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

129suggest that the YycG kinase might in vivo respond to membrane fluidityvia its TM domains.[1] Fabret and Hoch (1998), J Bacteriol., 180(23):6375-83.[2] Dubrac et al. (2008), Mol Microbiol.,70(6):1307-22.[3] Fukushima et al. (2011), Mol Microbiol., 79(2):503-22.MPV3-FGStructure and function of the PorB porin from disseminatingN. gonorrhoeaeK. Zeth* 1 , V. Kozjak-Pavlovic 2 , M. Faulstich 2 , O. Kepp 2 , T. Rudel 21 University of Tübingen, ZMBP, Tübingen, Germany2 University of Würzburg, Department of Microbiology, Würzburg,GermanyThe outer membrane of Gram-negative bacteria is permeabilized by a largenumber of porin channels for the uptake of small molecules. Porins are themajor outer membrane proteins of proteobacteria and mitochondria. Someporins of the Neisseriaceae clade can insert and permeabilize the innermitochondrial membranes of mammalians cells during infection and maylead to mitochondrially induced apoptosis. Porins of the serotype A(PorB IA) of N. gonorrhoeae are associated with disseminating gonococcaldisease and mediate the rapid invasion into host cells in a phosphatesensitive manner. To understand these functions on the basis of structuraldata we analyzed the structure of PorB IA isolated from wildtype N.gonorrhoeae. The structure in complex with ATP and phosphate solved atthe resolution 3.1 Å displays a surplus of positive charges inside thechannel with a potential for substrate transfer. ATP is coordinated bypositively charged residues via aromatic, sugar and pyrophosphate moietyatoms. A short b-bulge inserted into the b2-strand nearby the location ofATP and the long L3 loop narrows the barrel diameter significantly andtogether support substrate specificity. Phosphate ions known to interferewith bacterial uptake after host cell contact are present, one of which iscoordinated by two Arg residues nearby the ATP at the extraplasmicchannel exit. In vivo assays of bacteria carrying mutations at residuescomplexing phosphate molecules confirm the importance of these residuesfor host cell invasion. Interestingly, the structure also comprises a smallpeptide sequence as remnant of a periplasmic protein which physicallylinks porin molecules to the peptidoglycane layer. Although similar insequence to related Neisseriacea, PorB IA shows differences which moreclearly deviate from the non-apoptotic porin from N. sicca or N. lactamica.Models of these PorB channels are compared in structure and possiblefunctional implications are outlined and tested.MPV4-FGMethicillin Resistance inStaphylococcus aureusDepends onß-O-GlcNAcylation of Wall Teichoic AcidsS. Brown 1 , G. Xia* 2 , L. G. Luhachack 3 , J. Campbell 1 , T. Meredith 1 , C. Chen 1 ,V. Winstel 2 , C. Gekeler 2 , J. E. Irazoqui 3 , A. Peschel 2 , S. Walker 11 Department of Microbiology and Immunobiology, Harvard Medical School,Boston MA, USA2 Cellular and Molecular Microbiology Section, Interfaculty Institute ofMicrobiology and Infection Medicine, University of Tübingen, Tübingen.Germany3 Department of Pediatrics, Massachusetts General Hospital, Harvard MedicalSchool, Boston, USAStaphylococcus aureuspeptidoglycan is densely functionalized withanionic polymers called wall teichoic acids (WTAs), which are requiredfor proper cell division. Preventing WTA polymer synthesis sensitizesmethicillin resistantS. aureus(MRSA) to beta-lactams. Here we describethe discovery and biochemical characterization of a novelglycosyltransferase, TarS, that attaches b-O-N- Acetylglucos-amine (b-O-GlcNAc) residues toS. aureusWTAs. We show that b-O-GlcNAcylation ofWTAs is required for the beta-lactam resistant phenotype in MRSA. Theb-O-GlcNAc residues play a specific structural role in mediating resistancesince neither a-O-GlcNAc modifications nor b-O-glucosyl modificationsconfer resistance. We propose that b-O-GlcNAcylated WTAs scaffoldpeptidoglycan biosynthetic complexes containing the resistanttranspeptidase PBP2a. The b-O-GlcNAc transferase identified here, TarS,is a new target for inhibitors that sensitize MRSA to beta-lactams.MPV5-FGIdentification of -haemolysin encoding genes in StreptococcusanginosusD. Asam* 1 , S. Mauerer 1 , E. Walheim 2 , B. Spellerberg 11 University Hospital Ulm, Institute for Medical Microbiology and Hygiene,Ulm, Germany2 University Ulm, Ulm, GermanyStreptococcus anginosus is a commensal of the oral cavity, thegastrointestinal and the female urogenital tract. It has clinical significancein abscess formation and has been suggested to play a pathogenic role inpatients with cystic fibrosis. An interesting feature of these bacteria is theinconsistent phenotype regarding Lancefield antigens as well ashaemolytic activity. While a considerable percentage of S. anginosusstrains display a prominent ß-haemolytic phenotype, the correspondinggenes have not been identified yet. In different streptococcal species the ß-haemolysin is a modified short peptide (SLS) that is related to Class Ibacteriocins. It is encoded in the sag gene cluster including genes for thecorresponding posttranslational modifications and transport machinery.By random chromosomal integration of the pGhost9:ISS1 transpositionvector we generated a plasmid-based mutant library of the haemolyticStreptococcus anginosus strain ATCC 12395. This library was screenedfor mutants showing a loss of the ß-haemolytic phenotype on blood agarplates and non-haemolytic mutants were selected for further investigation.By sequencing the insertion sites of these mutants we identified thus far 10different mutations sites in a gene cluster of 9 kb harbouring 9 openreading frames, with significant similarities to the sag (SLS associatedgene) gene cluster of Streptococcus pyogenes that encodes the haemolysinStreptolysin S (SLS). ORFs corresponding to all of the 9 sag genes (sagAto sagI) could be identified. Similarities of the deduced amino acids of theputative S. anginosus sag gene cluster to the Sag-proteins of S. pyogenesrange from 37 % (sagF) to 81 % (sagD). To further investigate the S.anginosus haemolysin, a functional haemolysin assay with culturesupernatants and whole bacteria was carried out. Haemolytic activity wasonly observed with whole cells, not in the supernatant, indicating that likeSLS of S. pyogenes, the S. anginosus haemolysin is able to lyseerythrocytes only in cell-associated form. But contrary to S. pyogenes,adding of FCS to the cell-free supernatant of S. anginosus did not increasehaemolytic activity.In summary we were able to identify an SLS-like gene cluster as thegenetic basis of S. anginosus ß-haemolysin production and could furthercharacterize the ß-haemolysin.MPV6-FGAlternative pathways of phagosomal escape of StaphylococcusaureusF. Glowinski 1 , K. Paprotka 2 , M. Grosz 3 , B. Sinha 3 , M. Fraunholz* 21 Max Planck Institute for Infection Biology, Dept. Molecular Biology, Berlin,Germany2 University of Würzburg , Chair of Microbiology, Biocenter, Würzburg,Germany3 Univeristy of Würzburg, Institute of Hygiene and Microbiology, Würzburg,GermanyStaphylococcus aureus is efficiently taken up by non-professionalphagocytes. Subsequently, certain bacterial strains are able to escape thephagoendosome in an agr-controlled process. We have recently identifiedthat expression of S. aureus -toxin, an agr-effector encoded by RNAIII,can augment phagosomal escape in presence of -toxin. Here we show thatexpression of phenol soluble modulin (PSM) but not PSM also leads toescape of -toxin positive S. aureus. By contrast, the membranedestructiveactivity of PSM is supported by another phospholipase. Thus,alternative pathways exist for phagosomal escape of S. aureus, whichmight present different strategies to avoid lysosomal disinfection inpresence or absence of -converting phages.MPP1-FGCharacterization of a novel genomic island in a monophasicvariant of Salmonella TyphimuriumS. Simon*, A. Flieger, W. RabschRobert Koch Institute, National Reference Centre for Salmonella and otherEnteric Pathogens and Division of Bacterial Infections, Wernigerode,GermanyBesides the core genome, bacterial chromosomes harbour numerousaccessory genes acquired by horizontal gene transfer. Organised in clusterspredominantly at tRNA loci, these genes are recognised as genomic islands(GEIs). Depending on their gene content the elements are termedpathogenicity, symbiosis, metabolic, fitness or resistance islands [1]. Herewe describe a novel 18.4 kb genomic island adjacent to the thrWtRNAlocus of an endemic monophasic variant of Salmonella Typhimurium. 454sequencing and subsequent BLAST analyses revealed 27 open readingframes and a significantly lower G+C content compared to the closelyrelated S. Typhimurium LT2 genome. Homologies covering large parts ofthe island have been found to several E. coli and Shigella nucleotidesequences [2]. Protein BLAST analyses revealed a number of phagerelatedproteins, indicating that the island might be of phage-origin.Transcripts were detected for 24 ORFs. Further we showed that the islandcan be excised from the chromosome and form a circular intermediatewhich is mobilised under certain conditions. Broth mating experimentsresulted in the successful conjugational transfer of the 18.4 kb island fromthe donor to an appropriate S. Typhimurium recipient strain. To elucidatethe function of the island, we focussed on ORF 10 since it is predicted tocode for a T3SS effector. Our experiments suggest that its product is trulysecreted but probably not via the SPI1-, SPI2- or flagella-T3SS describedBIOspektrum | Tagungsband 2012