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

85FUP033A proteome reference map of Aspergillus nidulans and newputative targets of the AnCF complexK. Tuppatsch* 1,2 , O. Kniemeyer 1 , P. Hortschansky 1 , A.A. Brakhage 1,21 Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knoell Institute (HKI), Department Molecular and Applied Microbiology, Jena,Germany2 Friedrich Schiller University Jena, Institute of Microbiology, Jena, GermanyThe mould Aspergillus nidulans is a well suited model organisms forfilamentous fungi and is closely related to many Aspergillus species ofindustrial and medical interest. With the completion and publication of theA. nidulans genome it is feasible to study gene expression and proteinproduction on a global scale. A variety of transcriptome studies have beenalready carried out for A. nidulans. By contrast, only little information isavailable about the dynamic changes of the proteome of A. nidulans uponenvironmental changes, stress conditions or genetic modifications.Furthermore, no proteome map for A. nidulans has been published so far.For this reason, we established the first 2-D reference map for theintracellular protein fraction of A. nidulans strain TNO2A7. After 2D-gelelectrophoretic separation, visualisation of proteins by Coomassie stainingand image analysis with Delta 2D, 435 spots representing 364 differentproteins were identified by MALDI-TOF-MS/MS analysis. Qunatitativeproteomic analysis of a hapC deletion mutant revealed many proteins withdifference in abundance in comparison to the wild type. Two proteins, aconserved hypothetical protein and a guanine nucleotide dissociationinhibitor, were found to be putative, so far uncharacterised targets of theAnCF complex. Knock-out and double knock-out strains of thecorresponding genes are in progressFUP034Interaction of the phytopathogenic fungus Verticilliumlongisporum with the antagonistic soil bacterium PseudomonasfluorescensK. Nesemann*, S. Braus-Stromeyer, P. Tarazona Corrales, C. Hoppenau,H. Irmer, G. BrausInstitut für Mikrobiologie und Genetik, Universität Göttingen, Abteilungfür molekulare Mikrobiologie und Genetik, Göttingen, GermanyThe filamentous soil-borne fungus V. longisporum possessesphytopathogenic properties and is responsible for increasing economicallosses in the cultivation of oilseed rape (Brassica napus). Antagonisticbacteria like Pseudomonas fluorescens can be used as biological controlagents to reduce the infection intensity of saprophytic fungi in the absenceof appropriate fungicides. We analyse the interaction between these threeorganisms on a molecular and genetic level to explore the potential of P.fluorescens as a biocontrol tool for V. longisporum. Initial experimentssuggested that the impact of the bacterium on fungal growth is highlymedium and strain-dependent. We focused on potential bacterialmycotoxins. Biosurfactants (glucolipids or cyclic lipopeptids) andphenazines produced by P. fluorescens are acting against resting structuresof fungal pathogens. They are operating synergeticly by integrating thebiosurfactants into the cell membrane increasing its permeability for thetoxic phenazines to enter into the cytoplasm. The bacteria produce avariety of additional antifungal secondary metabolites like 2,4-diacetylphloroglucinol, hydrogen cyanide or pyrrolnitrin which are allcontrolled by the two-component system gacS-gacA. P. fluorescensmutants defective in the production of single secondary metabolites testedwere still able to inhibit the germination of fungal spores. Knockouts of thegeneral regulator gacA of P. fluorescens lost the ability to inhibitgermination of the fungus. Our future focus will be the fungal response tothe presence of the bacterium.HMV001Deglycosylation of polyphenolic C-glucosides by a human gutbacteriumA. Braune*, M. BlautGerman Institute of Human Nutrition Potsdam-Rehbruecke,Gastrointestinal Microbiology, Nuthetal, GermanyDietary polyphenols, such as (iso)flavonoids, have been implicated in theprevention of age-related chronic disorders including cancer andcardiovascular diseases [1]. Polyphenols are present in plant-derived foodand food supplements, predominantly in their glycosidic form, either as O-glycosides or as C-glycosides. In contrast to the O-glycosides, ingestedC-coupled glycosides resist cleavage by human enzymes and can mostlikely only be deglycosylated by gut bacteria. A rod-shaped Gram-positivebacterium, strain CG19-1, capable of deglycosylating the isoflavonepuerarin (daidzein 8-C-glucoside) to daidzein was isolated from humanfeces [2]. Comparative 16S rRNA gene sequence analysis indicated thatthe strictly anaerobic isolate is a new species of the Lachnospiraceae.Strain CG19-1 also converted polyphenolic C-glucosides other thanpuerarin. The xanthone C-glucoside mangiferin was deglycosylated tonorathyriol. Several C-glucosides of the flavones luteolin and apigeninwere cleaved to their aglycones, which were further degraded to thecorresponding hydroxyphenyl propionic acids. Strain CG19-1 alsoconverted (iso)flavonoid O-glucosides, but at rates that were lower thanthose observed for the C-glucosides. The isoflavone O-glucosides wereconverted to their aglycones, while the flavone O-glucosides underwentdeglycosylation and subsequent degradation to hydroxyphenyl propionicacids. Thus, strain CG19-1 may affect the bioavailability and, thereby, theeffects not only of polyphenolic O-glucosides but also of C-glucosidespreviously assumed to be stable in the human body. The mechanism of theC-glucosyl cleavage remains to be elucidated. For this purpose,identification of the involved enzyme(s) from strain CG19-1 is in progress.[1] Crozier A, Jaganath IB, Clifford MN (2009) Nat Prod Rep 26: 1001-43[2] Braune A, Blaut M (2011) Environ Microbiol 13: 482-91HMV002Application of real-time PCR, T-RFLP and direct sequencing forthe identification of polybacterial 16S rRNA genes in ascitesS. Krohn* 1,2 , J. Hartmann 1 , A. Brodzinski 1 , A. Chatzinotas 2 , S. Böhm 1 , T. Berg 11 University Hospital Leipzig, Division of Gastroenterology andHepatology, Leipzig, Germany2 Helmholtz Centre for Environmental Research - UFZ, Department ofEnvironmental Microbiology, Leipzig, GermanyQuestion: Spontaneous bacterial peritonitis (SBP) is a serious complicationin cirrhotic patients with a mortality rate up to 50%. However, earlydiagnosis and antibiotic treatment can improve clinical outcome. Due tothe limited detection rates of culture-dependent bacterial identification inpatients with clinical SBP diagnosis, we evaluated 16S rRNA geneamplification for the rapid detection of bacterial DNA in ascites andfurther characterized polybacterial samples by terminal restrictionfragment length polymorphism (T-RFLP) and direct sequencing.Methods: 98 ascitic fluid samples from 43 patients undergoing severaldiagnostic paracenteses were studied. To avoid cross hybridization ofbacterial broad range primers with the human DNA background weselectively isolated bacterial DNA of all samples with a commerciallyavailable isolation kit (MolYsis). 16S rRNA genes were amplified by realtimepolymerase chain reaction (PCR) and directly sequenced. Using theweb-based tool RipSeq (iSentio), mixed chromatograms were immediatelyinterpreted. T-RFLP analysis characterized polymicrobial samples bydisplaying their bacterial diversity patterns.Results: Bacterial DNA (bactDNA) was detected in 57/98 (58%) of theascitic fluid samples. 22/43 patients (51%) underwent several paracenteses(mean 3.5; range 2-6) from which 5/22 patients (23%) showed positivebactDNA in ascites throughout all paracenteses and 4/22 (18%) patientswere bactDNA negative. In the remaining 13 patients at least one positiveascites sample could be detected (mean number of positive samples 2.3;range 1-4). A single paracentesis was performed in 21/43 (49%) patients.BactDNA positive ascites was observed in 12/21 (57%) samples whereas9/21 (43%) ascitic fluids were PCR negative. Using TRFLP, multiple T-RF were detected in positive ascites potentially indicating the presence ofseveral distinct strains. Direct sequencing with 16S rRNA gene basedprimers showed mixed chromatograms which revealed gram positive aswell as gram negative organisms.Conclusion: A mixed bacterialDNA content can bedetected in ascites viaPCR targeting the 16S rRNA genes and T-RFLP analysis. Directsequencing of PCR products and analysis of mixed chromatograms usingRipSeq may offer a rapid tool to identify the most abundant sequencetypes.HMV003Analysis of the intestinal microbiota using SOLiD 16SrRNAgene sequencing and SOLiD shotgun sequencingK. Förster-Fromme* 1 , S. Mitra 2 , T. Scheurenbrand 2,3 , S. Biskup 3 , D. Boehm 3 ,D.H. Huson 2 , S.C. Bischoff 11 Universität Hohenheim, Institut für Ernährungsmedizin, Stuttgart, Germany2 University of Tübingen, Center for Bioinformatics, Tübingen, Germany3 CeGaT GmbH, Tübingen, GermanyMetagenomics seeks to understand microbial communities andassemblages by DNA sequencing. Technological advances in nextgeneration sequencing technologies are fuelling a rapid growth in thenumber and scope of projects aiming to analyze complex microbialenvironments such as marine, soil or the gut. Recent improvements inlonger read lengths and paired-sequencing allow better resolution inprofiling microbial communities. While both 454 sequencing and Illuminasequencing have been used in numerous metagenomic studies, SOLiDsequencing is not commonly used in this area, as it is believed to moresuitable in the context of reference-guided projects. To investigate theperformance of SOLiD sequencing in a metagenomic context, wecompared taxonomic profiles of both Sanger and SOLiD mate-pairsequencing reads obtained from the bacterial 16S rRNA gene that wasamplified from microbial DNA extracted from a human fecal sample.Additionally, from the same fecal sample, complete genomic microbialBIOspektrum | Tagungsband 2012