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

197Through intensive investigations aiming at increased production, C.glutamicum has become a model organism for systems biology as well [2].We will present a targeted approach for direct quantification of keyenzymes from the central carbon metabolism in C. glutamicum rawextracts by high performance liquid chromatography coupled tandem massspectrometry [LC-MS/MS, 3]. Focusing on glycolysis, TCA, anaplerosisand glyoxylate shunt our method provides a quantitative overview of theenzymes building the core metabolic pathways in C. glutamicum. Ametabolic labeling strategy with the stable nitrogen isotope 15 N is used toovercome measurement errors originating from sample handling and trypticdigestion of protein extracts by isotope dilution mass spectrometry [IDMS, 4].Sampling batch cultivations of C. glutamicum in microtiter plates, ourstudy comprises proteome adaptations to different growth phases andalternative carbon sources. Results show massive reconstitutions of proteinlevels well agreeing to known changes of metabolic fluxes. Furthermore,we conducted time resolved measurements of protein expression aftermetabolic switch from glycolytic to gluconeogenetic carbon sources underindustrial relevant conditions in stirred tank reactors. Significant changesin protein levels could be detected within 15 min after substrate pulse.In conclusion we will present a rapid and reliable methodology forquantitative analysis of protein expression and dynamics providing newinsights into metabolic regulation of C. glutamicum.[1] Eggeling, L., Bott, M.,Handbook of Corynebacterium glutamicum, Academic Press, Inc., BocaRaton, FL 2005.[2] Wendisch, V. F., Bott, M., Kalinowski, J., Oldiges, M., Wiechert, W., EmergingCorynebacterium glutamicum systems biology. J Biotechnol 2006, 124, 74-92.[3] Lange, V., Picotti, P., Domon, B., Aebersold, R., Selected reaction monitoring for quantitativeproteomics: a tutorial. Mol Syst Biol 2008, 4, 222.[4] Mayya, V., K Han, D., Proteomic applications of protein quantification by isotope-dilution massspectrometry. Expert Rev Proteomics 2006, 3, 597-610.RSP011Unusual reactions involved in cyclohexanecarboxylate formationduring crotonate fermentation in Syntrophus aciditrophicusL. Ebelt* 1 , J.W. Kung 1 , A. Schmidt 2 , M. Boll 11 Universität Leipzig, Institut für Biochemie, Leipzig, Germany2 Universität Konstanz, Dept für Biologie - Mikrobielle Ökologie,Konstanz, GermanyThe obligately anaerobic Deltaproteobacterium Syntrophus aciditrophicuscan feed on crotonate as its sole carbon and electron source without asyntrophic partner. The main products of the fermentation pathway areacetate and cyclohexanecarboxylate [1]. The reducing equivalents formedduring crotonate oxidation to acetate are recycled by concomitantreduction of crotonate in reverse -oxidation-like reactions of the benzoyl-CoA degradation pathway. The transiently formed benzoyl-CoA isbelieved to serve as electron acceptor for recycling redox equivalentsyielding six-electron reduced cyclohexanecarboxyl-CoA. We demonstratethat disproportionation reactions of cyclohexa-1,5-diene-1-carboxyl-CoA(1,5-dienoyl-CoA) and cyclohex-1-ene-1-carboxyl-CoA (1-monoenoyl-CoA) are involved in benzoyl-CoA and cyclohexanecarboxyl-CoAformation. These reactions are most likely catalyzed by tungstencontaining class II benzoyl-CoA reductases [2]. The cyclohexanecarboxyl-CoA is converted into the end product cyclohexanecarboxylate by athioesterase or a CoA transferase. The endergonic reductivedearomatization of benzoyl-CoA to 1,5-dienoyl-CoA by NADH (G°’ =+58 kJ mol -1 ) can be explained by an electron bifurcation mechanism. Wepropose that this reaction is driven by the concomitant reduction of 1,5-dienoyl-CoA to 1-monoenoyl-CoA and/or 1-monoenoyl-CoA tocyclohexanecarboxyl-CoA by NADH to (G°’ < -50 kJ mol-1).[1] Mouttaki, H. et al (2008): Use of benzoate as an electron acceptor bySyntrophusaciditrophicusgrown in pure culture with crotonate. Env Microbiol 10(12):3265-3274.[2] Kung, J.W. et al (2010): Reversible Biological Birch Reduction at an Extremely Low RedoxPotential. Proc Nat Acad Sci 132:9850-9856.RSP012Mutational analysis of the transcriptional regulator AlsR ofBacillus subtilisC. Frädrich*, E. HärtigTU Braunschweig, Mikrobiologie, Braunschweig, GermanyAcetoin formation in Bacillus subtilis requires acetolactate synthase and -decarboxylase encoded by the alsSD operon. The alsSD expression isactivated in response to fermentative growth conditions, addition ofacetate, low pH in the growth medium and aerobic stationary growth. Thetranscriptional regulator AlsR is essential for alsS-lacZ reporter geneexpression under all growth conditions tested. The AlsR regulator is amember of the LysR-type transcriptional regulators (LTTR) and composedof two domains: an N-terminal DNA binding domain with a winged HTHmotif and a C-terminal regulatory domain which is involved in co-inducerbinding and oligomerization.To identify functional relevant amino acid residues for effector-bindingand oligomerization we mutagenized the alsR gene in the C-terminalregulatory domain and tested the activity of the produced AlsR mutantproteins in an in vivo complementation system. Here, mutated alsR geneswere integrated into the amyE locus of a B. subtilis alsR knock out mutantstrain and expressed under the control of the xylose-inducible xylApromoter. AlsR activity was monitored by ß-galactosidase activitiesderived from an AlsR-dependent alsS-lacZ reporter gene fusion. SeveralAlsR mutants tested showed reduced alsS-lacZ expression in vivo.In addition, we produced and purified the AlsR mutant proteins asTrx/Strep-AlsR fusion proteins and after cleavage with the HRV-3Cprotease we finally obtained pure AlsR protein. We analyzed the in vitrobinding ability by EMSA analyses and performed in vitro transcriptionstudies with the purified AlsR mutant proteins. The amino acid exchangefrom serine at position 100 of AlsR to alanine inactivated the AlsR proteinfor transcriptional activation in vivo and in vitro. Compared to the wildtype protein, the AlsRS100A mutant protein has a defect in DNA-proteincomplex formation. Whereas, wild type AlsR formed 3 different migratingcomplexes, AlsRS100A is no longer able to form the slowest migratingcomplex III in EMSA analyses. Therefore, we deduced complex III as thetranscriptional active form. A model of transcriptional active complexformation of AlsR is given.RSP013Interconnectivity between two histidine kinase / responseregulator systems in Escherichia coliS. Behr*, L. Fried, T. Kraxenberger, K. JungLudwig-Maximilians-Universität, Biology I - Microbiology, München, GermanyBacteria use two-component systems (TCSs) to encounter fluctuatingenvironmental conditions. A membrane-bound histidine kinase (HK)senses a stimulus and transduces it into a cellular signal viaphosphorylation. The transfer of this phosphoryl group to a responseregulator (RR) with DNA-binding properties mediates the inert reaction,generally an alteration in gene expression (1). Based on the limited numberof TCSs in Escherichia coli (30/32 HK/RR) it is necessary to coordinatecellular adaptions in order to respond to a multitude of environmentalsignals. To this end many so called auxiliary proteins have been describedrecently (2). These proteins can be involved in sensing, scaffolding orconnecting TCSs and evolved to an emerging field of bacterial signaltransduction.Although many TCSs in Escherichia coli are well characterized, theYehU/YehT and YpdA/YpdB TCSs are largely unknown. Both belong tothe group of LytS/LytR-like TCSs comprising of a HK with GAF-domainand a RR with LytTR-DNA-binding domain. Based on bioinformaticaldata these two TCSs share an amino acid identity of more than 30%. Theyare wide-spread and co-occure in many -proteobacteria (3).The characterization of the YehU/YehT and the YpdA/YpdB systemsrevealed reversed transcriptional effects on target genes. Using thebacterial adenylate cyclase-based two-hybrid system YehS was uncoveredas hub connecting the two TCSs via protein-protein interactions. Surfaceplasmon resonance measurements with purified YehS and the RRsconfirmed the interactions and suggest an interconnectivity betweenYehU/YehT and YpdA/YpdB.1) Stock et al. (2000): Two-component signal transduction. Annu Rev Biochem 69:183-2152) Jung et al. (2011): Histidine kinases and response regulators in networks. Curr. Opin. Microbiol. In press3) Szklarczyk et al. (2011): The STRING database in 2011: functional interaction networks of proteins,globally integrated and scored. Nucleic Acids Res.39:561-568RSP014Identification of Marinobacter adhaerens HP15 genes required forthe interaction with the diatom Thalassosira weissflogii by In vivoexpression technologyI. Torres-Monroy*, M. UllrichJacobs University Bremen, Molecular Life Science Research Center,Bremen, GermanyAggregate formation by living cells and organic matter in the ocean is animportant mechanism that mediates sinking of organic carbon. Diatombacteriainteractions play an important role during this process by inducingsecretion of different extra-cellular polysaccharides, which increase thesize of marine aggregates. To study cell-to-cell diatom-bacteriainteractions, a bilateralin vitromodel system has been establishedconsisting of the diatom Thalassosira weissflogii and the marine bacteriumMarinobacter adhaerens HP15. The bacterium was previously shown tospecifically attach to T. weissflogii cells, to induce transparentexopolymeric particle formation, and to increase aggregation. In addition,it has been shown that M. adhaerens HP15 is genetically accessible, itsgenome has been sequenced, and several bacterial genes potentiallyimportant during the interaction are currently being investigated. However,genes specifically expressedin vivoare still unknown. The aim of this workwas to establish anIn Vivo Expression Technology (IVET) screening toidentify bacterial genes specifically induced when M. adhaerens HP15interacts with T. weissflogii. The IVET vector was constructed by cloningthe full-size promoterlesslacZ gene downstream of a promoterless pyrBgene, which encodes an essential growth factor fundamental forpyrimidines biosynthesis. A site-directed mutagenesis approach was usedto generate apyrB-deficient mutant in M. adhaerens HP15. This mutantwas unable to grow in the absence of uracil and in presence of the diatom,BIOspektrum | Tagungsband 2012