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

The gene cluster in the genome of the anammox bacterium CandidatusKuenenia stuttgartiensis that contains the catalytic subunits of nitratereductase (narGH) covers almost the full natural repertoire of electroncarriers, apparently mediating electron flow and bifurcation associated withthe RET. This includes genes encoding six putative heme-containingproteins and two putative blue-copper proteins and a putative anchor to themembrane showing homology to a cytochrome bd oxidase subunit (cydA).In order to understand the metabolic processes involved in energyconservation in anammox bacteria, respiratory membrane-bound enzymecomplexes, including the NAR system, were separated by Blue Native -PAGE and identified by specific in-gel activity assays and LC-MS/MSanalysis. The in-gel activity assays resulted in a single band showing NARactivity, when using reduced methyl viologen as artificial electron donor.Additionally, protein correlation profiling using LC-MS/MS data fromconsecutive Blue Native gel slices enabled the identification of many moreprotein complexes involved in energy conservation and RET of anammoxbacteria.[1] Strous, M. et al (2006): Deciphering the evolution and metabolism of an anammox bacterium froma community genome. Nature 440: 790-794.[2] Jetten, M.S.M. et al (2009): Biochemistry and molecular biology of anammox bacteria. Crit RevBiochem Mol Biol 26: 1-20.[3] Wessels, J.C.T. et al (2009): LC-MS/MS as an alternative for SDS-PAGE in blue native analysisof protein complexes. Proteomics 17:4221-4228.AMP043Monoterpene degradation in Castellaniella defragrans:Mutants, enantioselectivity and a first view on thegenomeF. Lüddeke 1 , J. Petasch* 1 , S. Klages 2 , R. Reinhardt 2 , T. Schweder 3 ,J. Harder 11 Department of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, Germany2 Max Planck Institut for Molecular Genetics, Berlin, Germany3 Institute of Pharmacy, Department of Biopharmaceutical, Ernst-Moritz-Arndt-University, Greifswald, GermanyCastellaniella defragrans is a betaproteobacterium metabolizing severalmonoterpenes by oxygen or nitrate respiration. After the establishment of agenetic system we have started tocreate a number of mutants lacking genesof the myrcene degradation pathway: the unique linalool dehydrataseisomerase(LDI, (1)), the geraniol dehydrogenase (GeDH) and bothgenes.Initial physiological investigations of C. defragrans Δldi Δgedh revealed aphenotype with growth on the monocyclic phellandrene (like the wild type),but no growth on the acyclicmyrcene. These observations indicated that thecyclic monoterpenes are not degraded via myrcene and that an independentactivation reaction for the degradation of cyclicmonoterpenes exists.However, the analysis of our mutants suggested also that myrcene may be abyproduct of this unknown activation reaction. To disclose the proteinsinvolved, wehave initiated a genomic and comparative proteomic study ofthe anaerobic monoterpene degradation pathway in C. defragrans. Initialresults will be presented.The stereospecificity of the linalool dehydratase-isomerase has beeninvestigated with myrcene as educt. Product analyses by chiral GC revealedthe formation of S-(+)-linalool. R-(-)-linalool was not detected. This mayhave potential applications in the white biotechnology.AMP044Thiosulfate reduction by thiosulfate reductase PhsABC ofSalmonella enterica serovar Typhimurium is driven bythe proton potential and reversibleL. Stoffels* 1,2 , M. Krehenbrink 2 , B. Berks 2 , G. Unden 11 Institute for Microbiology and Wine Research, Johannes-Gutenberg-University, Mainz, Germany2 Department of Biochemistry, University of Oxford, Oxford, UnitedKingdomThiosulfate is a common inorganic sulfur species in the biosphere in soilsand marine environment. In the colon and cecum thiosulfate is formed fromsulfide and from methanethiol that are produced in significant amounts bycolonic bacteria. The enteric bacteria Salmonella, Proteus and Citrobacterhave the capacity to utilise thiosulfate as a respiratory electron acceptor. Themembrane-bound thiosulfate reductase PhsABC of Salmonella entericacatalyses the terminal step of thiosulfate respiration (menaquinol +thiosulfate -> menaquinone + sulfide + sulfite). Under standard conditions,this reaction is strongly endergonic (ΔE 0’ = -328 mV). Thiosulfate reductionwith hydrogen, formate or glycerol as electron donors is depended on thepresence of a proton motive force (pmf) across the membrane. In thiosulfaterespiration only the reaction catalyzed by PhsABC, and within PhsABCreaction only the menaquinol dependent reaction was sensitive to dissipationof pmf. Upon heterologous expression in Escherichia coli mutants, onlymenaquinone but not the more electro-positive demethylmenaquinoneserved as an efficient electron donor for thiosulfate reduction. Bioinformaticanalysis suggests that the transmembrane protein PhsC of PhsABC containsfour conserved His residues that are arranged in pairs typical for heme bbinding, reminiscent of reverse redox-loop enzymes. The endergonicreaction, pmf dependence and presence of two putative heme b groups intransmembrane arrangement suggests that thiosulfate reduction by PhsABCis driven by pmf in a reverse redox-loop mechanism. PhsABC also catalysedthe reverse reaction (oxidation of sulfide + sulfite to thiosulfate) whenelectron acceptors like TMAO or napthoquinone analogs were present. Incontrast to thiosulfate respiration, sulfite/sulfide oxidation was pmfindependentand also took place with demethylmenaquinone.AMP045Induction of (1-methylalkyl)succinate synthaseexpression by n-alkanes and other hydrocarbons in strainHxN1K. Webner*, F. Widdel, O. GrundmannDepartment of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, GermanyThe Betaproteobacterium strain HxN1 is able to degrade the n-alkaneshexane, heptane and octane under nitrate-reducing conditions. Due to thechemical stability of alkanes, a first activation step is necessary for thedegradation. The enzyme (1-methylalkyl)succinate synthase (Mas) activatesthe n-alkanes by addition of a secondary alkyl radical to fumarate, analogousto the activation of toluene by benzylsuccinate synthase (Bss).Based on enzymatic data from protein purification, the substrate range ofHxN1 was reinvestigated, identifying that also pentane is a growth substrate,but with a significantly lower rate. On the other site western blot analysiswas applied to examine expression of the large subunit MasD in relation tothe presence of potential inductors. These experiments clearly demonstratedthat the expression of (1-methylalkyl)succinate synthase is not the reason forthe narrow substrate range. Interestingly, some additional hydrocarbonswhich cannot be metabolized by HxN1, as well as substituted hydrocarbonsthat do not require activation by (1-methylalkyl)succinate synthase, inducedthe expression. The only obvious similarity of all these hydrocarbons is afree methyl-group, suggesting a pivotal role of this group for expression of(1-methylalkyl)succinate synthase. However, caproate (n-hexanoate), whichhas a „free” methyl-group, strongly represses the (1-methylalkyl)succinatesynthase expression. Additional specifications of the inductors and possibleinhibitors are currently under investigation.AMP046Anaerobic degradation of naphthalene and 2-methylnaphthalene by marine sulfate-reducing bacteriaG. Chen* 1 , F. Musat 1 , R. Rabus 1,2 , F. Widdel 11 Department of Microbiology, Max Planck Institute for MarineMicrobiology, Bremen, Germany2 Institute for Chemistry and Biology of the Marine Environment (ICBM),University of Oldenburg, Oldenburg, GermanyNaphthalene and 2-methylnaphthalene as typical aromatic hydrocarbons areof great concerns due to their toxicity and recalcitrance. Anaerobicdegradation of naphthalene and 2-methylnaphthalene were observed inanoxic habitats and microcosms under conditions of sulfate reduction, nitratereduction and methanogenesis. 2-Methylnaphthalene degradation occurs inanalogy to anaerobic toluene degradation by addition of fumarate to themethyl group. However, the activation mechanism of anaerobic naphthalenedegradation is still unclear. In this study, anaerobic degradation ofnaphthalene and 2-methylnaphthalene was investigated with three marinesulfate-reducing bacteria, strains NaphS2, NaphS3 and NaphS6. Thesestrains are able to utilize both naphthalene and 2-methylnaphthalene.Previous substrate tests showed that naphthalene-grown cells were notinduced to utilize 2-methylnaphthalene, indicating that these strains do notactivate naphthalene via methylation [1]. In order to examine whether 2-methylnaphthalene-grown cells were induced to utilize naphthalene,spektrum | Tagungsband 2011