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

206Ser46~P complex. Additionally, B. subtilis possesses the carbon-fluxregulator Crh. Like HPr, Crh can be phoshorylated at the regulatory siteSer~46. However, the influence of Crh on CCR is weak. Recent studiesshowed that Crh senses the metabolic state of B. subtilis, therebycontrolling flux through the toxic methylglyoxal pathway (2).Different carbohydrates form a hierarchy in their ability to triggerphosphorylation of HPr and Crh by HPrK/P. Upon utilization of preferredcarbon sources HPr and Crh are predominantly phosphorylated. Incontrast, the non-phosphorylated forms prevail in the presence ofsecondary substrates (3, 4).Here we are focusing on conditions leading to dephosphorylation of HPrand Crh at Ser~46. Both, Crh and HPr accumulate in their nonphosphorylatedforms upon entry in the stationary phase. We demonstratethat phosphorylation as well as dephosphorylation of Crh is carried out bythe single enzyme, HPrK/P. In contrast, it turned out thatdephosphorylation of HPr depends on a different enzyme, namely aphosphatase of the PP2C family. The physiological consequences of theinvolvement of this phosphatase are discussed.(1) Görke, B. and J. Stülke (2008). Carbon catabolite repression in bacteria: many ways to make themost out of nutrients.Nature Reviews Microbiology6, 613-624.(2) Landmann, J.J., Busse, R.A., Latz, J.H., Singh, K.D., Stülke, J. and B. Görke (2011). Crh, theparaloque of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis inBacillus subtilis.Molecular Microbiology 82(3), 770-787.(3) Singh, K.D., Schmalisch, M.H., Stülke, J. and B. Görke (2008). Carbon catabolite repression inBacillus subtilis: Quantitative analysis of repression exerted by different carbon sources.Journal ofBacteriology 190, 7275-7248.(4) Landmann, J.J., Werner, S., Hillen, W., Stülke, J. and B. Görke (2011). Carbon source control ofthe phosphorylation state of the Bacillus subtilis carbon-flux regulator Crh in vivo.FEMS MicrobiolLetters.RSP055Novel structures of PII signal transduction proteins fromoxygenic phototropic organismsV.R. Chellamuthu* 1,2 , M. Hartmann 2 , K. Forchhammer 11 University of Tuebingen, Department of Microbiology/ OrganismicInteractions, Tuebingen, Germany2 MPI for Developmental Biology, Protein Evolution, Tuebingen, GermanyPII proteins constitute one of the most widely distributed families of signaltransduction proteins, whose representatives are present in archaea,bacteria and plants. They play a pivotal role to control the nitrogenmetabolism in response to the central metabolites ATP, ADP and 2-oxoglutarate (2-OG). These signals from energy status, carbon andnitrogen metabolism are integrated and transmitted to the regulatorytargets (key enzymes, transporters and transcription factors). In oxygenicphototrophic organisms, from cyanobacteria to higher plants, thecontrolling enzyme of arginine synthesis, N-acetyl-glutamate kinase(NAGK), is a major PII target, whose activity responds to the cellular 2-OG and energy status via PII signalling. Novel crystal structures of PIIsignal transduction proteins from oxygenic phototrophs in the presence ofsignaling metabolites and in complex with NAGK give deeper insightsinto their control mechanism and sheds light on the evolutionaryadaptation of PII signal transduction.RSP056Hierarchy of Selenoprotein Gene Expression in the ArchaeonMethanococcus maripaludisM. Rother* 1 , T. Stock 2 , M. Selzer 3 , S. Connery 4 , D. Seyhan 2 , A. Resch 51 Technische Universität Dresden, Institut für Mikrobiologie, Dresden, Germany2 Institute for Molecular Biosciences, Goethe-University Frankfurt, Frankfurt,Germany3 Universität Bayreuth, Department of Ecological Microbiology, Bayreuth,Germany4 School of Crystallography, Birbeck College London, London, United Kingdom5 Deparment of Microbiology, Immunobiology and Genetics, University ofVienna, Vienna, AustriaProteins containing selenocysteine are found in members of all threedomains of life, Bacteria, Eukarya and Archaea. A dedicated tRNA(tRNA sec ) serves as a scaffold for selenocysteine synthesis. However,sequence and secondary structures differ in tRNA sec from the differentdomains. An Escherichia coli strain lacking the gene for tRNA sec couldonly be complemented with the homolog from Methanococcus maripaludiswhen a single base in the anticodon loop was exchanged demonstratingthat this base is a crucial determinant for archaeal tRNA sec to function in E.coli. Complementation in trans of M. maripaludis JJ mutants lackingtRNA sec , O-phosphoseryl-tRNA sec kinase, or O-phosphoseryltRNAsec :selenocysteine synthase with the corresponding genes from M.maripaludis S2 restored the mutant’s ability to synthesize selenoproteins.However, only partial restoration of the wild-type selenoproteome wasobserved as only selenocysteine-containing formate dehydrogenase wassynthesized. Quantification of transcripts showed that disrupting thepathway of selenocysteine synthesis leads to down-regulation ofselenoprotein gene expression, concomitant with up-regulation of aselenium-independent backup system, which is not re-adjusted uponcomplementation. This transcriptional arrest was independent ofselenophosphate but depended on the “history” of the mutants and wasinheritable, which suggests that a stable genetic switch may cause theresulting hierarchy of selenoproteins synthesized.RSV1-FGSignal recognition and transmission by the CpxAR-twocomponent systemS. Hunke* 1 , V.S. Müller 1 , K. Tschauner 1 , P. Scheerer 1,21 Universität Osnabrück, Molekulare Mikrobiologie, Osnabrück, Germany2 Charité - Universitätsmedizin Berlin, Institut für Medizinische Physik undBiophysik (CC2), Berlin, GermanyTwo-component systems (TCS) are the predominant signalling systemsallowing bacteria to communicate with their environment [1]. In general, aTCS comprises of a sensor kinase (SK) and a response regulator (RR).Upon stimulation the SK is autophosphorylated and transfers thephosphoryl group to the RR which acts now a transcription regulator oftarget genes. To balance the response some SKs also dephosphorylate thephosphorylated RR. However, the mechanistic details in signal recognitionand transmission by TCS are still only poorly understood.The Cpx-TCS is a well established model ubiquitous in Gram-negativebacteria that integrates a broad variety of different signals including saltstress, pH stress, lipids and misfolded proteins that cause envelope stress[2]. The Cpx-TCS is made up of the SK CpxA, the RR CpxR and theaccessory protein CpxP. CpxP is known to shut off the Cpx-TCS byinhibiting CpxA autophosphorylation [3] and to promote degradation ofmisfolded pilus subunits [4]. Recent structural and functional studiesprovide first insight into how CpxP inhibits CpxA and serves as sensor formisfolded pilus subunits, pH and salt [5]. Now, we have used membrane-SPINE [6] to demonstrate not only the direct interaction between CpxPand CpxA under non-stress conditions but also the release of CpxP fromCpxA under certain stress conditions in vivo.Other signals are CpxP-independent recognized by CpxA as a misfoldedvariant of the maltose binding protein that activates phophotransfer toCpxR [2] and lipids that inhibit dephosphorylation of activated CpxR.Hence, independent entry points for the Cpx-TCS exist that result inspecific activities of CpxA. In addition, we will present a structurehomology model of the catalytic domain of CpxA in complex with CpxRthat we have proved for critical residues in the interface between bothproteins in vivo. Thus, we have now the system and methods in hand togain a deeper understanding of signal recognition and transmission overthe membrane in a TCS in general.1. A.M. Stock, V.L. Robinson and P.N. Goudreau, Annu. Rev. Biochem.69(2000), p. 183.2. S. Hunke, R. Keller and V.S. Müller, FEMS Microbiol. Lett (2011) doi: 10.1111/j.1574-6968.2011.02436.x.3. R. Fleischer, K. Jung, R. Heermann and S. Hunke, J. Biol. Chem.282(2007), p. 8583.4. D.D. Isaac, J.S. Pickner, S.J. Hultgren and T.J. Silhavy, PNAS102(2005), p. 17775.5. X. Zhou, R. Keller, Volkmer, R., Krauss, N., Scheerer, P. and S. Hunke, J. Biol. Chem.286(2011), p.9805.6. V.S. Müller, P.R. Jungblut, T.F. Meyer and S. Hunke, Proteomics11(2011), p. 2124.RSV2-FGMechanism of signal transfer by the tandem hamp domainfrom Natronomonas pharaonisJ. Natarajan*, J. SchultzUniversity of Tübingen, Department of Pharmaceutical Biochemistry,Tübingen, GermanyChemotaxis and phototaxis in bacteria share most steps in signaltransducing mechanisms. External stimuli are converted via a HAMPdomain (Histidine kinases, Adenylyl cyclases [ACs], Methyl-acceptingchemotaxis proteins [MCP] and Phosphatases) into a conformationalresponse of the output machinery, mostly kinases or kinase control unitswhich impinge on the flagellar motor to affect swimming behaviour.Hitherto, more than 12,000 HAMP domains are annotated in the EMBLdata bank; only a few have been functionally characterized (1).The phototransducer from Natronomonas pharaonis (NpHtrII) belongs tothe group of MCP´s. It receives its signal from the light excited sensoryrhodopsin (SR II) which forms a complex with the transducer Htr II (2).SRII initiates a sliding of the second transmembrane helix of HtrII (3)resulting in a conformational change in the HAMP domain (rotation andtranslation) which sets off the signalling cascade. HtrII, unlike many of thewell studied MCP’s has a tandem HAMP domain (HAMP HtrI and II).We study signal conversion through such a tandem HAMP domain bygenerating chimeras with Tsr, the E. coli serine receptor, and themycobacterial AC Rv3645 as a reporter enzyme. Such chimeras have beenshown to be regulated by serine using the HAMP domain of either Tsr orof Rv3645 (4). After sequence comparisons with positively operatingHAMP domains two chimeras with mutated Htr II tandem HAMP weregenerated, which responded differently to the serine signal between sensorand output-domains i.e an inversion of signal was observed. Themechanistic differences in these two chimeras, which lead to completelyopposite output, are being actively investigated.1. Hulko, M., F. Berndt, M. Gruber, J.U. Linder, V. Truffault, A. Schultz, J.Martin, J.E. Schultz, A.N. Lupas,M. Coles. Cell,2006,126, 929-940.2. Wegener, A. A., Chizhov, I., Engelhard, M. & Steinhoff, H. J. J. Mol. Biol,2000,301, 881-891.BIOspektrum | Tagungsband 2012