VAAM-Jahrestagung 2012 18.–21. März in Tübingen

231feeding with contaminated powdered infant formula (PIF) [1]. PIFtherefore is strictly recommended to be “sakazakii-free” which is definedas the absence of any colony forming unit in 30 samples of 10g of PIF [2].Recent studies e.g. [3] noticed a cross-resistance of Cronobacter strainspointing to common regulation mechanisms for coping with heat and dryresistance which both play a key role in the production of PIF.Investigating these mechanisms, we are going to develop a modifiedproduction process in cooperation with the Food Process Engineeringsection of our institute and the Milchwerke “Mittelelbe” GmbH, anindustrial producer of PIF.[1] Friedemann M (2008), Bundesgesundheitsbl Gesundheitsforsch Gesundheitsschutz 51, 664[2] Besse NG, Leclercq A, Maladen V (2006), J AOAC Int 89, 1309[3] Dancer GI, Mah JH, Rhee MS, Hwang IG, Kang DH (2009), J. Appl. Microbiol. 107, 1606SSP011Characterization of Staphylococcus aureus persister cells upondaptomycin treatmentS. Lechner* 1 , W. Eisenreich 2 , I. Maldener 3 , A. Herbig 4 , K. Nieselt 4 , R. Bertram 11 Universität Tübingen/IMIT, Mikrobielle Genetik, Tübingen, Germany2 Technische Universität München/Chemie, Biochemie, Garching, Germany3 Universität Tübingen/IMIT, Organismische Interaktionen, Tübingen, Germany4 Universität Tübingen/ZBIT, Integrative Transkriptomik, Tübingen, GermanyBacterial cultures contain a subpopulation of dormant cells, persisters.These non-growing cells are phenotypic variants of the wild type that aretolerant but not resistant to antibiotics. As persistence is a transientphenotype it is inherently difficult to study the molecular mechanismsassociated with this kind of bacterial dormancy.Growth phase, strain background, and genotype are critical for theformation of Staphylococcus aureus persister cells. According to ourresults, S. aureus cells in stationary growth phase are generally lessvulnerable by antibiotics than exponential cultures presumably due toelevated persister levels. We previously identified parameters for theselection of S. aureus persister cells. Biphasic killing kinetics, highlyindicative of persisters, were observed by exponential-phase cells treatedwith 10- and 100-fold MIC of tobramycin and ciprofloxacin, respectively.Under stationary growth phase cells challenged with 100-fold MIC ofdaptomycin showed an initial reduction of viable cell counts within thefirst hour (99.98%) followed by a plateau of surviving cells with a ratherslowly decrease in the amount of CFUs. S. aureus SA113 stationary-phasepersisters selected by daptomycin treatment are currently being analyzedfor alterations in transcriptional and metabolic patterns by microarray and13 C isotopologue profiling, and in morphology via transmission electronmicroscopy.The new findings may aid in revealing persister genes in S. aureus as wellas in deciphering physiologic and cellular states of S. aureus persisters.SSP012The influence of supercritical CO 2 on sulphate reducing andmethanogenic enrichment cultures from hydrocarbon reservoirsin GermanyJ. Frerichs* 1,2 , C. Gniese 3 , C. Ostertag-Henning 1 , M. Krüger 11 Bundesanstalt für Geowissenschaften und Rohstoffe, Geochemie derRohstoffe, Hannover, Germany2 BGR-Hannover Geomikrobiologie, Germany3 TU Bergakademie Freiberg, Institut für Biowissenschaften, Freiberg, GermanyLarge-scale solutions are needed to reduce the emissions of greenhousegases such as CO 2 or CH 4 in consequence of their global warmingpotential. Carbon capture and storage (CCS) offers one option for reducingsuch emissions with the storage of CO 2 within depleted gas and oil fields.Our study is focusing on the direct influence of high CO 2 concentrationson the autochthonous microbial population and environmental parametersat such sites.The reservoir within the Schneeren-Husum formation was investigated forits chemical properties, activity profile and microbial communitycomposition via T-RFLP, clone libaries and quantitative-PCR (qPCR).Even within one reservoir differences between two well heads wereobserved in the inducible activity after substrate addition. Also qPCRanalysis showed two distinct communities with varying copy numbers ofseveral bacterial and archaeal genes (16S rRNA, dsrA, mcrA etc.).Pyrosequencing data gave insights into the reservoir community in a directcomparison of produced well head fluids and deep reservoir samples(down hole sampling).High CO 2 had a negative effect on methane and sulphide production inexperiments conducted with amended original fluids and enrichmentcultures. In a second step original fluids (amended with substrate) from thereservoir were incubated for several weeks under near in situ temperature(~50°C) with supercritical CO 2 (100 bar). In this experiment the viabilityof microorganisms together with community changes were investigatedusing quantitative PCR, DGGE and CARD-FISH. In conclusion thisexperiment provides information on possible microbiological changes inthe reservoir during and after storage of CO 2.SSP013Biogenesis of PHB granules in Ralstonia eutropha H16 and inmutants with overexpressed or deleted PhaM and PhaP5 proteinsA. Wahl* 1 , N. Schuth 1 , S. Nußberger 2 , D. Pfeiffer 1 , D. Jendrossek 11 Universität Stuttgart, Institut für Mikrobiologie, Stuttgart, Germany2 Universität Stuttgart, Biologisches Institut - Abteilung Biophysik,Stuttgart, GermanyPoly(3-hydroxybutyrate), PHB, is the most commonpolyhydroxyalkanoate and is important for many bacterial species as acarbon and energy source during times of starvation. Prior to this work, atwo-hybrid approach was applied to screen for uncharacterized proteinswith the ability to interact with PHB synthase (PhaC1) and other PHBrelatedproteins of R. eutropha. As a result, two new proteins - PhaM andPhaP5 - were identified that are involved in biosynthesis of PHB. Bothproteins showed interactions with other PHB-associated proteins and witheach other and colocalized with PHB granules in vivo (as fusion with eYfp).A phaM mutant accumulated only one or two large PHB granules insteadof several medium-sized PHB granules of the wild type, and distribution ofgranules to daughter cells was disordered. This phenotype was reversibleby substitution of phaM in trans. When PhaM was constitutivelyoverexpressed the cells formed many small PHB granules that wereassociated with the cell pole-facing side of the nucleoid region. PurifiedPhaM revealed strong but sequence-independent DNA-binding ability inEMSA experiments in vitro.A phaP5 mutant showed no significant effect on size and localization ofaccumulated PHB granules. However, when PhaP5 was constitutivelyoverexpressed, cells formed smaller PHB granules than the wild type andthe granules were organized in tight bundles always associated to both cellpoles. This phenotype is similar to that of a phaP1-4 mutant. Inconclusion, PhaM and PhaP5 determine number, surface to volume ratio,subcellular localization and distribution to daughter cells of PHB granulesin R. eutropha H16. Subcellular localization of PHB granules in R.eutropha depends on a concerted expression of at least three PHB-granuleassociatedproteins, namely PhaM, PhaP5 and PHB synthase PhaC1.SSP014Killing of Biothreat agents on metallic copper surfacesP. Bleichert* 1 , C. Espirito Santo 1,2 , G. Grass 11 Bundeswehr Institute of Microbiology, Department of Medical BiologicalSpecial Diagnostics and High Security, Munich, Germany2 Universidade de Coimbra, Departamento de Ciências da Vida e Insititutodo Mar (IMAR), Coimbra, PortugalCurrently there is an increasing interest in metallic copper (Cu) surfacesdue to their antimicrobial properties. Using surfaces that might diminishsurface related contamination is of great interest in order to improvehygiene. Dry Cu surfaces demonstrated that at both laboratory conditionsand hospital trials a wide variety of microorganisms get inactivated. Themechanism by which microbes are killed by dry Cu surfaces is still notfully understood. Nonetheless, a microbe faced with Cu surfaces, is rapidlyinactivated within minutes by a quick and high copper uptake. Bacterialspecies able to evade the host immune system are among the most seriouslethal microbial challenges to human health. This group of pathogenscomprises biothreat species classified by the Center for Disease andControl and Prevention (CDC) as bacterial select agents with the potentialto be misused as bioterroristic weapons.We investigated the killing effectiveness of Cu surfaces against Gramnegativebacteria that cause high morbidity and mortally rates in humans(Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei,Francisella tularensis and Yersinia pestis). The pathogens’ inactivationkinetics validates efficient and rapid inactivation on dry Cu surfaces. Ascontrol surface we used stainless steel that is known not to beantimicrobial. Furthermore we tested the ability of Cu surfaces to affectthe membrane-integrity of bacteria after different Cu-exposition times withso called LIVE/DEAD ® staining. By this technique we demonstrated thatCu surfaces damage the membranes of Gram-negative bacteria.These results can be expected to help reinforcing the idea of applying Cusurfaces, for improving hygiene and to aid in the war against nosocomialandother infections.SSP015Unravelling the function of multiple PHB depolymerases in R.eutrophaA. Sznajder*, D. Leuprecht, D. JendrossekUniversität Stuttgart, Inst. f. Mikrobiologie, Stuttgart, GermanyPoly(3-hydroxybutyrate) (PHB) is a biodegradable thermoplast that isproduced by fermentation of the Gram-negative “Knallgasbakterium”Ralstonia eutropha in an industrial scale ( 50.000t/a). More than a dozenof proteins that are relevant for PHB metabolism have been previouslyidentified. Nevertheless many problems especially in understanding theprocesses of intracellular PHB degradation remain unsolved. Up to now,BIOspektrum | Tagungsband 2012