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

201RSP029Mutational Analysis within the Periplasmic PAS Domain ofthe Escherichia coli Sensor Kinase DcuSC. Monzel*, G. UndenJohannes Gutenberg-Universität Mainz, Institut für Mikrobiologie undWeinforschung, Mainz, GermanyE. coli utilizes C 4-dicarboxylates as a carbon source or as an electronacceptor under aerobic and anaerobic conditions, respectively. Metabolicregulation is effected by the two-component system DcuSR, consisting ofthe membrane-embedded sensor histidine kinase DcuS and the responseregulator DcuR. Sensing of C 4-dicarboxylates generates a signal that leadsto an autophosphorylation of a conserved histidine residue in the kinasedomain of DcuS.Studying the periplasmic PAS domain (PAS P) of DcuS revealed both, ONmutantswith a distinct fumarate-independent expression of a dcuB-lacZreporter gene fusion and OFF-mutants showing a considerable loss ofDcuS-activation by the effector. ON- and OFF-mutations located outsidethe binding pocket, were studied in more detail. For these definded sitesthe chemical and sterical requirements were probed by site-directedmutagenesis, introducing variable residues at one and the same position. Inaddition the impact of ON- and OFF-mutations on oligomerization andsubcelluar localization were tested, using the bacterial two-hybrid system(BACTH) and in vivo fluorescence microscopy.[1] Zientz et al. (1998) J Bacteriol 178(24):7241-7247[2] Scheu et al. (2010) J Bacteriol 192(13):3474-3483RSP030The transmembrane domain of the sensory histidine kinaseDcuS: role in dimerizationP.A. Steinmetz*, G. UndenInstitut für Mikrobiologie und Weinforschung, AG Unden, Mainz, GermanyBacteria are capable of adjusting to changing environmental conditions. Toensure quick adaptation among various conditions, sensors detect stimuliand regulators trigger the cellular response. Two-component systems arewidespread in bacteria, consisting of a sensory histidine kinase and aresponse regulator. The DcuS/DcuR two-component regulatory system ofEscherichia coli senses C 4-dicarboxylates and stimulates the expression ofgenes for anaerobic fumarate respiration [1]. The oligomeric state of thesensor DcuS is supposed to be an important parameter for its function [2].Transmembrane domains (TMDs) of membrane proteins have crucial rolesas interaction sites. Dimerization sites in the TMDs of DcuS wereanalyzed. A tandem SxxxGxxxG motif was identified in TMD2 of DcuS.A combination of bacterial two-hybrid system (BACTH) and GALLEX [3]interaction studies with DcuS variants suggest a role of the GxxxG motifand the TMD in the dimerization of DcuS.(1) Zientz E., Bongaerts J., Unden G. (1998) J. Bacteriol 180: 5421-5425(2) Scheu PD, Liao YF, Bauer J, Kneuper H, Basché T, Unden G, Erker W. (2010) J Bacteriol.192(13):3474-83.(3) Schneider D, Engelman DM. (2003) J Biol Chem. 31;278(5):3105-11.RSP031HtrA-mediated control of nitrate/nitrite assimilation in S.coelicolorR. Amin*, J. Reuther, A. Bera, W. Wohlleben, Y. MasUniversity of Tübingen, IMIT, Microbiology and Biotechnology, Tübingen,GermanyStreptomyces usually grow in nutritional limiting environment oftenlacking essential elements for growth. Streptomyces coelicolor, a modelorganism for studying the regulation of nitrogen metabolism exhibits aspecific regulatory network. In this control system, OmpR-liketranscriptional regulation GlnR plays a central role by controlling thetranscription of at least 14 genes 9 of which are directly implicated innitrogen assimilation. During this study, we identified a new GlnR targetgene SCO2958 named htrA. In silico analysis revealed the presence of twodistinct domains in HtrA sequence: an N-terminal uroporphyrinogen-IIIsynthase (HemD)-like enzymatic domain and a C-terminal DNA bindingdomain. Complementation experiments with a hemin auxotroph E.colihemD mutant strain showed that HtrA has no HemD activity.Physiological studies of a S. coelicolor htrA::Tn5062 mutant showed thatHtrA is involved in regulation of nitrite reduction. By electrophoreticmobility shift assays the functionality of the HtrA DNA binding domainwas confirmed and found that HtrA binds in front of the genes narK(putative nitrate extrusion protein), nirB (nitrite reductase), nirA (putativenitrite/sulphite reductase), and nasC (putative nitrate reductase), which areassociated with nitrate/nitrite assimilation. Furthermore, a cooperativebinding of HtrA together with GlnR to the nirB promoter was observed,suggesting that HtrA may act as a “GlnR-helper protein”.RSP032Posttranslational modification of global response regulatorGlnR links nitrogen metabolism and transcription of GlnRtarget genes in S. coelicolorA. Bera*, Y. Ahmed, R. Amin, W. WohllebenUniversity of Tübingen, IMIT, Microbiology and Biotechnology, Tübingen,GermanyTranscriptional regulation of nitrogen assimilation genesinStreptomycescoelicoloris mediated by a global response regulator GlnR.GlnR was shown to act as both a transcriptional repressor and activatorunderN-limiting conditions. The GlnR-induced genesincludeglnAandglnIIwhich encode key enzymes of ammoniumassimilation: glutamine synthetases GSI and GSII, respectively. GlnRrepresses expression of thegdhAgene encoding the glutamatedehydrogenase GDH, which is able to assimilate ammonium intoglutamate only under conditions of high ammonium concentrations.Therefore a repression under nitrogen-limiting conditions is reasonable.Finally, GlnR controls reactions for the uptake of ammonium and theutilization other nitrogen sources like nitrate/nitrite or urea. GlnR regulatestranscription of the amtB operon,nirBDencoding a nitrite reductaseandureAencoding a urease [1]. The mechanism for this repressor/activatorfunction of GlnR was unknown and itself regulation was not investigatedtill now. We were able to show how the nitrogen status of the cell isconnected to the control ofglnRexpression and GlnR activity. Western blotanalyses provided evidence that GlnR undergoes posttranslationalmodification viaSer/Thrphosphorylation andLysacetylation inS.coelicolorM145. LC-MS/MS analyses revealed that underN-excess fourserine residues and three threonineresidues were phosphorylated.Additionally two lysine residues were acetylated. The pattern of themodification underN-limited conditions differed significantly (noacetylation and only two phosphorylated serine residues). This kind ofregulation is surprising and somehow unusual since GlnR belongs toOmpR-like family and as might be expected it should interact with till nowunknown cognate histidine kinase. Various acetylation andphosphorylation patterns influence GlnR´s DNA binding activity.Acetylation seems to completely abolish the binding of GlnR to promoterregions of its target genes. Regulation via acetylation seems to bedepending on concentration of nitrogen source however phosphorylation isfine-tuning regulation and depends on type of theN-source. To ourknowledge this is the first report aboutLysacetylation andSer/Thrphosphorylation of the response regulator in actinobacteria.1. Tiffert, Y., Supra, P., Wurm, R., Wohlleben, W., Wagner, R.,Reuther, J.,Mol.Microbiol.67(2008) p.861-880RSP033Quorum sensing in Pseudomonas putida colonies under flowconditionsB. HenseHelmholtz Zentrum München, Institute of Biomathematics and Biometry,Neuherberg, GermanyBacterial communication via release and sensing of signal molecules(autoinducer, AI) has been mainly investigated in batch cultures. Hereusually coordinated response of the whole population is induced in a celldensity dependent manner (quorum sensing, QS). However, most bacterialive heterogeneously distributed in aggregates or biofilms attached tosurfaces. Under these conditions, functionality of the signalling system isless well understood and more difficult to approach experimentally. Wethus use a combined experimental/mathematical modelling strategy toinvestigate the induction dynamics of the PpuI/R QS system inPseudomonas putida IsoF. Induction of AI controlled expression ofagfpgene was followed with high spatio-temporal (single cell or colonylevel) resolution. The influence of flow respectively addition of externalAI was examined. Main results were: Mass transfer (flow) delays theinduction behaviour, probably by removal of AIs. A compartmentation ofyet unkown origin occurs, limiting the influence of AI from outside thecolony. AI regulation promoted intra- as well as intercolonialheterogeneity. Summarized, there were fundamental differences betweenthe AI functionality in cell aggregates and planktonic batch cultures, whichhave been analysed before [1]. These differences have consequences forthe ecological functionality of autoinducers.[1] Fekete A, Kuttler C, Rothballer M, Hense BA, Fischer D, Buddrus-Schiemann K, Lucio M,Müller J, Schmitt-Kopplin P, Hartmann A. (2010) FEMS Microbiol. Ecol. 72, 22-34.RSP034An essential role for cyclic dinucleotide signaling in Bacillus subtilisF. Mehne* 1 , K. Gunka 1 , A. Garbe 2 , V. Kaever 2 , J. Stülke 11 University of Göttingen, Dept. of General Microbiology, Göttingen, Germany2 Hannover Medical School, Institute of Pharmacology, Hannover, GermanyCyclic dinucleotides (c-di-AMP and c-di-GMP) act as second messengersin several bacterial species. In the last decade these messengers haveBIOspektrum | Tagungsband 2012