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

differentially expressed if compared to data at pH 6.9 (fold change ≥2). AtpH 4.0 the response was less pronounced with about 120 genes beingdifferentially expressed. 48 genes reacted to both extreme pH values, with16 genes being up-regulated at pH 8.0 and down-regulated at pH 4.0. Thetwo-component system RegSR seems to be involved in the regulation ofseveral of these genes. In the presence of 80 mM NaCl and similar to otherbacteria, B. japonicum exhibits an up-regulation of genes involved insynthesis of osmotic protectants, e.g., trehalose and of genes encodingtransport systems. After heat shock for 15 min at 43°C, 654 genes weredown-regulated and 279 genes were up-regulated. This included the wellknownheat shock genes. Several hundred genes were differentiallyexpressed after cultivation for 48 hours at 35.2°C. Four genes were upregulatedunder all tested stress conditions. Therefore, these genes might beinvolved in the general stress response of B. japonicum. One of the genes(gscR) is likely to encode a transcriptional regulator involved in generalstress response. To test this hypothesis, we created a gscR mutant andanalysed its transcriptome under various stress conditions.SRP004γ-eccompensates for the loss of glutathione in EscherichiacoliC. Schulte*, L.I. LeichertAG Redox Proteomics, Medicine Proteom-Center, Ruhr-University,Bochum, GermanyPartially reduced oxygen forms very toxic Reactive Oxygen Species (ROS).The presence of too much ROS in cells is called oxidative stress. Theglutathione (GSH) system is an important system that protects cells fromoxidative stress. GSH is the main thiol-redox buffer in many organismsincluding the model organism Escherichia coli. It is thought to protect cellsagainst the negative effects of ROS, such as damage of DNA, lipids, orproteins, by maintaining the thiol-redox state of cells. A change in the ratioof reduced and oxidized (GSSG) glutathione has also been observed inseveral diseases. A ΔgshB E. coli mutant strain, with a disruptedbiosynthesis of glutathione, however, shows no apparent growth phenotypeunder standard conditions, when compared to wildtype. This suggested to usthat other Low Molecular Weight Thiols (LMWT) in E. coli could becompensating for the loss of GSH in this mutant. Our HPLC analysesconfirmed the absence of GSH and showed an increased level of γ-glutamylcysteine(γ-EC),a GSH-precursor, in the ΔgshB-mutant. Enzymatic testswith glutathione reductase revealed that γ-EC, unlike other LMWTcommonly found in E. coli, including cysteine and homocysteine, is asubstrate of this enzyme with a K mof 604 μM. Although degradation andredox stability experiments showed that glutathione is more stable whencompared to γ-EC in vitro, stress-experiments showed an equivalentresistance of the ΔgshB-mutant against 3 mM H 2O 2 stress and an even betterresistance against 125 μM paraquat stress when compared to the wildtype.We also detected protein modifications by γ-EC in the mutant comparable toprotein-glutathionylation in the wildtype, which is known to serve as aprotection system against protein damage under oxidative stress. Theseexperiments suggest that γ-EC can partially assume the function ofglutathione in E. coli.SRP005Identification of redox regulated proteins uponperoxynitrite stress in Escherichia coliC. Lindemann*, N. Lupilova, L. LeichertMedicine Proteom-Center, Ruhr-University, Bochum, GermanyPeroxynitrite is a reactive nitrogen species (RNS) that is generated in cells ofthe mammalian immune system to fight off pathogens. Reactive nitrogenspecies are known to damage a wide range of biomolecules. We arespecifically interested in protein modifications that occur in bacteria that aresubjected to peroxynitrite stress. It has been shown that tyrosins aremodified by peroxynitrite and form nitro-tyrosin. With Nitro-tyrosin specificantibodies we could detect a peroxynitrite-concentration-dependent increasein modified tyrosins in Escherichia coli. But Peroxynitrite also targetscysteines. This can lead to a modification of the thiol redox state by theformation of disulfides, S-nitrosylation and S-hydroxylation. Because thesethiol modifications are reversible in vivo and could therefore play a potentialrole in redox-signalling, we additionally investigated the consequence ofperoxynitrite on the thiol-redox proteome in E. coli. Thus, we used an ICAT(isotope coded affinity tag) based method, which allows us to investigate thethiol redox state of proteins in vivo. With this method, we were able toidentify several proteins that are significantly more oxidized in E. coli upontreatment with 1 mM peroxynitrite: the glutathione-dependent formaldeyhdedehydrogenase (FrmA), the asparagine synthetase (AsnB), the malic enzyme(MaeB) and YjgF, a protein of unknown function. Deletion strains in genesencoding these proteins showed a significant defect in cell growth and cellsurvival under peroxynitrite stress, indicating a direct or indirect role of theidentified genes in cell defense mechansims against reactive nitrogenspecies.SRP006Sensing of osmotic stress by salt dependent proteinnucleicacid interaction in the cyanobacteriumSynechocystis sp. PCC 6803B. Roenneke*, J. Novak, K. MarinDepartment for Biochemistry, University of Cologne, Cologne, GermanyUnder osmotic stress conditions most bacteria accumulate compatiblesolutes by uptake or de novo synthesis. Whereas the osmotic stress responseby regulation of gene expression was investigated extensively understandingof the immediate response by biochemical activation of enzymes is scarce.In the cyanobacterium Synechocystis sp. PCC6803 synthesis of the maincompatible solute glucosylglycerole (GG) is triggered by salt stress in atranscription independent manner. The key enzyme is the glucosylglycerolephosphatesynthase (GgpS) for which a novel mechanism of the activityregulation was found. The protein is inhibited by binding to the backbone ofnucleic acids by an electrostatic interaction. Liberation of GgpS is saltdependent and activates the enzyme. Inhibition of GgpS occurs by noncompetitive inhibition indicating inhibitor binding apart from the substratebinding pocket. In order to identify the interaction site or nucleic acidbinding biotinylation of the protein in absence and presence of nucleic acidswas performed and a subsequent analysis by mass spectrometry. Residuescovered by nucleic acids are protected against biotinylation and theaccording peptides show no specific increase in mass. Residues putativelyinvolved in inhibitor binding were exchanged by site directed mutagenesisof the ggpS gene and the impact of these modifications on nucleic acidbinding and enzyme activity will be discussed.SRP007Systemic analysis of bacterial aconitase deletion mutantsreveals a strong selection pressure for secondarymutations inactivating citrate synthaseM. Baumgart*, N. Mustafi, A. Krug, M. BottInstitute of Bio- And Geosciences (IBG), Department of Biotechnology,Research Center Jülich, Jülich, GermanyAconitase, a 4Fe-4S cluster containing protein, catalyses the second step ofthe tricarboxylic acid cycle, the reversible isomerisation of citrate toisocitrate [1]. In the past years it was shown that the aconitase gene acn ofthe Corynebacterium glutamicum, a member of the actionbacteria, is subjectto a complex expression control by four different transcriptional regulators[2-5]. In order to better understand the causes for this elaborate regulation, aC. glutamicum ∆acn mutant was analysed regarding growth, proteome,transcriptome, and secretion of organic acids. The mutant was glutamateauxotrophic,showed a strong growth defect and secreted large amounts ofacetate. Importantly, none of these phenotypes could be complemented byplasmid-encoded aconitase, suggesting the presence of a secondarymutation. In fact, a point mutation within the gltA gene encoding citratesynthase was identified, which caused degradation of this protein and analmost complete lack of its enzymatic activity. Subsequently, 27 further,independent ∆acn clones were isolated and 15 of them were found to containmutations in the gltA gene causing loss of citrate synthase activity. Elevatedintracellular citrate concentrations were considered to be the main cause ofthis selection pressure. Citrate toxicity was subsequently investigated bycitrate pulse experiments with a C. glutamicum strain overexpressing thecitrate carrier CitH. In fact, rapid citrate uptake by cells not adapted to thissubstrate elicited a complete, though temporary growth inhibition.According to these results, the tight control of aconitase synthesis mighthave evolved due to the necessity to avoid toxic citrate levels on the onehand and the excessive synthesis of a labile protein requiring both iron andsulphur on the other hand.[1] Baumgart, M. And M. Bott (2010): Biochemical characterisation of aconitase fromCorynebacterium glutamicum. J Biotechnol :doi:10.1016/j.jbiotec.2010.1007.1002.spektrum | Tagungsband 2011