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

141layer that specifically detects carbohydrate-protein binding interactions(mannoside - ConA), as well as real time interaction of carbohydrates withdifferent E. coli strains in solution. Binding to the Cantilever surfacecauses mechanical surface stress, that is transduced into a mechanical forceand cantilever bending. The degree and duration of cantilever deflectioncorrelates with the interaction‘s strength. In this study we presentcarbohydrate-based cantilever biosensors as a robust, label-free, andscalable method to analyze carbohydrate-protein and carbohydrate-bacteriainteractions. The cantilevers thereby exhibit specific and reproducibledeflection with a high sensitivity range of over four orders of magnitude.OTP015Antibiotics Screening 2.0 - Tools for in silico Genome Miningfor Natural Product Biosynthesis PathwaysK. Blin 1 , M.H. Medema 2 , R. Marc 3 , O. Kohlbacher 3 , R. Breitling 2,4 , E. Takano 2 ,T. Weber* 11 IMIT / Universität Tübingen, Mikrobiologie/Biotechnologie - SecondaryMetabolite Genomics, Tübingen, Germany2 Groningen Biomolecular Sciences and Biotechnology Institute / University ofGroningen, Microbial Physiology, Groningen, Netherlands3 ZBiT / Universität Tübingen, Applied Bioinformatics, Tübingen, Germany4 University of Glasgow, Institute of Molecular, Cell and Systems Biology,Glasgow, United KingdomMicroorganisms are a rich source for natural products of which many havepotent antimicrobial or antitumor activity. While in the past, functionalscreening approaches directed directly to the substances or to putativetargets were the main approaches for the identification and isolation ofnovel compounds, the easy availability of whole genome sequence data ofputative producers nowadays offers great possibilities to assess the geneticpotential of the strainsin silico.For such analyses of genomic data novel, sophisticated tools are requiredwhich allow the prediction of putative biosynthetic products. Therefore,several tools were developed in our group:The Open Source annotation platform CLUSEAN 1 is a versatile tool forthe analysis of single biosynthetic gene clusters as well as whole genomesequences. CLUSEAN contains generic modules for automated BLAST orHMMer analyses as well as specialized tools for the domain assignmentand specificity prediction of modular polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS).Included into CLUSEAN is NRPSpredictor 2,3 . This tool allows theprediction of substrate specificities of adenylation domains of NRPSenzymes and thus the prediction of the peptide products. Here, we presentthe new version NRPSpredictor 2 which contains updated models for theamino acids and now allows prediction up to the amino acid level.All of these tools are now also integrated into the antibiotics and secondarymetabolites analysis shell antiSMASH 4 . This pipeline contains most toolsthat are currently available for the analysis of secondary metabolite geneclusters, including CLUSEAN and NRPSpredictor2. antiSMASH is eitheravailable as a standalone application or as a web based service.URLs for downloading/using the software:CLUSEAN: http://redmine.secondarymetabolites.org/projects/cluseanNRPSpredictor2: http://nrps.informatik.uni-tuebingen.deantiSMASH: http://antismash.secondarymetabolites.org/1. Weber, T., et al. (2009). J. Biotechnol. 140, 13-17.2. Rausch et al., (2005) Nucleic Acids Res. 33, 5799-58083. Röttig, M., et al. (2011) Nucleic Acids Res. 39, W362-3674. Medema, M.H., et al. (2011) Nucleic Acids Res. 39, W339-W346.OTP016Relative protein quantification using 36 S- or 34 S- sulfateF.-A. Herbst* 1 , N. Jehmlich 1,2 , M. Taubert 1 , T. Behr 1 , J. Seifert 1 , F. Schmidt 1,2 ,M. von Bergen 11 UFZ - Helmholtz Centre for Environmental Research, Proteomics, Leipzig,Germany2 Ernst-Moritz-Arndt-University Greifswald, Department of FunctionalGenomics, Greifswald, GermanyTo uncover changes in the proteome and to draw conclusions from this it iscrucial to quantify as accurate as possible. One of the favored methods isthe metabolic introduction of stable isotopes. Currently in use are heavylabeled amino acids or substrates to directly compare the intensities ofassociated peptide pairs of two or more different conditions during a singlemeasurement [1]. Even though these techniques have proven to befeasible, they have drawbacks as well. The addition of amino acids mightinfluence the proteome or they get metabolized, resulting in anunpredictable spread of the label. The labeling of the whole proteome by13 C or 15 N labeled substrates usually results in incorporation patterns whichare hard to predict and therefore bioinformatically complicated [2].Here we show the potential of utilizing heavy sulfur isotopes for relativeprotein quantification. Sulfur is an essential element for microorganismsand is part of methionine and cysteine, so it can be used as universal labelfor quantitative proteomic studies. The fact that sulfur containing aminoacids are encountered infrequently is a mixed blessing. Although only asmall fraction of measurable peptides will give quantitative information,the incorporation patterns are well predictable in comparison to carbon ornitrogen labeling strategies. So far the relative proteomic change of P.putida with benzoate as carbon source was elucidated using 36 S-labeledsulfate [3]. It could be shown that this technique leads to the relativequantification of many relevant proteins. Due to the high costs and lowavailability of 36 S-sulfur or -sulfate, we further investigated the usage of34 S-labeled sulfate. As most tryptic peptides contain only one sulfur atom,the mass shift of 2 Da corresponding to the 34 S-label is not enough to fullyseparate the isotopic patterns with routine resolutions. We are showing thatthe in silico separation of the isotopic pattern for relative quantification ispossible, taking the monoisotopic peak as reference to simulate the correctdistributions. The proteomic change in P. fluorescens during naphthalenedegradation will be presented from a label switch experiment using 34 S-sulfate to first confirm the suitability of 34 S as universal label and secondto identify relevant physiological changes besides the known.1. Beynon, R.J. and J.M. Pratt,Metabolic labeling of proteins for proteomics.Molecular & cellularproteomics : MCP, 2005.4(7): p. 857-72.2. Jehmlich, N., et al.,Decimal place slope, a fast and precise method for quantifying 13C incorporationlevels for detecting the metabolic activity of microbial species.Molecular & cellular proteomics : MCP,2010.9(6): p. 1221-7.3. Jehmlich, N., et al.,Sulphur-(36) S stable isotope labeling of amino acids for quantification(SULAQ).Proteomics, 2011.OTP017Development of a functional screening method for novel[NiFe]-hydrogenases from metagenomesN. Rychlik*, M. PernerUniversity Hamburg - BZF, Molecular Biology of Microbial Consortia,Hamburg, GermanyThe interconversion between molecular H 2 and protons and electrons isextremely interesting for biotechnological applications because H 2 is oneof the most promising renewable fuels. This reaction is catalyzed byenzymes called hydrogenases ( H 2 2 H + + 2 e - ). The direction of thisreaction depends on the redox potential of the components able to interactwith the enzyme. One biotechnological application for hydrogenases is infuel cells, where energy becomes available through the oxidation of H 2.Alternatively, hydrogenases are applicable for the biological H 2 productionin electrochemical cells. One of the most crucial challenges in thesebiotechnological applications is to resolve the problem associated with theoxygen sensitivity of hydrogenases.In hydrothermal deep sea vent habitats, hot hydrothermal fluids enrichedwith reduced inorganic compounds e.g. H 2 emit from the ground. As theascending hydrothermal fluids come in contact with cold, oxygenatedambient seawater, mixing processes constitute habitats with steep physicochemicalgradients, e.g. habitats with high concentrations of H 2 andoxygen. With respect to these abiotic conditions, the rich energy sourceprovided by H 2 oxidation and the large numbers of diverse H 2-oxidizingmicroorganisms, hydrothermal vents facilitate ideal conditions for seekingoxygen tolerant hydrogenases.To identify and study novel oxygen tolerant hydrogenases we usedmetagenomic material from these habitats and constructed broad-hostrange fosmid libraries. Since heterologous expression of functionalhydrogenases in the standard host Escherichia coli is difficult becausecomplex interactions of maturation- and assembly proteins are oftenneeded, we are establishing function based screenings with alternativeheterologous hosts. Therefore, two new deletion mutants are currentlybeing constructed: These are the -proteobacterium Shewanella oneidensisMR-1 and the -proteobacterium Wolinella succinogenes. Both organismspossess a single [NiFe]-hydrogenase and are promising candidates forestablishing this functional screening method. A [NiFe]-hydrogenasedeletion mutant of Shewanella oneidensis MR-1 (hyaB) was developedsuccessfully and we here report our first results of the conducted functionalscreen.OTP018Fate of elemental sulfur in coastal sediments andhydrothermal ventsP. Pjevac*, S. Lenk, M. MußmannMax Planck Institute for Marine Microbiology, Molecular Ecology,Bremen, GermanyZero-valence sulfur (ZVS) species such as elemental sulfur (S 0 ) andpolysulfides are central intermediates in sulfur cycling at redox clines inmarine and freshwater sediments. We found significant amounts of ZVS atthe sediment surface of tidal flats in the German Wadden Sea. Also, largeS 0 precipitates are covering the surface at a hydrothermal system in theManus Basin/Papua-New Guinea. It is generally unknown, howmicroorganisms in these environments metabolize dissolved andparticulate ZVS under different oxygen regimes. To investigate thebacterial community utilizing ZVS, we sampled native S 0 fromgeochemically diverse systems in the Manus Basin. Moreover, we exposedS 0 slabs as colonization surfaces in both coastal sediments and athydrothermal vents for a period of 2-6 weeks. To identify key S-cyclingBIOspektrum | Tagungsband 2012