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

194conditions tested. Its 2D structure - as validated by enzymatic probing -consists of five independent stem-loops.Overexpression or knockdown of scr5239 results in distinct macroscopicphenotypes. The scr5239 overproduction strain can not express the agaraseDagA and therefore cannot use agar as carbon source (Fig. 1A).Interestingly, the level ofdagAmRNA is not influenced by scr5239.Nevertheless, we identified a direct and specific interaction of the dagAmRNA with scr5239 using competitive gel mobility shift assays andchemical probing. The interaction occurs in the coding region of theagarase mRNA ~ 40 nt downstream of the start codon possibly blockingtranslation [2].DagA, however, is not the only target of scr5239. Currently, we investigatepossible further targets of scr5239 regulation in the central metabolism ofS. coelicolor.1. Vockenhuber M-P, Sharma CM, Statt MG, Schmidt D, Xu Z, et al. (2011) Deep sequencingbasedidentification of small non-coding RNAs in Streptomyces coelicolor. RNA Biol 8.2. Vockenhuber M-P & Suess B, (2011) Streptomyces coelicolor sRNA scr5239 inhibits agaraseexpression by direct base pairing to dagA coding region. Microbiology, acceptedRSV013Two hybrid histidine kinases utilize inter- and intra-proteinphosphorylation to regulate developmental progression inMyxococcus xanthusA. Schramm*, B. Lee, T. Jeganathan, P.I. HiggsMax Planck Institute Marburg, Ecophysiology, Marburg, GermanySignal transduction in bacteria is primarily mediated by histidine-aspartate(His-Asp) phosphorelay [often termed two-component signal transduction(TCST)] proteins. In the paradigm two-component system, the signalingprocess is mediated by two proteins: a histidine protein kinase (HPK) anda response regulator (RR). Signal perception by the HPK leads tophosphorylation of an invariant histidine residue, which then serves as aphosphoryl donor for the aspartic acid residue in the RR. RRphosphorylation modulates a cellular response. In addition to these simpletwo-component systems, the highly adaptable histidine kinase and receivermodules can be arranged into more sophisticated signaling systems whichprovide additional sites for regulation, and integration of disperse signalsinto a common response. These more complex systems are favored inmicroorganisms with complex lifestyles.Upon nutrient limitation, Myxococcus xanthus undergoes a developmentalprocess in which the cells of the swarming community aggregate intomulticellular fruiting bodies and then differentiate into environmentallyresistant spores. M. xanthus encodes a large repertoire of His-Aspphosphorelay proteins, many of which are involved in complex signalingpathways. Several of these complex signaling systems have been shown tobe negative regulators of developmental progression, because therespective mutants develop earlier than the wild type.In this study, we demonstrate that two orphan HyHPK, EspA and EspC,intimately function together in a single signaling system. Using acombination of genetic, biochemical, and bioinformatic analyses, wedemonstrate that EspC’s kinase region does not act as a phosphor donorfor the Esp system. Interestingly however, EspA’s kinase performs intraandinter-molecular phosphorylation of both its own and EspC’s receiverdomain, which together control developmental progression. Additionally,we demonstrate that the Esp system regulates developmental progressionby controlling the proteolytic turnover of MrpC an importantdevelopmental transcription factor. We speculate that this regulateddegradation of MrpC ensures a gradual accumulation of MrpC which isnecessary for coordinated fruiting body formation.RSV014Studies of an Fnr-like transcriptional regulator inGluconobacter oxydans 621HS. Schweikert*, S. Bringer, M. BottForschungszentrum Jülich GmbH, IBG-1: Biotechnologie, Jülich, GermanyThe strictly aerobic -proteobacterium Gluconobacter oxydans is used fora wide variety of industrial applications such as vitamin C synthesis. Onespecial characteristic is the incomplete oxidation of substrates like sugarsor sugar alcohols in the periplasm. Despite its industrial importance,knowledge of the metabolism of G. oxydans and its regulation, especiallyconcerning the sugar metabolism, is still very scarce. Transcriptionalregulators participating in energy and redox metabolism have not beendescribed yet. In silico analysis of the genome sequence revealed 117genes from 38 different regulator families coding for putativetranscriptional regulators (TRs).Concerning the genomic proximity to important genes of the carbonmetabolism or the potential function of the members of the TR repertoire,we selected candidates for further examination. As a consequence of TRdeletion mutant studies we have chosen an Fnr-like regulator, GOX0974,for further characterisation. Fnr (fumarate-nitrate reduction regulator) inEscherichia coli is a switch between aerobic and anaerobic respiration.However, G. oxydans is strictly aerobic and so far no biochemistry foranaerobic respiration has been identified. With regard to these facts, adifferent function of GOX0974 is very likely that does not involve theswitch to anoxic metabolism. The characterisation of this regulatorincluded microarray and physiological analyses of GOX0974 to identifytarget genes and phenotype. Additionally the biochemistry of this proteinwas studied, such as regulator activity and spectral properties. As a result,these experiments gave evidences for a new function of an Fnr-likeprotein.The experimental studies of this regulator are the first presented of atranscriptional regulator from Gluconobacter oxydans.Prust, C., Hoffmeister, M., Liesegang, H., Wiezer, A., Fricke, W.F., Ehrenreich, A., Gottschalk, G.,Deppenmeier, U.: Complete genome sequence of the acetic acid bacterium Gluconobacteroxydans.Nat Biotechnol 2005, 23:195-200.RSV015EIIA Ntr of the nitrogen phosphotransferase system regulatesexpression of the pho regulon via interaction with histidinekinase PhoR in Escherichia coliD. Lüttmann*, B. GörkeInstitut für Mikrobiologie und Genetik, Allgemeine Mikrobiologie, Göttingen,GermanyIn addition to the phosphotransferase system (PTS) dedicated to sugartransport, many Proteobacteria possess the paralogous PTS Ntr . In the PTS Ntrphosphoryl-groups are transferred from PEP to protein EIIA Ntr via thephosphotransferases EI Ntr and NPr. The PTS Ntr has been implicated inregulation of diverse physiological processes in different species (1). In E.coli PTS Ntr plays a role in potassium homeostasis. In particular, EIIA Ntrincreases expression of the genes encoding the high-affinity K + transporterKdpFABC. To this end, EIIA Ntr binds to and stimulates activity of histidinekinase KdpD, which in turn controls expression of kdpFABC (2). Here weshow that the genes belonging to the phosphate (pho) regulon are likewiseregulated by PTS Ntr . The pho regulon is activated by the two-componentsystem PhoR/PhoB under conditions of phosphate starvation (3). However,maximum expression of the pho genes requires EIIA Ntr . The data reveal adirect interaction between EIIA Ntr and PhoR that ultimately stimulatesphosphorylation of response regulator PhoB. Thus, the PTS Ntr modulatesthe activity of two central sensor histidine kinases by direct interaction.(1) Pflüger-Grau und Görke, 2010 Trends Microbiol.18:205-14(2) Lüttmann et al., 2009 Mol.Microbiol.72:978-94(3) Hsieh and Wanner, 2010 Curr Opin Microbiol.13:198-203RSV016A Pseudomonas putida bioreporter strain for the detection ofalkylquinolone-converting enzymesC. Müller*, S. FetznerWestfälische Wilhelms-Universität Münster, Institut für MolekulareMikrobiologie und Biotechnologie, Münster, GermanyPseudomonas aeruginosa is an opportunistic pathogen which regulates itsvirulence via a complex quorum sensing (QS) network. This networkincorporates N-acylhomoserine lactones as well as 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ). PQS and HHQ belong to the over 50 different 2-alkyl-4(1H)-quinolone (AQ) compounds which are produced by P.aeruginosa. They differ mainly in the length and degree of saturation ofthe alkyl chain and the presence or absence of a hydroxyl substituent at theC3-position [1]. Both PQS and HHQ act as the effectors of the LysR-typetranscriptional regulator PqsR and operate as autoinducers in QS [2, 3].We constructed the bioreporter strain P. putida KT2440 [pBBR1-pqsR-P pqsA::lacZ] which constitutively expresses the pqsR gene, whereas the lacZreporter gene is fused to the PqsR-responsive pqsA promoter. Therefore, -galactosidase activity is a function of the PqsR-stimulated transcription,which is dependent on the concentration of HHQ or PQS. The bioreporterstrain is highly sensitive for HHQ (EC 50 1.44 ± 0.23 M) and PQS (EC 500.14 ± 0.02 M).To test whether the bioreporter strain can be used for the detection of AQdegradingenzymes, the hod gene coding for 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase was expressed in the bioreporter. Hod is anenzyme involved in the quinaldine (2-methylquinoline) degradationpathway of Arthrobacter nitroguajacolicus Rü61a and catalyzes thecleavage of 1H-3-hydroxy-4-oxoquinaldine to carbon monoxide and N-acetylanthranilate. It has been shown that Hod is also active towards thestructurally related PQS, forming carbon monoxide and N-octanoylanthranilate [4].P. putida KT2440 [pBBR1-pqsR-P pqsA::lacZ] harboring pME6032-hod wascultivated in the presence of different PQS concentrations. The coexpressionof hod significantly decreased the -galactosidase activity incomparison to the corresponding control strain which contained thepME6032 vector. These results provide proof of principle that thebioreporter strain will be useful for the screening of AQ-convertingenzymes.[1] Lépine F, Milot S, Déziel E, He J, Rahme LG (2004) J. Am. Soc. Mass. Spectrom. 15:862-869[2] Wade DS, Calfee MW, Rocha ER, Ling EA, Engstrom E, Coleman JP, Pesci EC (2005) J.Bacteriol. 187:4372-4380[3] Xiao G, Déziel E, He J, Lépine F, Lesic B, Castonguay MH, Milot S, Tampakaki AP, StachelSE, Rahme LG (2006) Mol. Microbiol. 62:1689-1699BIOspektrum | Tagungsband 2012