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

142bacteria in situ, we used 16S rRNA clone libraries, 454-tag sequencing andCARD-FISH. S 0 slabs incubated in oxic sediment were mainly colonizedby epsilonproteobacteria that were related to Sulfurimonas andSulfurovum. Sulfate formation supported that epsilonproteobacteria areimportant ZVS-oxidizing organisms not only in hydrothermal systems andin OMZs but also in temperate marine sediments. S 0 slabs from the anoxicsediment were colonized mainly by probably S-disproportionatingDesulfocapsa. We will compare the diversity of S 0 -utilizing organismsfrom coastal sediments and from hydrothermal systems to look fordifferences and commonalities. Our data will provide detailed insights intothe bacterial community involved in biogeochemical cycling of zerovalence sulfur species in different habitats.OTP019Synthetic microbial production pathways for benzoyl-CoAderivedmetabolitesJ. Mock*, J. HeiderUniversität Marburg, Mikrobiologie, Marburg, GermanyBenzoyl-CoA is a key intermediate in several metabolic pathways. Forinstance it is a precursor for polyketide synthesis in plants andmicroorganisms or an intermediate in aromatic hydrocarbon degradation.Since benzoyl-CoA normally does not occur in microbial metabolism, anydesigned benzoyl-CoA-dependent microbial pathway requires an initiationmodule to synthesise this intermediate. We made use of a benzoatetransporter and a benzoate-CoA ligase from the betaproteobacteriumAromatoleum aromaticum, which are involved in anaerobic benzoatedegradation, to create recombinant benzoyl-CoA-producing bacterialstrains.Our first efforts to couple benzoyl-CoA to the synthesis of a product aimedat the polyketide biphenyl, which is synthesised from benzoyl-CoA andthree malonyl-CoA by biphenyl synthase of the rowan berry Sorbusaucuparia 1 . The initial host strain of E.coli did not produce any detectableproducts, probably because of limited malonyl-CoA supply. Therefore, weshifted to the related Shimwellia (formerly Escherichia) blattae, since thisspecies can be grown on malonate 2 , which should release the metabolicbottleneck. First results indicate that a metabolite is indeed produced byrecombinant cultures fed with benzoate and malonate, which will befurther characterized.Another more extended possible biosynthetic pathway starting frombenzoyl-CoA is pursued for the production of (R)-benzylsuccinate, anaromatic compound of potential interest for production of biodegradablepolymers. (R)-benzylsuccinate is an intermediate of anaerobic toluenedegradation and is usually synthesised from toluene and fumarate via aglycyl radical enzyme 3 . We will try to establish a synthetic pathway forthis compound from benzoyl-CoA and the mixed acid-fermentationproduct succinate by reversing the -oxidation pathway involved inbenzylsuccinate degradation. The first step of this reverse pathway is thecondensation of benzoyl-CoA and succinyl-CoA to benzoylsuccinyl-CoAby a new type of thiolase. First results on establishing this reaction will beshown.1 Liu B, Raeth T, Beuerle T, Beerhues L. (2007). Biphenyl synthase, a novel type III polyketidesynthase. Planta. 225, 1495-503.2 Priest F G, Barker M. (2010). Gram-negative bacteria associated with brewery yeasts:reclassification of Obesumbacterium proteus biogroup 2 as Shimwellia pseudoproteus gen. nov., sp.nov., and transfer of Escherichia blattae to Shimwellia blattae comb. nov.Int Journal Systematic andEvol Microbiology.60,828-8333 Leutwein C, Heider J. (2002). (R)-Benzylsuccinyl-CoA dehydrogenase of Thauera aromatica, anenzyme of the anaerobic toluene catabolic pathway.Arch Microbiol.178, 517-524,OTP020Influence of the initial dissolved H 2 concentration on the reductivedechlorination of 1,2,3-trichlorobenzene by Dehalococcoides sp.strain CBDB1S. Hartwig* 1 , G. Sawers 2 , U. Lechner 11 Martin-Luther-University Halle, Biology/Microbiology AG Lechner,Halle (Saale), Germany2 Martin-Luther-University Halle, Biology/Microbiology AG Sawers, Halle(Saale), GermanyThe genus Dehalococcoides comprises strictly anaerobic bacteria thatconserve energy exclusively by organohalide respiration. They only usehalogenated organic compounds such as 1,2,3-trichlorobenzene (TCB) aselectron acceptor and hydrogen as electron donor. Moreover, they only useacetate as a carbon source. The genome of Dehalococcoides sp. strainCBDB1 encodes 32 homologous cobalt-dependent reductivedehalogenases (Rdh), 5 types of multi-subunit hydrogenases (Hyd) and aputative formate dehydrogenase (Fdh) as one of the most abundantproteins in strain CBDB1 [1].The influence of varying the dissolved H 2 concentration on thestoichiometric dechlorination of 1,2,3-TCB to 1,3-dichlorobenzene (DCB)was monitored in Dehalococcoides sp. strain CBDB1 using gaschromatography. Initial dissolved H 2 concentrations from 1 M to 10 Mwere applied to cultures containing 50 M 1,2,3-TCB. The completedechlorination of50 M 1,2,3-TCB was achieved within 48 hours using an initial H 2concentration of 10 M. We could demonstrate that the rate of reductivedechlorination of 1,2,3-TCB to 1,3-DCB increased with increasing initialdissolved H 2 concentrations. Moreover, the hydrogen uptake rate alsoincreased with increasing dissolved H 2 concentrations. Hydrogenconsumption was also observed at an elevated redox potential (between -350 and -110 mV), whereas under these conditions reductivedechlorination did not occur. This indicates that hydrogen oxidation andreductive dechlorination can be uncoupled. Using qRT-PCR it could bedemonstrated that the genes encoding the catalytic subunits of the Fdh andthe periplasmatic hydrogenase Hup were highly expressed. Expression ofthese genes was also influenced in response to different initial dissolved H 2concentrations.Acknowledgement: This work is supported by the DFG (research unit FOR1530)[1] Kube et al. (2005) Genome sequence of the chlorinated compound-respiring bacteriumDehalococcoides species strain CBDB1. Nat Biotechnol 23, p.1269-1273OTP021Heterologues Regulation of Reductive Dehalogenase GeneExpression by a MarR-type RegulatorL. Segler* 1 , G. Sawers 2 , U. Lechner 11 Martin-Luther-University Halle, Biology/Microbiology AG Lechner,Halle (Saale), Germany2 Martin-Luther-University Halle, Biology/Microbiology AG Sawers, Halle(Saale), GermanyThe anaerobic bacterium Dehalococcoides sp. CBDB1 belongs to thephylum Chloroflexi and is unusual in that it is able to dechlorinatedifferent chloroaromatic compounds such as 1,2,3-trichlorbenzene (TCB)and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Dechlorination takesplace in a process called organohalide respiration and is catalysed byreductive dehalogenases (Rdh) and driven by hydrogen as electron donor.The genome of strain CBDB1 contains 32 genes encoding putative Rdhs[1]. The Rdh enzymes consist of a catalytic subunit RdhA together with aputative membrane-anchor subunit RdhB and these are encoded by rdhABoperons. The rdhAB operons are closely associated with genes encodingeither MarR-type or two-component system (TCS) transcriptionalregulators. The role of these regulators in the transcriptional control of therespective rdhAB genes is unclear.Because Dehalococcoides bacteria are not genetically tractable and aredifficult to work with, a heterologous system to study the function of theMarR protein CbdbA1625 in controlling expression of the rdhA genecbdbA1624 was developed. A heterologous in vivo system in Escherichiacoli was established using a single-copy reporter-lacZ fusion comprisingthe intergenic region (IR) between cbdbA1624 and the divergentlytranscribed cbdbA1625, encoding a MarR-type regulator. We analysed theactivity of the promoter of cbdbA1624 and that of cbdbA1625. Bothpromoters were functional in E. coli MC4100. The effect of multicopycbdbA1625 on expression of both reporter-lacZ fusions was analysed. TheMarR-type regulator had a negative effect on expression of bothpromoters. This work demonstrates that heterologous expression systemsprovide a powerful approach to dissect the transcriptional regulation of rdhgene expression.Acknowledgement: This work is supported by the DFG (research unit FOR1530)[1] Kube et al.(2005) Genome sequence of the chlorinated compound-respiring bacteriumDehalococcoides species strain CBDB1. Nat Biotechnol 23, p.1269-1273.OTP0224-Sulfoacetophenone Baeyer-Villiger-type Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1M. Weiss* 1 , K. Denger 1 , T. Huhn 2 , D. Schleheck 11 Microbial Ecology, Department of Biological Sciences, Konstanz, Germany2 Organic Chemistry, Department of Chemistry, Konstanz, GermanyThe xenobiotic laundry surfactant Linear Alkylbenzene Sulfonate (LAS)(3 x 10 6 tons per year worldwide, [1]) is completely degraded byheterotrophic bacterial communities [2]. However, until now, noinformation on the enzymes and genes involved has been established.3-(4-Sulfophenyl)butyrate (3-C4-SPC) is a biodegradation intermediate ofLAS, and 3-C4-SPC is mineralized by Comamonas testosteroni KF-1 [3,4] through a pathway that involves 4-sulfoacetophenone (SAP) and aninducible Baeyer-Villiger-type monooxygenase (BVMO) to yield 4-sulfophenylacetate (SPAc) from SAP. The hydrolysis of SPAc to 4-sulfophenol (SP) and acetate is catalysed by an esterase. This SPAcesterasewas purified to homogeneity and the corresponding gene in C.testosteroni KF-1 identified by peptide-mass fingerprinting. A predictedBVMO gene was located directly upstream to the SPAc-esterase gene.This candidate gene was over expressed in Escherichia coli and purified.The recombinant enzyme catalyzed the NADPH-dependent oxygenation ofSAP to SPAc, which was hydrolyzed after the addition of purified SPAcesterase,yielding SP and acetate. Thus, the first two genes and enzymesinvolved in the complete degradation pathway for LAS have beenidentified and characterized.References:[1], Knepper, T. P., D. Barceló, and P. de Voogt (eds.)., 2003. Elsevier, Amsterdam, chapter 1.BIOspektrum | Tagungsband 2012