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

1461. Ye, L.D., Schilhabel, A., Bartram, S., Boland,W., and G. Diekert. 2010. Reductive dehalogenation ofbrominated ethenes by Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S. Environ.Microbiol. 12, 501-5092. Nijenhuis, I., Andert, J., Beck, K., Kästner, M., Diekert, G., and H. H. Richnow. 2005.Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillummultivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin. Appl.Environ. Microb. 71 (7), 3413-3419OTP038Screening halophilic and halotolerant bacteria from saline soil,mud, brine and salt sediments of Urmia lake in IranF. Jookar Kashi*, M.A. Amoozegar, P. OwilaShahed University, Biology, Tehran, Islamic Republic of IranHypersaline lakes, with salinity ranges at or near saturation are extremeenvironments; yet, they often maintain remarkably high microbial celldensities and are biologically very productive ecosystems. To adapt tosaline conditions, bacteria have developed various strategies to maintaincell structure and function. Studies of such bacteria are of greatimportance, as they may produce compounds of industrial interest. Weemployed culture-dependent techniques to study microbial diversity inUrmia Lake , a unique hypersaline lake (24.6% salinity) in northwest Iran.The samples were collected in November 2010 into sterile bottles andstored in ice boxes in the laboratory ,pH, moisture content, and Na+ , K+ ,Ca2+ , Mg2+ , and Cl content of the salt and sediment samples weremeasured according to standard methods. Screening bacteria from salinesoil, mud, brine and salt sediments of Urmia lake led to the isolation of280 moderately halophilic and 40 extremely halophilic bacteria amongwhich there were 191 gram-positive rods, 99 gram- negative rods and 30gram-positive cocci. PCR Amplification of 16S rDNA of isolates wascarried out by using universal primers and products were sequencedcommercially.These gene sequences were compared with other genesequences in the GenBank databases to find the closely related sequences.Most of the isolates belonged to different species of genus Bacillus.OTP039Generating mutated variants of the unique 5-chloromuconolactone dehalogenase from Rhodococcus opacus1CP and their comparison with the wildtype enzyme to elucidatecatalytic relevant residuesJ.A.D. Gröning* 1 , C. Roth 2 , S.R. Kaschabek 1 , N. Sträter 2 , M. Schlömann 11 TU Bergakademie Freiberg, Environmental Microbiology, Freiberg, Germany2 University of Leipzig, Center for Biotechnology and Biomedicine, Institute forStructural Analytics of Biopolymers, Leipzig, Germany5-Chloromuconolactone dehalogenase ClcF plays an unique role in 3-chlorocatechol degradation by R. opacus 1CP. The variant of a so calledmodified ortho-cleavage pathway in that actinobacterium differs from theone typically found in proteobacteria by the inability of chloromuconatecycloisomerase ClcB2 to convert 2-chloro-cis,cis-muconate into transdienelactone.Instead, ClcB2 behaves like a muconate cycloisomerasecatalyzing cyclization of 2-chloro-cis,cis-muconate to 5-chloromuconolactone. Further dechlorination to cis-dienelactone isperformed by ClcF an enzyme showing high similarity to(methyl)muconolactone isomerases. Although these enzymes are typicallyinvolved in (methyl)catechol degradation their biochemical ability tocatalyze dechlorination of chloromuconolactones has been recently reported.As a first step to elucidate the mechanism of dechlorination as well as toidentify residues, relevant for activity, mutational analysis of recombinantClcF was made. Properties of variants were compared to wildtype ClcF aswell as to muconolactone isomerase MLI and methylmuconolactoneisomerase MMLI from (methyl)catechol-degrading Cupriavidus necatorJMP134 in respect of changes in product formation (cis-/transdienelactone),kinetic parameters, and the ability to convertmuconolactone. Using an E. coli / pET expression system and a three-steppurification procedure turned out to be a well suited strategy to obtainrecombinant proteins in high purity. A considerable extent ofspecialization of ClcF for its new physiological function in strain 1CP isindicated by an extremely low activity of that enzyme to convertmuconolactone into 3-oxoadipate enollactone which represents the originalfunction of (methyl)muconolactone isomerases. A similar picture wasobtained by comparison of specificity constants towards 5-chloromuconolactone of ClcF (1.4 M -1 s -1 ), MLI (0.6 M -1 s -1 ), andMMLI (0.06 M -1 s -1 ).OTP040Identification of amino acids involved in substrate binding ofPHB depolymerase PhaZ7 of Paucimonas lemoigneiS. Hermawan* 1 , T. Papageorgiou 2 , D. Jendrossek 11 Institut für Mikrobiologie, Stuttgart, Germany2 Turku Centre for Biotechnology, Turku, FinlandThe extracellular PHB depolymerase PhaZ7 of P. lemoignei is uniqueamong extracellular PHB depolymerases due to its specificity foramorphous native PHB granules (nPHB). The structure of PhaZ7 wassolved first at 1.9 Å [1] and recently at 1.4 Å [2]. PhaZ7 is a single-domainglobular protein with an / hydrolase fold and a catalytic triad consistingof S136, E242, and H306. Analysis of PhaZ7 structure showed a highsimilarity to lipase LipA of Bacillus subtilis except for the presence of anadditional domain in PhaZ7 that is absent in LipA. This lid-like domaincontained many hydrophobic amino acid residues suggesting their possibleinvolvement in nPHB binding. Since the PhaZ7 structure has no accessiblesubstrate entry to the catalytic site we suggest that conformational changesmust take place upon substrate binding. The effects of mutations ofselected hydrophobic amino acids of the PhaZ7 lid-like domain on activityand nPHB binding ability were investigated. Our results showed thatmutations of Y105, Y176, Y189, Y190 and W207 to alanine or glutamateresulted in reduced nPHB depolymerase activity. Interestingly, a lag-phaseof several minutes in the depolymerase reaction was observed beforemaximal activity was determined. Binding assays with nPHB revealed areduced binding ability of these PhaZ7 muteins compared with wild typePhaZ7. The structure of Y105D and Y190D muteins were determined andrevealed changes in the 280-290 region and in the 248-251 region.Recently, the structure of inactive PhaZ7 S136A mutein bound to 3-hydroxybutyrate (3-HB) trimer has also been determined. It showed that 3-HB trimer is bound to a groove surrounded by Y189/Y190, Y105 andY176. This result is consistent with our mutagenesis results. Additionally,similar to the structure of the Y105D and Y190D muteins, the 280-295region and the 248-253 region of S136A mutein bound to 3-HB trimerwere missing indicating some flexibility of these regions. Hence, ourhypothesis that hydrophobic amino acid residues of the PhaZ7 lid-likedomain are involved in substrate binding and that conformational changesupon substrate binding occur was confirmed. Our results afford newinsights into the mechanism of biopolymer binding to PHB depolymerasesand enzymatic PHB hydrolyis.1. A. C. Papageorgiou, S. Hermawan, C. B. Singh, and D. Jendrossek. 2008. J. Mol. Biol.382:1184-94.2. S. Wakadkar, S. Hermawan, D. Jendrossek, and A. C. Papageorgiou. 2010. Acta Crystallogr. Sect .FStruct. Biol. Cryst. Commun.66:648-54.OTP041Genome-guided analysis of physiological and morphologicaltraits of the metabolically versatile fermentative acetateoxidizer Thermacetogenium phaeumD. Oehler* 1 , A. Poehlen 2 , R. Daniel 2 , G. Gottschalk 2 , B. Schink 11 Universität Konstanz, Biology, Konstanz, Germany2 Universität Göttingen, Biology, Göttingen, GermanyFermentative conversion of acetate to CO2 and hydrogen becomespossible if the hydrogen partial pressure is kept low by a methanogenicpartner, but the energy gained from this process is very low. Thisrelationship is called syntrophy.Thermacetogenium phaeum, isolated froma thermophillic anerobic methanogenic reactor, is able to grow on varioussubstrates to form acetate as sole product, and in coculture with amethanogenic bacterium,Thermacetogenium phaeumis able to grow onacetate. It was shown previously that the Wood-Ljungdahl pathway is usedin both modes of living, but the mechanism of energy conservation isunknown.To extend our knowledge on the biochemistry and physiology of thisinteresting organism, we completely sequenced the genomeofThermacetogenium phaeum.The strain has one circular chromosome ofthe size of 2.93 Mb; the G+C content of the DNA is 53.88mol%. Themanual annotation of the 3215 CDS encoded by the genome gave a deeperinsight into the physiology of the organism.All genes necessary for the Wood-Ljungdahl pathway were found but incomparison to the H + -dependent acetogen Moorella thermoacetica and theNa + -dependent acetogen Acetobacterium woodii no indications ofcytochromes, sodium dependence, or of RNF-complexes were found aspotential energy conserving mechanisms. It was reported thatThermacetogenium phaeum is a sulfate reducing bacteria but neither thegenome sequence nor physiological experiments could confirm this result.As a sign of heavy phage attack in the past a lot of CRISPR sequences arepresent in the genome, and also a complete prophage was found.OTP042Construction of Rubber Oxygenase A variants (RoxA), adiheme-dioxygenase from Xanthomonas sp. 35YJ. Birke*, N. Hambsch, D. JendrossekInstitut für Mikrobiologie, AG Jendrossek, Stuttgart, GermanyThe extracellular diheme-dioxygenase RoxA (Rubber oxygenase A) fromXanthomonas sp. 35Y is able to cleave natural rubber, the primary productis ODTD (12-oxo-4,8-dimethyltrideca-4,8-diene-1-al) [1]. The cleavagemechanism of this reaction is unknown. Heterologous expression of RoxAin Escherichia coli, Bacillus subtilis or Pseudomonas putida was notsuccessful, therefore an overexpression of RoxA from a broad-host rangerhamnose inducible plasmid was established in its natural host strainXanthomonas sp. 35Y [2]. However, it was not possible to obtainrecombinant RoxA with either a strep-tag or his-tag at the C-terminus. TheBIOspektrum | Tagungsband 2012