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

the clone library analyses indicated bacterial sequences which were mostclosely related to Aerococcus viridans, Pantoea agglomerans andAcinetobacter spp. which are well known as causatives of differentrespiratory diseases. These results underline the necessity of improvedventilation and last but not least adequate breathing protection at specialworkplaces.EMP013Structure and function of an m-xylene degrading, sulfatereducingenrichment culture revealed by molecular andstable isotope tracer techniquesD. Bozinovski 1 , S. Herrmann 2 , M. von Bergen 1 , H. Hermann Richnow 2 ,J. Seifert* 1 , C. Vogt 21 Department of Proteomics, Helmholtz Center for Environmental Research-UFZ, Leipzig, Germany2 Department of Isotope Biogeochemistry, Helmholtz Center forEnvironmental Research (UFZ), Leipzig, GermanyA meta-xylene degrading, sulfate-reducing mixed culture originally enrichedfrom ground water of a hydrocarbon contaminated field site was investigatedin this study. Xylene-isomers belong to the group of BTEX compounds(benzene, toluene, ethylbenzene, xylene) and as toxic and commonsubstances they all represent a big threat to humans and the environment.The aim of the study was to get valuable insights into the anoxic degradationof such compounds following the incorporation of 13 C within the proteins ofthe microbial community (Protein-SIP) [1]. Stable isotopes such as 13 C serveas tracers which can be detected in the biomass and the metabolic endproducts.For 13 C-labeling, we grew the culture using m-xylene labeled with 13 C atboth methyl groups ( 13 C-content of meta-xylene: 25 atom%). Controlcultures were grown with non-labeled m-xylene, acetate and benzoate,respectively. Protein analyses were carried out by 1-DE gels and UPLCOrbitrap-MS/MS.Labeled and non-labeled m-xylene was metabolized in similar rates withsulfate as electron acceptor. Two species dominated the enrichment cultureunder all cultivation conditions, as revealed by Terminal RestrictionFragment Length Polymorphism (T-RFLP) analyses. One phylotype isaffiliated to members of the genus Desulfobacterium, the other is related toEpsilonproteobacteria. The Desulfobacterium phylotype is believed todegrade m-xylene. The metabolic function of the Epsilonproteobacterium isnot yet known.About 110 proteins were identified and the majority belonged to members ofDeltaproteobacteria. Proteins of the following metabolic pathways werefound: xylene degradation, sulfate reduction and C1 metabolism. Thepreliminary protein analyses of both 12 C- and 13 C- xylene samples revealedthat the majority of Deltaproteobacteria peptides contained approximately20 atom% 13 C, indicating that both methyl-groups were predominantlyassimilated by the Deltaproteobacterium. The time course of13 C-incorporation will be tracked by a time-series experiments in the near future[1] Jehmlich, N. et al (2010): Protein stable istope probing (Protein-SIP). Nat. Protoc. 5 (12), 1957-1966.EMP014Insights into an anoxic benzene degrading consortiumprovided by protein based stable isotope probing(Protein-SIP)M. Taubert* 1 , M. von Bergen 1 , C. Vogt 2 , H.-H. Richnow 2 , J. Seifert 11 Department of Proteomics, Helmholtz Center for Environmental Research-UFZ, Leipzig, Germany2 Department of Isotope Biogeochemistry, Helmholtz Center forEnvironmental Research (UFZ), Leipzig, GermanyMicrobial communities play a key role in the Earths biogeochemical cycles,performing a huge variety of complex converting and degradative processesunder oxic and anoxic conditions, e.g. the degradation of benzene. Benzeneis a major environmental contaminant of anthropogenic source, belonging tothe group of BTEX compounds (benzene, toluene, ethylbenzene, xylene). Itis highly stable due to resonance stabilization of the π electron system,turning its degradation into a biochemical challenge especially under anoxicconditions. Benzene is posing a threat to human health and environment dueto its toxic and carcinogenic effects. Although it is a widespread pollutant,knowledge about its degradation under anoxic conditions is still sparse. Oneof the reasons is a lack of suitable methods for analysing complex microbialcommunities. To open new ways for the analysis of microbial communities,we expanded the classical stable isotope probing (SIP) methods tometaproteomic analysis [1]. This method is based on the analysis ofmetabolization of substrates labeled with nonradioactive heavy isotopes (e.g.13 C), and the subsequent incorporation of the label into proteins. Highresolution mass spectrometry is used to detect the heavy isotopeincorporation on peptide level, together with the identification of peptidesand subsequently of proteins. This allows a direct linkage betweentaxonomic and functional information as well as metabolic conditions, henceoffering a powerful tool to study trophic structures of microbialcommunities.Object of our research is a benzene degrading, sulfate reducing culture froma contaminated aquifer near Zeitz, Saxonia-Anhalt. First clues on taxonomiccomposition of the culture have been acquired by DNA-SIP experiments [2].In our recent Protein-SIP study either 13 C 6-labeled benzene or 13 C-labeledcarbonate is used to trace the carbon flux within the microbial culture,allowing an overview of the usage of these carbon sources. A time resolvedpicture of the metabolization and utilization of the labeled carbon wasachieved by sampling at several times during cultivation. Extensive analysisof the metaproteome also allowed the identification of proteins possiblyinvolved in sulfate reduction and aromatic hydrocarbon degradation.Combining metaproteomic information on phylogeny and metabolic activitywill enable us to draw a more detailed picture of the process of anaerobicbenzene degradation.[1] Jehmlich et al. (2010): Protein-stable isotope probing (Protein-SIP). Nature Protocols. 5 (12),1957-1966.[2] Herrmann et al. (2009): Functional characterization of an anaerobic benzene-degrading enrichmentculture by DNA stable isotope probing. Environ Microbiol. 12(2):401-411.EMP015Effects of a genetically modified potato line with alteredstarch metabolism on carbon fluxes within the plant-soilsystem and on microbial community structure andfunction in the rhizosphereS. Gschwendtner* 1 , J. Esperschütz 1 , M. Reichmann 2 , M. Müller 2 ,M. Schloter 31 Department of Soil Ecology, Techincal University Munich, Neuherberg,Germany2 Bavarian State Institute for Agriculture, Freising, Germany3 Department of Terrestrial Ecogenetics, Helmholtz Center Munich,Neuherberg, GermanyFrom the two potato starch components amylose and amylopectin, thesecond one is of greater interest for industry. To avoid the costly process ofseparating, genetic engineers developed a potato cultivar, which containsonly amylopectin by blocking amylose production through insertion of anartificial gene with antisense orientation to the starch synthase gene. Despitethe use of a tuber-specific promoter, it cannot be excluded that the geneticmodification might affect the whole plant metabolism, resulting in modifiedroot exudation pattern and thus in altered microbial community structure inrhizosphere. While most rhizosphere microorganisms provide benefits totheir host plant, this in turn may reduce plant growth and health.Hence, to assess potential effects of genetically modified (GM) amylopectinaccumulatingpotato line #1332 (Solanum tuberosum L.) on carbontransformation within the plant-rhizosphere system with special focus onchanges in rhizosphere community pattern, greenhouse and field studieswere conducted. Besides the parental variety ‘Walli’, a second nontransgenicpotato cultivar was planted, in order to relate possible GMdependenteffects to natural variation among different plant genotypes.Rhizosphere samples were taken at young leaf developmental and atflowering stage of potatoes. For investigation of carbon fluxes within theplant-rhizosphere system and microbial community structure, 13 C stableisotope probing in combination with phospholipid fatty acid analysis waschosen. To get a more detailed insight into rhizosphere microbialpopulations, abundance pattern of the potato pathogen Phytophthorainfestans, of plant beneficial microbes (Pseudomonas spp., Trichodermaspp.), and of functional groups involved in soil mineralization processeswere examined using quantitative real-time PCR.Our results revealed that the genetic modification did affect neither carbonfluxes from plant into soil nor microbial community structure and activity inthe rhizosphere. Furthermore, no difference in abundance pattern ofphylogenetic groups and functional genes under investigation between theGM line and its parental variety was observed. Nevertheless, the nontransgenicpotato cultivars varied significantly regarding to all parametersunder investigation, and also plant developmental stage affected carbonspektrum | Tagungsband 2011