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

cations. Besides the catalase dependent damage of proteins the H 2O 2dependent decomposition of DNA was analysed. To address the variety andthe extent of H 2O 2 induced oxidative modifications at the proteome level thecatalase mutant was applied as a tool in LC-MS/MS studies. Interestingly,numerous oxidative modifications were found even for wild type cells underin vivo conditions during fermentation of C. glutamicum in controlledbioreactors. In particular enzymes of central metabolic pathways wereidentified as targets. Our results underline the continuous formation of ROSand unravel their deleterious effects on the physiological performance of C.glutamicum in spite of the presence of a highly active catalase enzyme.SRV008On the multitude of mechanisms that establish high-levelheavy metal resistance in an aggregate formingbacteriumG. Sturm*, J. GescherDepartment of Microbiology, Albert-Ludwigs-University, Freiburg,GermanyIn the last decades chromium has become a wide spread pollutant in theenvironment. This is mainly due to anthropogenic factors, namely an ofteninadequate toxic waste management in leather tannery, dye-, car- and steelindustry.Consequently chromium has become the most important heavymetal pollutant in the European Union. The toxicity of chromium isdependent on its oxidation state. Cr(VI) is the most toxic and bioavailableform, whereas Cr(III) is only sparsely soluble and therefore less toxic.In this study the chromate resistance strategy of a new Leucobacter species(L. chromiiresistens) was investigated [1]. This species is capable oftolerating more than 300 mM chromate and shows a distinct correlationbetween the chromate concentration in the medium and the production ofaggregates. Formation of these aggregates accompanies with the enhancedproduction of extracellular polymeric substances (EPS), mainly extracellularDNA (eDNA) and sugars. Extracellular DNA was shown to be essential forthe structural integrity of the aggregates. Inhibition of aggregate formationvia DNaseI treatment resulted in an almost complete loss of resistanceagainst potassium chromate. Our hypothesis regarding the role of EPSproduction and cell aggregation is that these factors result in decreasedCr(VI) uptake and therefore reduce intracellular Cr(VI) concentrations.Besides aggregate formation, Leucobacter chromiiresistens produces acarotene-related pigment in the membrane as a response to chromium stress.Carotenes are known to function as radical quenchers in photosyntheticorganisms. In L. chromiiresistens they might protect the cell from lipidperoxide formation triggered by chromium radicals. Last but not least wecould measure a soluble cytoplasmic chromate reductase activity. NAD(P)Hserves as electron donor for this enzyme.We suggest that aggregate formation, carotene production and chromatereductase expression serve in an orchestrated way to protect the cell fromoxidative stress caused by chromium(VI) or chromium radicals.[1] Sturm, G. et al: Leucobacter chromiiresistens sp. nov., a novel chromate-resistant strain in thegenus Leucobacter. Int J Syst Evol Microbiol.SRV009Osmotic stress response in Bacillus subtilis - integrationof the fluxome with the regulatory networksM. Kohlstedt* 1 , J. Becker 1 , C. Korneli 1 , P.K. Sappa 2 , H. Meyer 3 , S. Maaß 4 ,M. Lalk 3 , U. Mäder 2 , E. Bremer 5 , M. Hecker 4 , U. Völker 2 , C. Wittmann 11 Institute of Biochemical Engineering, Braunschweig, Germany2 Institute for Genetics and Functional Genomics, Functional Genomics,Greifswald, Germany3 Institute of Pharmacy, Pharmaceutical Biology, Greifswald, Germany4 Institute for Microbiology, Microbial Physiology and Molecular Biology,Greifswald, Germany5 Institute for Molecular Microbiology, Marburg, GermanyBacillus subtilis is one of the major industrial working horses inbiotechnology. In industrial production environments it typicallyexperiences high osmolarity, making this an important parameter to beinvestigated. The specific osmotic stress response of Bacillus has beenelucidated in detail [1] but information about the integration into theregulatory network of B. subtilis is still incomplete. Protection against anosmotic challenge is primarily conferred by a specific adaptational responsethat controls the uptake, synthesis and accumulation of osmoprotectivesubstances. In addition to the uptake of compatible solutes, e.g. glycinebetaine, Bacillus subtilis is able to synthesize amino acids de novo especiallyglutamate and proline to counteract the external osmotic pressure.Furthermore, this specific osmoadaptation response is integrated with theSigB-dependent general stress response, because genes such as opuD andopuE are subject to overlapping control by SigB.In the present work, the response of Bacillus subtilis 168 trp + to osmoticstress was assessed by a polyomics approach, integrating the fluxome asfunctional network output of Bacillus subtilis with its cellular componentsinvolving metabolome, proteome and transcriptome analysis. For thispurpose, cells were grown in glucose-limited chemostats at NaClconcentrations up to 1.2M. At metabolic steady-state, samples wereanalyzed for systems-wide metabolome, transcriptome, proteome andfluxome analysis. This should unravel regulatory interactions between thedifferent functional layers of the cell [2].[1] Kempf, B. And E. Bremer (1998): Uptake and synthesis of compatible solutes as microbial stressresponses to high-osmolarity environments, Arch. Microbiol, 170:319-330.[2] Kohlstedt, M. et al (2010): Metabolic fluxes and beyond - systems biology understanding andengineering microbial metabolism, Appl. Microbiol. Biotechnol. 88:1065-1075.SRV010Post-transcriptional activation of the SacP phosphatasecounteracts phosphosugar stress in enterobacteriaK. Papenfort* 1 , D. Podkaminski 1 , C.K. Vanderpool 2 , J. Vogel 11 Institute for Molecular Infection Biology, Julius-Maximilians-University,Würzburg, Germany2 Department of Microbiology, University of Illinois at Urbana-Champaign,Urbana, USAQuestion: The small regulatory RNA SgrS is well known to counteractphosphosugar stress, a process that involves the post-transcriptionaltargeting of the ptsG mRNA, coding for the major glucose transporter [1].Bacterial non-coding RNAs have now been established to control theexpression of multiple target genes rather than single transcripts. In thisstudy we aimed to elucidate the target profile of SgrS in the model pathogenSalmonella Typhimurium.Methods: To investigate the role of SgrS in S. Typhimurium we made useof a pulse-expression approach that combines tightly controlled expressionof an sRNA from an inducible promoter with whole genome microarraysanalysis [2].Results: Our analysis revealed an extended SgrS regulon, displaying alarger set of repressed mRNA targets, but also up-regulation of a singletranscript, termed sacP. Interestingly, sacP is the 2nd gene of a polycistronicmessenger, however SgrS mediated gene activation is limited to sacP anddoes not render the expression of other members of this operon.Mechanistically, this up-regulation involves RNA duplex formation of SgrSwith distal parts of the preceding pldB mRNA and requires the action of theRNA chaperone Hfq and the RNase E ribonuclease. Biocomputational andbiochemical analysis have shown that SacP belongs to the group of HADphosphatasesthat display high affinity towards phosphorylated sugarsubstrates, including Glucose-6-phosphate [3]. Indeed, under phosphosugarstress conditions, post-transcriptional up-regulation of SacP by SgrS iscritical for cellular replication, suggesting that SacP activation is required todecrease the intracellular amount of phosphorylated sugars.Conclusions: We present a sophisticated mechanism of discoordinateoperon expression that leads to induction of the conserved sugarphosphatase SacP. SacP is required to dephosphorylate accumulated sugarcompounds and required for counteraction of phosphosugar stress inbacteria.[1] Vanderpool and Gottesman (2004): Mol. Microbiology 54(4):1076-89.[2] Papenfort et al (2006): Mol. Microbiology 62(6):1674-88.[3] Kuznetsova et al (2006): JBC; 281(47):36149-61.SRV011The Phage-Shock Protein LiaH of Bacillus subtilisD. Wolf* 1 , M. Reineck 1 , B. Voigt 2 , T. Mascher 11 Department I/Synth. Biologie, Ludwig-Maximilians-University, Munich,Planegg-Martinsried, Germany2 Ernst-Moritz-Arndt-University, Greifswald, GermanyThe LiaRS two-component system (TCS) is part of the cell envelope stressresponse in Bacillus subtilis, which is triggered by compounds that affect theintegrity of the cell wall [1, 2]. The main target of the response regulatorLiaR is the liaI promoter, resulting in a strong induction of the liaIH operon,which encodes a small putative membrane protein and a member of thespektrum | Tagungsband 2011