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

232nine putative PHB depolymerases have been postulated to exist in R.eutropha. However, except for PHB depolymerase PhaZa1 [1] only little isknown about subcellular localization and in vivo activity of the respectivegene products. In this contribution, fusions of candidate phaZ genes witheyfp were generated and conjugatively transfered to R. eutropha HF39.Localization of expressed fusion proteins was determined under conditionpermissive for PHB accumulation and PHB mobilization. Colocalizationof PhaZa1-eYfp with PHB granules was confirmed for PhaZa1 and wasalso found for PhaZa3, PhaZa4 and PhaZa5 but not for PhaZa2. Fusionswith 3HB-oligomer hydrolases (PhaZb, PhaZc) were homogenouslydistributed in the cytoplasm and a colocalization with PHB granules wasnever observed. Moreover, PhaZd1, a putative PHB depolymerase with sofar highest in vitro depolymerase activity with nPHB granules [2], did alsonot colocalize with PHB in vivo. While it is reasonable to assume soluble3HB-oligomer hydrolases, because 3HB oligomers are water-soluble, thefunction of soluble PHB depolymerases remains unclear.[1] K. Uchino, T. Saito, D. Jendrossek, Appl. Environ. Microbiol. 2008;74(4):1058-1063.[2] T.Abe, T. Kobayashi, T. Saito, J. Bacteriol. 2005;187(20):6982-6990.SSP016Elucidation and studies of a new protein involved in anaerobicphosphite oxidationD. Simeonova*, A. Schmidt, B. SchinkUniversity of Konstanz, Biology, Konstanz, GermanyProtein identification is based on the availability of genomic data. Using“bottom-up” proteomics approaches the identification of proteins is oftenstraightforward. In the absence of genomic data it is highly complex orunfeasible and/or typically requires “de novo”- identification approaches.Here we present the identification approach and some preliminary studiesof a new enzyme involved in the anaerobic phosphite oxidation processbyDesulfotignum phosphitoxidans(strain FiPS-3), a strictly anaerobic andsulfate-reducing bacterium [1].In the presense of phosphite as e-donnor we found a specifically expressedprotein of a molecular mass around 40 kDa on SDS-PAGE gels. Furtherproteomic and genetic studies revealed that this is a new protein which wehave identified as a putative NAD(P)-dependent epimerase/dehydratase[2], with calculated MW mass of 35.8 kDa. The protein was found in themembrane and in the soluble protein fractions ofD. phosphitoxidans. Inaddition we have found that 20% of the total phosphite oxidizing activitywas in the washed membrane fractions ofD. phosphitoxidans. Theestimated molecular weight on 6% native PAGE of this protein is about140 kDa, which suggests that the protein of interest is a homotetramer.This corresponds to the specific differentially phosphorylated pattern thatthis protein showed on 2D SDS PAGE. A more detailed functionalcharacterization of the new protein is presently carried out.[1] Schink B, Thiemann V, Laue H, Friedrich MW. Desulfotignum phosphitoxidans sp. nov., a new marinesulfate reducer that oxidizes phosphite to phosphate. Arch Microbiol 2002 May 177 (55): 381-391.[2] Simeonova D.D.,SusneaI., Moise A., Schink B., Przybylski M. (2009) “Unknown-genome”-proteomics:A new NAD(P)-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR andhigh resolution mass spectrometry. Mol Cell Proteomics 8 (1): 122-131.SSP017Effect of Salt and Matric stress on Growth, Cell SufaceProperties, Membrane Composition and Gene Expression ofPseudomonas putida mt-2M. Schweigert*, J.A. Müller, H.J. HeipieperHelmholtz Centre for Environmental Research, EnvironmentalBiotechnology, Leipzig, GermanyWithin the framework of the EU-project BACSIN (Bacterial Abiotic Stressand Survival Improvement Network) effects of different environmentalstressors on ubiquitously occurring and metabolically versatilemicroorganisms are investigated in order to enable biotechnologicalapplications for bioremediation or biotransformation. The ubiquitouslyoccurring bacterium Pseudomonas putida fulfills these requirements.Therefore, adaptive mechanisms of P. putida mt-2 to salt (sodiumchloride) and matric stress [polyethylene glycol 6000 (PEG 6000)] wereinvestigated on the physiological and transcriptional level. Changes in thephysiology of the cell were recorded by the analysis of growth, cellenvelope hydrophobicity (contact angle measurements) and the charge ofthe cell envelope (zeta potential measurements). Global transcriptionalchanges were monitored via DNA-microarrays. The experiments lead tothe following results:(i) Salt-stressed P. putida mt-2 grew at lower water activities compared tomatric-stressed cells. This suggests that adaptive strategies are moreeffective during exposition to high salt concentrations. (ii) NaCl had aneffect on the fatty acid composition, the hydrophobicity and the surfacecharge of the cell envelope whereas the matric stressor PEG 6000 had noinfluence. With increasing salt concentrations the cell envelope becamemore hydrophobic, more charged and more rigid. (iii) With the help of theDNA-microarray technology general insights in the household of the cellwere obtained. The metabolic activity was restructured due to the influenceof the stressor. Generally, several enzymes of the citric acid cycle, thearginine fermentation, the lipid and the pentose phosphate pathway weredown regulated, whereas enzymes of the lactic acid fermentation(lctP,lldD) and aerobic compound degrading enzymes were up regulated.(iv) Finally, taurine or a similar aliphatic sulphate was identified as apossible compatible solute based on the up-regulation of aliphatic sulphatetransport systems.SSP018Bacterial Interaction Leading to Pattern FormationG. Poxleitner* 1 , A. Boschini 2 , E. Hebisch 1 , J. Rädler 1 , E. Frey 2 , M. Leisner 11 Ludwig-Maximilians-Universität, Lehrstuhl für Experimentalphysik, München,Germany2 Ludwig-Maximilians-Universität, Arnold Sommerfeld Center für TheoretischePhysik, München, GermanyBacterial communities represent complex and dynamic ecological systems.Different environmental conditions as well as bacterial interactions havedetermining influence on establishment and conservation of bacterialdiversity and can lead to so-called pattern formation. Stable coexistence ofseveral bacterial strains is often only possible under well-definedconditions.To study the development of bacterial populations we use time-lapsemicroscopy to investigate the colicin E2 system of threeEscherichiacolistrains labeled with different fluorescent proteins. Combinations ofthese strains, with distinct growth parameters, lead to either instable,metastable or stable coexistence. Besides growth rate and colicinproduction, coexistence was mainly influenced by lag time variations. Inaccordance with the results, two main strategies lead to survival: sensitivestrains need short lag phases and rapid growth rates, while toxin producingstrains even with extended lag phases and slower growth rates can prevail.Specific growth parameters enable cyclic dominance, where the colicinproducingstrain kills the sensitive strain, outgrows the resistant one. Thisin turn has a growth advantage over the first.SSP019RecA-mediated LambdaSo prophage induction in Shewanellaoneidensis MR-1 biofilmsL. Binnenkade* 1 , J. Gödeke 2 , K. Thormann 1 , *L. Binnenkade 1 , J. Gödeke 2 ,K. Thormann 11 Max Planck Institute for Terrestrial Microbiology, Ecophysiology, Marburg,Germany2 Twincore - Zentrum für Experimentelle und Klinische InfektionsforschungGmbH -, Pathophysiologie bakterieller Biofilme, Hannover, GermanyThe respiratory versatile -proteobacterium Shewanella oneidensis MR-1has emerged as a model system for biofilm formation of environmentalbacteria. Our laboratory recently demonstrated that extracellular DNA isan important structural component in all stages of biofilm formation, andthat deletion of prophages (LambdaSo, MuSo1, MuSo2) correlates with asignificant reduction in cell lysis and eDNA release. In order tocharacterize LambdaSo prophage induction in S. oneidensis MR-1 biofilmsin time and space, we generated MR-1 strains carrying venus astranscriptional fusion to regulatory and assembly genes in the LambdaSoprophage genome. Biofilm development under hydrodynamic conditionsand prophage induction was monitored by confocal laser scanningmicroscopy. Our results strongly indicate that induction of prophageLambdaSo occurs 24 hours after initial attachment. Interestingly,significant fluorescence correlated with a filamentous morphology of cellsthat were evenly distributed in the biofilm, but absent in microcolonies.Similar filamentous structures that were mutually exclusive to cellsexhibiting Venus fluorescence were also visible after staining eDNA,suggesting induction of cell lysis after filamentation. Since activation ofthe RecA-mediated SOS-response in E. coli induces filamentation andLambda prophage induction, we determined whether recA is responsiblefor LambdaSo induction in S. oneidensis MR-1 biofilms. Deletion of recAcompletely abolished venus expression during all stages of biofilmdevelopment, indicating suppression of LambdaSo induction. Addition ofhydrogen peroxide to planktonic cultures strongly increased bothfilamentation and prophage induction, and moreover, considerablehydrogen peroxide levels were detected in biofilm associated cells. Basedon these results, we hypothesize that LambdaSo induction is under controlof RecA under biofilm conditions and that oxidative stress may be a directstimulus.BIOspektrum | Tagungsband 2012