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

183Expression of sulfatases was studied with R. baltica SH1 T grown ondifferent sulfated polysaccharides. Transcriptome-wide gene expressionstudies applying a well-established microarray platform [3, 4] revealed astrong functional link between tested substrates and active sulfatases.Besides, further potential functions mediated by sulfatases could bededuced from the expression profiles. The transcriptomic approach wascombined with a phylogenetic assessment of sulfatase genes found in eightdraft genomes of cultured strains representing five different species of thegenusRhodopirellula [5, 6]. More than 1100 sulfatase sequences revealed172 clusters of orthologous and (rare) paralogous genes. Phylogeneticanalysis of theRhodopirellula sulfatases resulted in 17 major groups, ofwhich only six included sulfatases of known function as derived from theUniProtKB database. Considering potential applications in medicine andbiotechnology, sulfatases can be considered a promising hotspot in futureresearch relating to the physiologically diverse PVC superphylum.1 . Glöckner FO, et al. (2003) PNAS100:8298-83032. Thrash JC, Cho J-C, Vergin KL, Morris RM, Giovannoni SJ (2010)J. Bacteriol.192:2938-29393. Wecker P, Klockow C, Ellrott A, Quast C, Langhammer P, Harder J, Gloeckner FO (2009)BMCGenomics10:4104. Wecker P, Klockow C, Schueler M, Dabin J, Michel G, Gloeckner FO (2010)Microb. Biotech.3:583-5945. Winkelmann N, Harder J (2009)J. Microbiol. Meth.77:276-2846. Winkelmann N, et al. (2010) Appl. Environ. Microbiol. 76: 776-785PSP032Solvent tolerance in Pseudomonas sp. strain VLB120- Biofilms vs. Planktonic Cells-K. Schmutzler*, J. Volmer, B. Halan, K. Buehler, A. SchmidTU Dortmund University, Laboratory of Chemical Biotechnology,Dortmund, GermanyOne of the key bottlenecks in biocatalysis involving toxic and / or organicsubstances is the stability of the chosen host organism. In the recent yearsa couple of strains belonging to the Pseudomonas genus have beendescribed showing specific properties interesting for the conversion oftoxic reactants [1], such as high solvent tolerance, metabolic versatility,and a high metabolic capacity for redox cofactor regeneration [2]. Anotherinteresting feature of several Pseudomonas species is their ability to formbiofilms. Solvent tolerance in planktonic cells is highly related to theexistence of RND efflux pumps, especially TtgGHI in P. putida DOT-T1Eand SrpABC in P. putida S12 [3-4]. The tolerance phenomena in biofilmgrowing cells are in part attributed to the existence of extracellularpolymeric substances (EPS). EPS are excreted by biofilm growingorganisms and form a sticky frame work giving the biofilm its threedimensional structure [5].Here, we compare different mechanisms of Pseudomonas sp. strainVLB120 responsible for the excellent solvent tolerance of this strain [6-7]in planktonic growing cultures as well as in biofilm growing cells with theaim to obtain a stable solvent tolerant phenotype for redox biocatalysiswith toxic reactants.Regarding planktonic cells, different adaptation procedures with differentorganic solvents (e.g. toluene, 1-octanol) were tested and the resultingsolvent tolerant phenotypes have been characterized and compared to nonsolventtolerant phenotypes. In a second step, genetic engineering wasused to create knock-out mutants to overcome critical aspects of adaptedsolvent tolerant phenotypes such as poor reproducibility, tediousadaptation procedures, and low stability.Biofilms of Pseudomonas sp. strain VLB120 have been cultivated in aspecifically designed flow-cell and the influence of the solvent styrene wasinvestigated. It became obvious, that although cells suffered severedamage upon the solvent shock, the biofilm organisms recovered andadapted to high concentrations of styrene [8]. Concomitantly the excretionof EPS was boosted upon the addition of this organic solvent.[1] Ramos, J.L.et al., 2002, Annu Rev Microbiol.56: p. 743-68.[2] Blank, L.M.et al., 2008, Febs J.275(20): p. 5173-5190.[3] Rojas, A.et al., 2001, J Bacteriol.183(13): p. 3967-73.[4] Kieboom, J.et al., 1998, J Biol Chem.273(1): p. 85-91.[5] Rosche, B.et al., 2009, Trends Biotechnol.27(11): p. 636-43.[6] Halan, B.et al., 2010, Biotechnol Bioeng.106(4): p. 516-527.[7] Park, J.B.et al., 2007, Biotechnol Bioeng.98(6): p. 1219-29.[8] Halan, B.et al., 2011, Appl Environ Microbiol.77(5): p. 1563-1571.PSP033The novel subtilase SprP influences the lifestyle ofPseudomonas aeruginosaA. Pelzer* 1 , M. Laschinski 1 , F. Rosenau 2 , K.-E. Jaeger 1 , S. Wilhelm 11 Institute for Molecular Enzyme Technology, Heinrich-Heine-UniversityDuesseldorf, Juelich, Germany2 Institute of Pharmaceutical Biotechnology, Ulm University, Ulm,GermanyP. aeruginosa is a very undemanding organism that is ubiquitouslydistributed. The bacterium can be found in wet or humid surroundings,ranging from soil to human and produces a huge variety of extracellularproteins. Hence, there exists a big potential for enzymes with suitableproperties for biotechnological application. Several proteases belong to thearsenal of secreted enzymes. Some of these proteases like Elastase andProtease IV are well characterized but others exist of which nothing isknown so far (Hoge et al., 2010). Proteases in general are highly relevantfor technical enzyme applications. Subtilases for example are typicaldetergent proteases and are defined as serine proteases that belong to thepeptidase_S8 family. These subtilases are encoded as preproenzymescarrying a signal peptide which drives their translocation through thecytoplasmic membrane and a propeptide acting as a folding mediatorrequired to give the protease its final native conformation.By homology, we have identified the open reading frame PA1242 in thegenome sequence of P. aeruginosa PAO1 encoding a so far hypotheticalprotein as a putative member of the E-H-S family of subtilases. The geneproduct of PA1242 (sprP) contains a predicted signal sequence and apeptidase S8 domain. Sequence analysis revealed the presence of anadditional element in the domain organization of the protease. SprPcarries, beside its signal peptide and the S8 domain, a domain of unknownfunction (DUF) between both elements. After the identification of SprP,the gene was cloned, expressed in E. coli and the protease activity wasmeasured with established protease substrates.Often, proteases have an impact on different physiological processes likeprotein processing and activation, secretion of other proteins andpathogenicity of the host bacterium. A P. aeruginosa sprP-negative mutantwas constructed and different phenotypes were tested to elucidate thephysiological role of SprP.We were able to illustrate an eminent role ofSprP by characterization of different phenotypes. Deletion of sprP causesan increased biofilm formation and pyoverdine biosynthesis, theaccumulation of cell aggregates during growth, and a reduced growthunder anaerobic conditions.R. Hoge, A. Pelzer, F. Rosenau & S. Wilhelm, (2010) Weapons of a pathogen: Proteases and theirrole in virulence of Pseudomonas aeruginosa. In: Current Research, Technology and EducationTopics in Applied Microbiology and Microbial Biotechnology.A. M. Vilas (ed). Formatex ResearchCenter, pp. 383-395.PSP034Will not be presented!PSP035The structure of the NADH: ubiquinone oxidoreductase fromVibrio chloeraeM. Casutt 1 , G. Vohl* 1 , T. Vorburger 2 , J. Steuber 2 , G. Fritz* 11 University of Freiburg, Department of Neuropathology, Freiburg, Germany2 University of Hohenheim, Department of Microbiology, Stuttgart, GermanyVibrio choleraemaintains a Na + -gradient across the cytoplasmic membrane(1,2). The generated sodium motive force is essential for substrate uptake,motility, pathogenicity, or efflux of antibiotics. This gradient is generatedby an NADH:ubiquinone oxidoreductase (NQR) that is related to the RNFcomplex of archea and bacteria. NQR is an integral membrane proteincomplex consisting of six different subunits, NqrA-NqrF (3,4). In order toget insights into the redox-driven Na + -transport mechanism we haveisolated and crystallized the NQR of Vibrio cholerae (5). The crystals ofthe entire membrane complex diffract so far to 3.7 Angstrom and providefirst detailed structural information in this respiratory enzyme.(1) Türk K, Puhar A, Neese F, Bill E, Fritz G, Steuber J NADH oxidation by the Na + -translocatingNADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit. (2004) JBiol Chem 279:21349-55(2) Juárez O, Morgan JE, Nilges MJ, Barquera B. Energy transducing redox steps of the Na+pumpingNADH:quinone oxidoreductase fromVibrio cholerae. (2010) PNAS 107:12505-10.(3) Casutt MS, Nedielkov R, Wendelspiess S, Vossler S, Gerken U, Murai M, Miyoshi H, MöllerHM, Steuber J. Localization of Ubiquinone-8 in the Na + -pumping NADH:Quinone Oxidoreductasefrom Vibrio cholerae. (2011) J Biol Chem 286:40075-82(4) Casutt MS, Huber T, Brunisholz R, Tao M, Fritz G, Steuber J. Localization and function of themembrane-bound riboflavin in the Na + -translocating NADH:quinone oxidoreductase (Na + -NQR)fromVibrio cholerae. (2010) J Biol Chem 285:27088-99.(5) Casutt MS, Wendelspiess S, Steuber J, Fritz G. Crystallization of the Na + -translocatingNADH:quinone oxidoreductase from Vibrio cholerae. (2010) Acta Cryst F66:1677-9.PSP036Proteome assessment of an organohalide respiring species:Dehalococcoides sp. CBDB1C.L. Schiffmann* 1 , L. Adrian 2 , M. von Bergen 1,3 , J. Seifert 11 UFZ - Helmholtz Centre for Environmental Research, Proteomics, Leipzig,Germany2 UFZ - Helmholtz Centre for Environmental Research, IsotopeBiogeochemistry, Leipzig, Germany3 UFZ - Helmholtz Centre for Environmental Research, Metabolomics, Leipzig,GermanyChlorinated hydrocarbons that were released into the environment are dueto their toxic and cancerogenic potential a threat to nature and humanhealth. The ability of anaerobic bacteria belonging to Dehalococcoidesspp. to use a broad range of chemicals from this class as terminal electronacceptors shows potential for bioremediation use. The strictly anaerobicDehalococcoides sp. CBDB1 utilizes a wide range of electron acceptorswith the help of its reductive dehalogenase enzymes. In the sequencedgenome are 32 different reductive-dehalogenase-homologous (rdh) geneoperons annotated [1]. The high number of rdh clusters sparks a specialinterest in the differences between the gene products. Strong substratespecificities of the encoded rdh genes can explain this. To analyse theBIOspektrum | Tagungsband 2012