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

vinelandii. The proteins are encoded by the nif-genes (nitrogen-fixation).These nif-genes were also found in some other bacteria, for exampleGeobacter sulfurreducens and Wolinella succinogenes. We show that thesebacteria expresses the nif-genes if there is only little or no ammoniaavailiable. The expression of nitrogenase could be followed by a reductionassayof acetylene to ethylene using gas chromatography. The proteins werepurified and identified by mass spektroscopy.Baar, C. et al. (2003): Complete genome sequence and analysis of Wolinella succinogenes, PNAS,100, 11690-11695.Einsle, O. et al.(2002): Nitrogenase MoFe-Protein at 1.16 Å Resolution: A central ligand in the FeMo-Cofactor. Science, 297, 1696 - 1670.Stubal, J. C. et al. (2009): Genome sequence of Azotobacter vinelandii, an obligate aerobe specializedto support diverse anaerobic metabolic processes, Journal of Bacteriology, 191, 4534-4545.AMP014Assessment of molybdenum oxidoreductases in Wolinellasuccinogenes: key enzymes for formate-dependentanaerobic respiration with nitrate, polysulfide andarsenateO. Klimmek*, T. Heß, J. SimonInstitute of Microbiology & Genetics, University of Technology, Darmstadt,GermanyMolybdopterin-containing oxidoreductases are widely used in anaerobicmicrobial metabolism, for instance in the catabolic turnover of energysubstrates such as formate, nitrate, polysulfide, thiosulfate, tetrathionate,dimethyl sulfoxide, trimethylamine N-oxide, chlorate, selenate and arsenate.In many cases, such proteins form complexes with an iron-sulphur clusterprotein and a quinone/quinol-reactive membrane protein and typically thiscomposition is reflected in the corresponding gene clusters. However, it ishard to predict the substrate specificity of a particular molybdoenzyme fromits primary structure, especially in case of enzymes that catalyse the turnoverof sulphur-containing compounds. The genome of theEpsilonproteobacterium Wolinella succinogenes encodes eleven distinctmolybdopterin oxidoreductases comprising periplasmic nitrate reductase,polysulfide reductase, dimethyl sulfoxide reductase and at least two formatedehydrogenases (1). Three of the remaining enzymes are predicted to formmembrane-bound complexes with an iron-sulphur protein and a membraneanchor of the widespread PsrC/NrfD family. The molybdopterin-containingsubunit of these complexes is thought to be located in the periplasm due tothe presence of Tat-type signal peptides. Corresponding gene deletionmutants were constructed and characterized concerning growth by anaerobicrespiration and conversion of various typical molybdoenzyme substrates. Itturned out that one of the so far uncharacterised enzymes sustained growthby arsenate respiration while another one served as an alternative polysulfidereductase.[1] Baar et al (2003): Complete genome sequence and analysis of Wolinella succinogenes, Proc NatlAcad Sci USA. 100 (20): 11690-5.AMP015First steps into production of (R)-benzylsuccinate via ananaerobic microbial synthetic pathwayJ. Mock*, J. Kölschbach, J. HeiderDepartment of Microbiology, Philipps-University, Marburg, GermanyThe denitrifiying bacteria Aromatoleum aromaticum, Thauera aromaticaand the iron(III)-reducing species Geobacter metallireducens degradetoluene under anaerobic conditions. The first intermediate of the pathway is(R)-benzylsuccinate, an aromatic compound of potential interest in theproduction of polymers, due to its two reactive carboxyl groups. We attemptto redesign the fermentation metabolism of standard bacteria such asEscherichia coli to establish the production of this intermediate as abiotechnological process. Synthesis of benzylsuccinate may either be startedfrom the fermentation intermediate fumarate and exogenous toluene or fromthe fermentation product succinate and exogenous benzoate. We started ourfirst efforts with the production of benzylsuccinate from benzoate, using thetoluene pathway in reverse, since most steps can be catalyzed under aerobicconditions. To enter this reverse pathway, the precursor benzoate must betransported into the cytosol via a membrane-bound permease and activatedto benzoyl-CoA. Subsequently, benzoylsuccinyl-CoA is formed frombenzoyl-CoA and succinyl-CoA followed by several further steps tobenzylsuccinate as end product. First results on establishing benzoyl-CoAformation and characterizing the benzoylsuccinyl-CoA thiolase of Thaueraaromatica will be shown.AMP016Anaerobic metabolism of toluene in denitrifying, Fe(III)and sulfate-reducing bacteriaM. Hilberg*, S. Kölzer*, J. HeiderDepartment of Microbiology, Philipps-University, Marburg, GermanyAnaerobic degradation of toluene is initiated by an unusual addition reactionof the methyl group of toluene to the double bond of a fumarate cosubstrateto form the first intermediate (R)-benzylsuccinate. This reaction is catalyzedby the glycyl radical enzyme benzylsuccinate synthase, encoded in thetoluene-inducible bss-operon. We produced all three subunit containing (R)-benzylsuccinate synthase (BssA, B and C) and the activating enzyme (BssD)of the denitrifying bacteria Thauera aromatica in Escherichia coli andpresent a complete cofactor characterization via Mössbauer-, EPR-, andUV/vis- spectroscopy.(R)-benzylsuccinate is then further degraded via several steps to benzoyl-CoA and succinyl-CoA in a modified ß-oxidation pathway. In an early stepof this pathway benzylsuccinyl-CoA is oxidized to phenylitaconyl-CoA bybenzylsuccinyl-CoA dehydrogenase, which apparently interacts withelectron transferring flavoprotein. Genetical and biochemical analysis ofthese enzymes in anaerobic toluene-degrading bacteria with differentphysiology gives insight into conservedness and differences of energymetabolism between denitrifying, Fe(III) and sulfate-reducing bacteria.AMP017Benzene degradation under anaerobic conditionsK. Nicolaisen*, N. Abu Laban, R. Diesveld, R.U. MeckenstockInstitute of Groundwater Ecology, Helmholtz Center Munich, Neuherberg,GermanyThe iron-reducing culture BF and the sulphate-reducing culture BPL wereanalyzed in order to identify the first step in benzene degradation underanaerobic conditions. Growth analysis of strain BPL showed that phenol andtoluene, which would be intermediates during hydroxylation respectivelymethylation, are not used as substrates by this strain. Also by proteomicanalysis of benzene grown cells no putative enzymes for these reactionscould be identified [1]. Based on metabolite analysis with stable isotopelabelledbenzene or bicarbonate buffer direct carboxylation to benzoate isproposed as initial reaction [2]. Combined proteomic and genomic analysisof strain BF led to the identification of a putative anaerobic benzenecarboxylase (Abc) consisting of several subunits, which show 43% and 37%sequence identity to phenylphosphate carboxylase subunit PpcA and PpcDand 67% to 3-octaprenyl-4-hydroxybezoate carboxy-lyase (UbiD/ubiX) ofAromatoleum aromaticum strain EbN1 [2].[1] Abu Laban et al (2009): FEMS Microbiol. Ecol. 68, 300-311.[2] Abu Laban et al (2010): Environ. Microbiol. 12(10), 2783-2796.AMP018Metal reduction without outer membrane cytochromes inShewanella oneidensisC. Bücking*, J. GescherDepartment of Microbiology, Albert-Ludwigs-University, Freiburg,GermanyRespiratory metal reduction is a highly abundant form of microbial energygeneration. Still, the solubility of metal oxides is often low under neutralconditions. Therefore, Shewanella species have established the formation ofan extended respiratory chain delivering electrons through the periplasm andonto the cell surface to the insoluble electron acceptor. Surface localizedouter membrane cytochromes (OMC) are believed to catalyze the finalreduction step.In previous work, we constructed a deletion mutant devoid of any OMCproteins [1]. This mutant contains an arabinose inducible promotor in frontof known key genes for metal reduction: mtrA and mtrB.Surprisingly, this mutant retained some low level metal reducing activity.We used this ability for a directed evolution approach selecting for fastergrowth with ferric citrate as terminal electron acceptor. After severalgenerations, we could isolate a suppressor strain which shows reproduciblyspektrum | Tagungsband 2011