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

68CEP013Role of RodA in Staphylococcus carnosus TM300T. Roth*, J. Deibert, S. Reichert, D. Kühner, U. BertscheUniversity of Tübingen, IMIT - Mikrobielle Genetik, Tübingen, GermanyBacteria appear in various shapes like rods, mycetes, cocci, and manymore. The cell shape of every bacterium is determined by its cell wall ormurein (peptidoglycan), as the murein sacculus provides stability againstthe internal turgor pressure. For peptidoglycan biosynthesis the interactionof several proteins is required, amongst them are the so called SEDSfamilyproteins (e.g. RodA, FstW).SEDS stands for shape, elongation, division, and sporulation, and most ofthe proteins are essential and can therefore not be deleted. In rod-shapedbacteria like Bacillus subtilis or E. coli the protein RodA is required forlateral growth of the organism. Yet, a deletion mutant of rodA in E. coli isknown, which is viable in minimal medium as enlarged cocci, albeit with alow growth rate.In Staphylococcus carnosus (S. carnosus) there are three genes alignedwhich encode proteins of the SEDS family, one of them being rodA. Sinceit has never been observed that bacteria of this genus grow in other shapesthan cocci, we tried to investigate the role of rodA in S. carnosus. We wereable to completely delete the rodA gene and obtain a slow growing butviable mutant. There seems to be no difference in shape or diameter whenviewed by electron microscopy. However, the peptidoglycanbiosynthesizing machinery is disordered. We could show in a pulsefeeding experiment, followed by fluorescent vancomycin labeling that thelocalization of newly synthesized peptidoglycan is altered compared to thewild type strain. In addition HPLC analysis of digested petidoglycanrevealed differences in the muropeptide pattern. Together with ourBacterial-Two-Hybrid experiments, where we obtained an interactionbetween RodA and all of the four native PBPs of S. aureus,these resultsindicate that RodA indeed plays a role during cell growth of staphylococci,even though these bacteria do not elongate.CEP014Penicillin binding protein 2x of Streptococcus pneumoniae: therole of different domains for cellular localizationK. Peters* 1 , C. Stahlmann 2 , I. Schweizer 1 , D. Denapaite 1 , R. Hakenbeck 11 University of Kaiserslautern, Department of Microbiology,Kaiserslautern, Germany2 Helmholz Institute for Pharmaceutical Research, Department of DrugDelivery, Saarbrücken, GermanyPenicillin-binding protein 2x (PBP2x) is one of the six PBPs in S.pneumoniae involved in late steps of peptidoglycan biosynthesis. PBP2xcatalyse a penicillin-sensitive transpeptidation reaction. The PBP2xdomain architecture is organized in an N-Terminal PBP dimerizationdomain, a central transpeptidase domain (TP) and two PASTA (Penicillinbinding protein And Ser/Thr protein kinase Associated) domains in its C-terminal region [1]. Mutations in the TP domain of PBP2x that interferewith beta-lactam binding are crucial for the development of high levelpenicillin-resistance which involves other PBPs as well. The PASTAdomains of bacterial Ser/Thr protein kinases exhibit low affinity for betalactamantibiotics and are likely to sense peptidoglycan precursors.As revealed by immunofluorescence techniques localization of PBP2x atthe septum has confirmed its role in the division process [2]. However,immunostaining has the disadvantage that cells need to be fixed and haveto undergo a damaging cell wall permeabilization treatment. Greenfluorescence protein (GFP) fusions can overcome these problems andallow the visualization of fusion proteins in living cells.To investigate the role of PBP2x during growth and division of S.pneumoniae cells, an N-terminal GFP-PBP2x fusion was constructed usinga Zn-inducible promoter. Upon induction with Zinc, a GFP-PBP2x signalwas observed at the septum in S. pneumoniae cells. Furthermore, thenative copy of the pbp2x gene could be deleted in these cells withoutaffecting cell growth and morphology, showing that GFP-PBP2x isfunctional. This conditional mutant of pbp2x could be grown for five toseven generations in the absence of inducer before depletion of PBP2x wasapparent, resulting in distinct phenotypes including significant changes incell morphology before a complete halt in growth was observed. In orderto better understand the role of the various domains of PBP2x forlocalization at the septum, different mutant constructs in the TP and the C-terminal domain were constructed and characterised. The data show thatthe PASTA domain is required for localization at the septum.1. E. Gordon, N. Mouz, E. Duee and O. Dideberg, J. Mol. Biol. 299 (2000), p. 477-485.2. C. Morlot, A. Zapun, O. Dideberg and T. Vernet, Mol. Microbiol. 50 (2003), p. 845-55.CEP015Inter- and intramycelial DNA-translocation during StreptomycesconjugationL. Thoma*, E. Sepulveda, J. Vogelmann, G. MuthUniversität Tübingen, Mikrobiologie/Biotechnologie, Tübingen, GermanyThe Gram positive soil bacterium Streptomyces transfers DNA in a uniqueprocess involving a single plasmid-encoded protein TraB and a doublestrandedDNA molecule. TraB proteins encoded by different Streptomycesplasmids have a highly specific DNA binding activity and interact onlywith a specific plasmid region, the clt locus, but do not bind to unrelatedplasmids. They recognize characteristic 8 bp direct repeats (TRS, TraBRecognition Sequence) via a C-terminal wHTH motif. Exchange of the 13aa helix H3 of TraB pSVH1 against H3 of TraB pIJ101 was sufficient to switchspecificity of clt recognition 1 . Binding of TraB to clt is non-covalently anddoes not involve processsing of the plasmid DNA. In addition to theplasmid localized clt, TraB pSVH1 also binds to clt-like sequences on thechromosome, indicating a chromosome mobilization mechanismindependent of plasmid integration.TraB pSVH1 assembles to hexameric ring structures with a central 31 Åchannel and forms pores in lipid bilayers. Structure and DNA bindingcharacteristics of TraB indicate that TraB is derived from a FtsK-likeancestor protein suggesting that Streptomyces adapted the FtsK/SpoIIIEchromosome segregation system to transfer DNA between two distinctStreptomyces cells 1 .In adaptation to the mycelial growth, Streptomyces conjugation alsoinvolves subsequent spreading of the transferred plasmid within therecipient mycelium. Whereas primary transfer from the donor to therecipient seems to depend on a single protein (TraB), plasmid spreadingvia septal crosswalls requires five to six plasmid encoded Spd-proteins inaddition to TraB. None of the Spd proteins has any similarity to afunctionally characterized protein. Bacterial two-hybrid analyses, in vivocrosslinking and pulldown assays revealed interactions of TraB and manySpd proteins. Biochemical analyses of purified proteins revealedpeptidoglycan-binding activities for TraB, SpdB2, Orf108, SpdA, TraRand DNA-binding activities for TraB, SpdB2, TraR and SpdA. SpdArecognizes a conserved palindromic DNA motif inside the spdA codingregion 2 . SpdB2 was shown to form pores in planar lipid bilayers.These data suggest a large DNA translocation complex at the septalcrosswalls with TraB acting as the motor protein and SpdB2 probablyforming a channel structure.1, Vogelmann, J., Ammelburg, M., Finger, C., Guezguez, J., Linke, D., Flötenmeyer, M., Stierhof,Y., Wohlleben, W., and Muth, G.Conjugal plasmid transfer in Streptomyces resembles bacterialchromosome segregation by FtsK/SpoIIIE., EMBO J.2011, 30:2246-542, Sepulveda, E., Thoma, L., and Muth, G., A short palindromic DNA motif is involved inintramycelial plasmid spreading during Streptomyces conjugation, submittedCEP016A highly sensitive enzymatic assay for lytic transglycosylasesand their product 1,6-anhydro-N-acetylmuramic acidB. Naegele*, A. Schneider, J. Hirscher, C. MayerUniversität Tübingen, Biotechnology, Microbiology, AG Mayer, Tübingen,GermanyThe peptidoglycan (or murein) is a huge, net-shaped glycopeptidemacromolecule that surrounds and stabilizes the bacterial cell. It consistsof glycan strands composed of two alternating amino sugars N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Thelatter of both is unique to bacteria. During cell growth large amounts ofMurNAc-containing fragments (muropeptides) are released from thebacterial cell wall. Fragments carrying a 1,6-anhydroMurNAc moiety attheir reducing-end are generated by a special type of muramidases, thelytic transglycosylases (LTs), catalyzing an intramoleculartransglycosylation reaction. In E. coli LTs are the main cell wall lyticenzymes, in other bacteria such as B. subtilis the occurance of theseenzymes remains unclear.Since it is difficult to analyze LTs and 1,6-anhydroMurNAc, a novel assaywas developed to identify and characterize unknown LTs and determinetheir specificity. After digestion of purified peptidoglycan with LTs 1,6-anhydroMurNAc was released by total hydrolysis and re-N-acetylation ofthe samples. In a second step a highly sensitive enzymatic assay wasapplied which is based on radioactive phosphorylation of 1,6-anhydroMurNAc with anhydroMurNAc-kinase AnmK of E. coli.1 Theenzyme specifically converts 1,6-anhydro-MurNAc to MurNAc-6P whichis then detectable down to femtomolar amounts by TLC.1. Uehara, T. et al, 2005, J. Bacteriol. 187:3643-9BIOspektrum | Tagungsband 2012