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

120proteins are excreted. On the contrary, the most abundant cytoplasmicproteins were not found in the secretome. These results suggest that thereexists a selection mechanism in the excretion of cytoplasmic proteins. Thepresence or absence of prophages had little influence on the secretomepattern. Furthermore we could show in the atl mutant that secondarypeptidoglycan hydrolases were increased both in the secretome as well thecorresponding genes were transcriptionally up-regulated suggesting acompensatory mechanism for the atl mutation. As the major autolysinbinds at the septum site, we assume that the proteins are preferentiallyreleased at and during septum formation.MPP046Relaxed substrate specificity of bacterial phospholipidflippases - alanyl- phosphatidylglycerol confers wild type leveldaptomycin resistance in the presence of lysylphosphatidylglycerolflippases in Staphylococcus aureusC. Slavetinsky*, C. Ernst, A. PeschelUniversity of Tubingen, Interfaculty Institute of Microbiology andInfection Medicine (IMIT), Cellular and Molecular Microbiology Section,Tübingen, GermanyThe Multiple Peptide Resistance Factor (MprF) of Staphylococcus aureusis a bifunctional enzyme with two separable functional domains thatsynthesize positively charged lysyl- phosphatidylglycerol (Lys-PG) andfacilitate Lys-PG flipping into the outer leaflet of the membrane, resultingin repulsion of cationic antimicrobial peptides encountered duringcolonization and infection of the human host or competing microorganisms(Peschel et al.,2001, Ernst et al., 2009). The impact of MprF- mediatedLys-PG production on CAMP resistance has been confirmed with MprFhomologs from major human pathogens, such as Listeria monocytogenes,Bacillus anthracis, Mycobacterium tuberculosis, and also with MprFhomologs from Rhizobium tropici and Bacillus subtilis.Interestingly, some MprF proteins synthesize zwitterionic alanylphosphatidylglycerol(Ala-PG), such as MprF homologs fromEnterococcus faecium, Clostridium perfringens, or Pseudomonasaeruginosa. The Impact of the production of zwitterionic Ala-PG onsusceptibility to antimicrobial peptides has so far only been studied in thegram- negative pathogen P. aeruginosa, which alanylates 6% of thephospholipids, leading to select phenotypes, such as reduced susceptibilityto cromium ions, protamine sulphate and cefsulidin (Klein et al., 2009).We expressed the Ala-PG producing MprF of C. perfringens in a S. aureusmprF deletion mutant and show that Ala-PG integrates effectively in thephospholipid biosynthetic pathways of S. aureus, leading to the productionof more than 60 % Ala-PG. The production of Ala-PG in S. aureus enabledus to investigate the impact of zwitterionic Ala-PG on CAMPsusceptibility in a gram positive pathogen and led to the unexpectedobservation that Ala-PG is as effective in conferring a basic level ofresistance to the CAMP- like antibiotic daptomycin, as Lys-PG, as long asLys-PG flippases are present, indicating that Lys-PG flippases have broadrange specificity for aminoacyl- phospholipids.MPP047Functional genome analysis of Paenibacillus larvae, the causativeagent of the American Foulbrood of honey bees (AFB)M. Djukic* 1 , E. Brzuszkiewicz 1 , A. Fünfhaus 2 , E. Genersch 2 , R. Daniel 11 Georg-August-University Goettingen, Goettingen Genomics Laboratory,Goettingen, Germany2 Institute for Bee Research, Hohen Neuendorf, GermanyPaenibacillus larvae is a rod-shaped and spore-forming Gram-positivebacterium causing American Foulbrood of honey bees. First P. larvae hasbeen described as Bacillus larvae in 1906. Recently, it was shown that thespecies P. larvae comprises different genotypes differing in virulence atthe individual insect and at the colony level [1]. P. larvae is able to infecthoney bees and honey bee larvas via the spores, but only kills the latter.The way of infection and killing is still poorly understood. It has beenshown, that approximately 10 infectious spores from virulent strains aresufficient to cause mortality [2].Raw-sequencing of the P. larvae str. 08-100 (ERIC I) and str. 04-309(ERIC II) genomes were done by using 454-pyrosequencing. The obtainedsequences were assembled and analyzed. Subsequently, contigs weresorted and remaining gaps closed. The genome size of P. larvae str. 04-309 (ERIC II) and the GC content are approximately 4.05 Mb and 45 %,respectively, while the genome size of P. larvae str. 08-100 is about 4.51Mb and has a GC content of 44 %. The annotation of the genomesequences provided new important insights into genes involved inpathogenesis.[1] Genersch et al., Int. J. Syst. Evol. Microbiol. 56, 501-511 (2006)[2] Brodsgaard et al., Apidologie 29, 569-578 (1998)MPP048Transcriptome and proteome analyses of P. aeruginosa PAO1expressing the biofilm-inhibiting SDR BpiB09 reveal asignificant effect on QS-controlled genesC. Utpatel* 1 , P. Bijtenhoorn 1 , A. Thürmer 2 , E. Brzuszkiewicz 2 , R. Daniel 2 ,B. Voigt 3 , M. Hecker 3 , C. Vollstedt 1 , W.R. Streit 11 University of Hamburg, Biozentrum Klein Flottbek - Mikrobiologie &Biotechnologie, Hamburg, Germany2 University of Göttingen, Institute of Microbiology and Genetics -Göttingen Genomics Laboratory, Göttingen, Germany3 University of Greifswald, Institute of Microbiology - Division ofMicrobial Physiology and Molecular Biology, Greifswald, GermanyIn Pseudomonas aeruginosa, quorum sensing-regulated gene expressioncontributes to the formation and maintenance of biofilms and theirtolerance to conventional antimicrobials. Therefore QS and QS-relatedgene expression are promising targets for the development of newantimicrobial drugs. Here we report on a genome wide transcriptomeanalysis using next generation sequencing RNA-seq and proteome analysisof PAO1 cells expressing the recently published novel and metagenomederivedshort-chain dehydrogenase/reductase (SDR) BpiB09 1 . Expressionof BpiB09 resulted in a significantly reduced pyocyanin production,decreased motility, poor biofilm formation and decreased paralysis ofnematodes. HPLC-MS analyses correlated these phenotypes with thealmost complete absence of synthesized autoinducers in PAO1. Ourgenome wide comparative transcriptome and whole-cell-protein proteomeanalysis of P. aeruginosa PAO1 expressing BpiB09 identified significanteffects on most of the quorum sensing controlled genes like lasI, rhlI, pqsRand pqsABCD. A least 38 of these well-known QS-regulated genes werestrongly (>10-fold) down-regulated in their expression profiles. As well asignificant number of genes and ORFs were detected that had been linkedto QS-phenotypes in PAO1 and that were less than 10-fold but at least 4-fold altered in their expression level. Altogether these were 80 genes/ORFsand among those we found the hcnB and hcnC genes involved in hydrogencyanide synthesis, the aprD and aprE genes involved in alkaline proteasesecretion as well as lecB and lasA. Additionally a defined subset of so farnot QS-linked genes was affected. These data were supported by 2Dproteomeanalyses of PAO1 cells. Altogether, our data suggest that thedirect expression of SDR in PAO1 and/or the exogenous addition ofBpiB09 to growing PAO1 cells have profound effects on PAO1 geneexpression and might be a useful tool for the development of novel antibiofilmstrategies.1 Bijtenhoorn, P., Mayerhofer, H., Müller-Dieckmann, J., Utpatel, C., Schipper, C., Hornung, C., Szesny,M., Grond, S., Thürmer, A., Brzuszkiewicz, E., Daniel, R., Dierking, K., Schulenburg, H., & W. R. Streit(2011) A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosaBiofilm Formation and Virulence on Caenorhabditis elegans. PLoS ONE 6(10)MPP049Comparative global transcriptome analysis ofCandidaalbicansandCandida dubliniensisallows new insights intochlamydospore developmentK. Palige* 1 , J. Linde 2 , F. Citiulo 3 , D.J. Sullivan 4 , S. Rupp 5 , J. Morschhäuser 6 ,B. Hube 3 , P. Staib 11 Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute (HKI), JRG Fundamental Molecular Biology of PathogenicFungi, Jena, Germany2 Hans Knöll Institut, Systems Biology/Bioinformatics, Jena, Germany3 Hans Knöll Institut, Microbial Pathogenicity Mechanisms, Jena, Germany4 University of Dublin, School of Dental Science and Dublin Dental Hospital,Trinity College, Dublin, Ireland5 University of Stuttgart, Institute of Interfacial Engineering, Stuttgart, Germany6 University of Würzburg, Institute for Molecular Infection Biology, Würzburg,GermanyCandida albicans and Candida dubliniensisare highly related pathogenicyeast species displaying differences in their epidemiology and in somephenotypic characteristics, including virulence-associated traits. During invitro growth on certain nutrient-poor media, both share the species-specificability to produce chlamydospores, large spherical, thick-walled cells withunknown function. Interestingly however, onlyC. dubliniensisformspseudoyphae with abundant chlamydospores on Staib agar (syn.Guizotiaabyssinicacreatinine agar), on whichC. albicansgrows as a budding yeast.In order to get new insights into chlamydospore development, wecompared the global transcriptional profile of both species during growthin Staib medium by DNA microarray analysis and RNA sequencing. As ameans to narrow down the putative set of chlamydospore- versuspseudohyphae-specific genes, the analysis of aC. albicansnrg1mutant wasalso included in this study.C. albicansmutants in this global repressor offilamentation have previously been demonstrated to produce not onlypseudohyphae but also abundant chlamydospores in Staib medium, similarasC. dubliniensis. At present, individual identified genes are functionallycharacterized inC. albicansandC. dubliniensis, for their putative role inchlamydospore development but also with respect to other phenotypicBIOspektrum | Tagungsband 2012