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

the corresponding cell extracts. The enzyme is currently purified andcharacterized for its biochemical features and the presence of metals. Asindicated by the enzymes analyzed here, A. aromaticum may be a modelsystem for the coexistence of molybdenum- and tungsten-enzymes in thesame cell. In addition, detailed genome evaluation revealed hints for theexistence of metal-specific isoenzymes for molybdate or tungstate transportand for molybdenum or tungsten insertion during molybdenum-cofactorbiosynthesis.AMP027The Role of adhE 2 in the solventogenic ClostridiumacetobutylicumD. Hönicke*, L. Ziyong, J. Lesiak, D. Krauße, W. Liebl, A. EhrenreichDepartment of Miciobiology, Technical University Munich, Freising,GermanyThe large genus Clostridium encompasses species like Clostridiumacetobutylicum that are able to ferment starch and sugars into solvents.Especially butanol is an important bulk chemical with a wide range ofindustrial applications for example as biofuel due to its superior propertiescompared to ethanol and biodiesel.The aldehyde/alcohol dehydrogenase (AADH) is mainly involved in butanolformation and possibly plays an important role in the switch fromacidogenesis to solventogenesis. During sequencing of the C.acetobutylicum ATCC 824 genome two open reading frames (ORFs)encoding for a bifunctional aldehyde/alcohol dehydrogenase (AADH) wereidentified. Both are carried by the pSOL1 megaplasmid. The 2,577-bpadhE2 (CAP0035) is located about 47 kb away from the adhE (CAP0162)which has been shown to be the gene responsible for the two final steps ofbutanol production in solventogenic cultures. Because the adhE2 isspecifically expressed, as a monocistronic operon, under the condition of ahigh NADH/NAD + ratio, it is assumed to be responsible for the butanolproduction in alcohologenic cultures.For further researches we generated a mutant of the gene adhE2 using theClosTron technique, a clostridial insertional inactivation system that basedon the selective retargeting of a group II intron. Within batch fermentationsof the mutant we took samples for quantitative analysis which include thedetermination of the substrate/product concentrations and transcriptome timeseries. Using an oligonucleotide-based microarray we obtained an overviewof the transcript levels in the adhE2-mutant.AMP028Monitoring of a biogas producing microbial communityby its metaproteomeA. Hanreich* 1 , R. Heyer 2 , D. Benndorf 2 , E. Rapp 3 , U. Reichl 3 , M. Klocke 11 Department of Bioengineering, Leibniz Institute for AgriculturalEngineering, Potsdam, Germany2 Otto-von-Guericke-University, Magdeburg, Germany3 Max Planck Institute for Dynamics of Complex Technical Systems,Magdeburg, GermanyFor the production of biogas, various agricultural wastes but also crops canbe utilized by a complex microbial consortium consisting of fermentativebacteria and methanogenic archaea. Here, we present a metaproteomicapproach to investigate the metabolic activities of methanogens within abiogas producing community.A robust method for protein extraction and separation from biogas reactorsamples was developed including (i) a phenol extraction step, (ii) a methodfor estimation of protein quantities in presence of interfering substances, and(iii) paper-bridge loading for first dimension isoelectric focusing leading toefficient removal of contaminants. After two-dimensional gelelectrophoresis, major protein spots were analyzed by nanoHPLC onlinecoupled to tandem mass spectrometry in order to identify major proteins.Attention was directed to the extraction of intracellular proteins as thisprotein fraction promises to give further insight into the pathway ofmethanogenesis. From approximately sixty analyzed spots, almost a thirdcould be mapped to the methanogenic pathway.Key enzymes of methanogenesis like methyl-coenzymeM reductase werefound to be expressed in high amounts by different members of the family ofMethanosarcinaceae, which can produce methane from acetate as well as bythe reduction of CO 2. Interestingly, also members of theMethanomicrobiaceae, which only use the hydrogenotrophic pathway ofmethanogenesis, were identified. These results suggest that methane is atleast to a certain part produced from CO 2 and H 2. This claim is supported bythe findings of several house-keeping enzymes of Anaerobaculumhydrogeniformans. This syntrophic organism produces H 2 and can onlygrow, if H 2 is removed by other organisms, for example by methanogenicarchaea.Our study proves the feasibility of the extraction and characterization of themetaproteome of complex biogas producing communities in principle andgives valuable insights into active metabolic pathways. In future, monitoringof protein expression patterns may act as a valuable tool for the estimationof the metabolic activity of a microbial community helpful for processoptimization.AMP029Disproportionation and possible electron bifurcationreactions involved in crotonate fermentation bySyntrophus aciditrophicusA. Schmidt*, S. Mosler, K. Kuntze, M. BollFaculty of Biosciences, Pharmacy and Psychology, University of Leipzig,Leipzig, GermanyThe fermenting Deltaproteobacterium Syntrophus aciditrophicus is able todegrade crotonate in absence of a syntrophic partner with acetate andcyclohexanecarboxylate representing the main fermentation products[Mouttaki 2008]. The reducing equivalents formed during crotonateoxidation to acetate are recycled in reverse reactions of the benzoyl-CoAdegradation pathway yielding cyclic mono-/dienoyl-CoA compounds, whichmight be further reduced to cyclohexanoyl-CoA. In this work we studied theunknown formation of cyclohexanecarboxylate from the proposedfermentation intermediates cyclohex-1-ene-1-carboxyl-CoA (monoenoyl-CoA)/cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA). Cell-freeextracts from S. aciditrophicus grown on crotonate disproportionated both,monoenoyl-CoA to dienoyl-CoA plus benzoyl-CoA, and dienoyl-CoA tobenzoyl-CoA plus monoenoyl-CoA. Such disproportionation reactions areassigned to activities of W-containing class II benzoyl-CoA reductases(BCR) as described previously for Geobacter metallireducens [2]. Thecyclohexanoyl-CoA formed is then converted to cyclohexanecarboxylateeither by a thioesterase or a CoA transferase. In the presence of externalelectron donors such as dithionite or NADH, the benzoyl-CoA formedduring the disproportionation reactions was converted to reduced cyclicproducts. The endergonic reductive dearomatization of benzoyl-CoA todienoyl-CoA by NADH (ΔG°’ = +58 kJ mol -1 ) can only be explained by anelectron bifurcation mechanism. We propose that this reaction is driven bythe concomitant reduction of dienoyl-CoA by NADH to monoenoyl-CoA(ΔG°’ < -50 kJ mol -1 ). The combined action of disproportionation andelectron bifurcation reactions enables an extended recycling of reducingequivalents in S. aciditrophicus during growth on crotonate.[1] Mouttaki, H. et al (2008): Use of benzoate as an electron acceptor by Syntrophus aciditrophicusgrown in pure culture with crotonate. Env Microbiol 10(12):3265-3274.[2] Kung, J.W. et al (2010): Reversible Biological Birch Reduction at an Extremely Low RedoxPotential. Proc Nat Acad Sci 132:9850-9856.AMP030Anaerobic degradation of p-methylbenzoate by thedenitrifying strain pMbN1 involves a novel type ofbenzoyl-CoA reductaseS. Lahme* 1,2 , C. Eberlein 3 , M. Boll 3 , H. Wilkes 4 , R. Rabus 1,21 Department of General and Molecular Microbiology, Institute forChemistry and Biology of the Marine Environment (ICBM), Oldenburg,Germany2 Department of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, Germany3 Institute of Biochemistry, University of Leipzig, Leipzig, Germany4 Organic Geochemistry, German Research Center for Geosciences (GFZ),Potsdam, GermanyIn anaerobic bacteria a large variety of aromatic compounds is converted tothe central intermediate benzoyl-CoA, which serves as substrate fordearomatizing benzoyl-CoA reductases (BCRs). However, common BCRsdo not accept p-methylbenzoyl-CoA as a substrate, which is probably thereason why known aromatic compound degrading anaerobes cannot utilizep-methylbenzoate. The newly isolated denitrifying α-proteobacterium strainpMbN1, belonging to the genus Magnetospirillum, uses p-methylbenzoateor benzoate as sole carbon source, which enabled a first study of theunknown p-methylbenzoate degradation pathway.spektrum | Tagungsband 2011