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

177homogeneity. Both run as dimers in gel filtration chromatography but theyalso form higher aggregates in denaturing and non-denaturingelectrophoresis. The absorption maxima were 552, 525, and 410 nm(reduced: 420nm) for Igni_0955 and 554, 521 and 409 nm (reduced 419nm) for Igni_1369. Hemochrome spectra showed 7 hemes/subunit, whilemass spectroscopy resulted in 8 hemes for both proteins in accordancewith the prediction from sequence. Igni_1369 is one of the most abundantproteins in Ignicoccus cells (5%) but its function is unknown.A survey of multiheme proteins in Archaea showed that they occur insome but not all of the Desulfurococcales, the Archaeoglobi and theMethanomicrobia, and in the species Pyrobaculum calidifontis andNatrialba magadii. An overview of the distribution of various types of c-type cytochromes in Archaea will be discussed.PSP006Studies on the interaction of the O-demethylase components of theanaerobe Acetobacterium dehalogenans using two-hybrid systemsH.D. Nguyen*, S. Studenik, G. DiekertFriedrich-Schiller-University Jena, Institute for Microbiology, Jena,GermanyThe anaerobe acetogen Acetobacterium dehalogenans utilizes the methylgroup of phenyl methyl ethers, which are products of lignin degradation, asa carbon and energy source. The O-demethylation reaction in which themethyl group of the substrate is transferred to tetrahydrofolate is mediatedby the key enzymes, the O-demethylases, in the methylotrophicmetabolism. Different O-demethylases are induced in response to differentphenyl methyl ethers formed upon fungal lignin degradation.The O-demethylase complex consists of four enzymes: a methyltransferaseI (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. Themethylated corrinoid protein is subsequently demethylated and the methylgroup is transferred to tetrahydrofolate by MT II. The inactivated form ofthe corrinoid protein, cob(II)alamin, which may be generated byinadvertent oxidation, is reduced by the activating enzyme in an ATPdependent reaction.To catalyze the complete O-demethylase reaction, an interaction of at leastthree of the four proteins components is required. Protein-proteininteractions were investigated using bacterial and yeast two-hybridsystems. First results indicate that CP, as methyl group carrier during theO-demethylation process interacts with all other proteins of the O-demethylase complex. This finding supports the crucial role of CP in themethylotrophic metabolism of Acetobacterium dehalogenans.PSP007Design of a bacterial electron transport module: Interaction ofmembrane-bound NiFe-hydrogenase with cytochromes b and cO. Klimmek*, M. Kern, M. Hirschmann, F. Keul, J. SimonTU Darmstadt, Department of Biology, Darmstadt, GermanyMany bacteria employ membrane-bound NiFe-hydrogenases (MBHs) thatserve in hydrogen gas uptake and electron transport in anaerobicrespiratory chains. MBHs possess a heterodimeric protein complex thatcontains the active site of hydrogen turnover and three iron-sulfur clusters.This entity is located at the periplasmic side of the membrane and linked tothe membrane via a quinone-reactive dihaem cytochrome b. In contrast,soluble heterodimeric NiFe-hydrogenases from, for example, sulfatereducers are periplasmic enzymes that interact with multihaemcytochromes c.The MBH complex of the Epsilonproteobacterium Wolinella succinogenes(HydABC) is the key enzyme of anaerobic respiration using hydrogen gasas electron donor. The enzyme is anchored to the membrane by both thedihaem cytochrome b HydC and a C-terminal transmembrane helicalregion of the iron-sulfur subunit HydA [1,2]. In the absence of bothanchors, active hydrogenase was found almost exclusively in theperiplasmic cell fraction [1].The aim of this work was to identify amino acid residues involved inHydA-HydC interaction. Furthermore, the ability of W. succinogenesMBH to reduce cytochromes c was investigated using purified HydABC orcell fractions containing the periplasmic HydAB complex. Advised by coevolutionarydependency studies based on information theory, engineeringof HydAB was performed in order to optimize cytochrome c reduction byhydrogen gas, thus designing a novel periplasmic electron transfer networkin W. succinogenes.[1] Gross et al. (1998) Arch Microbiol 170: 50-58[2] Gross et al. (2004) J Biol Chem 279: 274-281PSP008The methylotrophic metabolism of Desulfitobacterium spp.M. Vogel, S. Studenik*, G. DiekertFriedrich-Schiller-University Jena, Institute for Microbiology, Jena, GermanyDesulfitobacterium spp. are strictly anaerobic bacteria first isolated fromenvironments contaminated with halogenated compounds. In 2004, it wasshown, that at least two strains of Desulfitobacterium hafniense (DCB-2and PCE-S) are able to use phenyl methyl ethers, which are degradationproducts of lignin, as electron donors. By then, only acetogens had beenreported to convert these compounds under anoxic conditions. In contrastto acetogenic bacteria, Desulfitobacterium hafniense is not able to use CO 2as electron acceptor.We currently investigate the metabolic pathways involved in the phenylmethyl ether consumption of Desulfitobacterium hafniense DCB-2. Keyenzymes are the O-demethylases, inducible enzyme systems first describedfor acetogens. On the basis of the genome sequence 17 putative O-demethylase operons were identified. Recent studies concentrate on theheterologous expression of putative O-demethylase genes and thecharacterization of the corresponding gene products.PSP009Temporal and spatial effects of adaptation, a new mechanismrelying on posttranslational modification of key enzymes indegradative microorganismsS. Leibeling* 1 , J.L. Zilles 2 , C.J. Werth 2 , R.H. Müller 1 , H. Harms 11 Helmholtz Centre for Environmental Research GmbH - UFZ,Environmental Microbiology, Leipzig, Germany2 University of Illinois at Urbana-Champaign, Civil and EnvironmentalEngineering, Urbana, United StatesA vast spectrum of organic chemicals is steadily released to theenvironment by the industry and consumers. Despite the xenobioticcharacter of these chemicals, the main process responsible for mitigatingtheir impact is pollutant degradation by microorganisms. The capability ofmicroorganisms to adapt to environmental pollutants and to couple theirdegradation to growth has been attributed to genetic mechanisms likemutation and recombination of genes. However, other mechanisms mayalso explain adaptative responses of microorganisms. Herein, we presentevidence for a mechanism improving the activity of degradative enzymesby posttranslational modification.The soil bacterium Delftia acidovorans MC1 was used; it degradesphenoxyalkanoate herbicides like 2,4-dichlorophenoxyacetate (2,4-D) and(RS)-2-(2,4-dichlorophenoxy-)propionate ((RS)-2,4-DP). Key enzymes forthe initial degradation step are -ketoglutarate-dependent dioxygenases,which determine the microorganism’s substrate specificity, e.g. the (R)-2,4-DP/-ketoglutarate dioxygenase (RdpA) attacks the R-enantiomer of(R)-2,4-DP but not 2,4-D. The latter is cleaved by 2,4-D/- and (S)-2,4-DP/-ketoglutarate dioxygenases (TfdA and SdpA, respectively). Westudied adaptation in long-term cultivation experiments with mutant strainsbearing only RdpA. Noteworthy, cultivation in the presence of (R)-2,4-DPand 2,4-D led to improved degradation of 2,4-D (K m) and its utilization forbiomass formation. This was accompanied by a change in the enzymepattern, as made visible by 2D gel electrophoresis, showing line-ups ofRdpA forms varying in their pI and number. Since there is only one rdpAgene in the genome of D. acidovorans and no mutations were found,posttranslational modification is a likely explanation for the appearance ofRdpA variants. Particularly plausible are charge relevant carbonylationreactions since they alter the proteins’ pI, as observed in our study.Carbonylation is induced by reactive oxygen species (ROS), which areknown side products of oxygenase reactions which, in turn, causecarbonylation of the enzyme itself and other proteins in its vicinity.Carbonyl groups were identified through Western blotting via theirspecific reactions with dinitrophenylhydrazine. Our study of D.acidovorans adaptation was extended to a two-dimensional microfluidicpore network, which simulates subsurface pore spaces. Here, initial growthon (R)-2,4-DP and adaptation on 2,4-D was observed via reflected DICmicroscopy in the pore network. Effluent collected during adaptation iscurrently analyzed for the appearance of RdpA variants. Our researchprovides insight into adaptational capabilities of microbial strains inbiotopes with limited genetic diversity, and defines growth properties atlimiting substrate concentrations which are relevant for treatment ofcontaminants in soil and groundwater.BIOspektrum | Tagungsband 2012