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

230SSP006Initial proteome analysis of a novel Antarctic haloarchaeal biofilmG. Losensky* 1 , M. Dyall-Smith 2 , F. Pfeifer 1 , S. Fröls 11 Technische Universität Darmstadt, Institut für Mikrobiologie und Genetik,Darmstadt, Germany2 Charles Sturt University, School of Biomedical Sciences, Wagga Wagga,AustraliaAlthough biofilm formation is the predominant modus vivendi ofmicroorganisms in nature it is only poorly characterized in terms ofarchaea. It was recently shown that several haloarchaea are able to adhereto surfaces and form biofilms. Since the underlying mechanisms are stillunknown we used a proteomic approach for an initial investigation offactors involved in biofilm formation of the novel Antarctic isolate t-ADLDL24 which is able to form densely packed multilayer biofilms.Static liquid cultures of t-ADL DL24 were cultivated for 28 days in petridishes before planktonic and adherent cells were harvested separately.Cytoplasmic and membrane fractions were separated by sedimentation.Comparative protein analysis using SDS-PAGE yielded differential proteinpatterns for planktonic and adherent cells. MS-analyses were performed toidentify the protein sets of both phenotypes. Altogether 801 differentproteins were identified in cytoplasmic and membrane fractions ofadherent cells, corresponding to 23 % of the predicted ORFs, whereas atotal of 678 proteins were detected in planktonic cells (20 %). While anoverlap of 573 proteins was found in both phenotypes, 228 Proteins weresolely detected in adherent cells and 105 proteins were associated withplanktonic lifestyle. Categorisation of the phenotype specific proteinsaccording to their cluster of orthologous groups of proteins (COG)provided first insights into the biological processes contributing to biofilmformation of t-ADL DL24. Evidence for an adjustment of energymetabolism in biofilm cells was found, especially proteins indicating achangeover from aerobic to anaerobic energy conversion and multipletransport proteins. Concerning information processing a couple oftranscriptional activators as well as components of signal transductionsystems and stress responses were exclusively detected in adherent cells.Furthermore a number of biofilm specific proteins related to cellularprocesses like synthesis of the cell envelope, type IV pili and biofilmmatrix were identified.The results of this first proteome analysis demonstrate that the biofilmlifestyle goes along with fundamental cellular rearrangements on theprotein level affecting diverse biological processes in haloarchaea.SSP007The role of myo-inositol-1-phosphate synthase in theadaptation of the thermoalkaliphile Caldalkalibacillusthermarum to supraoptimal temperatureF. Kalamorz* 1 , A. Carne 2 , G.M. Cook 31 Institute of Biology / Microbiology, Division of Molecular Microbiology,Halle (Saale), Germany2 Otago School of Medical Sciences, Department of Biochemistry, Dunedin,NZ, New Zealand3 Otago School of Medical Sciences, Department of Microbiology andImmunology, Dunedin, NZ, New ZealandMicroorganisms encounter rapid and dramatic changes in theirenvironment on a regular basis. One of the most likely and threatening ofthese changes is an increase in temperature, impairing protein function,membrane and cell envelope integrity and the performance of chemicalprocesses. Bacteria have evolved several systems to counteract transientand persisting increases in temperatures, universally called the heat shockresponse for short term, transient mechanisms, and heat adaptation forprolonged survival at supraoptimal temperatures. We studied theadaptation of the thermoalkaliphile Caldalkalibacillus thermarum tosupraoptimal temperature. Cultures of C. thermarum incuabted at theoptimal growth temperature of 60C and then exposed to the supraoptimaltemperature of 70C showed a significant increase in the expression ofmetabolic enzymes, including a 256-fold increase in myo-inositol-1-phosphate synthase (mccI) protein levels. This enzyme catalyzes theconversion of glucose-6-phosphate to myo-inositol-1-phosphate and isinvolved in the synthesis of the thermoprotectant di-myo-inositol-1,3’-phosphate (DIP). The gene encoding myo-inositol-1-phosphate synthase inC. thermarum is part of the mccXIC operon encoding a putative CDPalcoholphosphatidyltransferase (mccC), another enzyme of the DIPsynthesis pathway.A degenerated CIRCE element overlaps the start codon of mccX, and thekinetics of mccI induction after exposure to 70C indicate that this operonis regulated by HrcA, the negative regulator of class I heat shock responsein Gram-positive bacteria.Further, expression of enzymes involved in the degradation of myoinositol-1-phosphateto D-glyceraldehyde 3-phosphate and acetyl-coA wasfound to be upregulated at 70C. At the same time, the activity ofphosphofructokinase Pfk, an enzyme of the upper part of glycolysis,showed a 20-fold decrease. This suggests that exposure of C. thermarum tosupraoptimal temperature leads to a re-routing of glucose-6-phosphatefrom glycolysis into inositol synthesis and degradation.SSP008S-bacillithiolation protects against hypochlorite stress in Bacillussubtilis as revealed by transcriptomics and redox proteomicsB. Khanh Chi* 1 , K. Gronau 1 , U. Mäder 2 , B. Hessling 1 , D. Becher 1 ,H. Antelmann 11 University of Greifswald, Institute for Microbiology, Greifswald, Germany2 University of Greifswald, Interfaculty Institute for Genetics and FunctionalGenomics, Greifswald, GermanyProtein S-thiolation is a post-translational thiol-modification that controlsredox-sensing transcription factors and protects active site Cys residues ofessential enzymes against irreversible overoxidation to sulfonic acids. InBacillus subtilis the MarR-type repressor OhrR was shown to senseorganic hydroperoxides via formation of mixed disulfides with the redoxbuffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation[1]. We have studied changes in the transcriptome and redox proteomecaused by the strong oxidant hypochloric acid in B. subtilis [2]. Theexpression profile of NaOCl stress is indicative of disulfide stress asshown by the induction of the thiol- and oxidative stress-specific Spx,CtsR and PerR regulons. Thiol redox proteomics identified only fewcytoplasmic proteins with reversible thiol-oxidations in response to NaOClstress that include GapA and MetE. Shotgun-LC-MS/MS analysesrevealed that GapA, Spx and PerR are oxidized to intramoleculardisulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrRrepressor, two methionine synthases MetE and YxjG, the inorganicpyrophosphatase PpaC, the 3-D-phosphoglycerate dehydrogenase SerAand the putative bacilliredoxin YphP. S-bacillithiolation of the OhrRrepressor leads to up-regulation of the OhrA peroxiredoxin that conferstogether with BSH specific protection against NaOCl. S-bacillithiolation ofMetE, YxjG, PpaC and SerA causes hypochlorite-induced methioninestarvation as supported by the induction of the S-box regulon. Themechanism of S-glutathionylation of MetE has been described inEscherichia coli also leading to enzyme inactivation and methionineauxotrophy. In summary, our studies discover an important role of theBSH redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of key enzymesof the methionine biosynthesis pathway.[1] Lee, J. W., Soonsanga, S., and Helmann, J. D. (2007) A complex thiolate switch regulatestheBacillus subtilisorganic peroxide sensor OhrR. Proc Natl Acad Sci U S A, 104, 8743-8748.[2] Chi BK, Gronau K, Mäder U, Hessling B, Becher D, Antelmann H. (2011)S-BacillithiolationProtects Against Hypochlorite Stress inBacillus subtilisas Revealed by Transcriptomics and RedoxProteomics. Mol Cell Proteomics 10, M111.009506.SSP009How bacteria take care of their kids: Evidence for controlledand fair distribution of PHB granules to daughter cells inRalstonia eutropha H16D. Pfeiffer*, D. JendrossekInstitut für Mikrobiologie, Universität Stuttgart, Stuttgart, GermanyRalstonia eutropha H16 has become the model organism for studyingmetabolism of poly(3-hydroxybutyrate) (PHB), an importantbiodegradable biopolymer that is sustainable produced worldwide in thescale of 10 5 t/a from renewable resources such as sugars [1].R. eutrophacells usually accumulate about a dozen PHB granules during growth athigh C/N-ratios. While biochemistry and molecular biology of PHBaccumulation and PHB biodegradation have been investigated in greatdetail during the last two decades only little is known whether and howsubcellular localization of PHB granules is controlled by the bacteria. Weaddressed this question by performing a two-hybrid approach to screen forproteins with the ability to interact with proteins of the PHB granulesurface [2,3]. Two novel Pha proteins were identified which controlsubcellullar localization of PHB granules and ensure almost equaldistribution of PHB granules to daughter cells after cell division asrevealed by fluorescence microscopy and transmission electronmicroscopy. A revised model for PHB granule formation will be proposed.[1] Reinecke, F., Steinbüchel, A. (2009). J. Mol. Microbiol. Biotechnol.16:91-108[2] Pfeiffer D., Jendrossek D. (2011).Microbiology.157:2795-807.[3] Pfeiffer D., Wahl A., Jendrossek D. (2011).Mol Microbiol.82:936-51.SSP010Food Safety: Is there a positive relationship between heatresistance and dehydration stress of infant pathogen Cronobacter?S. Baumann, C. Halloin, C. Heck, J. Rudat*Karlsruhe Institute of Technology (KIT), Chemical and ProcessEngineering, Karlsruhe, GermanyCronobacter bacteria, formerly classified as Enterobacter sakazakii, havebeen implicated in several incidents as the cause of meningitis andenterocolitis with high mortality rates in premature infants resulting fromBIOspektrum | Tagungsband 2012