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

69CEP017Unique wall teichoic acid glycosylation of the borderlineStaphylococcus aureus strain PS187 is required for hostpathogen interactionV. Winstel* 1 , P. Sanchez-Carballo 2 , C. Liang 3 , T. Dandekar 3 , O. Holst 2 ,A. Peschel 1 , G. Xia 11 Interfaculty Institute of Microbiology and Infection Medicine, Cellularand Molecular Microbiology Division, Tuebingen, Germany2 Research Center Borstel, Leibniz-Center for Medicine and Biosciences,Division of Structural Biochemistry, Borstel, Germany3 University of Würzburg, Biozentrum, Bioinformatik, Würzburg, GermanyStaphylococccus aureus is a major human pathogen causing severediseases including endocarditis, pneumonia and sepsis. Important surfacepolymers are wall teichoic acids (WTA) known to play a crucial role in anumber of processes including host pathogen interaction, biofilmformation, resistance to antimicrobials and phage adsorption. Using agenome sequencing approach S. aureus strain PS187 was found to be aborderline S. aureus isolate sharing almost all classical surface proteinadhesins required for host pathogen interaction. Of note one formerlyidentified nasal colonization factor, WTA, is changed in PS187 to a uniqueWTA consisting of repetitive units of polyglycerolphosphate (GroP)substituted with D-ala or N-acetylgalatosamine (GalNAc) revised by NMRand renamed as C-type WTA. Based on the genome sequencing approacha novel WTA biosynthesis gene cluster encoding for a unique WTAglycosyltransferase designated as TagN was discovered. Genetic mutantsin PS187 lacking C-type WTA and only the GalNAc modification wereconstructed and used for biochemical analysis. Lectin overlay, WTAPAGE and 1H-NMR analysis clearly demonstrate TagN acts as a WTAGalNAc glycosyltransferase. Based on this C-type WTA glycosylation wasfound to play a crucial role in phage infection as well as in surviving athigh temperatures. Of clinical relevance C-type WTA glycosylation isrequired for interaction with human epithilial cells indicating PS187 WTAis essential for the colonization process although the WTA structure isdifferent if compared to other S. aureus strains. Hence inhibition of S.aureus WTA glycosylation can be a promising strategy to avoid nasalcolonization.CEP018Communication and Heterogeneity among Microcystis coloniesK. Makower*, E. DittmannUniversity of Potsdam, Microbiology, Potsdam, GermanyBloom formation of the cyanobacterial genus Microcystis represents aworldwide phenomenon reflecting enormous ecological success of thesephototrophic bacteria under certain environmental conditions. Asophisticated and diverse formation of intriguing colonial morphotypesreflects heterogeneity as well as genetic plasticity among Microcystis cellsand is influenced by a comprehensive intercellular communication. In arecently initiated project heterogeneity of Microcystis colonies shall beinvestigated both, from the molecular basis and the physiological effects.Effects of known impact factors on Microcystis colony size like high-lightconditions, as well as certain cell surface proteins and other peptides arebeing systematically monitored and characterized. Insights into themolecular basis of Microcystis colony formation shall be gathered byinvestigating fluorescence labeled Microcystis knockout strains, deficientin the production of the cell-cell-interaction affecting proteins andpeptides, respectively. Furthermore new sequencing approaches aresupposed to clarify genetic conformity or varying genetic compositionwithin cells of Microcystis colonies. In addition to the molecularcharacterization of colonies ecological aspects such as vertical migrationproperties and enzyme gradients within cell assemblies might give furtherindications as to the biological benefits ofMicrocystis’sophisticated colonyformation.CEP019The C-terminal domain confers binding partner specificity toBacillus subtilis DivIVAS. Halbedel* 1 , S. van Baarle 2 , I. Nazli Çelik 3 , M. Bramkamp 2 ,L.W. Hamoen 31 Robert Koch-Institut, FG11 - Bakterielle Infektionen, Wernigerode, Germany2 Universität Köln, Institut für Biochemie, Köln, Germany3 Newcastle University, Center for Bacterial Cell Biology, Newcastle uponTyne, United KingdomDivIVA proteins are curvature sensitive membrane binding proteins thatrecruit other proteins to the poles and the division septum. They comprisean N-terminal lipid binding domain fused to less conserved C-terminalcoiled coil domains that vary in length and sequence among the differentgram positive species. We used bacterial two hybrid analyses to test whichpart of B. subtilis DivIVA is responsible for the interaction to MinJ andRacA. This approach identified short C-terminal truncations of DivIVAthat selectively have lost the ability to interact with MinJ and RacA,suggesting that C-terminus of DivIVA is crucial for binding partnerrecruitment. Complementation experiments of the B. subtilis divIVAbackground with chimeric DivIVA proteins that consist of N-terminalstretches of B. subtilis DivIVA and corresponding C-terminal portions ofDivIVA from Listeria monocytogenes furthermore demonstrated that thecomplete C-terminal coiled coil domain is required for MinJ and RacAbinding. Our analyses provide evidence that the C-terminal domain of B.subtilis DivIVA is the structural unit that provides the docking site towhich MinJ and RacA bind. Fusion of the DivIVA-like lipid bindingdomain to a less conserved C-terminal protein recruitment module thatserves a species-specific cellular function therefore appears to be theunifying architectural feature of DivIVA proteins.CEP020Identification of DivIVA interaction partners in ListeriamonocytogenesK.G. Kaval*, S. HalbedelRobert Koch-Institut, FG11 - Bakterielle Infektionen, Wernigerode, GermanyCell division, a vital process in all organisms, involves the division of theparent cell into two or more daughter cells, so as to maintain growth andproliferation. DivIVA is a well conserved protein involved in this processin various Gram-positive bacteria, having an N- terminal lipid bindingdomain (LBD) connected to a C-terminal coiled coil domain (CTD) via aflexible linker. The CTD is postulated to confer diverse morphogeneticfunctions to DivIVA orthologues in different bacterial species, by allowingit to bind to different interaction partners. Previous work showed asimilarity in the phenotype of divIVA and secA2 deletion mutants ofListeria monocytogenes, indicating a possible interaction between thesetwo proteins either directly or via some other intermediates. The accessorysecretion ATPase SecA2 allows for the translocation of virulence relatedautolysins and thus contributes to full virulence of L. monocytogenes.Bacterial two hybrid assays were used to test for direct interactionsbetween listerial DivIVA, SecA and SecA2. However, these experimentsshowed only self-interactions but no direct interactions between theseproteins, which hinted to the presence of other intermediary interactionpartners. Affinity tagged constructs of the respective genes were cloned forthe purpose of carrying out affinity pull-down assays using the respectiveaffinity tagged proteins, to identify and characterize these binding partners.This approach will help us to identify so far unknown genes that play arole in SecA2-dependent protein secretion, cell division and virulencepathways of L. monocytogenes. Current progress of these experimentswould be presented on this poster.CEP021Cell wall modifications as a mechanism of antibiotic selfresistanceR. Pozzi*, H.-J. Frasch, E. StegmannUniversität Tübingen, Mikrobiologie/biotechnologie, Tübingen, GermanyInvestigations into mechanisms of antibiotic self-resistance inactinomycetes are important to understand the emergence of antibioticresistance in pathogens and to acquire fundamental knowledge useful forthe development of high producer strains by metabolic engineering. Animportant target for lantibiotics and glycopeptides is the bacterial cell wall.We are currently interested in the study of the self-immunity mechanism inMicrobispora ATCC PTA-5024 and Amycolatopsis balhimycina bothsynthesizing antibiotics interfering with the bacterial cell wall.Microbispora is the producer of NAI-107, the first example of a class Ilantibiotic produced by actinomycetes. It inhibits the incorporation oflipid-II in the nascent peptidoglycan by binding to the pyrophosphatemoiety. This novel lantibiotic has attracted attention as a potential drugcandidate because of its antibacterial profile that cover Gram-positiveresistant pathogens like glycopeptide-intermediate S. aureus (GISA) andvancomycin-resistant enterococci (VRE) [1]. A. balhimycina produces thevancomycin-type glycopeptide balhimycin which binds to the D-Ala-D-Ala ending cell wall precursors. The most common resistance mechanismof bacteria against glycopeptides is to reprogram the murein syntheticmachinery resulting in resistant cell wall precursors ending on D-Ala-D-Lac.To understand the self-resistance of the producer and the mode of action ofthe antibiotic it is important to analyse the cell wall composition of theproducer under production and non-production conditions. In contrast tothe model organism Streptomyces coelicolor M145, both strainsMicrobispora and A. balhimycina do not have a monoglycine interbridgebut they present a direct linkage between peptide chains. Mature A.balhimycina peptidoglycan contains mainly tri- and tetrapeptides and onlytraces of the D-Ala-D-Ala ending pentapeptides that are binding sites forthe antibiotic produced. Both A. balhimycina wild type and a nonproducingmutant strain synthesize mainly peptidoglycan precursorsending with D-Lac indicating a constitutive synthesis of a resistant cellwall [2]. HPLC-MS analyses of Microbispora cell wall precursors reveal amass peak of 1193.4 Da. This value corresponds to the precursor UDP-BIOspektrum | Tagungsband 2012