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

as the sole source of energy for growth. Carbon monoxide dehydrogenase/acetyl-coenzyme A (acetyl-CoA) synthase (CODH/ACS) catalyzes COoxidation as well as acetyl-CoA synthesis/cleavage, and is, therefore, thekey enzyme for growth on CO or acetate. The M. acetivorans genomecontains two copies of a six-gene operon encoding CODH/ACS-isoforms(designated Cdh1 and Cdh2), which share 95 % amino acid sequenceidentity,and encodes a single stand-alone CdhA subunit, designated CdhA3.To address the role of these CODH/ACS-isoforms in M. acetivorans, thecomplete set of cdh disruption mutants was constructed and phenotypicallyanalyzed. To address differential cdh-expression, reporter strains wereconstructed carrying fusions of the individual cdhA promoters and uidA,both in the wild-type strain background and in the single cdh mutants. Bothanalyses, of cdh gene expression and of the mutant phenotypes, will bepresented and argue for a clear functional hierarchy and regulatory cross-talkof the CODH/ACS-isoforms.AMP005Thiosulfate dehydrogenase from Allochromatiumvinosum: an unusual acidophilic c-type cytochromeK. Denkmann* 1 , I. Pereira 2 , R. Zigann 1 , F. Grein 1 , C. Dahl 11 Institute for Microbiology and Biotechnologies, Friedrich-WestphalianWilhelms-University, Bonn, Germany2 Institute of Chemical and Biological Technology, New University ofLisbon, Oeiras, PortugalEvidence is emerging that c-type cytochromes with an unusual axial His/Cyscoordination of the heme iron play a pivotal role in sulfur-based energymetabolism [1]. We identified the acidophilic tetrathionate-formingthiosulfate dehydrogenase from the purple sulfur bacterium Allochromatiumvinosum [2] as another probable member of this exciting group of proteins.The corresponding gene (tsdA, YP_003442093) was identified on the mainA. vinosum chromosome (NC_013851) on the basis of the previouslydetermined N-terminal amino acid sequence. The identity of the gene wasconfirmed by experiments with an A. vinosum ΔtsdA in frame deletionmutant. This strain completely lost the ability to produce tetrathionate fromthiosulfate while the production of sulfate via the thiosulfate-oxidizing Soxmultienzyme complex was unaffected. The tsdA gene starts with a sequenceencoding a typical Sec-dependent signal peptide. The mature enzyme is asoluble periplasmic monomeric 25.8-kDa cytochrome c. Homolgous genesare present in a number of α-, β-, γ- and ε-proteobacteria including humanpathogens like Campylobacter jejuni. The rather wide-spread occurrence ofthe gene agrees with reports of tetrathionate formation not only byspecialized sulfur oxidizers but also by many chemoorganoheterotrophs thatuse thiosulfate as a supplemental but not as the sole energy source. Theamino acid sequence deduced from the A. vinosum tsdA gene contains twopossible Cys-X 2-Cys-His heme binding motifs. Comparative sequenceanalysis provides indication for axial coordination of the two heme irons bymethionine (Met 222 or Met 236) and cysteine (Cys 123). Recombinant TsdAproduced in E. coli was indiscernible from the native A. vinosum proteinregarding specific activity, pH optimum and UV-Vis spectrum. Toinvestigate the role of conserved Cys 123 for catalysis and heme coordination,mutant forms of the protein in which this residue was replaced by eitherglycine, histidine or serine were also produced. All these were essentiallyinactive, thereby proving the importance of Cys 123 for catalysis. EPRspectroscopic characterization of the wild type protein yielded signals thatcan be provisionally attributed to a His/Cys-ligated heme.[1] Grein et al (2010) Biochemistry 49, 8290-8299.[2] Hensen et al (2006) Mol Microbiol 62, 794-810.AMP006Application of anaerobic fluorescence proteins for in vivoreporter systems in clostridiaF. Schulz*, T. Lütke-EverslohInstitute of Biological Sciences, Department of Microbiology, University ofRostock, Rostock, GermanyFluorescent proteins such as the green fluorescence protein and itsderivatives strictly require oxygen similar to luciferase-based reportersystems, which excludes these gentle in vivo reporters for applications inanaerobes. Recently, novel flavin mononucleotide (FMN)-based fluorescentproteins harboring light-oxygen-voltage domains were engineered for noninvasivereporter systems applicable for both aerobic and anaerobicconditions in Escherichia coli and Rhodobacter capsulatus (Drepper et al.,Nat. Biotechnol. 25:443-445). We have optimized these fluorescence-basedreporters for Clostridium acetobutylicum and this study provides suitableapplications for monitoring gene expression in members of the genusClostridium. Since this group of anaerobic bacteria, which contains bothimportant pathogenic strains and apathogenic species of biotechnologicalimpact, severely lacks a good choice of genetic tools for modifying geneexpression, we generated a basic plasmid portfolio to monitor geneexpression in clostridia. For this, we constructed several E. coli-Clostridiumshuttle vectors according to a new modular plasmid system comprisingdifferent origins of replication for the use in various clostridial species(Heap et al., J. Microbiol. Methods 78:79-85). Furthermore, we provide anovel high-throughput application for analyzing and engineering geneexpression in C. acetobutylicum in a 96-well microtiter plate scale.AMP007Studies on the interaction of the O-demethylasecomponents of the anaerobe AcetobacteriumdehalogenansH.D. Nguyen* 1 , S. Studenik 1 , G. Diekert 11Institute for Microbiology, Department of Applied and EcologicalMicrobiology, Friedrich-Schiller-University, Jena, GermanyThe anaerobe homoacetogen Acetobacterium dehalogenans utilizes themethyl group of phenyl methyl ethers, which are products of lignindegradation, as a carbon and energy source. The O-demethylation reaction inwhich the methyl group of the substrate is transferred to tetrahydrofolate ismediated by the key enzymes, the O-demethylases, in the methylotrophicmetabolism. Different O-demethylases are induced in response to differentphenyl methyl ethers.The O-demethylase complex consists of four enzymes: a methyltransferase I(MT I), a methyltransferase II (MT II), a corrinoid protein (CP) and anactivating enzyme (AE). The methyl group is transferred from the phenylmethyl ether to the super-reduced corrinoid protein by MT I. The methylatedcorrinoid protein is subsequently demethylated and the methyl group istransferred to tetrahydrofolate by MT II. The inactivated form of thecorrinoid protein, cob(II)alamin, which may be generated by inadvertentoxidation, is reduced to active cob(I)alamin by the activating enzyme in anATP dependent reaction. To investigate the reaction mechanism of theenzyme system we purified and currently characterize the four proteincomponents. The investigation also includes protein-protein interactionstudies using biochemical methods and electron microscopy.AMP008Propionic acid metabolism during biowaste digestiondominantdegraders and their oxidation pathwaysM. Felchner-Zwirello* 1,2 , J. Winter 1 , C. Gallert 11 Institute of Bioengineering and Biotechnology of Waste Water, KarlsuheInstitute of Technology, Karlsruhe, Germany2 Department of Analytical Chemistry, Gdansk University of Technology,Gdansk, GermanyAnaerobic digestion is known as a solution for biowaste utilization withbiogas production and its potential is estimated to share at least 25 % of thebioenergy produced in European Union in the future [1]. It’s complexity andsensitivity requires however an effort in maintaining the performancewithout any failure. The process is often disrupted by i.e. organic overloadwhat leads to volatile fatty acids accumulation, especially propionic acid(PA), pH drop and digester upset [2]. The diversity of microorganism groupstaking part in biowaste conversion into biogas makes it difficult to manageand describe. The need for analyzing microorganisms’ communities inanaerobic digesters is essential to understand the process and facilitate stableecosystem by finding optimal conditions [3]. However, there is still too littleinformation about involved bacteria. Identification and description of PAdegraders can be done by i.e. the metabolic degradation pathwayidentification and Fluorescence in-situ hybridization (FISH). A combinationof a Dani 3950 headspace sampling unit (HS), a Varian 431 gaschromatograph (GC) and a Varian 210 mass spectrometer (MS) has beenapplied to quantify and specifically identify metabolites of PA oxidation.The use of 1- 13 C-labeled PA as a carbon source for microorganisms allowsdifferentiation between two known pathways (methyl-malonyl-CoA and C-6-dismutation) resulting in CO 2 and acetic acid (AC) production.Appearance of the 13 C-moiety either in the carboxyl and methyl moiety ofAC can be detected by MS. The method was successfully applied forspektrum | Tagungsband 2011