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

221produced in terrestrial ecosystems [2]. The goal of the present project is toprovide a closer insight towards the structure and function of thesecommunities by identifying metabolically active species, interactions andmetabolic networks. For the detection of metabolic key players protein-SIPis applied, a method based on the metabolic incorporation of isotopicallylabeled substrates, e.g. with 13 C, 15 N or 36 S, into the proteome ofmicroorganisms [3].Protein-SIP experiments were performed in which soil from a tobaccofield in Germany was incubated with leaf litter from either 15 N-labeledtobacco or 13 C-labeled corn plants as substrate over 14 days. The microbialgrowth within the approaches was monitored by measuring the biologicaloxygen demand. Immediate oxygen consumption was measured in the leaflitter-soil incubations and sampling took place three times in the first threedays and three times within the remaining 11 days. The samples wereconducted to two protein extraction steps: one for the extracellular andanother one for the intracellular proteome. Proteins were separated by 1-dimensional SDS gel electrophoresis and peptides were analyzed by UPLCOrbitrap MS/MS measurements. For protein identification themetagenome sequence of the soil from the tobacco field was conducted.454 pyrosequencing resulted in about 390 Mb distributed over about871,000 reads with an average length of 450 bp. MG-RAST analysisshowed that a large proportion of the functional genes belong to bacterialproteins (~97%) and to eukaryotic proteins (~2%). In addition to theassessment of the phylogeny of organism in the soil the metagenome willfacilitate the identification rate of the metaproteome approach andtherefore will increase the number of proteins for which the 13 C and 15 Nincorporation patterns can be determined.1. Yadav V, Malanson G (2007) Progress in soil organic matter research: litter decomposition, modelling,monitoring and sequestration. Progress in Physical Geography 31: 131-1542. Zhang Q, Zak JC (1998) Potential Physiological Activities of Fungi and Bacteria in Relation to PlantLitter Decomposition along a Gap Size Gradient in a Natural Subtropical Forest. Microb Ecol 35: 172-1793. Jehmlich N, Schmidt F, Taubert M, Seifert J, Bastida F, von Bergen M, Richnow HH, Vogt C (2010)Protein-based stable isotope probing. Nat Protoc 5: 1957-1966SMP032Horizontal gene transfer in wastewater irrigated soils in theMézquital Valley, MexicoM. Broszat* 1,2 , T. Sakinc 1 , Y. López Vidal 3 , J. Huebner 1 , E. Grohmann 11 University Medical Centre Freiburg, Department of Infectious Diseases,Freiburg, Germany2 University Freiburg, Institute of Microbiology, Freiburg, Germany3 Universidad Nacional Autónoma de México, Departamento de Microbiologíay Parasitología, Mexico City, MexicoThe Mézquital Valley (60 km north of Mexico City) is the world´s largestwastewater (WW) irrigation area. There, untreated WW from Mexico Cityis reused for crop irrigation. This practise might pose risks for fieldworkers and consumers of agricultural products, because of the presence ofpharmaceuticals, pathogens and antibiotic resistance genes in the WW. Weperformed soil column experiments with two different types of soil (soilirrigated with WW for 100 years and rain-fed soil) to investigate thespread of resistance genes by horizontal gene transfer (HGT) in WWirrigated soils. To visualize plasmid transfer an Enterococcus faecalisdonor harbouring a mobilizable broad host range plasmid labeled with theGreen Fluorescent Protein (GFP) [1] and a second non-mobilizableplasmid labelled with the Red Fluorescent Protein (RFP) [2] were added torain-fed and 100 years-irrigated soil, each in soil columns of 20 cm heightand 15 cm diameter. The mobilizable plasmid contains a replication originfor Gram-positive and Gram-negative bacteria, the gfp gene under controlof the inducible nisin promoter and the pIP501 origin of transfer. Soilcolumns were irrigated once a week, in total three times. At each irrigation10 9 donors were applied to the columns with one pore volume of artificialrainwater (for rain-fed soil) or WW (for WW-irrigated soil). Duringirrigation leachate water was collected. Furthermore pore water wassampled at 4 heights with suction cups. Soil samples from the top weretaken daily. After 4 weeks soil samples were taken from different heights(every 2.5 cm). Bacteria in soil and water which have acquired themobilizable resistance plasmid via plasmid transfer are detectable throughtheir green fluorescence while their donors are identified by their greenand red fluorescence. Transfer rates for both types of soil and in water willbe presented. The soil column experiment will help assess the risk posedby HGT of resistance determinants in WW-irrigated soil.[1] Arends, K., Schiwon, K., Sakinc, T., Huebner, J., and Grohmann, E. A GFP-labelled monitoring tool toquantify conjugative plasmid transfer between G+ and G- bacteria. Appl. Environ. Microbiol. (accepted)[2] Paprotka, K., Giese, B., Fraunholz, M. J. 2010. Codon-improved fluorescent proteins in investigation ofStaphylococcus aureus host pathogen interactions. J. Microbiol. Methods. 83: 82-86.SMP033Understanding factors which shape the community ofnitrifiers: structural and functional analysesA. Meyer* 1 , M. Schloter 2 , A. Focks 31 Technische Universität München, soil ecology, Neuherberg, Germany2 HelmholtzZentrum München, Enviromental Genomics, Neuherberg, Germany3 Wageningen University, Aquatic Ecology and Water Quality Management,Wageningen, NetherlandsUnderstanding factors which drive the ecology of microbial communitiesinvolved in nitrogen turnover is of central importance for sustainable landuse. As a model system grassland sites treated with different land useintensities were studied: (I) intensely used meadows, (II) intensely usedmown pastures and (III) extensively used pastures. Samples were taken inspring and in the summer to investigate the seasonal as well as the land useintensity effect. In the last years it was found that in many soils ammoniaoxidizingarchaea (AOA) are more abundant than ammonia-oxidizingbacteria. However, till now the contribution of AOA to total ammoniaturnover rates are not clear. In order to address this question we estimateda theoretical potential nitrification rate (PNR) based on the actualmeasured abundances of archaeal and bacterial ammonia monooxygenasegenes (amoAAOA andamoAAOB) and hypothetical maximum oxidationrate constants. This approach offers the possibility to estimate not only thetheoretical PNR values but also the respective contributions of AOA andAOB. A comparison between the theoretical and the measured PNR valuesshows that they fit quiet well together. In order to assess the correlationbetween the observed temporal changes in nitrification activities, but alsothe found variability between the single grassland plots, a diversity analysisbased onamoAAOA genes was performed. The results showed that the singletreatments are statistically well separated but surprisingly no clear differencesbetween the two time points could be found. Summarizing, our results strike outthat AOAs deliver a high ammonium turnover potential to the soils. Changes innitrification potentials are seemingly not due to AOA diversity, but driven bythe activity state of the AOAs, which probably has changed between spring andsummer. Based on the above results, we assume that diversity of theamoAAOAgene is shaped by long-term changes in environmental parameters, whereas theactivity is probably driven by seasonal changes of environmental conditions.SMP034Metagenomic and metatranscriptomic analysis of German soilsamplesH. Nacke* 1 , C. Fischer 1 , A. Thürmer 2 , R. Daniel 1,21 Institute of Microbiology and Genetics, Department of Genomic andApplied Microbiology, Göttingen, Germany2 Institute of Microbiology and Genetics, Göttingen Genomics Laboratory,Göttingen, GermanyPhylogenetic, transcriptomic, and functional analyses of microbialcommunities present in soil samples from the German BiodiversityExploratories Schorfheide-Chorin, Hainich-Dün, and Schwäbische Albwere performed (see www.biodiversity-exploratories.de). Theexperimental procedure included the isolation of whole genomic DNAfrom the A horizon and B horizon of selected forest and grassland sites.The prokaryotic diversity present in the different samples was assessed bypyrosequencing of amplicons using hypervariable regions of 16S rRNAgenes as target. Differences in prokaryotic community compositionsbetween A- and B-horizons as well as between forest and grasslandsamples were detected. Additionally, we extracted total RNA from soilsamples, enriched mRNA, and used it for the synthesis of cDNA.Pyrosequencing of the generated cDNA and subsequent sequence analysisallowed to assess soil microbial gene expression profiles.Metagenomic small-insert and large-insert libraries were constructed usinggenomic DNA extracted from the different soil samples. Comparativescreening of the libraries for key microbial functions, such as cellulolytic,hemicellulolytic, and lipolytic activities was carried out. Several clonesexpressing cellulase-, hemicellulase-, and lipase/esterase- activity wereobtained during function-driven screening of the libraries. Genes encoding(hemi)cellulolytic or lipolytic activity were recovered from thecorresponding clones and sequenced. So far, analyzed (hemi)cellulolyticenzymes were assigned to glycosidhydrolase families 9 and 11. Thirty-fiveof the 37 analyzed lipases/esterases grouped into superfamilies I, IV, V,VI, and VIII of lipolytic enzymes. The remaining two represent putativelynovel families. Biochemical characterization of (hemi)cellulolytic andlipolytic enzymes was carried out.SMP035Characterization of Paenibaciilus polymyxa RCP6 isolatedfrom root nodules of Blue peaA. AeronKurukshetra University, Department of Microbiology, Kurukshetra, IndiaQuestion: Clitoria purpurea L. (blue pea) is a slender climber legumeknown for its beautiful bluish-crimson coloured flowers. This is foundBIOspektrum | Tagungsband 2012