VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

Correspondingly, P. aeruginosa mutants defect in citE were not capable togrow on itaconate. In mammals, (S)-citramalyl-CoA/ (R)-3-hydroxy-3-methylglutaryl-CoA lyase is probably involved in itaconate degradation aswell. The source of itaconate and the role of this pathway in themitochondrial metabolism remains to be shown.PSP030Gene regulation of Geobacter metallireducens underdifferent growing conditionsM.S. Granitsiotis*, S. Marozava, T. Lueders, R.U. MeckenstockInstitute of Ground Water Ecology, Helmholtz Center for EinvronmentalHealth, Neuherberg, GermanyAromatic hydrocarbons concentration in groundwater are often exceedingthe electron acceptor availability of oxygen dissolved in groundwater, whichresults in a change from oxic to anoxic conditions. Contaminant degradationhas, therefore, to proceed anaerobically. However, it is totally unknown howgene regulation functions under in situ conditions. On chemostatexperiments showed that under carbon limiting conditions all catabolicpathways are expressed and different carbon sources are utilizedsimultaneously.We are interested in how genes are regulated in the model organismGeobacter metallireducens under in situ conditions. We cultivated G.metallireducens with different carbon sources (phenol, toluene, benzylalcohol, benzoate, acetate) and under excess and limiting carbon sources(batch culture and retentostat). The expression levels of different catabolicgenes were quantified by RT-qPCR and transcriptome approaches. Our workwill contribute to elucidate what microbes are really doing in theenvironment.[1] Ihssen, J. and T. Egli (2005): Global physiological analysis of carbon- and energy-limited growingEscherichia coli confirms a high degree of catabolic flexibility and preparedness for mixed substrateutilization. Environmental Microbiology 7(10): 1568-1581.[2] Kovarova-Kovar, K. and T. Egli (1998): Growth kinetics of suspended microbial cells: Fromsingle-substrate-controlled growth to mixed-substrate kinetics. Microbiology and Molecular BiologyReviews 62(3): 646-666.[3] Sorek, R. and P. Cossart (2009): Prokaryotic transcriptomics: a new view on regulation,physiology and pathogenicity. Nat Rev Genet 11(1): 9-16.RGV001The role of c-di-GMP in phototactic motility ofSynechocystis sp. PCC 6803 cellsA. Wilde, S. De Causmaecker*Institute for Micro- and Molecular Biology, Justus-Liebig-University,Giessen, GermanyThe cyanobacterium Synechocystis sp. PCC 6803 exhibits flagellarindependent„twitching motility” that allows bacteria to move over moistsurfaces using type IV pili. Mutants that lost type IV pili are non-motile. Inorder to use optimal light qualities and quantities for photosynthesis, theyare able of directed movement along a light gradient. Regulation ofphototactic motility is complex and involves many different gene products,including amongst others different photoreceptors, the RNA chaperone Hfqand adenylate cyclases.Here we demonstrate a biological function of theCph2 photoreceptor in motility. Wild-type Synechocystis cells fail to movetowards blue light, whereas Dcph2 mutant cells show blue-light inducedmotility. Accordingly, Cph2 is responsible for inhibiting cyanobacterialphototaxis towards blue light. Apart from possessing two distinctphotosensory modules, Synechocystis Cph2 differs from most otherphytochromes by harbouring two GGDEF and one EAL domains as effectorregions instead of histidine kinase domains. GGDEF and EAL domains werefound to be involved in the turnover of c-di-GMP, a novel second messengermolecule involved in motility and sessility behaviour in bacteria. Signallingproteins with GGDEF domains synthesize c-di-GMP from two GTPs.Cleavage of c-di-GMP is carried out by EAL domains that exhibitphosphodiesterase activity. We show here that overexpression of the C-terminal GAF domain together with the associated GGDEF domain leads toinhibition of motility, suggesting that light induced changes of the c-di-GMPlevel in Synechocystis cells regulate phototactic responses. In addition, weperformed experiments demonstrating that expression of Cph2 in E. colileads to changes in flagellar-based motility of these cells.RGV002Protein exchange dynamics and chemotaxis clusterstability in Escherichia coliS. Schulmeister*, V. SourjikCenter for Molecular Biology, University of Heidelberg, Heidelberg,GermanyChemotaxis enables bacteria to quickly find optimum growth conditions.Sensing attractants or repellents is based on a simple two-component signaltransduction system. The chemotaxis system of Escherichia coli isthoroughly studied and allows cells to move towards attractants and awayfrom repellents. Effectors are sensed by transmembrane receptors, which areorganized in clusters. The cluster core is composed of receptors, thehistidine kinase CheA and the adaptor protein CheW. All other chemotaxisproteins, like response regulator CheY and its phosphatase CheZ, as well asthe adaptation proteins CheR and CheB localize either to receptors or toCheA. Despite minimal complexity, this system demonstrates amazingperformance that remains partly unaccounted for, despite decades ofintensive research.We established fluorescence recovery after photobleaching (FRAP) tosystematically analyze the turnover of all chemotaxis proteins at bacterialreceptor clusters in vivo, and thereby filled one of the last gaps inquantitative understanding of the chemotaxis pathway. We could separateseveral classes of chemotaxis proteins, that can be assigned to theircharacteristic signaling function [1]. Moreover, we recently extended FRAPanalyses to investigate effects of temperature and pathway activity on clusterstability. Contrary to biochemical observations, temperature did not affectcluster stability in vivo. However, in accordance with biochemical studieswe observed that active clusters were indeed more stable, implying anadditional level of regulation in chemotaxis.[1] Schulmeister, S. et al (2008): Protein exchange dynamics at chemoreceptor clusters in Escherichiacoli. Proc Natl Acad Sci USA 105 (17):6403-6408.RGV003Helicobacter pylori as a new model organism forriboregulation in bacteria lacking the RNA chaperoneHfqS. Pernitzsch, G. Golfieri, C.M. Sharma*Research Center for Infectious Diseases, Julius-Maximilians-University,Würzburg, GermanyQuestion: Genome sequencing of Helicobacter pylori has revealed thepotential proteins and genetic diversity of this prevalent human pathogen,yet little is known about its transcriptional organization and non-codingRNA output. The microaerophilic, Gram-negative ε-proteobacterium waseven regarded as an organism without riboregulation as it lacks the RNAchaperone Hfq, a key player in small RNA (sRNA)-mediated regulation inmany bacteria. However, also Helicobacter has to cope with diversestresses, e.g. pH fluctuations or changes in nutrient availability, duringinfection and colonization of the human stomach. Therefore, we reasonedthat it might also use sRNAs as an additional layer for regulation of geneexpression during stress or virulence.Methods: Massively parallel cDNA sequencing (RNA-seq) has beenrevolutionizing the analysis of transcriptomes from both eukaryotes andprokaryotes. Recently, we have developed a novel differential approach(dRNA-seq) selective for the 5’ end of primary transcripts that allowed us topresent a global map of H. pylori transcriptional start sites (TSS) and itsoperon structure [1]. We discovered hundreds of TSS within operons, andopposite to annotated genes, suggesting that the complexity of geneexpression from the small H. pylori genome is increased by uncoupling ofpolycistrons and by genome-wide antisense transcription. Furthermore, wealso discovered around 60 small RNAs including the ε-subdivisioncounterpart of the regulatory 6S RNA and associated pRNAs, and potentialregulators of cis- and trans-encoded target mRNAs. Now we aim at afunctional characterization of abundant sRNAs and antisense RNAs alongwith their potential role in Helicobacter virulence as well as theidentification of associated RNA-binding proteins and new regulatorymechanisms. For example, microarray-based analyses of global wholetranscriptomechanges of sRNA deletion or overexpression mutants willfacilitate to identify direct mRNA targets. A first example of a classicaltrans-acting sRNA which represses one of the chemotaxis receptors inHelicobacter will be presented.Conclusion: Based on the transcriptome dataset, we are now using H. pylorias a new model organism for sRNA-mediated regulation in bacteria withoutspektrum | Tagungsband 2011