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

229chemical constituents are avaliable.This adaption continues to the stage ofsporulation where the structure and size of the sporulating entity is vital tothe next stage in the life cycle pathway.Two species that show thisadaption well are xanthomonas and clostridia in the strains xanthomonascampestris pv campestris and clostridia felsineum pv felsineum.It can beseen both in structural analysis of the pathway products of these twoexamples by various methods how this adaption occurs.This process ofadaption gives a deep insight into the microbial organisms selfmodification within the life cycle pathway.SSP002Membrane-active antimicrobial peptides can trigger thetransition of bacteria into a dormant stageM. Berditsch* 1 , T. Vladimirova 1 , T. Jäger 1 , P. Wadhwani 2 , A.S. Ulrich 1,21 Karlsruhe Institute of Technology, Institute of Organic Chemistry/Chairof Biochemistry, Karlsruhe, Germany2 Karlsruhe Institute of Technology, Institute of Biological Interfaces (IBG-2), Karlsruhe, GermanyQuestion: One of the survival strategies of bacterial populations is theproduction of a small number of dormant persister cells that grow in aform of small-colony variants (SCVs) [1]. These are tolerant to antibioticsand represent an attached bacterial growth in microbiofilm mode. Thetransition into the dormant stage occurs during the SOS response and ismodulated in E.coli by the membrane associated TisB peptide, whichdecreases proton motive force and ATP biosynthesis [2]. We observed thatantimicrobial peptides (AMPs), which interact with the bacterial plasmamembrane and perturb its integrity [3], can have a similar effect as TisBand lead to the generation of undesirable persister cells. Since these cellsreduce their metabolic activity, we suggest that cell respiration may serveas a possible indicator of the transition of cells into the dormant stage.Method: We developed a microdilution alamarBlue respiration assayfor monitoring the Bacterial Respiration (BR) as a cellular response tounfavourable conditions. The respiration activity of four bacterial strainswas measured as a difference in the absorption between reduced andoxidized forms of the redox indicator alamarBlue during 3 hours afterinoculation. The microtiter plates were then incubated for 24 hours at 37°Cto evaluate the ability of cells to grow.Results: By monitoring of BR, we observed three different kinds ofrespiration activity with respect to bacterial growth: activation, correlation,or shutdown, - depending on the combination of bacteria and AMP.Shutdown of initial BR at sub-MIC and subsequent growth at higherpeptide concentrations was observed for S.aureus upon exposure to MAP,PGLa, Magainin and a combination of PGLa/Magainin. In those cases,single cells of S.aureus were found to survive and form SCVs, which werevisible by magnification and were detectable via dye reduction.Conclusion: These findings suggest that exposure of S.aureus to thesemembrane-active AMPs induces a transition of single cells into thedormant stage, which can enhance risk of persistent infections.1. R. Singh, et al., 2009.J Med Microbiol., 58(8): 1067-73.2. T. Doerr et al., 2010.PLoS Biology, 8(2): 1-8.3. M. Hartmann et al., 2010.Antimicrob Agents Chemother., 54(8): 3132-3142.SSP003About a mechanism of stress dependent enzyme activityregulation via interaction with nucleic acids - salt dependentGG-synthesis in Synechocystis sp. PCC 6803B. Roenneke*, K. MarinUniversität zu Köln, Institut für Biochemie, Köln, GermanyUnder osmotic stress the synthesis and accumulation of the compatiblesolute glucosylglycerol in the cyanobacterium Synechocystis sp. PCC 6803is facilitated by the activation and fine tuning of the key enzyme of thepathway the Glucosylglycerole phosphate synthase (GgpS). At low saltconcentrations GgpS is inhibited via an electrostatic interaction withnucleic acids (Novak et. al 2010). Upon a sudden increase of the saltconcentration GgpS is liberated and present in its active form. During saltacclimation the largest fraction of GgpS is rebound by nucleic acids whilethe remaining active molecules ensure the ongoing GG-Synthesisaccording to the external salt concentration and growth rate.Biotinylation assays revealed the possible binding site for nucleic acids an-helix near the active centre of GgpS. Four positively charged aminoacids might contribute to the interaction, since an exchanged of theseamino acids against alanine caused a reduced affinity for the binding andhad an impact on enzyme activity. We found that binding to nucleic acidsleads to a conformational change of the protein and characterised thebinding and inhibition kinetics.Additionally, we compared regulatory features of GgpS enzymes orsimilar trehalose phosphate synthases (TPS) originating from organismsadapted to different environments with respect to fluctuations of the saltconcentration. We found proteins that do or do not bind to nucleic acidsand are or are not inhibited accordingly. The structural differences and theimpact of the regulatory circuit of osmolyte synthesis will be discussed.SSP004MscCG of Corynebacterium glutamicum - a mechanosensitivechannel with dual function in osmotic stress response andglutamate productionM. Becker*, K. Börngen, R. KrämerUniversität zu Köln, Institut für Biochemie, Köln, GermanyCorynebacterium glutamicum is a gram-positive, biotin auxotroph andapathogenic soil bacterium with exceptional importance for the industrialproduction of various amino acids, especially L-glutamate. Themechanism of glutamate export is not fully understood so far, although C.glutamicum has been used for the industrial production of amino acids fordecades. Glutamate excretion can be induced by several differenttreatments, all altering the cell envelope. Recently, evidence was providedthat the small MS channel protein MscCG of C. glutamicum is linked toglutamate excretion under glutamate production conditions. MscCGbelongs to the MscS-type family of mechanosensitive channels,functioning as emergency valves upon an osmotic downshift. The proteinis a close relative of the mechanosensitive channel MscS from E. coli (286AA) concerning its N-terminal and pore region. In addition, MscCGcarries a long C-terminal domain of 247 amino acids including a fourthtransmembrane domain. The electrophysiological analysis of MscCGshowed the typical pressure dependent gating behavior of a stretchactivatedchannel with a current/voltage dependence indicating a stronglyrectifying behavior. To unravel the dual function of MscCG as amechanosensitive channel and as a glutamate exporter, the wellcharacterized E. coli homolog of MscCG, MscS, was used and expressedin a mscCG strain. We also generated selected C-terminal truncations ofMscCG in C. glutamicum, gain-of-function and loss-of-function constructsof both E. coli MscS and C. glutamicum MscCG, as well as fusionconstructs of these two proteins, and we have investigated the properties ofthese constructs with respect to mechanosensitive efflux, electricalconductance, gating properties, as well as glutamate excretion. Variousrecombinant forms of MscCG were shown to be closely similar withrespect to conductance, but we found significantly differences concerningglutamate excretion. The results of these experiments argue for MscCGbeing both a relevant mechanosensitive channel in C. glutamicum uponhypoosmotic stress as well as the major efflux pathway for glutamateexcretion in response to particular physiologic conditions. Moreover, theresults obtained indicate the C-terminal domain of MscCG being ofsignificant impact for function and/or regulation of MscCG activity.SSP005Insights into biofilm formation by initial proteome analysisof a novel Antarctic haloarchaeal isolateS. Fröls* 1 , G. Losensky 1 , M. Dyall-Smith 2 , F. Pfeifer 11 TU Darmstadt, Institut für Mikrobiologie und Genetik , Darmstadt, Germany2 Charles Sturt University, School of Biomedical Sciences, Wagga Wagga,AustraliaThe formation of biofilms initiated by the adhesion to surfaces turns out tobe the most dominant mode of life of microorganisms in nature. Thescreening and characterisation of various haloarchaea strains and isolatesdemonstrated that the ability for adhesion is widely distributed within thegenera Halobacterium salinarumand Haloferax. Further characterisationshowed that the adhesive strains were able to form biofilms. Based on thisobservation we tested seven novel isolates from water samples of ahypersaline Antarctic deep lake by a fluorescence-based assay foradhesion. In comparison to the other haloarchaea the novel Antarcticisolate DL24 showed the highest adhesion signal and was chosen for moredetailed analyses. Isolate DL24 is a rod shaped, motile, extremelyhalophilic archaeon, representing a new genus of the Halobacteriaceae.Thecells are able to adhere on glass and plastic surfaces forming biofilms ofdense packed multi cell layers with towering macrocolonies up to 50 m inheight. Extra polymeric substances (EPS) were composed of free nucleicacids and glycoconjugates.To identify factors involved in adhesion a proteome analyses of wholeprotein fractions were carried out by mass spectrometry. By comparativeanalysis 220 proteins were exclusively identified in the protein fraction ofadherent cells. Among these were transcriptional regulators, differentfactors of two-component systems, a glycosyltransferase, a surfaceglycoprotein as well as proteins required for a functional type IV pilisystem. The largest group of proteins corresponded to transport systemssuch as sugar, ion and multidrug transporters. In addition proteinsdemonstrating a transition from aerob to anaerobic energy conversion werefound. This initial proteome analyses showed distinct differences betweenthe adherent and planktonic lifestyle belong to environmental response,transcriptional differentiation, adhesion and biofilm maturation.BIOspektrum | Tagungsband 2012