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

63extracellular domain is secreted through the outer membrane by a novelautotransport mechanism, termed type Ve secretion [1]. Compared toclassical (type Va) autotransporters, Intimin and Invasin have an invertedtopology, with the C-terminal passenger being exported through an N-terminal -barrel pore [2]. In addition, these proteins have an N-terminalperiplasmic domain with homology to LysM. We show that theperiplasmic domain of Intimin, but not the corresponding, smaller domainof Invasin, binds to peptidoglycan, and that Ca 2+ ions enhance this binding.Furthermore, the Intimin periplasmic domain mediates dimerisation. TheC-terminal passenger domains of Invasin and Intimin contains an array ofrepeated immunoglobulin (Ig)-like domains [3,4]. We have identified afurther Ig domain at the N-terminus of the passenger, which may beinvolved in passenger export. In addition, we have produced and refoldedthe -barrel translocator domain of Invasin for crystallisation trials. Thestructure of this domain would confirm our topology model and offerinsight into this new mechanism of autotransport.[1] Leo JC, Grin I, Linke D (2011):Type V secretion: mechanism(s) of autotransport through thebacterial outer membrane. Phil Trans R Soc B, in press.[2] Oberhettinger P, Schütz M, Heinz N, Leo JC, Berger J, Autenrieth IB, Linke D (2011): Intiminand Invasin are members of a family of autotransporters that export their C-terminus to the bacterialcell surface. Under revision.[3] Hamburger ZA, Brown MS, Isberg RR, Bjorkman PJ (1999) Crystal structure of Invasin: abacterial integrin-binding protein. Science 286, 291-295.[4] Luo Y, Frey EA, Pfuetzer RA, Creagh AL, Knoechel DG, Haynes CA, Finlaz BB & StrynadkaNCJ (2000) Crystal structure of the enteropathogenic Escherichia coli intimin-receptor complex.Nature 405, 1073-1077.CEV008The cell envelope as target of a novel antimicrobial peptideM. Wenzel* 1 , A.I. Chiriac 2 , B. Albada 3 , A. Otto 4 , A. Knüfer 3 , D. Becher 4 ,L. Hamoen 5 , H.-G. Sahl 2 , N. Metzler-Nolte 3 , J.E. Bandow 11 Ruhr University Bochum, Microbial Biology, Bochum, Germany2 University of Bonn, Pharmaceutical Microbiology, Bonn, Germany3 Ruhr University Bochum, Bioinorganic Chemistry, Bochum, Germany4 University of Greifswald, Microbial Physiology and Molecular Biology,Greifswald, Germany5 University of Newvastle, Institute for Cell and Molecular Biosciences,Newcastle, United KingdomCationic hexapeptide MP196, composed of alternating arginine andtryptophane [3,4], is a promising new antibacterial agent with excellentactivity against Gram positive bacteria whereas non-toxic to human cells.The mechanism of action of this peptide was studied by proteomicinvestigation of the bacterial stress response, which has been proven to bea useful tool in elucidating antibiotic targets [1,2]. This approach revealedstrong similarities of MP196 with potassium ionophore valinomycin aswell as cell wall biosynthesis-inhibiting bacitracin. More specifically, weobserved strong induction of both membrane stress-induced PspA and cellwall stress-induced LiaH proteins, suggesting a novel or combined cellenvelope-related mechanism of action. Further, we investigated theinfluence of MP196 on membrane integrity and cell wall biosynthesis byseveral cell-based assays, such as radioactive precursor incorporation,potassium efflux, and membrane potential measurements.Taken together, our results suggest, that MP196 treatment results in energyand, therefore, nutrient limitation caused by impaired membrane functions.[1] Bandow JE et al., Antimicrob. Agents Chemother., 2003, 47:948-55[2] Wenzel and Bandow, Proteomics, 2011, 11:3256-68[3] Strøm MB et al., J. Med. Chem., 2003, 46:1567-70[4] Chantson JT et al., ChemMedChem., 2006, 1:1268-74CEV009Processive movement of MreB-associated cell wall biosyntheticcomplexes in bacteriaJ. Dominguez-Escobar*, A. Chastanet, A.H. Crevenna, R. Wedlich-Söldner, R. Carballido-LópezMax-Planck-Institute of Biochemistry, Cellular Dynamics and CellPatterning, Martinsried, FranceThe rod-shaped model gram positive bacterium Bacillus subtilis expressesthree isoforms of the prokaryotic actin: MreB, Mbl and MreBH. All threeproteins are thought to polymerize into dynamic filamentous helicalstructures underneath the cell membrane and together with the cell wall(CW) control cell morphogenesis. The prevailing model postulates thatmembrane-associated MreB filaments spatially organize elongationspecificpeptidoglycan-synthesizing complexes along sidewalls.We have used Total Internal Reflection Fluorescence microscopy(TIRFM) to quantitatively characterize the in vivo distribution anddynamics of fluorescently-labelled MreB proteins and visualize thedynamic relationship between MreB isoforms and CW synthesis proteinsin Bacillus subtilis cells. We show that during exponential growth MreBproteins do not form helical structures. Instead, together with othermorphogenetic factors (MreC, MreD, PBPH, PBP2a and RodA), theyassemble into discrete patches that processively move along peripheraltracks perpendicular to the cell axis. We show with Fluorescence RecoveryAfter Photobleaching (FRAP) experiments that patch motility is not drivenby MreB polymerization. Patch motility arrest using CW inhibitorsvancomycin and phosphomycin, strongly suggest that the motive force forMreB patches is provided by peptydoglycan (PG) synthesis itself. We alsoprovide evidence that MreB determines rod shape by restricting mobility ofelongation complexes.We propose that 1) CW elongation complexes insert new PG along trackslargely normal to cell long axis, 2) complexes motility is powered by PGpolymerization, and 3) MreB acts as a polymeric clamp to restrict the diffusionof CW complexes and allow processive movement in correct orientation.CEV010A shortcut pathway to UDP-MurNAc through peptidoglycanrecycling in PseudomonasJ. Gisin* 1,2 , A. Schneider 3 , B. Nägele 3 , C. Mayer 3,21 Universität Konstanz, Molekulare Mikrobiologie, Konstanz, Germany2 Universität Konstanz, Graduiertenschule Chemical Biology, Konstanz,Germany3 Interfakultäres Institut für Mikrobiologie und Infektionsmedizin UniversitätTübingen, Biotechnologie/Mikrobiologie, Tübingen, GermanyIn almost all bacteria, the essential cell wall component peptidoglycan issynthesized by a conserved pathway that represents a major target forantibiotics. Synthesis of the soluble cell wall precursor UDP-MurNAcwithin the cytoplasm involves the essential and highly conserved proteinsMurA and MurB. Inhibition of MurA by the antibiotic fosfomycininterferes with peptidoglycan synthesis, causing growth arrest andeventially cell lysis.Studying peptidoglycan recycling in Pseudomonas, we now identified analternative pathway for UDP-MurNAc synthesis. MurNAc recovered fromthe own cell wall or scavenged from the environment is directly fed intopeptidoglycan synthesis. The pathway involves an anomeric kinase thatATP-dependently phosphorylates MurNAc at the C1 position.Subsequently, an uridyltransferase generates UDP-MurNAc fromMurNAc--1-phosphate. Mutants in the coding genes accumulated therespective recycling intermediates and showed an increased susceptibilityto fosfomycin, indicating the relevance of this pathway for UDP-MurNAcbiosynthesis and intrinsic fosfomycin resistance. The pathway is conservedin all Pseudomonas strains and many other gram negative bacteriaincluding important pathogens.CEV011Identification and in vitro analysis of the GatD/MurT enzymecomplexcatalyzing lipid II amidation in S. aureusD. Münch* 1 , T. Roemer 2 , S.H. Lee 2 , M. Engeser 3 , H.-G. Sahl 1 , T. Schneider 11 Universität Bonn, Pharmazeutische Mikrobiologie, Bonn, Germany2 Merck Research Laboratories, Department of Infectious Diseases,Kenilworth, NJ, United States3 Universität Bonn, Kekulé Institute for Organic Chemistry andBiochemistry, Bonn, GermanyThe peptidoglycan of Staphylococcus aureus is characterized by a highdegree of crosslinking and almost completely lacks free carboxyl groups,due to amidation of the D-glutamic acid in the stem peptide. Amidation ofpeptidoglycan has been proposed to play a decisive role in polymerizationof cell wall building blocks, correlating with the crosslinking ofneighboring peptidoglycan stem peptides. Mutants with a reduced degreeof amidation are less viable and show increased susceptibility tomethicillin.We identified the enzymes catalyzing the formation of D-glutamine inposition 2 of the stem peptide. We provide biochemical evidence that thereaction is catalyzed by a glutamine amidotransferase-like protein and aMur ligase homologue, encoded by SA1707 and SA1708, respectively.Both proteins, for which we propose the designation GatD and MurT, arerequired for amidation and appear to form a physically stable bi-enzymecomplex.To investigate the reaction in vitro we purified recombinant GatD andMurT His-tag fusion proteins and their potential substrates, i.e. UDP-MurNAc-pentapeptide, as well as the membrane-bound cell wallprecursors lipid I, lipid II and lipid II-Gly 5. In vitro amidation occurredwith all bactoprenol-bound intermediates, suggesting that in vivo lipid IIand/or lipid II-Gly 5 may be substrates for GatD/MurT. Inactivation of theGatD active site abolished lipid II amidation.Both, murT and gatD are organized in an operon and are essential genes ofS. aureus. BLAST analysis revealed the presence of homologoustranscriptional units in a number of gram-positive pathogens, e.g.Mycobacterium tuberculosis, Streptococcus pneumonia and Clostridiumperfringens, all known to have a D-iso-glutamine containing PG. A lessnegatively charged PG reduces susceptibility towards defensins and mayplay a general role in innate immune signaling.BIOspektrum | Tagungsband 2012