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

70MurNAc-L-Ala-D-Glu-LL-Dap-D-Ala-D-Ala indicating thatMicrobispora protect itself not by synthesizing resistant peptidoglycan.Castiglione, F.; Lazzarini, A; Carrano, L.; Corti, E.; Ciciliato, I.; Gastaldo, L.; Candiani, P.; Losi,D.; Marinelli, F.; Selva, E; Parenti, F.; Chemistry and Biology,2008, 15, 22Schäberle, T.F.; Vollmer, W.; Frasch, H.J.; Hüttel, S.; Kulik, A.; Röttgen, M.; von Thaler, A.K.,Wohlleben, W.; Stegmann, E.; Antimicrob Agents Chemother,2011, 55(9)CEP022Interaction and Localisation of the Ser/Thr kinase PknB and theessential two component system YycFG of Staphylococcus aureusP. Hardt* 1 , M. Türck 2 , S. Donat 3 , K. Ohlsen 3 , G. Bendeas 4 , H.-G. Sahl 1 ,G. Bierbaum 2 , T. Schneider 11 Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie,Pharmazeutische Mikrobiologie, Bonn, Germany2 Institut für Medizinische Mikrobiologie, Immunologie und Parasitologie,Medizinische Mikrobiologie, Bonn, Germany3 Institut für Molekulare Infektionsbiologie, Würzburg, Germany4 Institut für Pharmazie, Institut für pharmazeutische Chemie, Bonn, GermanyProkaryotic signal transduction pathways regulate cellular functions inresponse to environmental cues and enable bacteria to react immediately tochanging conditions like antibiotic stress. Besides two-componentregulatory systems (TCS), one-component regulatory systems (OCS)represent one of the most abundant signaling systems in prokaryotes.These OCS include eukaryotic-like serine/threonine kinases (ESTKs) andphosphatases (ESTPs), which are increasingly recognised as importantregulators of major processes such as cell wall metabolism and division,virulence/ bacterial pathogenesis and spore formation. One suchESTK/ESTP-couple has recently been identified in Staphylococcus aureusdesignated PknB/YloO [1]. The extracellular sensor part of the kinasecontains three daisy-chained PASTA-domains assumed to be capable ofbinding peptidoglycan subunits, suggesting that PknB monitors thecoordinated assembly of peptidoglycan biosynthesis and cell division. Thesignal recognised by PknB has not been identified so far.To further investigate the role of PknB we analysed the interplay with theessential YycFG TCS on the molecular level and show phosphorylation ofthe response regulator YycF. The YycFG system is involved in the controlof peptidoglycan metabolism in S. aureus and both, PknB-GFP and YycG-GFP co-localize at the septum, the site of active cell wall biosynthesis incocci. This makes an interaction with the cell wall precursor lipid II andsubunits thereof, very likely. Determination of the binding parameters toselected lipid II variants, including amidated lipid II and subunits, usingquartz crystal microbalance (QCM) biosensor technique will shed lightonto the signal recognized by PknB.[1] Donat S, Streker K, Schirmeister T, Rakette S, Stehle T, Liebeke M, Lalk M, Ohlsen K. (2009).Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB inStaphylococcus aureus. J Bacteriol. 191(13):4056-69.CEP023New insights into the regulation of the phage shock system in E. coliH. Osadnik*, T. BrüserLeibniz Universität, Institut für Mikrobiologie, Hannover, GermanyThe phage shock system is a membrane stress sensor and effector systemof E. coli, comprising seven genes in three operons. Some of the inducingsignals include addition of 10% ethanol, osmotic upshift, severe heatshock, misfolded membrane proteins and disturbance of lipid biogenesis.Stress leads to up-regulation of the systems’ genes, namely the geneencoding for the phage shock protein A (PspA), a protein with largecoiled-coil domains. Essential parts of the system, especially homologuesof PspA, are conserved and widespread among bacteria, archaea andplastids of higher plants, where the protein seems to be responsible forthylakoid membrane formation and organization.Since PspA production is strongly induced whenever the integrity of theinner membrane is at stake, PspA is thought to exhibit a membranestabilizing function via direct binding to the inner membrane leaflet. Whileit is well established that PspA forms multimeric complexes in vivo to doso, its mechanism of action is still poorly understood, as well as theregulation of the system itself.The cellular PspA-level is mainly regulated by a negative feedback-likeinteraction of PspA with the systems’ activator protein PspF. The integralmembrane proteins PspB and PspC relay stress signals via directinteraction with PspA, leading to the activation of PspF and thereforehigher PspA-levels.With our new data we provide improved and refined insights into theregulatory aspects of the Psp-regulon, leading to a better understanding ofa complex membrane stress system.CEP025Recovery of cell wall fragments in Bacillus subtilis:Characterisation of D-Glu-mDAP carboxypeptidase andMurNAc-6P etheraseA. Duckworth*, A. Schneider, S. Unsleber, C. MayerIMIT, Biotechnologie/Mikrobiologie, Tübingen, GermanyIn E. coli and other Gram negative bacteria, the peptidoglycan fragmentsreleased during cell growth and division are efficiently reutilised andrecycled. In contrast, cell wall recycling in the Gram positive bacterium B.subtilis has not been thoroughly studied to date. However, more than 30autolysins have been identified in this organism that are responsible forcleavage of peptidoglycan and release fragments into the medium duringdifferent developmental processes. We are characterising the cell wallturnover products of B. subtilis and are investigating an operon of sixgenes (ybbI, ybbH, ybbF, amiE, nagZ, ybbC) involved in peptidoglycanrecycling. NagZ and AmiE have been functionally characterised recently(Litzinger et al. 2010. J Bacteriol.;192(12):3132-43). NagZ is an Exo-GlcNAc'ase that cleaves the glycosidic bond between non-reducingGlcNAc and MurNAc residues of GlcNAc-MurNAc-peptides(muropeptides), and AmiE subsequently cleaves the MurNAc-peptidebond. Here we report the characterisation of YbbC and YbbI. YbbC wasshown to cleave the products of the AmiE reaction, such as L-Ala-D-GlumDAPtripeptide. NagZ, AmiE and YbbC, which are secreted, areinvolved in the sequential digest of muropeptides in the cell wallcompartment. The resulting amino acids mDAP, L-Ala-D-Glu dipeptideand amino sugar monomers MurNAc and GlcNAc are imported into thecytoplasm by individual transporters. YbbI is a MurNAc-6P etherase asrevealed by the Morgan-Elson assay. MurNAc is phosphorylated toMurNAc-6P by a PTS transporter and then converted to GlcNAc-6P by thecytoplasmic etherase YbbI. Thus, the ybbIHFEDC cluster is required forthe recycling of cell wall fragments in B. subtilis.CEP026The role of Lipoprotein STM 3690 in the biogenesis of thetrimeric autotransporter adhesin SadA in SalmonellaI. Grin* 1 , A. Felipe-Lopez 2 , G. Sauer 1 , H. Schwarz 1 , M. Hensel 2 , D. Linke 11 MPI für Entwicklungsbiologie, Proteinevolution, Tübingen, Germany2 Universität Osnabrück, Mikrobiologie, Osnabrück, GermanyQuestion: Salmonella is a major agent in human food-borne diseases.Among the proteins expressed on the surface of the cells, adhesins,proteins which allow the bacteria to stick to biotic and abiotic surfaces, arekey virulence factors.From the large family of adhesion proteins, the trimeric autotransporteradhesins (TAA) form a distinct subgroup. TAAs are non-fimbrial, nonpilus,homotrimeric adhesins which are widespread among proteobacteria.They have a modular domain structure of extended coiled-coil stretchesinterspersed with globular domains. The extracellular part of theautotransporter, which can be as large as 100 kDa and above, istransported over the outer membrane of gram-negative bacteria through itsown membrane anchor by an unknown mechanism. Several of theautotransporter operons in enterobacteria also contain a small periplasmiclipoprotein of unknown function upstream of the main TAA gene. Thelocation in the operon suggests a supporting role in the folding and exportof the passenger domain. The aim of the presented work was to gaininsight into the structure of the lipoprotein and to elucidate its function androle in the autotransport of SadA.Methods: Bioinformatics, Mass spectrometry, Immunofluorescence,FACS, Phage Display, X-Ray CrystallographyResults: Using GCView, a bioinformatics tool for visualizing genomiccontext for homology search results (1) we could show that the operon ofSTM 3690 and SadA is conserved in composition and genomic location inEnterobacteria.We were able to verify that STM 3690 is a periplasmic lipoprotein bysubcellular fractionation of bacterial cells expressing the protein.Furthermore we showed the correct lipid modification of the N-Terminusby mass spectrometry.Cells expressing either SadA and STM 3690 show a higher amount ofSadA on the surface compared to cells which express only SadA.We used Phage Diplay to screen for possible interaction partners of thelipoprotein. This was complemented by pulldowns as well as in vivocrosslinks in Salmonella.Conclusions: Preliminary data suggests a function as chaperone during theexport process.1) Grin I., Linke D. GCView: the genomic context viewer for protein homology searches. NucleicAcids Res. 2011, 39, W353-W356.BIOspektrum | Tagungsband 2012