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

kanamycin sensitive clones were isolated from chicken indicating that theloxP-rpsL-neo-loxP (LoxP) cassette could be deleted due to in vivo activepromoters. PCR analysis and sequencing demonstrated a range of insertsizes (1-2kb) suggesting that the screening is functional and the plasmidcould stably be maintained in the bacteria even after infection. Furtheranalysis of the DNA fragments' sequences revealed that 27 different APECgenes were induced inside the chicken during infection. With these resultsAPEC RIVET library could be adapted and the strategy showed to befunctional for the screening of host-induced APEC genes/ promoters inchickens.MPP002New insights into pyomelanin formation of AspergillusfumigatusJ. Macheleidt* 1,2 , K. Scherlach 3,2 , T. Heinekamp 1,2 , A. Brakhage 1,21 Department of Molecular and Applied Microbiology, Hans Knöll Institute(HKI), Jena, Germany2 Friedrich-Schiller-University, Jena, Germany3 Biomolecular Chemistry, Hans Knöll Institute (HKI), Jena, GermanyAspergillus fumigatus is an opportunistic fungal pathogen that causes severesystemic infections in immunocompromised patients. The biosynthesis ofdihydroxynaphthalene melanin is an important virulence determinant.Recently, an alternative type of melanin, pyomelanin, was described in A.fumigatus. Pyomelanin is produced by polymerisation of homogentisic acid,the central intermediate during degradation of phenylalanine and tyrosine.Four enzymes that are involved in tyrosine degradation are encoded in agene cluster in A. fumigatus. This cluster also contains a gene of unknownfunction that we designated hmgX. Disruption of hmgX preventedpyomelanin formation as well as growth of A. fumigatus on tyrosine as thesole carbon or nitrogen source, indicating that HmgX is essential forcomplete tyrosine decomposition. Ectopic integration of hmgX into thegenome of the ΔhmgX mutant strain restored the wild-type phenotype.Interestingly, a mutant in which no homogentisic acid was formed due todeletion of the gene encoding the dioxygenase HppD showed the samephenotype as the hmgX knock-out strain. Analysis of culture supernatants byHPLC confirmed that the hmgX mutant still converted tyrosine to p-hydroxyphenylpyruvate, whereas the next intermediate homogentisic acidwas not formed. These data as well as measurement of enzymatic activitiessuggest that HmgX is involved in the conversion of p-hydroxyphenylpyruvate to homogentisic acid that is catalysed by HppD.MPP003Strain-specific differences in pili formation and theinteraction of Corynebacterium diphtheriae with host cellsL. Ott*, M. Höller, J. Reinlaender, T.E. Schäffer, M. Hensel, A. BurkovskiInstitute for Microbiology, Friedrich-Alexander-University, Erlangen,GermanyCorynebacterium diphtheriae, the causative agent of diphtheria, is wellinvestigatedin respect to toxin production, while little is known about C.diphtheriae factors crucial for colonization of the host. In this study, weinvestigated strain-specific differences in adhesion, invasion andintracellular survival and analyzed formation of pili in different isolates.Adhesion of different C. diphtheriae strains to epithelial cells and invasionof these cells are not strictly oupled processes. Using ultrastructure analysesby atomic force microscopy, significant differences in macromolecularsurface structures were found between the investigated C. diphtheriae strainsin respect to number and length of pili. Interestingly, adhesion and piliformation are not coupled processes and also no correlation betweeninvasion and pili formation was found. Using RNA hybridization andWestern blotting experiments, strainspecific pili expression patterns wereobserved. None of the studied C. diphtheriae strains had a dramaticdetrimental effect on host cell viability as indicated by measurements oftransepithelial resistance of Detroit 562 cell monolayers and fluorescencemicroscopy, leading to the assumption that C. diphtheriae strains might useepithelial cells as an environmental niche supplying protection againstantibodies and macrophages.The results obtained suggest that it is necessary to investigate variousisolates on a molecular levelto understand and to predict the colonizationprocess of different C. diphtheriae strains.MPP004Streptomyces lividans inhibits the proliferation ofVerticillium dahliae on seeds and roots of ArabidopsisthalianaH. Meschke*, S. Rinkel*, S. HildgundApplied Genetics of Microorganisms, University of Osnabrueck,Osnabrueck, GermanyThe vascular disease in more than 200 dicotyledonous plants is due to theascomycete fungus Verticillium dahliae. As documented by videomicroscopy,the soil bacterium Streptomyces lividans strongly reduces thegermination of V. dahliae conidia, and the subsequent growth of the fungalhyphae. Quantification by the use of DNA-intercalating dyes andCalcofluor-staining revealed that during prolonged co-cultivation bacterialhyphae proliferate to a dense network, provoke a poor development of thefungal vegetative hyphae, and an enormous reduction of fungal conidia andmicrosclerotia. Additional studies allowed identifying proteins and thecorresponding genes of S. lividans, which play a role in this interaction.Upon individual application to seeds of the model plant Arabidopsisthaliana, either the Streptomyces spores, or the fungal conidia germinate ator within the mucilage, including its volcano-shaped structures. Theextension of hyphae from each individual strain correlates with the reductionof the pectin-containing mucilage-layer. Proliferating hyphae then spread toroots of the emerging seedlings. Plants, which arise in the presence of V.dahliae within agar or soil have damaged root cells, an atrophied stem androot, as well as poorly developed leaves with chlorosis symptoms. Incontrast, S. lividans hyphae settle in bunches preferentially at the outer layernear tips and alongside roots. Resulting plants have a healthy appearanceincluding an intact root system. A. thaliana seeds, which are co-inoculatedwith V. dahliae and S. lividans, have preferentially proliferating bacterialhyphae within the mucilage, and at roots of the outgrowing seedlings. As aresult, plants have considerably reduced disease-symptoms.MPP005Regulation of tyrosine degradation in the humanpathogenic fungus Aspergillus fumigatusS. Keller* 1 , I.D. Jacobsen 2 , T. Heinekamp 1 , A.A. Brakhage 11 Department of Molecular and Applied Microbiology, Hans-Knöll-Institute(HKI), Jena, Germany2 Project Microbial Pathogenicity Mechanisms, Hans-Knöll-Institute (HKI),Jena, GermanyThe saprotrophic fungus Aspergillus fumigatus is the most prevalentairborne fungal pathogen, causing severe disseminating infections withinvasive growth in immunocompromised patients. This mould possessesspecific physiological and molecular characteristics including thebiosynthesis of a distinct type of melanin, i.e. pyomelanin. This dark-brownpigment is formed by oxidative polymerization of homogentisate, anintermediate of the tyrosine degradation pathway. Interestingly, the genesinvolved in tyrosine catabolism are organized in a cluster in the genome ofA. fumigatus, suggesting that they are regulated in a common manner. Here,we present data on the functional characterization of hmgR, a gene localizedwithin the cluster, that encodes a putative transcription factor with aZn(II) 2Cys 6 DNA binding domain. Fluorescence microscopy showed that anHmgR-eGFP fusion protein concentrates in a region in the nucleus, which isnot yet identified in further detail. Analysis of hmgR deletion mutants andcomplemented strains revealed that HmgR is essential for tyrosine inducedtranscription of the cluster genes. These data strongly suggest the hypothesisthat HmgR is a transcriptional activator of the tyrosine degradation cluster.Interestingly, tyrosine degradation and pyomelanin formation seem to bedispensable for pathogenicity of A. fumigatus as deletion of hmgR did notaffect virulence in a mouse infection model.MPP006Characterisation of the PomA/B Stator Complex of theSodium-dependent Flagellum of Vibrio choleraeP. Halang*, T. Vorburger, J. SteuberInstitute for Microbiology, University of Hohenheim, Stuttgart, GermanyV. cholerae is a humanpathogen gram - bacteria which causes the acutediarrheal disease cholera mainly in third world countries. Its virulence isachieved by different virulence factors and the ability to colonize theintestine of its host. Vibrio is motile because of its single sodium ion-spektrum | Tagungsband 2011