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

versatility of strain Tol2 is reflected by the large number of genes related tocatabolic functions, e.g. >100 genes were assigned to aromatic compounddegradation pathways. Complete oxidation of the organic substrates isachieved via the Wood-Ljungdahl pathway. The large number (>400) ofgenes related to signal transduction suggests a fine tuned regulatory responseof strain Tol2 to changing environmental conditions. Considering the largenumber of mobile genetic elements (>160 transposase related genes), a highdegree of genome plasticity has to be assumed like previously observed forits close relative Desulfobacterium autotrophicum HRM2. The genomebased functional assignment was supported by comprehensive differentialproteomic analysis, allowing for a corroborated reconstruction of thecatabolic network of strain Tol2. While toluene degradation involves bss andbbs gene products, analogous to the pathway of denitrifiers, benzoatedegradation involves bam gene products as described for Geobactermetallireducens GS-15, although not all components of the strain GS-15bam gene clusters are present in the chromosome of strain Tol2. Genomeinspired, phenylalanine was identified as so far unknown growth substrate ofstrain Tol2. Based on the proteomic data, a degradation pathway differingfrom denitrifiers can be proposed. Overall, the formation of pathway relatedproteins is rather specific, demonstrating the regulatory adaptability of strainTol2.FGV002Genome mining of anti-inflammatory B. bifidum S17reveals multiple loci potentially involved in host-microbeinteractionsD. Zhurina*, C. RiedelInstitute for Microbiology and Biotechnology, University of Ulm, Ulm,GermanyBifidobacteria represent an important group of intestinal bacteria and somemembers are reported to suppress inflammation in vitro and in animalmodels of chronic intestinal inflammation. This makes them interestingalternatives for the treatment of intestinal inflammatory disorders.B. bifidum S17 was shown to strongly adhere to intestinal epithelial cells(IECs) and to display potent anti-inflammatory activity both in vitro and invivo. We thus sequenced and annotated the genome of this interestingprobiotic candidate strain (accession number CP002220). The completegenomic information of the B. bifidum S17 is contained on a single circularchromosome of 2,186,882 bp with an average GC content of 62%. A total of1,782 protein coding genes, 53 tRNA genes for all amino acids, and threerrn operons were identified. To 67% of the genes a function could beassigned based on similarities of the deduced amino acid sequence withproteins of known function. Approximately 10% of all genes are devoted tothe carbohydrate metabolism which allow B. bifidum S17 to metabolize awide range of substrates including human milk oligosaccharides, hostderived mucins and different polyols (e.g. sorbitol). Mobilome analysisrevealed the presence of a CRISPR system, which shares high similarity tothe CRISPR genes found in several Lactobacillus species and is thuspresumably horizontally acquired.S17 is able to strongly adhere to intestine epithelial cells. In line with thisobservation 3 clusters of cell-wall associated proteins with theircorresponding sortases were found. Moreover, 25 proteins with domainsinvolved in adhesion to extracellular matrix and host-derived glycans wereidentified.The striking feature of B. bifidum S17 is its ability to significantlyantagonize intestinal inflammation in vivo suggesting a powerfulimmunomodulatory capacities of this strain. Possible candidatescontributing to this effect were found in the genome of B. bifidum S17.These include a myosin cross-reactive protein, lactocepin, as well as severalpotentially glycosilated serin-rich proteins. Moreover two proteins withdomains involved in inhibition of macrophage migration and activation wereidentified indicating a possible cross-talk of B. bifidum S17 with the hostimmune system via these proteins.FGP001Complementation Studies to Identify Novel Thiol-Disulfide OxidoreductasesS. Nilewski*, A. Wiesner, L.I. LeichertMedicine Proteom-Center, Ruhr-University, Bochum, GermanyThiol-disulfide oxidoreductases play an important role in different cellularprocesses such as redox signaling and protein folding. We are interested inthe identification and characterization of novel thiol-disulfide oxidoreductasesfrom metagenomic datasets. We plan to concentrate on sequencedata which was obtained in the Global Ocean Sampling project, the largestmetagenomic project to date. To find new thiol-disulfide oxidoreductases,we plan to use the power of Escherichia coli genetics. E. coli hast wodistinct cellular compartments, the cytoplasm and the periplasm. In thesescompartments reside thiol-disulfide oxidoreductases with specific andopposite functions. Within the periplasm, the oxidase DsbA is responsiblefort he oxidation of protein thiols, while the reductases TrxB and Gor keepprotein thiols reduced in the cytoplasm. We will exploit the fact that thephenotypes of null-mutants in genes encoding those proteins can becomplemented by oxidases and reductases, respectively. We successfullyconstructed two complementation plasmids, one with an OmpA signalsequence for periplasmic destination of the protein of interest (pPC) and onefor the cytoplasmic destination without any leader sequence (pCC).Phenotypic experiments and protein expression tests revealed correctfunctionality of both vector systems. In the future both contructs can be usedto characterize metagenome derived potential oxidoreductases.FGP002Efficient, global scale quantification of absolute proteinamounts by integration of targeted mass spectrometryand 2-D gel-based proteomicsS. Maaß* 1 , S. Sievers 1 , D. Zühlke 1 , J. Kuzinski 2 , J. Muntel 1 , B. Heßling 1 ,J. Bernhardt 1 , R. Sietmann 1 , U. Völker 3 , M. Hecker 1 , D. Becher 11 Institute for Microbiology, Ernst-Moritz-Arndt-University, Greifswald,Germany2 Leibniz Institute for Farm Animal Biology, Research Unit NutritionalPhysiology, Dummerstorf, Germany3 Interfaculty Institute for Genetics and Functional Genomics, Department ofFunctional Genomics, Ernst-Moritz-Arndt-University, Greifswald, GermanySystems biology moved more and more in the focus of the life scienceresearch. For mathematical modeling and simulation of biological processesknowledge on absolute protein concentrations is mandatory. A newapproach for the absolute quantification of proteins at a global scale hasbeen developed and its applicability demonstrated using glucose starvationof the Gram-positive model bacterium Bacillus subtilis and the pathogenStaphylococcus aureus as proof of principle examples. For this purpose asubset of proteins was initially absolutely quantified by employing atargeted mass spectrometric method and isotopically labeled internalstandard peptides. Known concentrations of these anchor proteins were thenused to calibrate a 2-D gel allowing a calculation of the absolute amount ofall detectable proteins in the 2-D gel. With this technique we were able toabsolutely quantify more than 400 cytosolic proteins in a pH-range from 4-7providing protein concentrations of central metabolic enzymes. This newstrategy is fast, cost-effective and applicable to any cell type, and thus ofvalue for a broad community of labs with experience in 2-D gel basedproteomics and interest in quantitative approaches.FGP003Characterization of the response of Staphylococcusaureus to the host cell environment: Enrichment andanalysis of secreted S. aureus proteins by isolation ofphagosomesK. Surmann*, P. Hildebrandt, H. Pförtner, V.M. Dhople, F. Schmidt,U. VölkerInstitute for Genetics and Functional Genomics, Department of FunctionalGenomics, Ernst-Moritz-Arndt-University, Greifswald, GermanyS. aureus is a pathogen that causes a broad range of human diseases [1]. Itsvirulence is predominantly caused by secretion of various virulence factorslike superantigens, hemolytic toxins, adhesins and enzymes which are allknown to interfere with host cell signaling or survival. Although S. aureushas been widely recognized as an extracellular pathogen there is growingevidence that S. aureus can also invade into and persist in non-professionalphagocytic cells [2]. However, the study of adaptation of S. aureus uponinternalization by proteomic approaches is severely compromised by thevery low number of bacteria recoverable from host cells. Recently, weintroduced a newly developed workflow that combines a pulse-chase SILACapproach [3], GFP supported enrichment of bacterial proteins byfluorescence activated cell sorting (FACS) and gel-free mass spectrometryanalysis (MS) for monitoring of the proteome of S. aureus RN1HGinternalized by human epithelial cells [4]. Using this workflow we identifiedspektrum | Tagungsband 2011