität Marburg.<br />

Lars Leichert von <strong>der</strong> University<br />

of Michigan, Ann Arbor, USA übernahm<br />

am 1. Mai 2008 die Juniorprofessur<br />

für Redox-Proteomics<br />

an <strong>der</strong> Universität Bochum.<br />

Klaus Erb von <strong>der</strong> Firma Boehringer<br />

Ingelheim Pharma, Biberach<br />

übernahm am 13. Juni 2008<br />

eine außerplanmäßige Professur<br />

an <strong>der</strong> Universität Würzburg.<br />

Sven Hammerschmidt von <strong>der</strong><br />

LMU München übernahm am 1.<br />

Juli 2008 eine W3-Professur für<br />

Allgemeine und Molekulare Genetik<br />

an <strong>der</strong> Universität Greifswald.<br />

Christiane Dahl von <strong>der</strong> Universität<br />

Bonn wurde im Juli 2008 <strong>zur</strong><br />

apl.-Professorin ernannt.<br />

Fuli Li vom Max-Planck-Institut<br />

für terrestrische Mikrobiologie<br />

Marburg übernahm am 1. September<br />

2008 die Professur für<br />

Mikrobiologie am Qingdao Institute<br />

of Bioenergy and Bioprocess<br />

Technology (QIBEBT) in Qingdao,<br />

China.<br />

Thorsten Dittmar von <strong>der</strong> Florida<br />

State University, Tallahasee, Florida,<br />

USA übernahm am 1. September<br />

2008 die W2-Leitung <strong>der</strong><br />

Selbständigen Nachwuchsgruppe<br />

<strong>der</strong> Max-Planck-Gesellschaft<br />

für Marine Geochemie am Institut<br />

für Chemie und Biologie des<br />

Meeres <strong>der</strong> Universität Oldenburg.<br />

Ute Hentschel von <strong>der</strong> Universität<br />

Würzburg übernahm am 1.<br />

September 2008 die W2-Professur<br />

für Chemische Ökologie am<br />

Lehrstuhl für Botanik II an <strong>der</strong> Universität<br />

Würzburg<br />

Julia Bandow von <strong>der</strong> Firma Pfizer,<br />

Ann Arbor, Michigan, USA<br />

übernahm am 1. Oktober 2008<br />

die Juniorprofessur für Mikrobielle<br />

Antibiotikaforschung an <strong>der</strong><br />

Universität Bochum.<br />

Michael Friedrich vom Max-<br />

Planck-Institut für terrestrische<br />

Mikrobiologie Marburg übernahm<br />

am 1. Oktober 2008 die W2-Professur<br />

für Mikrobiologie an <strong>der</strong><br />

Universität Bremen.<br />

Helge Küster von <strong>der</strong> Universität<br />

Bielefeld übernahm am 1. Oktober<br />

2008 die W3-Professur für<br />

Genetik an <strong>der</strong> Leibniz Universität<br />

Hannover.<br />

Karin Römisch von <strong>der</strong> Universität<br />

Trento, Italien übernahm am<br />

1. Oktober 2008 die W3-Professur<br />

für Mikrobiologie an <strong>der</strong> Universität<br />

des Saarlandes.<br />

Monika Ehling-Schulz von <strong>der</strong><br />

Technischen Universität München<br />

übernahm am 1. November 2008<br />

eine Universitätsprofessur für<br />

Lebensmittelmikrobiologie an <strong>der</strong><br />

Veterinärmedizinischen Universität<br />

Wien.<br />

Petra Dersch von <strong>der</strong> Technische<br />

Universität Braunschweig nahm<br />

am 1. Dezember 2008 einen Ruf<br />

an das Helmholtzzentrum für<br />

Infektionsforschung (HZI) in<br />

Braunschweig an.<br />

Ann-Kristin Müller von <strong>der</strong> Universität<br />

Würzburg übernimmt am<br />

1. Januar <strong>2009</strong> eine Juniorprofessur<br />

am Hygiene-Institut des Universitätsklinikums<br />

Heidelberg.<br />

Pensionierungen/<br />

Emeritierungen:<br />

Helmut Tschäpe vom Robert<br />

Koch-Institut, Fachgebiet Bakterielle<br />

Infektionen, Außenstelle<br />

Wernigerode, wurde am 1.<br />

November 2006 pensioniert.<br />

Volker Brade, Direktor des Instituts<br />

für Medizinische Mikrobiologie<br />

an <strong>der</strong> Universität Frankfurt/Main<br />

wurde am 29. Februar<br />

2008 in den Ruhestand verabschiedet.<br />

Wolfgang Buckel vom Laboratorium<br />

für Mikrobiologie an <strong>der</strong> Universität<br />

Marburg wurde am 31.<br />

März 2008 pensioniert und ist<br />

seit 1. April 2008 Max Planck Fellow<br />

am Max-Planck-Institut für<br />

terrestrische Mikrobiologie, Marburg.<br />

Cornelius G. Friedrich vom Lehrstuhl<br />

für Technische Mikrobiologie<br />

<strong>der</strong> Fakultät Bio- und Chemieingenieurwesen<br />

an <strong>der</strong> Technischen<br />

Universität Dortmund<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

wurde an 31. August 2008 pensioniert.<br />

Klaus Hantke vom Institut für<br />

Mikrobiologie/Organismische<br />

Interaktionen <strong>der</strong> Universität<br />

Tübingen wurde am 31. August<br />

2008 pensioniert.<br />

Jürgen Weckesser vom Institut<br />

für Mikrobiologie an <strong>der</strong> Universität<br />

Freiburg wurde am 30. September<br />

emeritiert.<br />

Alfred Pühler vom Lehrstuhl für<br />

Genetik <strong>der</strong> Universität Bielefeld<br />

wurde am 30. September 2008<br />

entpflichtet und führt seine wissenschaftlichen<br />

Ar<strong>bei</strong>ten als<br />

Senior Research Professor am<br />

Centrum für Biotechnologie <strong>der</strong><br />

Universität Bielefeld fort.<br />

Isolde Röske vom Institut für<br />

Mikrobiologie an <strong>der</strong> Universität<br />

Dresden wurde am 30. September<br />

2008 emeritiert.<br />

Peter M. H. Kroneck vom Fachbereich<br />

Biologie an <strong>der</strong> Universität<br />

Konstanz wurde am 30. September<br />

2008 pensioniert.<br />

Mark Achtman vom Max-Planck-<br />

Institut für Molekulare Genetik<br />

wurde am 30. September 2008<br />

pensioniert. Er leitet jetzt die<br />

Ar<strong>bei</strong>tsgruppe Environmental<br />

Microbial Genomics an <strong>der</strong> Universität<br />

Cork, Irland.<br />

Wissenschaftliche Preise<br />

2008 (sofern nicht bereits<br />

in BIOspektrum gemeldet)<br />

Nassos Typas von <strong>der</strong> Freien Universität<br />

Berlin erhielt am 4.<br />

Dezember 2007 den Ernst-Reuter-Preis<br />

für seine Dissertation<br />

über “Deciphering the way SigmaS-containing<br />

RNA polymerase<br />

(E-SigmaS) targets its promoters<br />

in Escherichia coli“.<br />

Christina Pesavento von <strong>der</strong><br />

Freien Universität Berlin erhielt<br />

am 16. Januar 2008 den Katharina-Heinroth-Preis<br />

<strong>der</strong> Gesellschaft<br />

Naturforschen<strong>der</strong> Freunde<br />

zu Berlin für ihre Diplomar<strong>bei</strong>t<br />

über “Regulation und Funktion<br />

SigmaS-abhängiger GGDEF- und<br />

EAL-Domänen-Proteine in Escherichia<br />

coli“.<br />

Sascha Thewes von <strong>der</strong> Freien<br />

Universität Berlin erhielt am 15.<br />

Februar 2008 den Forschungs-<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

preis 2008 für den wissenschaftlichen<br />

Nachwuchs <strong>der</strong> DGHM-<br />

Fachgruppe „Eukaryontische<br />

Krankheitserreger“ und am 11.<br />

April 2008 den ERA-NET Patho-<br />

GenoMics PhD Award 2008 für<br />

seine Doktorar<strong>bei</strong>t „Molekularbiologische<br />

Untersuchungen <strong>zur</strong><br />

Invasivität von Candida albicans“.<br />

Laila P. Partida-Martinez vom<br />

Hans-Knöll-Institut, <strong>VAAM</strong>-Promotionspreisträgerin<br />

2008,<br />

erhielt am 20. Februar 2008 den<br />

DECHEMA-Doktorandenpreis für<br />

Naturstoffforschung für ihre Doktorar<strong>bei</strong>t<br />

„Discovery of endofungal<br />

bacteria: new insights into<br />

toxin biosyntheses and bacterialfungal<br />

symbiosis“.<br />

Herbert Schmidt von <strong>der</strong> Universität<br />

Hohenheim erhielt im<br />

April 2008 den Forschungspreis<br />

<strong>der</strong> Seeligerstiftung.<br />

Jörg Hacker von <strong>der</strong> Universität<br />

Würzburg (seit 1. März 2008:<br />

Robert-Koch-Institut) erhielt am<br />

16. April 2008 den „Arthur-Burkhardt-Preis<br />

für Wissenschaftsför<strong>der</strong>ung“<br />

für seine außerordentlichen<br />

Beiträge <strong>zur</strong> Verknüpfung<br />

von Erkenntnissen aus<br />

Naturwissenschaft und Geisteswissenschaft.<br />

Rudolf K. Thauer vom Max-<br />

Planck-Institut für terrestrische<br />

Mikrobiologie Marburg erhielt am<br />

16. Mai 2008 die Carl-Friedrich-<br />

Gauß-Medaille <strong>der</strong> Braunschweigischen<br />

Wissenschaftlichen<br />

Gesellschaft für seine Ar<strong>bei</strong>ten<br />

über die biochemischen Grundlagen<br />

durch Mikroorganismen verursachter<br />

ökologischer Prozesse.<br />

Julia Frunzke, wissenschaftliche<br />

Mitar<strong>bei</strong>terin des Instituts für Biotechnologie<br />

1 am Forschungszentrum<br />

Jülich, erhielt am 4. Juni<br />

2008 einen <strong>der</strong> DSM Science &<br />

Technology Awards (North) 2008<br />

als Auszeichnung für ihre Doktorar<strong>bei</strong>t.<br />

Peter F. Zipfel von <strong>der</strong> Universität<br />

Jena und dem Leibniz-Institut für<br />

Naturstoff-Forschung und Infektionsbiologie<br />

e. V. (Hans-Knöll-<br />

Institut Jena) erhielt am 20. Juni<br />

2008 den Heinz-Spitzbart-Preis<br />

für seine Ar<strong>bei</strong>ten <strong>zur</strong> För<strong>der</strong>ung<br />

therapierelevanter Forschungsar<strong>bei</strong>ten<br />

auf dem Gebiet <strong>der</strong> gynäkologischen<br />

Infektiologie und am<br />

29. Oktober 2008 den Lecture<br />

PERSONALIA AUS DER MIKROBIOLOGIE 2008 237<br />

award <strong>der</strong> European Fe<strong>der</strong>ation<br />

of Immunology societies (EFIS)<br />

für seine herausragenden Ar<strong>bei</strong>ten<br />

im Bereich <strong>der</strong> Immunbiologie.<br />

Marion Eisenhut von <strong>der</strong> Universität<br />

Rostock erhielt am 4. Juli<br />

2008 den Joachim-Jungius-För<strong>der</strong>preis<br />

<strong>der</strong> Universität Rostock<br />

für ihre Promotionsar<strong>bei</strong>t über<br />

„Molecular characterization of 2phosphoglycolate<br />

metabolism in<br />

the cyanobacterial model strain<br />

Synechocystis sp. PCC 6803“.<br />

Katja Zuther, Christoph W. Basse<br />

und Jan Schirawski vom Max-<br />

Planck-Institut für terrestrische<br />

Mikrobiologie Marburg erhielten<br />

am 5. September 2008 den Wissenschaftspreis<br />

<strong>der</strong> Deutschsprachigen<br />

Mykologischen Gesellschaft<br />

für ihre Ar<strong>bei</strong>ten über die<br />

Tryptophan-abhängige Pigmentsynthese<br />

in Ustilago maydis.<br />

Oliver Kurzai von <strong>der</strong> Universität<br />

Würzburg erhielt am 23. September<br />

2008 den För<strong>der</strong>preis <strong>der</strong><br />

Deutschen Gesellschaft für Hygiene<br />

und Mikrobiologie für seine<br />

Ar<strong>bei</strong>t über die Frage, unter welchen<br />

Bedingungen Mikroorganismen<br />

harmlose Kommensalen sind<br />

o<strong>der</strong> Schleimhautbarrieren überwinden<br />

können.<br />

Alfred Pühler von <strong>der</strong> Universität<br />

Bielefeld erhielt am 7. Oktober<br />

2008 die DECHEMA-Medaille für<br />

seine Verdienste um die stetige<br />

Weiterentwicklung <strong>der</strong> Biotechnologie-Aktivitäten<br />

<strong>der</strong> DECHE-<br />

MA.<br />

Beate Henrichfreise von <strong>der</strong> Universität<br />

Bonn erhielt am 9. Oktober<br />

2008 den Promotionspreis<br />

<strong>der</strong> Paul-Ehrlich-Gesellschaft für<br />

Chemotherapie e.V. für ihre Ar<strong>bei</strong>ten<br />

<strong>zur</strong> Antibiotika-Multiresistenz<br />

<strong>bei</strong> Pseudomonas aeruginosa.<br />

Mohamed Marahiel von <strong>der</strong> Universität<br />

Marburg erhielt am<br />

15.Oktober 2008 die Max-Bergmann-Medaille<br />

für seine richtungsweisendenwissenschaftlichen<br />

Ar<strong>bei</strong>ten auf dem Gebiet<br />

<strong>der</strong> Mechanismen <strong>der</strong> nicht-ribosomalen<br />

Synthese von Peptidantibiotika.<br />

Marta Kovacs von <strong>der</strong> Universität<br />

Kaiserslautern erhielt am 30. Oktober<br />

2008 den DAAD-Preis 2008<br />

für ihre Ar<strong>bei</strong>ten über regulatori-<br />

sche RNAs in Streptococcus<br />

pneumoniae und soziales Engagement.<br />

Patrizia Spitalny von <strong>der</strong> Universität<br />

Regensburg, Lehrstuhl für<br />

Mikrobiologie, erhielt am 31. Oktober<br />

2008 den Kulturpreis Bayern<br />

<strong>der</strong> E.ON Bayern AG für ihre<br />

Dissertation „Analyses of the<br />

Archaeal Transcription Cycle reveal<br />

a Mosaic of Eukaryotic RNA<br />

Polymerase II and III-like Features“.<br />

Martin Vödisch von <strong>der</strong> Universität<br />

Jena und Thomas Stridde<br />

von <strong>der</strong> Thüringer Landeszeitung<br />

erhielten am 6. November 2008<br />

den von Promega verliehenen<br />

Wissenschafts- und Journalistenpreis<br />

für gute Veröffentlichungen<br />

rund um die Biologie.<br />

Daniela Näther von <strong>der</strong> Universität<br />

Regensburg erhielt am 8.<br />

November 2008 den Biotech-Promotionspreis<br />

<strong>der</strong> BIOPARK<br />

Regensburg GmbH für ihre Ar<strong>bei</strong>t<br />

<strong>zur</strong> Untersuchung <strong>der</strong> Flagellen<br />

von Pyrococcus furiosus.<br />

Christoph Schön von <strong>der</strong> Universität<br />

Würzburg erhielt am 14.<br />

November 2008 den Postdoktoranden-Preis<br />

für Mikrobiologie <strong>der</strong><br />

Universität Würzburg und <strong>der</strong><br />

Robert-Koch-Stiftung in Berlin für<br />

seine Ar<strong>bei</strong>t über die Genetik des<br />

krankheitserregenden Bakteriums<br />

Neisseria meningitidis.<br />

Andreas Kappler von <strong>der</strong> Universität<br />

Tübingen erhielt am 22.<br />

November 2008 den Biologie-<br />

Preis 2008 <strong>der</strong> Akademie <strong>der</strong> Wissenschaften<br />

zu Göttingen für seine<br />

bahnbrechenden Forschungen<br />

über die Geobiologie des globalen<br />

mikrobiellen Eisen-Kreislaufs.<br />

Matthias Frosch von <strong>der</strong> Universität<br />

Würzburg erhielt am 28.<br />

November 2008 den Aronson-<br />

Wissenschaftspreis für seine<br />

Ar<strong>bei</strong>t über die Entwicklung von<br />

Impfstoffen gegen den Erreger<br />

<strong>der</strong> bakteriellen Hirnhautentzündung.<br />

Dagmar Kock von <strong>der</strong> Universität<br />

Hannover erhielt am 18. Dezember<br />

2008 den Hans-Joachim-Martini-Nachwuchspreis<br />

für ihre<br />

Ar<strong>bei</strong>ten <strong>zur</strong> Quantifizierung von<br />

Mikroorganismen in sulfidischen<br />

Bergbauhalden.

238 PROMOTIONEN 2008<br />

Promotionen in <strong>der</strong> Mikrobiologie 2008<br />

Bayreuth, Universität<br />

Thi Thuy Ai: Function of the ATPdependent<br />

metalloprotease FtsH<br />

during sporulation of Bacillus subtilis<br />

Referent: Wolfgang Schumann<br />

Bärbel Fösel: Mikrobiologie <strong>der</strong> Stickstoffentfernung<br />

in den Biofiltern einer<br />

marinen Aquakultur mit geschlossenem<br />

Wasserkreislauf<br />

Referent: Harold Drake<br />

Oliver Kreß: Funktion von mikrobiellen<br />

Farbstoffen in <strong>der</strong> Kellerassel Porcellio<br />

scaber<br />

Referent: Ortwin Meyer<br />

Berlin, Freie Universität<br />

Johanna Heuveling: Regulationsmechanismen<br />

<strong>der</strong> Säurestressantwort<br />

von Escherichia coli auf transkriptionaler,<br />

posttranskriptionaler und proteolytischer<br />

Ebene und <strong>der</strong>en Rolle in<br />

<strong>der</strong> Kinetik des Systems<br />

Referentin: Regine Hengge<br />

Alexandra Koumoutsi: Functional<br />

genome analysis of the plant-growth<br />

promoting bacterium Bacillus amyloliquefaciens<br />

strain FZB42; characterizing<br />

its production and regulation of<br />

nonribosomal peptide synthetases<br />

Referent: Rainer Borriss<br />

Sivaraman Subramaniam: Role of<br />

Cpx pathway in Salmonella pathogenesis<br />

Referent: Rainer Borriss<br />

Berlin, Max-Planck-Institut für<br />

Infektionsbiologie<br />

Peter Braun: Der molekulare Mechanismus<br />

<strong>der</strong> Aminosäure-induzierten<br />

Inhibition chlamydialen Wachstums<br />

und die Rolle des bakteriellen Transporters<br />

BrnQ<br />

Referent: Thomas F. Meyer<br />

Daniel Becker: Salmonella as a research<br />

model for fundamental insights<br />

into therapeutic strategies<br />

Referent: Thomas F. Meyer<br />

Berlin, Humboldt-Universität<br />

Antje Gebler: Analyse des NiFe-Zentrums<br />

und <strong>der</strong> Kofaktoren im H2-Sen sor von Ralstonia eutropha H16<br />

Referentin: Bärbel Friedrich<br />

Torsten Schubert: Spezifische Reifungsfaktoren<br />

eines Tat-abhängigen<br />

translozierten Eisen-Schwefel-Proteins,<br />

<strong>der</strong> O2-toleranten, membrangebundenen<br />

NiFe-Hydrogenase (MBH)<br />

Referentin: Bärbel Friedrich<br />

Bielefeld, Universität<br />

Christelle Bahlawane: MucR, a central<br />

regulator of exopolysaccharide<br />

biosynthesis and motility in Sinorhizobium<br />

meliloti, Referentin: Anke<br />

Becker<br />

Karina Brinkrolf: Das trankriptionelle<br />

Regulationsnetzwerk von Corynebacterium<br />

glutamicum unter Einbeziehung<br />

des LacI/GalR-Regulators<br />

UriR<br />

Marcus Ludwig: The oxygen-tolerant,<br />

membrane-bound hydrogenase of<br />

Ralstonia species: variations in the<br />

active site structure, the Fe-S cluster<br />

composition and maturation<br />

Referentin: Bärbel Friedrich<br />

Referent: Alfred Pühler<br />

Lars Gaigalat: Aufklärung <strong>der</strong> transkriptionellen<br />

Regulation <strong>der</strong> Gene des<br />

Phosphoenolpyruvat:Phosphotransferase-Systems<br />

in Corynebacterium<br />

glutamicum ATCC13032<br />

Peter Hebbeln: Untersuchungen <strong>zur</strong><br />

Struktur und Funktion primär und<br />

sekundär aktiver Transporter für Übergangsmetallaktionen<br />

– ein neuartiger<br />

Mechanismus <strong>der</strong> Substrataufnahme<br />

in Prokaryoten<br />

Referent: Alfred Pühler<br />

Javier Serrania Vallejo: Transkriptomische<br />

Analysen <strong>zur</strong> Xanthanproduktion<br />

in Xanthomonas campestris pv.<br />

campestris Referentin: Anke Becker<br />

Referent: Thomas Eitinger<br />

Bochum, Universität<br />

Mathias Grote: A biophysical study Stephanie Hacker: Rolle von Phos-<br />

on the catalytic cycle of the enterobphatidylcholin <strong>bei</strong> <strong>der</strong> Bakterien-Pflanacterial<br />

maltose ABc-transporter zen-Interaktion<br />

MalFGK2-E<br />

Referent: Franz Narberhaus<br />

Referent: Erwin Schnei<strong>der</strong><br />

Sonja Brandt: The fungal phytochro-<br />

Rebecca Fleischer: Biochemische me FphA from Aspergillus nidulans<br />

Charakterisierung des Cpx-Signaltransduktionssystems<br />

aus Escherichia<br />

coli<br />

Referent: Erwin Schnei<strong>der</strong><br />

Referentin: Nicole Frankenberg-Dinkel<br />

Jens Kamerewerd: Funktionelle Analyse<br />

nicht-konventioneller Signaltransduktionswege<br />

<strong>bei</strong> <strong>der</strong> Morphogenese<br />

des Modellorganismus Sordaria<br />

macrospora<br />

Referent: Ulrich Kück<br />

Sylvia Kleta: Einfluss des probiotischen<br />

Escherichia coli Nissle 1917<br />

(EcN) auf die Infektion mit atypischen<br />

enteropathogenen E. coli (aEPEC) im<br />

porcinen in vitro-Modell<br />

Referent: Erwin Schnei<strong>der</strong><br />

Bonn, Universität<br />

Dina Raafat Gouda Fouad: Chitosan<br />

as an antimicrobial compound: Modes<br />

of a and resistance mechanismen<br />

Referent: Hans-Georg Sahl<br />

Vera Saß: Die molekulare Wirkung<br />

des humanen ß-Defensins hBD3 auf<br />

Staphylococcus aureus, Referent:<br />

Hans-Georg Sahl<br />

Braunschweig, Technische<br />

Universität<br />

Nina Diekmann: Mikrobielle Gemeinschaften<br />

auf Klimawärmetauschern<br />

Referenten: Dieter Jahn, Christoph Tebbe<br />

Thorben Dammeyer: Ferredoxin-<br />

Dependent Bilin Biosynthetic Enzymes<br />

from Cyanobacteria and Marine<br />

Viruses<br />

Referenten: Dieter Jahn, Nicole Frankenberg-Dinkel<br />

Claudia Pommerenke: Integrated<br />

systems biology platforms for bacterial<br />

metabolic and gene- regulatory<br />

networks<br />

Referenten: Dieter Jahn, Frank Klawonn<br />

Andreas Georg Grote: Datenbanksysteme<br />

und bioinformatische Werkzeuge<br />

<strong>zur</strong> Optimierung biotechnologischer<br />

Prozesse mit Pilzen<br />

Referenten: Dieter Jahn, Dietmar<br />

Schomburg<br />

Maurice Patrick Scheer: Computational<br />

Analysis and Interpretation of<br />

Prokaryotic High-throughput Expression<br />

Data<br />

Referenten: Michael Steinert, Frank Klawonn<br />

Brinkmann, Nicole: A biosafety study<br />

on the fate of DNA from a transplastomic<br />

tobacco cultivar and the<br />

interactions with bacteria from soil<br />

and insect larvae<br />

Referenten: Christoph Tebbe, Dieter<br />

Jahn<br />

Olga Shevchuk: D. discoideum as a<br />

model for host-pahogen interaction:<br />

Phagosomal proteome of Legionellainfected<br />

cells<br />

Referenten: Michael Steinert, Jürgen<br />

Bode<br />

Bremen, MPI für marine<br />

Mikrobiologie<br />

Susanne Liebner: Adaption, Spatial<br />

Variability, and Phylogenetic Characterization<br />

of Methanotrophic Communities<br />

in Permafrost Soils of the<br />

Lena Delta, Siberia<br />

Referent: Rudolf Amann<br />

Maya Shovitri: Bacterial communities<br />

in the burrow of the lugworm Arenicola<br />

marina and community changes<br />

on the intertidal muddy sediments<br />

by applying terminal restriction fragment<br />

length polymorphism<br />

Referent: Rudolf Amann<br />

Tran Nhu Hoa: New organic substrates<br />

for anoxygenic phototrophic bacteria<br />

Referent: Friedrich Widdel<br />

Lars Wöhlbrand: Proteomische<br />

Untersuchungen zum Aromaten-<br />

Abbau in „/Arotmatoleum aromaticum/“<br />

Stamm EbN1<br />

Referent: Friedrich Widdel<br />

Michael Richter: Computational Processing<br />

and Biological Interpretation<br />

of Genomes and Metagenomes of<br />

Marine Bacteria<br />

Referent: Rudolf Amann<br />

Frank Zielinski: Mutualism and Parasitism<br />

in Chemosymbiotic Mussels<br />

from the Logatchev Hydrothermal<br />

Vent Field on the Mid-Atlantic Ridge<br />

Referent: Rudolf Amann<br />

Elsabé Julies: Microbial ecology of<br />

anaerobic carbon mineralization in<br />

Namibian schelf sediments<br />

Referent: Bo Barker Joergensen<br />

Gunter Wegener: Methane Oxidation<br />

and Carbon Assimilation in Marine<br />

Sediments,<br />

Referent: Bo Barker Joergensen<br />

Marie-Lise Schläppy: Chemical environments,<br />

microbial processes, feeding<br />

and ventilation behavior in two<br />

species of Demosponges (Porifera)<br />

Referent: Bo Barker Joergensen<br />

Simone Böer: Investigation of the distribution<br />

and activity of microorganisms<br />

in coastal habitats<br />

Referentin: Antje Boetius<br />

Darmstadt, Technische Universität<br />

Sabrina Fröls: Responses of the<br />

hyperthermophilic Archaeon Sulfolobus<br />

solfataricus to UV-light<br />

Referentin: Christa Schleper<br />

Fabian Müller: Untersuchungen des<br />

aeroben Schwefelstoffwechsels von<br />

Acidianus ambivalens<br />

Referent: Arnulf Kletzin<br />

Susan Schmidt: Intrinsische Substratproteine<br />

<strong>der</strong> Transglutaminase<br />

von Streptomyces mobaraensis: Identifizierung<br />

und Charakterisierung<br />

Referent: Lothar Fuchsbauer<br />

Cordula Böhm: Die dreidimensionale<br />

Struktur nativer Editosomen aus afrikanischen<br />

Trypanosomen<br />

Referent: H. Ulrich Göringer<br />

Moritz Niemann: RNA editing in African<br />

trypanosomes requires a 3’ nucleotidyl<br />

phosphatase – the biochemical<br />

consequences of the exoUase activitiy<br />

of TbMP42<br />

Referent: H. Ulrich Göringer<br />

Düsseldorf, Universität<br />

Fabien Cottier: Roles of Msb2p and<br />

other putative sensors in environmental<br />

responses of Candida albicans<br />

Referent: Joachim F. Ernst<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Duisburg, Universität<br />

Felipe Leon-Morales: Influence of<br />

biofilms on the transport of colloids<br />

and contaminants through porous<br />

media<br />

Referent: Hans-Curt Flemming<br />

Sascha Broekman: Etablierung einer<br />

Methodik <strong>zur</strong> Darstellung des extrazellulären<br />

Proteoms in Biofilmen eines<br />

mucoiden Stammes von Pseudomonas<br />

aeruginosa<br />

Referent: Hans-Curt Flemming<br />

Arne Hoerskens: Biologische Nitratentfernung<br />

in Wasserkreisläufen von<br />

ausgewählten Industriebetrieben nach<br />

Anwendung von Aluminiumnitratsulfat<br />

Referent: Hans-Curt Flemming<br />

Erlangen, Universität<br />

Eva Hänßler: Regulation of glutamate<br />

dehydrogenase in Corynebacterium<br />

glutamicum<br />

Referent: Andreas Burkovski<br />

Janko Daam: Charakterisierung <strong>der</strong><br />

Interaktion des Transkriptionsregulators<br />

TetR mit induzierenden Oligopeptiden<br />

Referent: Wolfgang Hillen<br />

Frank Wolschendorf: Physiological<br />

functions of mycobacterial outer<br />

membrane channel proteins<br />

Referent: Michael Nie<strong>der</strong>weis<br />

Frankfurt, Universität<br />

Mariam Brenneis: Regulation <strong>der</strong><br />

Translation in Haloferax volcanii, Referent:<br />

Jörg Soppa<br />

Anke Baumann: Charakterisierung<br />

des Zellzyklusses von Halobacterium<br />

salinarum<br />

Referent: Jörg Soppa<br />

Iris Graf: Natürliche DNA-Transformationssysteme<br />

in mesophilen Bakterien:<br />

funktionelle Analysen, In-vivo-<br />

Lokalisation und Verbreitung<br />

Referentin: Beate Averhoff<br />

Cornelia Schwarzenlan<strong>der</strong>: Der<br />

DNA-Translokator in Thermus thermophilus<br />

HB27: Charakterisierung<br />

des DNA-Transports und funktionelle<br />

Analysen <strong>der</strong> Kompetenzproteine<br />

Referentin: Beate Averhoff<br />

Freiburg, Universität<br />

Joachim Hogg: Untersuchungen zu<br />

den an <strong>der</strong> initialen Reaktion des anaeroben<br />

Tolouolabbaus beteiligten Proteine<br />

aus Thauera aromatica und <strong>der</strong><br />

Glycerindehydratase aus Rhodospirillum<br />

rubrum<br />

Referent: Hans Hei<strong>der</strong><br />

Bin Ding: Anaerobic Catechol Degradaton<br />

in a Denitrifying Bacterium<br />

Referent: Georg Fuchs<br />

Mahalakshmi Krishnamurthy: Role<br />

of novel SMC-like protein in Bacillus<br />

subtilis<br />

Referent: Peter Graumann<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Sirko Schmeling: Vergleichende<br />

Untersuchungen zum anaeroben Phenolstoffwechsel<br />

in Bakterien<br />

Referent: Georg Fuchs<br />

Gießen, Universität<br />

Miriam Drath: Proteolyse in Synechocystis<br />

sp. PCC6803: Funktion <strong>der</strong><br />

Methionin-Aminopeptidase 2 und <strong>der</strong><br />

FtsH2-Protease für die Photosystem-<br />

II-Stressresistenz<br />

Referent: Karl Forchhammer<br />

Suhad Sanjaq: Enterobacter sakazakii<br />

– Risikoprofil und Untersuchungen<br />

zum Nachweis in Säuglingsnahrungen<br />

Referentin: Sylvia Schnell<br />

Göttingen, Universität<br />

Claudine Hames: Glycerolmetabolismus<br />

und Pathogenität von Mycoplasma<br />

pneumoniae, Referent: Jörg<br />

Stülke<br />

Sebastian Hübner: Untersuchung <strong>der</strong><br />

Spezifität von Antiterminationsproteinen<br />

in Bacillus subtilis<br />

Referent: Jörg Stülke<br />

Nirmala Padmanabhan: The Saccharomyces<br />

cerevisiae HtrA orthologue,<br />

Ynm3, is a chaperone-protease<br />

that aids survival un<strong>der</strong> heat stress<br />

Referent: Gerhard Braus<br />

Karen Laubinger: Analysis of Neuronal<br />

Diseases in the Model Organism<br />

Aspergillus nidulans<br />

Referent: Gerhard Braus<br />

Christoph Sasse: Cross-pathway control<br />

of the pathogenic fungus Aspergillus<br />

fumigatus: a manifold stress response<br />

system<br />

Referent: Gerhard Braus<br />

Jörg Schuldes: Charakterisierung und<br />

Nutzung <strong>der</strong> mikrobiellen Diversität<br />

extremer Habitate <strong>der</strong> Kamtschatka-<br />

Region<br />

Referent: Rolf Daniel<br />

Elke Schwier: Regulators of Ubiquitin<br />

Dependent Protein Degradation in the<br />

Filamentous Fungus Aspergillus nidulans:<br />

Insights into CsnB, DenA and<br />

CandA Function<br />

Referent: Gerhard Braus<br />

Kalpana Singh: The role of protein<br />

phosphorylations in the regulation of<br />

carbon catabolite repression in Bacillus<br />

subtilis<br />

Referent: Jörg Stülke<br />

Andrea Thürmer: Untersuchung <strong>der</strong><br />

Stressantwort von Picrophilus torridus<br />

mittels 2D-Gelelektrophorese und<br />

Charakterisierung ausgewählter Dehydrogenasen<br />

Referent: Wolfgang Liebl<br />

Michael Valdez: Isolation, Characterization<br />

and Identification of Extremely<br />

Thermophilic and Hyperthermophilic<br />

Prokaryotes from Philippine Hot<br />

Springs<br />

Referent: Wolfgang Liebl<br />

Nico Vogt: Governing fungal polar cell<br />

extension: Analysis of Rho GTPase<br />

and NDR kinase signalling in Neurospora<br />

crassa<br />

Referent: Gerhard Braus<br />

Greifswald, Universität<br />

Birgit Heinze: Evolutive Methoden für<br />

Gene mit hohem GC-Gehalt am Beispiel<br />

von Cholinoxidasen<br />

Referent: Uwe Bornscheuer<br />

Robert Kourist: Studies on the enzymatic<br />

synthesis of optically pure tertiary<br />

alcohols<br />

Referent: Uwe Bornscheuer<br />

Falko Hochgräfe: Studies on antioxidant<br />

strategies and protein thiol modifications<br />

in<br />

Bacillus subtilis and Staphylococcus<br />

aureus<br />

Referent: Michael Hecker<br />

Jan Pané-Farré: Structure, function<br />

and regulation of the SigB regulon in<br />

Staphylococcus aureus<br />

Referent: Michael Hecker<br />

Christian Kohler: Stoffwechselphysiologische<br />

Proteomanalysen in Staphylococcus<br />

aureus<br />

Referent: Michael Hecker<br />

Nguyen Van Duy: Transcriptomic and<br />

proteomic signatures of Bacillus subtilis<br />

in response to aromatic compounds<br />

Referenten: Michael Hecker, Haike<br />

Antelmann<br />

Nguyen Thi Thu Huyen: Genomewide<br />

responses and regulatory<br />

mechanisms to thiol-specific electrophiles<br />

in Bacillus subtilis<br />

Referenten: Michael Hecker, Georg<br />

Auling<br />

Halle, Universität<br />

Anja Poehlein: Das Selenoprotein<br />

PrpU als Vermittler zwischen oxidativem<br />

und reduktivem Glycin-Metabolimus<br />

von Eubacterium acidaminophilum<br />

Referent: Jan Andreesen<br />

Thomas Wolsch: Die Pyrrol-2-carboxylat-Monooxygenase<br />

von Arthrobacter<br />

spec. Py1, eine Flavin-abhängige<br />

Vier-Komponenten-MO<br />

Referent: Jan Andreesen<br />

Hamburg-Harburg, Technische<br />

Universität<br />

Moritz Katzer: Production and characterization<br />

of thermostable cellulases<br />

from metagenomic gene libraries<br />

Referent: Garabed Antranikian<br />

PROMOTIONEN 2008 239<br />

Hannover, Universität<br />

Dagmar Kock: Investigations for the<br />

quantification of microorganisms in<br />

sulfidic mine waste dumps, Axel<br />

Schippers<br />

Heidelberg, Universität<br />

Peter Tessarz: Studies on the mechanism<br />

and physiological role of the<br />

AAA+ chaperone ClpB/Hsp104<br />

Referent: Bernd Bukau<br />

Frie<strong>der</strong> Merz: The Mechanism of<br />

Action of the Ribosome-Associated<br />

Chaperone Trigger Factor<br />

Referent: Bernd Bukau<br />

Anna Rutkowska: The Function of<br />

Ribosoe-Associated Chaperone Trigger<br />

Factor in the Co-tranlational Protein<br />

Folding<br />

Referent: Bernd Bukau<br />

Hohenheim, Universität<br />

Sven Kreutel: Interaktion des Photosensors<br />

Ppr aus Rhodocista centenaria<br />

mit Proteinkomponenten <strong>der</strong> chemotaktischen<br />

Signaltransduktion<br />

Referent: Andreas Kuhn<br />

Jena, Universität<br />

Sandra Studenik: Anaerobe O-Demethylieerung<br />

in Acetobacterium dehalogenans.<br />

Untersuchungen zum ATPabhängigenAktivierungsmechanismus<br />

B12-abhängiger Methyltransferasen<br />

Referentin: Gabriele Diekert<br />

Nicole Knabe: Untersuchung von Signalkomponenten<br />

<strong>der</strong> sexuellen Entwicklung<br />

<strong>bei</strong> dem Basidiomyceten<br />

Schizophyllum commune<br />

Referentin: Erika Kothe<br />

Jena, Hans-Knöll-Institut<br />

Katrin Haupt: Rekrutierung von Komplementregulatoren<br />

<strong>der</strong> Faktor-H-Proteinfamilie<br />

als Mechanismen <strong>der</strong><br />

Immunevasion humanpathogener<br />

Erreger<br />

Referent: Peter F. Zipfel<br />

Sophia Poltermann: Funktion von<br />

Gpm1p <strong>bei</strong> <strong>der</strong> Komplementevasion,<br />

Dissemination und Virulenz <strong>der</strong><br />

humanpathogenen Hefe Candida albicans<br />

Referent: Peter F. Zipfel<br />

Al-Abdallah Qusai: Regulation ot the<br />

CCAAT-binding complex of Aspergillus<br />

nidulans un<strong>der</strong> oxidative stress<br />

and iron-depleting conditions<br />

Referent: Axel A. Brakhage<br />

Franziska Lessing: Untersuchungen<br />

zu den molekularen Mechanismen <strong>der</strong><br />

Stressantwort von Aspergillus fumigatus<br />

<strong>bei</strong> Interaktion mit Immuneffektorzellen<br />

Referent: Axel A. Brakhage<br />

Petra Spröte: Regulation and evolution<br />

of the penicillin biosynthesis gene<br />

cluster of Aspergillus nidulans<br />

Referent: Axel A. Brakhage

240 PROMOTIONEN 2008<br />

Uta Schmidt: Das multifunktionelle<br />

Signalprotein Topoisomerase IIß-Bindeprotein<br />

1 (TopBP1) und seine Funktion<br />

in <strong>der</strong> DNA-Schadenserkennung<br />

und Replikation<br />

Referent: Hans Peter Saluz<br />

Martina Werneburg: Chemo-Biosynthese<br />

von Aureothin und verwandten<br />

Pyronverbindungen<br />

Referent: Christian Hertweck<br />

Tina Zöllner: Chemische Derivatisierung<br />

des Makrolid-Antibiotikums Leucomycin<br />

Referent: Christian Hertweck<br />

Martin Siegemund: Analyse <strong>der</strong> Protein-Transphosphorylierungsaktivität<br />

<strong>der</strong> Phosphatindylinositol 3-Kinase<br />

Vps34p aus Candida albicans<br />

Referent: Uwe Horn<br />

Ricardo Almeida: The Exploitation of<br />

Host Iron Sources by Candida Albicans<br />

during Oral Infection, Referent:<br />

Bernhard Hube<br />

Jülich, Forschungszentrum<br />

Verena Engels: Genetic control of<br />

carbohydrate uptake and utilization<br />

in Corynebacterium glutamicum<br />

Referent: Volker Wendisch<br />

Ramon Diesveld: Nutzung heterologer<br />

Transporter <strong>zur</strong> Steigerung <strong>der</strong> L-<br />

Threonin-Bildung mit Corynebacterium<br />

glutamicum<br />

Referent: Hermann Sahm<br />

Vera Krajewski: Modifikation des Glucosestoffwechsels<br />

in Gluconobacter<br />

oxydans<br />

Referent: Hermann Sahm<br />

Christian-Gunnar Schultz: Posttranslationale<br />

Regulation <strong>der</strong> 2-Oxoglutarat-Dehydrogenase<br />

in Corynebacterium<br />

glutamicum<br />

Referent: Michael Bott<br />

Kaiserslautern, Universität<br />

Thomas Lux: Bacteriocine in Streptococcus<br />

pneumoniae<br />

Referentin: Regine Hakenbeck<br />

Alexan<strong>der</strong> Halfmann: Identifizierung<br />

des Regulons des Zwei-Komponenten<br />

Systems CiaRH von Streptococcus<br />

pneumoniae<br />

Referentin: Regine Hakenbeck<br />

Petra Becker: Genetische Determinanten<br />

für die Resistenz von Streptococcus<br />

pneumoniae gegen das neue<br />

Antibiotikum Vancoresmycin<br />

Referent: Bernhard Henrich<br />

Carsten Volz: Analyse genetischer<br />

Verän<strong>der</strong>ungen in einer Familie<br />

Piperacillin-resistenter Mutanten von<br />

Streptococcus pneumoniae<br />

Referent: Bernhard Henrich<br />

Karlsruhe, Universität<br />

Janina Purschwitz: Molekulare Untersuchung<br />

<strong>der</strong> Rolle von Rot- und Blaulichtrezeptoren<br />

in <strong>der</strong> Entwicklung von<br />

Apsergillus nidulans, Referent: Reinhard<br />

Fischer<br />

Frank Leitermann: Entwicklung und<br />

Optimierung eines biotechnologischen<br />

Prozesses <strong>zur</strong> Herstellung<br />

mikrobieller Rhamnolipide auf Basis<br />

nachwachsen<strong>der</strong> Rohstoffe<br />

Referenten: Christoph Syldatk, Clemens<br />

Posten<br />

Ivana Magario: Enzyme Reaction<br />

Engineering for the Conversion of<br />

emulsified Di-Rhamnolipid by free and<br />

immobilized Naringiniase from Penicillium<br />

decumbens, Referenten: Christoph<br />

Syldatk, Matthias Franzreb<br />

Sebastian Rühle: Grundlegende<br />

Untersuchungen <strong>zur</strong> biotechnologischen<br />

Kultivierung von Schwämmen –<br />

Massenbilanzierung <strong>bei</strong> Aplysina aerophoba,<br />

Referenten: Christoph Syldatk,<br />

Clemens Posten<br />

Kiel, Bundesforschungsinstitut<br />

für Ernährung und Lebensmittel<br />

Jochen Dietrich: Inaktivierung von<br />

Bakteriophagen <strong>der</strong> mesophilen und<br />

thermophilen Milchsäurebakterien<br />

Referent: Knut Heller<br />

Antje Hammon: Untersuchungen<br />

zum Vorkommen und <strong>zur</strong> Toxizität von<br />

Bacilllus cereus<br />

in Gewürzen<br />

Referent: Manfred Gareis<br />

Katrin Weidenbach: Identification of<br />

the specific nitrogen regulator NrpR<br />

and the general impact of the histone<br />

on transcription in Methanosarcina<br />

mazei strain Gö1<br />

Referentin: Ruth Schmitz-Streit, Göttingen<br />

Matthias Reher: Zuckerstoffwechsel<br />

von hyperthermophilen und extrem<br />

thermophilen Archaea<br />

Referent: Peter Schönheit<br />

Köln, Universität<br />

Martin Follmann: Untersuchungen<br />

zum Einfluss von pH-Variation und<br />

erhöhter CO2 Konzentration auf Stoffwechsel<br />

und Aminosäureproduktion<br />

mit Corynebacterium glutamicum<br />

Referenten: Reinhard Krämer, Karin<br />

Schnetz<br />

Vera Ott: Der Regulationsmechanismus<br />

des Osmosensors BetP aus<br />

Corynebacterium glutamicum, Referenten:<br />

Reinhard Krämer, Ulf-Ingo<br />

Flügge<br />

Elena Jolkver: Identification and characterization<br />

of carboxylate transporters<br />

in Corynebacterium glutamicum<br />

Referenten: Reinhard Krämer, Ulf-Ingo<br />

Flügge<br />

Sascha Carsten Thomas Nicklisch:<br />

EPR-based structural and functional<br />

characterization of the C-terminal<br />

domain of the osmoregulated glycine<br />

betaine transporter BetP from Corynebacterium<br />

glutamicum<br />

Referenten: Reinhard Krämer, Günter<br />

Schwarz<br />

Konstanz, Universität<br />

Katharina Styp von Rekowski: Zell-<br />

Zell-Interaktionen heterotropher Bakterien<br />

<strong>bei</strong> <strong>der</strong> Besiedelung and dem<br />

Abbau partikulären organischen Materials<br />

im Bodensee<br />

Referenten: Bodo Philipp, Bernhard<br />

Schink<br />

Leipzig, Helmholtz-Zentrum<br />

für Umweltforschung<br />

Florian Centler: Chemical organizations<br />

in natural reaction networks<br />

Referent: Peter Dittrich<br />

Charles Junghanns: Behandlung von<br />

Textilfarbstoffen durch aquatische Pilze<br />

Referent: Gerd-Joachim Krauß<br />

Claudia Martin: Biotransformation<br />

von Mikrokontaminanten durch aquatische<br />

Pilze<br />

Referent: Gerd-Joachim Krauß<br />

Lei Shi: Electro-bioremediation of<br />

hydrophobic organic contaminants:<br />

Effects, mechanisms and interactions<br />

Referent: Hauke Harms<br />

Frank Zielinski: Geobiological coupling<br />

of hydrothermal vent fluids with<br />

endosymbiotic primary producers of<br />

Bathymodiolus mussels from hydrothermal<br />

vents on the Mid-Atlantic Ridge<br />

Referentin: Nicole Dubilier<br />

Leipzig, Universität<br />

Bärbel Thiele: Untersuchungen zu<br />

dearomatisierenden und aromatisierenden<br />

Enzymen des anaeroben Aromatenstoffwechsels<br />

Referent: Matthias Boll<br />

Jörg Johannes: Charakterisierung von<br />

Enzymen des anaeroben mikrobiellen<br />

Abbaus von para-hydroxylierten aromatischen<br />

Verbindungen<br />

Referent: Matthias Boll<br />

Mainz, Universität<br />

Jens Pfannebecker: Entwicklung und<br />

Anwendung molekularbiologischer<br />

Methoden <strong>zur</strong> Art- und Stammidentifizierung<br />

pro- und eukaryotischer<br />

Organismen<br />

Referent: Helmut König<br />

Martin Müllner: Biochemische Charakterisierung<br />

des O2-Sensors NreB<br />

aus Staphylococcus carnosus<br />

Referent: Gottfried Unden<br />

Tanja Zaunmüller: Identifizierung von<br />

Hexosecarriern und Regulation des<br />

Phosphoketolasewegs in Oenococcus<br />

oeni<br />

Referent: Gottfried Unden<br />

Jens Krämer: Der C4-Dicarboxylatund<br />

Citratsensor DcuS aus Escherichia<br />

coli – Signalerkennung und Regulation<br />

Referent: Gottfried Unden<br />

Marburg, MPI für terrestrische<br />

Mikrobiologie<br />

Sandra Kittelmann: Identifizierung<br />

dehalorespirieren<strong>der</strong> Mikroorganismen<br />

in anoxischen Sedimenten mit<br />

Hilfe stabiler Isotope<br />

Referent: Michael Friedrich<br />

Yan Zheng: Identification of transcriptional<br />

regulators for the Ustilago<br />

maydis mig genes<br />

Referent: Christoph W. Basse<br />

Jana Glöckner: Metagenom-Analyse<br />

zum PVC-Superphylum im anoxischen<br />

Reisfeldboden<br />

Referent: Werner Liesack<br />

Dennis Gövert: Carbon isotope fractionation<br />

during the anaerobic degradation<br />

of acetate<br />

Referent: Ralf Conrad<br />

Julia Hin<strong>der</strong>berger: Ferredoxin<br />

Reduktion und Oxidation im Stoffwechsel<br />

von Chlostridium kluyvery,<br />

Referent: Rudolf K. Thauer<br />

David Kamanda-Ngugi: Transformation<br />

and mineralization of nitrogenous<br />

soil components in the gut of soil-feeding<br />

termites<br />

Referent: Andreas Brune<br />

Julian König: Die Identifikation von<br />

Ziel-Transkripten des RNA bindenden<br />

Proteins Rrm4 aus Ustilago maydis<br />

Referent: Michael Feldbrügge<br />

Chetsada Pothiratana: Functional<br />

characterization of the homeodomain<br />

transcription factor Hdp1 in Ustilago<br />

maydis<br />

Referent: Jörg Kämper<br />

Minita Shrestha: Dynamics of methane<br />

oxidation and composition of<br />

methanotrophic community in planted<br />

rice microcosms<br />

Referent: Ralf Conrad<br />

Ulrike Theisen: Nuclear Pore Behaviour<br />

in Interphase and „Open“ Mitosis<br />

of Ustilago maydis<br />

Referent: Gero Steinberg<br />

Marburg, Universität<br />

Peter Friedrich: Substratstereochemie<br />

und Untersuchungen zum Mechanismus<br />

<strong>der</strong><br />

4-Hydroxybutyryl-CoA-Dehydratase<br />

aus Clostridium aminobutyricum<br />

Referent: Wolfgang Buckel<br />

Marina Höing: Struturelle und funktionelle<br />

Untersuchungen an Substratbindeproteinen<br />

aus ABC-Transportern<br />

<strong>zur</strong> Aufnahme von kompatiblen<br />

Soluten<br />

Referent: Erhard Bremer.<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong>

Gloria E. Herrmann Twarz: Enzymes<br />

of two clostridial amino-acid fermentation<br />

pathways<br />

Referent: Wolfgang Buckel.<br />

Elamparithi Jayamani: A unique way<br />

of energy conservation in glutamate<br />

fermenting clostridia<br />

Referent: Wolfgang Buckel<br />

München, Technische Universität<br />

Susanne Kaditzky: Sucrose metabolism<br />

in lactobacilli and bifidobacteria<br />

Referenten: Rudi F. Vogel, Siegfried<br />

Scherer, Michael Gänzle<br />

Jürgen Behr: Mechanisms of hop inhibition,<br />

tolerance and adaption in Lactobacillus<br />

brevis, Referenten: Rudi F.<br />

Vogel, Angelika Görg, Elke Arendt<br />

Martina Fricker: Development of<br />

genotypic and phenotypic methods<br />

for the identification and differentiation<br />

of hazardous Bacillus cereus<br />

group strains<br />

Referent: Siegfried Scherer<br />

Cecilia Rebuffo-Scheer: Development<br />

of a rapid identification system<br />

for Listeria at the species, and Listeria<br />

monocytogenes at the serovar<br />

level by Artificial Neural Network analysis<br />

of Fourier Transform Infrared<br />

Spectra<br />

Referent: Siegfried Scherer<br />

Monica Karen Dommel: Molecular<br />

characterization of the genetic locus<br />

responsible for cereulide toxin production<br />

in emetic Bacillus cereus,<br />

Referent: Siegfried Scherer<br />

Angelika Sell: Virulence gene induction<br />

of enterohaemorrhagic Escherichia<br />

coli by Caenorhabditis elegans,<br />

Acanthamoeba castellanii and Arabidopsis<br />

thalina, Referent: Siegfried<br />

Scherer<br />

Isabel Koch: Immunevasionsmechnismen<br />

von Yersinia enterocolitica:<br />

Hemmung <strong>der</strong> Funktion natürlicher<br />

Killerzellen als neues pathogenetisches<br />

Prinzip bakterieller Krankheitserreger<br />

Referent: Siegfried Scherer<br />

Andreas Bauer: Entwicklung eines<br />

DNS-Mikroarrays <strong>zur</strong> verlässlichen<br />

Identifizierung von Escherichia-coli-<br />

Sicherheitsstämmen<br />

Referent: Karl-Heinz Schleifer<br />

Martin Pilhofer: Elucidation of the<br />

Cell Division Mechanism and Characterization<br />

of Tubulins in the Bacterial<br />

Phylum Verrucomicrobia<br />

Referent: Karl-Heinz Schleifer<br />

München, Universität<br />

Christiane Koller: Regulation des<br />

Säure-induzierten Cad-Systems von<br />

Escherichia coli durch den membranintegrierten<br />

Transkriptionsaktivator<br />

CadC und die Lysin-spezifische Permease<br />

LysP<br />

Referentin: Kirsten Jung<br />

BIOspektrum | <strong>Tagungsband</strong> <strong>2009</strong><br />

Stephanie Kögl: Die hyperosmotische<br />

Stressantwort von Escherichia coli –<br />

von <strong>der</strong> Proteomanalyse zu einzelnen<br />

Komponenten<br />

Referentin: Kirsten Jung<br />

Nina Stambrau: Der LuxP/Al-2- und<br />

LuxQ-abhängige Signaltransduktionsweg<br />

des Quorum Sensing-Systems<br />

von Vibrio harveyi<br />

Referentin: Kirsten Jung<br />

Xaver Sewald: Identifizierung des<br />

Rezeptors und Charakterisierung <strong>der</strong><br />

Aufnahme des vakuolisierenden Cytotoxins<br />

VacA von Helicobacter pylori<br />

in T-Zellen<br />

Referentin: Kirsten Jung<br />

Annika Schmid: Analyse von Regulatorproteinen<br />

des Yersinia-TypIII-Sekretionssystems:<br />

Bedeutung Typ III-spezifischer<br />

Chaperone und Kontrolle <strong>der</strong><br />

Aktivität <strong>der</strong> Phosphoenolpyruvatcarboxylase<br />

Referentin: Kirsten Jung<br />

Kajetan Vogl: Spezifische Anpassungen<br />

<strong>der</strong> Epibionten in dem phototrophen<br />

Konsortium „Chlorochromatium<br />

aggregatum“<br />

Referent: Jörg Overmann<br />

Delita Zul: Molecular ecology and cultivation<br />

dependent analysis of soil<br />

microbial communities<br />

Referent: Jörg Overmann<br />

Isabella Koch: Diversität und Physiologie<br />

neuartiger Acidobacteria aus<br />

Böden<br />

Referent: Jörg Overmann<br />

Katja Junge: Die Funktion <strong>der</strong> CDF-<br />

Transporter MamB und MamM <strong>bei</strong>m<br />

magnetosomalen Eisentransport in<br />

Magnetospirillum gryphiswaldense,<br />

Referent: Dirk Schüler<br />

Münster, Universität<br />

Julia Schumacher: Molecular studies<br />

on heterotrimeric G-protein-mediated<br />

signal transduction in the grey mould<br />

fungus Botrytis cinerea<br />

Referentin: Bettina Tudzynski<br />

Christane Bömke: Molecular analysis<br />

and evolution of gibberellin biosynthetic<br />

gene clusters in filamentous<br />

fungi<br />

Referentin: Bettina Tudzynski<br />

Yvonne Rolke: Signalling in the early<br />

pathogenesis of the ascomycete Claviceps<br />

purpurea: characterisation of<br />

the small GTPases Rac and Cdc42 and<br />

the PAK kinase Cla4<br />

Referent: Paul Tudzynski<br />

Nicole Lorenz: Evolutionary and functional<br />

analysis of the alkaloid biosynthesis<br />

in Claviceps species<br />

Referent: Paul Tudzynski<br />

Tim Stöveken: Bacterial wax ester<br />

synthases/acyl-CoA:diacylglycerol<br />

acyltransferases: Biochemical characterization<br />

and biotechnological<br />

application of a novel enzyme family<br />

Referent: Alexan<strong>der</strong> Steinbüchel<br />

Ahmed Sallam: Biodegradation of<br />

cyanophycin to dipeptides: technical<br />

processing and applications<br />

Referent: Alexan<strong>der</strong> Steinbüchel<br />

Hedda Merkens: Quercetin degradation<br />

in Streptomyces sp. FLA: Biochemical<br />

and spectroscipic characterization<br />

of quercetin-2,4-dioxygenase<br />

QueD and analysis of queDtranscription<br />

Referentin: Susanne Fetzner<br />

John Paluszynski: Investigation of<br />

antimicrobial agents and naturally<br />

secreted yeast killer toxins<br />

Referent: Friedhelm Meinhardt<br />

Oldenburg, Universität<br />

Antje Gittel: Community, structure,<br />

activity and ecophysiology of sulfatereducing<br />

bacteria in deep tidal flat<br />

sediments<br />

Referent: Heribert Cypionka<br />

Anne Berghoff: Eignung von Mikrokosmen<br />

<strong>zur</strong> Bewertung von Selbstreinigungsprozessen<br />

(Natural Attenuation)<br />

im Grundwasser eines ehemaligen<br />

Kokereigeländes<br />

Referent: Heribert Cypionka<br />

Jörg Fichtel: Bestimmung des Endosporenanteils<br />

an <strong>der</strong> mikrobiellen<br />

Lebensgemeinschaft in Wattsedimenten<br />

Referent: Jürgen Rullkötter<br />

Elke Freese: Charakterisierung des<br />

organischen Materials und Untersuchung<br />

<strong>der</strong> temperaturabhängigen<br />

Fettsäurezusammensetzung von Bakterien<br />

aus Sedimenten des Spiekerooger<br />

Rückseitenwatts, Referent<br />

Jürgen Rullkötter<br />

Osnabrück, Universität<br />

Knut Hamann: Systematic Analysis<br />

of Stimulus Perception of the Sensor<br />

Kinase KdpD of Escherichia coli<br />

Referent: Karlheinz Altendorf<br />

Anne-Eva Feuerbaum: In vivo Lokalisations-<br />

und Interaktionsstudien <strong>der</strong><br />

Sensorkinase KdpD aus Escherichia<br />

coli<br />

Referent: Karlheinz Altendorf<br />

Postdam-Rehbrücke, Deutsches<br />

Institut für Ernährungsforschung<br />

Gemma Hen<strong>der</strong>son: Development of<br />

bacterial food preparations with<br />

tumour-prophylactic potential<br />

Referent: Michael Blaut<br />

Potsdam, Alfred-Wegener-<br />

Institut für Polar- und Meeresforschung<br />

Susanne Liebner: Adaptation, spatial<br />

variability and phylogenetic characterization<br />

of methanotrophic communities<br />

in permafrost soils of the<br />

Lena Delta, Siberia<br />

Referent: Dirk Wagner, Rudolf Amann<br />

PROMOTIONEN 2008 241<br />

Regensburg, Universität<br />

Patrizia Spitalny: Analyses of the<br />

Archaeal Transcription Cycle reveal a<br />

Mosaic of Eukaryotic RNA Polymerase<br />

II and III-like Features<br />

Referent: Michael Thomm.<br />

Sebastian Grünberg: Untersuchungen<br />

<strong>zur</strong> Funktion <strong>der</strong> RNA-Polymerase-Untereinheit<br />

E’ und des Transkriptionsfaktors<br />

E<br />

Referent: Michael Thomm<br />

Daniela Näther: Untersuchung <strong>der</strong><br />

Flagellen von Pyrococcus furiosus<br />

Referent: Reinhard Wirth<br />

Stuttgart, Universität<br />

Karin Förster-Fromme: Der Citronellolstoffwechsel<br />

in Pseudomonaden –<br />

Funktionelle Zuordnung beteiligter<br />

Gene und <strong>der</strong>en Produkte<br />

Referenten: Dieter Jendrossek, Arnd<br />

Heyer<br />

Tübingen, Universität<br />

Stefanie Lohmiller: TonB-abhängige<br />

Substrataufnahme <strong>bei</strong> dem Gramnegativen<br />

Bakterium Caulobacter<br />

crescentus<br />

Referenten: Volkmar Braun, Klaus Hantke<br />

Julia Hullmann: Untersuchungen <strong>zur</strong><br />

Expression und Aktivität des Eisenrezeptors<br />

FhuA in ausgewählten Nullmutanten<br />

des Protein-Kontrollsystems<br />

in Escherichia coli K-12<br />

Referenten: Volkmar Braun, Klaus Hantke,<br />

Karl Forchhammer<br />

Claudia Unsin: Genetische und biochemische<br />

Analyse <strong>der</strong> Lysolipin-Biosynthese<br />

und -Resistenz in Streptomyces<br />

tendae Tü4042<br />

Referent: Wolfgang Wohlleben<br />

Yvonne Mast: Biosynthetische und<br />

regulatorische Aspekte <strong>der</strong> Pristinamycin-Produktion<br />

in Streptomyces<br />

pristinaespiralis<br />

Referent: Wolfgang Wohlleben<br />

Yvonne Tiffert: Regulation des Stickstoff-Metabolismus<br />

von Streptomyces<br />

coelicolor M145: Charakterisierung<br />

des globalen Transkriptionsfaktors<br />

GlnR<br />

Referent: Wolfgang Wohlleben<br />

Till Schäberle: Genetische und biochemische<br />

Analyse <strong>der</strong> Glykopeptid-<br />

Resistenz in dem Balhimycin-Produzenten<br />

Amycolatopsis balhimycina<br />

DSM 5908<br />

Referent: Wolfgang Wohlleben<br />

Daniela Eminger: Identifizierung und<br />

Charakterisierung des Aurantimycin-<br />

Biosynthesegenclusters in Streptomyces<br />

aurantiacus IMET 43917<br />

Referenten: Dirk Schwartz, Wolfgang<br />

Wohlleben

242 PROMOTIONEN 2008<br />

Emir Kulauzovic: Untersuchungen<br />

<strong>zur</strong> Rolle <strong>der</strong> Zellwandteichonsäure<br />

von Staphylococcus aureus <strong>bei</strong> Kolonisation<br />

und Infektion<br />

Referenten: Andreas Peschel, Friedrich<br />

Götz<br />

Christine Leoff: Secondary cell wall<br />

polysaccharides in Bacillus anthracis<br />

and Bacillus cereus strains<br />

Referenten: Elmar Kannenberg, Andreas<br />

Peschel<br />

Julia Buschmann: Aktivierung des<br />

angeborenen Immunsystems von Zellwandkomponenten<br />

aus Staphylococcus<br />

aureus<br />

Referent: Friedrich Götz<br />

Steffen Schlag: Dissimilatory nitrate<br />

and nitrite reduction in staphylococci:<br />

regulation and implication in biofilm<br />

formation<br />

Referent: Friedrich Götz<br />

Patrick Müller: Interactions of essential<br />

cell division and cell elongation<br />

proteins with murein synthases in<br />

Escherichia coli<br />

Referent: Friedrich Götz<br />

Ulm, Universität<br />

Beate Schaller: Charakterisierung<br />

von Virulenzregulatoren und Virulenzfaktoren<br />

aus Streptococcus agalactiae<br />

Referent: Dieter Reinscheid<br />

IMPRESSUM<br />

Verantwortlich für den Inhalt:<br />

Prof. Dr. Ulrich Kück<br />

Ruhr-Universität Bochum<br />

Fakultät für Biologie<br />

Lehrstuhl für Allgemeine & Molekulare Botanik<br />

D-44780 Bochum<br />

Tel.: +49 (0)234 - 32 26 212<br />

Fax: +49 (0)234 - 32 14 184<br />

ulrich.kueck@ruhr-uni-bochum.de<br />

Prof. Dr. Franz Narberhaus<br />

Ruhr-Universität Bochum<br />

Lehrstuhl für Biologie <strong>der</strong> Mikroorganismen<br />

Universitätsstraße 150<br />

D-44801 Bochum<br />

Tel.: +49 (0)234 - 32 28 100<br />

Fax: +49 (0)234 - 32 14 620<br />

Franz.Narberhaus@ruhr-uni-bochum.de<br />

Organisation:<br />

Jutta Vach<br />

Conventus<br />

Congressmanagement & Marketing GmbH<br />

Markt 8<br />

D-07743 Jena<br />

Tel.: +49 (0)3641-35 33 15<br />

Fax: +49 (0)3641-35 33 21<br />

jutta.vach@conventus.de<br />

Rita Fischer: Anpassung von Acinetobacter<br />

baylyi Stamm ADP1 an aromatische<br />

Substratbedingungen<br />

Referentin: Ulrike Gerischer<br />

Anke Lübeck: Analyse <strong>der</strong> Interaktion<br />

von Acinetobacter baumanii mit<br />

humanen epitelialen Zellen<br />

Referentin: Ulrike Gerischer<br />

Stephanie Würfl: Molekularbiologische<br />

Untersuchungen <strong>zur</strong> Regulation<br />

<strong>der</strong> Sporulation in Clostridium acetobutylicum<br />

Referent: Peter Dürre<br />

Niklas Nold: Untersuchungen <strong>zur</strong><br />

Regulation des sol-Operons in Clostridium<br />

acetobutylicum<br />

Referent: Peter Dürre<br />

Würzburg, Universität<br />

Stefanie Weibel: Kolonisierung von<br />

soliden Tumoren durch E. coli K12 im<br />

Mausmodell<br />

Referent: Werner Goebel<br />

Sonja Mertins: Einfluss des Kohlenstoff-Metabolismus<br />

auf die Aktivität<br />

des Virulenzfaktors PrfA von Listeria<br />

monocytogenes<br />

Referent: Werner Goebel<br />

Regina Stoll: Einfluss <strong>der</strong> Phosphoenolpyruvat-Phosphotransferasesyteme<br />

auf die Aktivität des Virulenzgenregulators<br />

PrfA von Listeria monocytogenes<br />

Referent: Werner Goebel<br />

Redaktion:<br />

Dr. Christine Schreiber<br />

Redaktion BIOspektrum<br />

Spektrum Akademischer Verlag GmbH<br />

Tiergartenstraße 17<br />

D-69121 Heidelberg<br />

Tel.: +49 (0)6221 - 487 8043<br />

Fax: +49 (0)6221 - 487 68043<br />

christine.schreiber@springer.com<br />

biospektrum@springer.com<br />

Verlag:<br />

Spektrum Akademischer Verlag GmbH<br />

Tiergartenstraße 17<br />

D-69121 Heidelberg<br />

Tel.: +49 (0)6221 - 487 8043<br />

Fax: +49 (0)6221 - 487 68043<br />

Spektrum Akademischer Verlag GmbH ist ein<br />

Imprint von Springer Science+Business Media<br />

Geschäftsführer:<br />

Dr. Ulrich Vest<br />

Anzeigen:<br />

top-ad Bernd Beutel<br />

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Tel.: +49 (0)6201-290 92 0<br />

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Thorsten Berg: Virulenzregulationskaskade<br />

und Chitobiose-Metabolismus<br />

in Vibrio cholerae<br />

Referent: Joachim Reidl<br />

Maria Bösl: Charakterisierung des<br />

Zwei-Partner-Sekretionssystems von<br />

Meningokokken<br />

Referent: Matthias Frosch<br />

Gerhard J. Hutter: Kulturunabhängige<br />

16S rRNA Analyse des subgingivalen<br />

bakteriellen Biofilms <strong>bei</strong> <strong>der</strong><br />

aggressiven Parodontitis<br />

Referent: Rudi F. Vogel<br />

Andrea Villwock: Bedeutung des<br />

Klasse A Scavenger Rezeptors für die<br />

Zytokinsekretion von humanen dendritischen<br />

Zellen nach Kontakt mit<br />

dem humanen Pathogen Neisseria<br />

meningitidis, Referent: Matthias<br />

Frosch<br />

Neelam Dabas: Control of nitrogen<br />

regulated virulence traits of the<br />

human fungal pathogen Candida albicans<br />

Referent: Joachim Morschhäuser<br />

Alexan<strong>der</strong> Hoffmann-Wolz: Bakterielle<br />

Adhärenz an Krytoschnitten<br />

humaner Darmbiopsien – Screening<br />

von Kohlenhydraten auf antibakterielle<br />

Wirkung<br />

Referent: Jörg Hacker<br />

Ulrich Lermann: Molekulare Untersuchungen<br />

<strong>zur</strong> Regulation und Funktion<br />

<strong>der</strong> sekretierten Aspartatproteasen<br />

von Candida albicans<br />

Referent: Joachim Morschhäuser<br />

Claudia Mack: Inhibition des programmierten<br />

Zelltodes und proinflammatorischer<br />

Signale durch das<br />

Cytomegalovirus-Protein M45<br />

Referent: Jörg Hacker<br />

Markus Mezger: Interaktion zwischen<br />

dem humanen Cytomegalievirus,<br />

Aspergillus fumigatus, dendritischen<br />

Zellen und neutrophilen Granulozyten<br />

Referent: Jörg Hacker<br />

Erik Weinmann: Ein neues Konjugationssystem<br />

in Legionella pneumophia<br />

Corby<br />

Referent: Jörg Hacker<br />

Sabrina Scholz: Analyse von zellulären<br />

molekularen Wechselwirkungen<br />

<strong>der</strong> PfCCp-Multiadhäsionsdomäne-<br />

Proteinfamilie und funktionale Charakterisierung<br />

von PfCCp4 in den<br />

Sexualstadien des Malariaerregers<br />

Plasmodium falciparum<br />

Referent: Jörg Hacker<br />

Jaroslaw Zdziarski: Bacterial genome<br />

plasticity and its role for adaptation<br />

and evolution of asymptomic bacteriuria<br />

(ABU) Escherichia coli strains<br />

Referent: Jörg Hacker<br />

Satz:<br />

TypoDesign Hecker GmbH<br />

Stralsun<strong>der</strong> Ring 13<br />

D-69181 Leimen<br />

Tel.: +49 (0)62 24-8 27 60<br />

Fax: +49 (0)62 24-82 76 20<br />

info@typodesign-hecker.de<br />

Abo-Service:<br />

Springer Customer Service Center GmbH<br />

Haberstraße 7<br />

D-69126 Heidelberg<br />

Tel.: +49 (0)6221-345 4304<br />

Fax: +49 (0)6221-345 4229<br />

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Druck:<br />

Stürtz GmbH, Würzburg<br />

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