VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

[4] Neuendorf, S. et al (2004): Biochemical characterization of different genotypes of Paenibacilluslarvae subsp. larvae, a honey bee bacterial pathogen. Microbiology. 150, 2381-2390.[5] O'Farrell, P. H.(1975): High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem.250, 4007-4021.MPP036Chitin-binding proteins of Paenibacillus larvae and theirrole in pathogenesisE. Garcia-Gonzalez*, L. Poppinga, E. GenerschState Institute for Bee Research, Department of Molecular Microbiologyand Bee Diseases, Hohen Neuendorf, GermanyAmerican foulbrood (AFB) is considered the most contagious anddestructive infectious disease in honeybees, caused by the Gram-positive,spore-forming bacterium Paenibacillus larvae [1]. Despite the growingimpact of this disease, molecular mechanisms involved in pathogenesis stillremain elusive. It has been shown that P. larvae spores ingested by youngbee larvae proliferate massively in the midgut lumen and that breaching theepithelium is one of the last steps in the disease process [2]. However, toachieve their way through the gut, the bacteria must first penetrate theperitrophic matrix, a chitin-rich protective layer of the larval gut. Therefore,we hypothesized that chitin-binding proteins play a major role in bothattachment and local degradation of the peritrophic matrix.Here, we present our data on two chitin-binding proteins secreted by P.larvae, which we identified as enhancin and a chitinase-like protein.Knowing that enhancins target insect intestinal mucin[3] while chitinasesdisrupt chitin, which both are the two major components of the peritrophicmatrix, we were prompted to functionally characterize them in infectedlarvae. We show an expression profile during P. larvae infection focused onthe production of chitinase and enhancin. Transcriptomic, proteomic andhistological studies are combined, both in vivo and in vitro, to elucidate therole of these chitin-binding proteins during P. larvae infection.[1] Genersch, E. et al (2006): Reclassification of Paenibacillus larvae subsp. pulvifaciens andPaenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int. J.Syst. Evol. Microbiol. 56, 501-11.[2] Yue, D. et al (2008): Fluorescence in situ hybridization (FISH) analysis of the interactionsbetween honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood ofhoneybees (Apis mellifera). Environ. Microbiol. 10, 1612-20.[3] Fang, S. et al (2009): Bacillus thuringiensis bel protein enhances the toxicity of Cry1Ac protein toHelicoverpa armigera larvae by degrading insect intestinal mucin. Appl. Environ. Microbiol. 75,5237-43.MPP037Molecular identification of bamboo-inhabiting anddegrading fungiD.S. Wei*, O. Schmidt, W. LieseDepartment of Biology, University of Hamburg, Hamburg, GermanyBamboo as a fast growing woody grass is of increasing interest for thesustainable production of a material with many potential applications forbuildings and industrial utilization. Bamboo has generally a low naturaldurability and is easily attacked by fungi during storage, transport,processing and final use. Little is known about the fungi inhabiting anddegrading the bamboo. Furthermore, for applications it is important to knowwhich fungi might cause harm to potential products. We therefore isolatedmany Deuteromycetes, Ascomycetes and Basidiomycetes inhabiting anddegrading the bamboo and identified them by molecular methods (rDNA-ITS sequencing). Construction of a database with the obtained ITSsequences from bamboo provides a future tool for a fast identification of thefungi even in early stages of colonization. Such knowledge is needed for abetter utilization of bamboo and sustainable protection measures.MPP038The tetraspanin FgPls1 is involved in fitness andpathogenicity of Fusarium graminearumL.N. Nguyen 1 , G.T.T. Le 2 , K. Lambou 3 , C. Barbisan 3 , C. Staerkel 2 ,C. Staerkel* 2,3 , M.-H. Lebrun 4 , W. Schäfer 21 Albert Einstein College of Medicine, New York, USA2 Phytopathology and Genetics, University of Hamburg, Hamburg, Germany3 Bayer Crop Science, Center National de la Recherche Scientifique, LyonCedex 09, Germany4 French National Institute for Agricultural Research, Thiverval-Grignon,FranceTetraspanins are a family of small membrane proteins specific to animalsand fungi. These proteins with characteristic secondary structures areinvolved in a broad range of biological processes. They behave as„molecular facilitators” interacting with other membrane proteins such asintegrins, adhesion proteins, metalloproteases and proteins with Ig domainsin animals. In fungi, three different families of tetraspanins werecharacterized. Pls1 is present in ascomycota and basidiomycota while Tsp2is unique to basidiomycota, and Tsp3 is unique to ascomycota. Pls1 nullmutants from plant pathogenic fungi such as Magnaporthe grisea, Botrytiscinerea, and Colletotrichum lindemuthianum are non pathogenic on plantsbeing defective in appressorium mediated penetration. In this study, weidentified FgPLS1, the functional orthologue of MgPLS1 in the wheat scabfungus F. graminearum. Null mutants obtained by targeted genereplacement displayed defects in pathogenicity and additional phenotypes(altered mycelium growth, highly reduced production of macroconidia) notobserved in other fungal PLS1 mutants. These results demonstrate that thisgene is important for vegetative growth, sporulation, and pathogenicity in F.graminearum. Therefore, although Pls1 tetraspanins control cellularfunctions involved in infection conserved among fungal plant pathogens,they have been recruited to control cellular functions involved in growth andsporulation specifically in F. graminearum.MPP039Genome sequencing of a vanA-negative, high-levelvancomycin resistant Staphylococcus aureus mutantreveals multiple genetic polymorphismsA. Berscheid* 1 , P. Sass 1 , A. Jansen 1 , M. Oedenkoven 1 , C. Szekat 1 ,G. Gottschalk 2 , G. Bierbaum 11 Institute of Medical Microbiology, Immunology and Parasitology,Friedrich-Westphalian Wilhelms-University, Bonn, Germany2 Institute of Microbiology and Genetics, Georg-August-University,Göttingen, GermanyThe glycopeptide antibiotic vancomycin remains the main therapeutic agentfor the treatment of serious infections caused by methicillin-resistant S.aureus (MRSA) that are refractory to other clinically used antibiotics.However, MRSA strains with reduced susceptibility to vancomycin haveemerged during the last decade. In times of increased antibiotic treatmentfailure, there is an obvious need to understand how bacteria respond to thepresence of antibacterial compounds and develop resistance.Characterization of clinical and laboratory vancomycin-intermediateresistant S. aureus strains (VISA) identified multiple, resistance-associatedchanges most probably due to stepwise mutations [1, 2]. In a previous study,the mutator strain S. aureus RN4220ΔmutS, a mutS gene deletion mutant ofthe parent strain RN4220, was subjected to a stepwise vancomycin selectionprocedure [3]. Multiple passaging in the presence of increasingconcentrations of vancomycin resulted in the generation of the mutant strainRN4220ΔmutS-VC40 that exhibits a significantly higher resistance level tovancomycin compared to the non-resistant parent, indicating full resistanceof strain VC40 (MIC: 64 μg/ml versus 2 μg/ml, respectively).In this study, the full genome sequence of strain S. aureus VC40 and itsparent strain S. aureus RN4220ΔmutS was determined which revealedmultiple genetic polymorphisms in genes related to cell wall metabolism,transport and gene regulation, including the two-component regulatorysystems VraSR and WalKR. Further in-depth analysis of strain VC40 bytranscriptomic and proteomic studies as well as mutational analyses willcomplete the genomic data in order to gain a better understanding of themechanisms underlying glycopeptide resistance development in S. aureus.[1] McAleese et al (2006): JBac 188:1120-1133.[2] Ohta et al (2004): DNA Res 11:51-56.[3] Schaaff et al (2002): AAC 46:3540-3548.spektrum | Tagungsband 2011