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

nearly the same growth rate as the wild type during ferric citrate reductionalthough OMCs are not produced. This reduction is strictly dependent onarabinose induction which triggers the production of MtrA and MtrB.We are currently investigating which proteins could have functionallyreplaced the OMCs. Candidates are the proteins of the DMSO reductase.This is the only other main protein complex which is also bound to the outermembrane of S. oneidensis. The results of an in vitro DMSO reductasemeasurement point in the same direction: The suppressor mutant showed anelevated DMSO reduction rate when the cells were pregrown on ferriccitrate. We hypothesize that parts of this DMSO reductase complex couldfunction as metal reductase module thereby interacting with MtrA and MtrB.This study displays the enormous respiratory versatility and geneticadaptability of S. oneidensis. It is furthermore the first evidence for an OMCindependent electron transport chain to ferric iron which will most probablyhave implications in basic and applied sciences.[1] Bücking, C. et al (2010): FEMS Microbiol Lett 306:144-51.AMP019Involvement of the Shewanella oneidensis decahemecytochrome MtrA in periplasmic stability of the β-barrelprotein MtrB.M. Schicklberger* 1 , C. Buecking 1 , B. Schütz 1 ,H. Heide 2 , J. Gescher 11 Faculty of Biology II, Department of Microbiology, Albert-Ludwigs-University, Freiburg i. Br., Germany2 Institute for Molecular Bioenergetics, Center of Biological Chemistry,Frankfurt, GermanyShewanella oneidensis MR-1 is a model organism for the elucidation ofmolecular mechanisms involved in dissimilatory iron reduction. The outermembrane ß-barrel protein MtrB is an integral component of the respiratorychain to ferric iron due to its formation of a membrane spanning complextogether with the periplasmic c-type cytochrome MtrA and the outermembrane c-type cytochrome MtrC [1]. We and others have found thatMtrB is not detectable in a ΔmtrA mutant [2, 3]. In this study the reason forthis MtrA dependence was investigated. An effect of mtrA expression onmtrB transcription was excluded using qPCR. Since heterologous expressionexperiments in E. coli also revealed an MtrA dependent MtrB production,we screened for periplasmic proteases in S. oneidensis MR-1 that are similarto ubiquitously distributed proteases in Gram-negative bacteria. A serineprotease(SO_3942) was detected in S. oneidensis MR-1 that is highlysimilar to E. coli DegP. Therefore, a conditional degP E. coli mutant wasconstructed and via western blot analysis, we showed that this mutant doesnot require MtrA for MtrB stability. It was possible to verify the detectedDegP sensitivity of MtrB in the absence of MtrA via the construction of aΔSO_3942 mutant in S. oneidensis. To our knowledge, this is the firstdescription of the necessity of an electron transfer protein (MtrA) for theperiplasmic stability of an outer membrane ß-barrel protein (MtrB). Sincemoduls similar to mtrA and mtrB can be found in a multitude ofproteobacteria it seems reasonable to assume that this novel mechanism ofß-barrel protein guidance through the periplasm is widely distributed aswell.[1] Ross et al (2007): Characterization of protein-protein interactions involved in iron reduction byShewanella oneidensis MR-1. AEM.[2] Hartshorne et al (2009): Characterization of an electron conduit between bacteria and theextracellular environment. PNAS.[3] Schicklberger et al: Involvement of the Shewanella oneidensis decaheme cytochrome MtrA inperiplasmic stability of the β-barrel protein MtrB. AEM accepted.AMP020Re-evaluation of the function of the F 420 dehydrogenase inelectron transport in Methanosarcina mazeiC. Welte*, U. DeppenmeierInstitute of Microbiology and Biotechnology, Friedrich-WestphalianWilhelms-University, Bonn, GermanyMethanosarcina mazei is a methanogenic archaeon that is able to grow onH 2/CO 2, methanol, methylamines, or acetate. Electrons derived from thedifferent substrates are utilized by both membrane-bound and cytoplasmicelectron transport pathways before they finally enter the core methanogenicrespiratory chain. A couple of redox-active proteins as well as smallproteinaceous and non-proteinaceous electron donors are involved inelectron transport and thus form the highly complex and branchedrespiratory chain of this methanogenic archaeon.In this study, knockout mutants of one of the core proteins in methanogenicrespiration were constructed: two genes encoding the membrane-bound F 420dehydrogenase were individually deleted (ΔfpoF and ΔfpoA-O) and thecorresponding knockout mutants analyzed. Both mutants exhibited severegrowth deficiencies with trimethylamine, but not with acetate ortrimethylamine + H 2 as substrate. Cell lysates of the fpo mutants showed astrong reduction of the F 420: heterodisulfide oxidoreductase activity althougha second enzyme involved in F 420H 2 oxidation, the soluble F 420 hydrogenase,was still present. This led to the conclusion that the predominant part ofcellular F 420H 2 oxidation in Ms. mazei is performed by F 420 dehydrogenaseand not by F 420 hydrogenase.Enzyme assays of cytoplasmic fractions of the two knockout mutantsrevealed that ferredoxin: F 420 oxidoreductase activity was essentially absentin the ΔfpoF mutant, but was present in the other mutant and the wildtype.Subsequently, the single FpoF protein was overproduced in Escherichia coliand purified for further characterization. Purified FpoF catalyzed theferredoxin: F 420 oxidoreductase reaction with high specificity (K m forreduced ferredoxin 0.5 μM) but low velocity (v max 225 mU mg -1 ) and waspresent in the Ms. mazei cytoplasm in considerable amounts. In summary,FpoF might have a dual function: first, to oxidize F 420H 2 as electron inputmodule of the membrane-bound F 420 dehydrogenase. Secondly, it mightparticipate in electron transfer from reduced ferredoxin to coenzyme F 420 inthe cytoplasm. Consequently, it might facilitate survival of the Ms. mazeiΔech mutant that lacks the membrane-bound ferredoxin-oxidizing Echhydrogenase.AMP021Biosynthesis of the [Fe]- hydrogenase cofactorM. Schick* 1 , X. Xie 2 , J. Kahnt 3 , U. Linne 2 , S. Shima 11Max Planck Institute for Biochemistry, Marburg, Germany2 Faculty of Chemistry, Philipps-University, Marburg, Germany3 Ecophysiology Group, Max Planck Institute, Marburg, GermanyHydrogenases catalyze the reversible activation of molecular hydrogen. Thethird type of hydrogenase, the [Fe]-hydrogenase, catalyzes the reversiblehydrogenation of methenyltetrahydrometanopterin (methenyl-H 4MPT + ) withH 2 to methylene-H 4MPT. This enzyme harbours a unique ironguanylylpyridinol(FeGP) cofactor in the active site, in which a low-spiniron(II) is coordinated with an acyl-carbon [C(O)-CH 2-pyridinol] and a sp 2 -hybridized nitrogen of the pyridinol ring as well as by two carbon monoxide(CO) and the sulfur of cysteine 176 of the protein (Hiromoto et al 2009). Inorder to elucidate the biosynthetic pathway of the FeGP cofactor, the acetateauxotroph Methanobrevibacter smithii and the autotrophicMethanothermobacter marburgensis were grown in the presence of differentstable isotopes. After cultivation, the FeGP cofactor was extracted andanalyzed by mass spectrometry and NMR spectroscopy. These dataindicated that six carbons are derived from C-1 of acetate, three carbons arefrom C-2 of acetate, five carbons are from C-1 of pyruvate and thus sevencarbons are derived from CO 2 (not bound to pyruvate C-1). Based on thelabeling patterns, the biosynthetic pathway of the FeGP-cofactor will bediscussed.Hiromoto T, Warkentin E, Moll J, Ermler U, Shima S. 2009. The crystal structure of an [Fe]-hydrogenase-substrate complex reveals the framework for H2 activation. Angew Chem Int Ed Engl48:6457-60AMP022In vitro reductive dearomatization of naphthoyl-Coenzyme A in a sulphate reducing enrichment cultureC. Eberlein* 1 , J. Johannes 2 , R. Meckenstock 2 , M. Boll 11 Institute of Biochemistry, University of Leipzig, Leipzig, Germany2 Helmholtz Center Munich, German Research Center for EnvironmentalHealth, Munich, GermanyPolyaromatic hydrocarbons (PAH) are harmful to the environment andhuman health; they are highly persistent due to the high resonance energy ofthe ring system and to the low bioavailability. Whereas the aerobicdegradation pathways have been studied in great detail, only little is knownabout enzymes involved in the anaerobic metabolism of PAHs. The initialactivation of naphthalene is considered to proceed either by carboxylation[2] or methylation [3]. In both cases 2-naphthoyl-CoA would be formed.Initial evidence was obtained that this key intermediate is dearomatized by areduction yielding 5,6,7,8-tetrahydronaphthoyl-CoA (THNCoA) [1], whichmay be further dearomatized in another reduction step. In this work wedemonstrate electron donor-dependent in vitro 2-naphthoyl-CoA reductasespektrum | Tagungsband 2011