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Jörg Simon - One of the best experts on this subject based on the ideXlab platform.
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significance of mccr mccc mccd mccl and 8 methylmenaquinone in sulfite respiration of Wolinella succinogenes
Biochimica et Biophysica Acta, 2019Co-Authors: Jakob Eller, Sascha Hein, Jörg SimonAbstract:Reduction of sulfite to sulfide is an essential step in the biogeochemical sulfur cycle. The Epsilonproteobacterium Wolinella succinogenes uses the copper-containing octahaem cytochrome c sulfite reductase MccA to respire sulfite. MccA is encoded by the first gene of the mcc gene cluster, whose transcription is apparently induced by the two-component regulatory system MccRS. It has been proposed that the iron‑sulfur protein MccC, the putative quinol dehydrogenase MccD, the copper chaperone MccL as well as menaquinone-6 (MK) and/or 8-methylmenaquinone-6 (8-MMK) are involved in the electron transport chain of W. succinogenes sulfite respiration. Here, non-polar W. succinogenes mutants were constructed that lacked MccC, MccD, MccL or the 8-MMK-producing MK methyltransferase MqnK. Each mutant possessed a frameshift-corrected mccR gene, thus inducing mcc expression in the presence of a mixture of fumarate and sulfite as terminal electron acceptors. Under these conditions, growth by sulfite respiration of cells lacking MccA, MccC or MccD was found to be abolished. However, cells lacking MccL or 8-MMK still coupled formate oxidation to sulfite reduction and grew by sulfite respiration to some extent. The results indicate that MccR, MccC, MccD, MccL and 8-MMK are essential or significant components of W. succinogenes sulfite respiration.
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clade ii nitrous oxide respiration of Wolinella succinogenes depends on the nosg c1 c2 h electron transport module nosb and a rieske cytochrome bc complex
Environmental Microbiology, 2017Co-Authors: Sascha Hein, Samantha Witt, Jörg SimonAbstract:Microbial reduction of nitrous oxide (N2 O) is an environmentally significant process in the biogeochemical nitrogen cycle. However, it has been recognized only recently that the gene encoding N2 O reductase (nosZ) is organized in varying genetic contexts, thereby defining clade I (or "typical") and clade II (or "atypical") N2 O reductases and nos gene clusters. This study addresses the enzymology of the clade II Nos system from Wolinella succinogenes, a nitrate-ammonifying and N2 O-respiring Epsilonproteobacterium that contains a cytochrome c N2 O reductase (cNosZ). The characterization of single non-polar nos gene deletion mutants demonstrated that the NosG, -C1, -C2, -H and -B proteins were essential for N2 O respiration. Moreover, cells of a W. succinogenes mutant lacking a putative menaquinol-oxidizing Rieske/cytochrome bc complex (QcrABC) were found to be incapable of N2 O (and also nitrate) respiration. Proton motive menaquinol oxidation by N2 O is suggested, supported by the finding that the molar yield for W. succinogenes cells grown by N2 O respiration using formate as electron donor exceeded that fumarate respiration by about 30 %. The results demand revision of the electron transport chain model of clade II N2 O respiration and challenge the assumption that NosGH(NapGH)-type iron-sulfur proteins are menaquinol-reactive. This article is protected by copyright. All rights reserved.
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tsdc a unique lipoprotein from Wolinella succinogenes that enhances tetrathionate reductase activity of tsda
Fems Microbiology Letters, 2017Co-Authors: Julia M Kurth, Jörg Simon, Anja Schuster, Waldemar Seel, Stefanie Herresthal, Christiane DahlAbstract:The diheme cytochromes c of the widespread TsdA family are bifunctional thiosulfate dehydrogenase/tetrathionate reductases. Here, biochemical information was collected about TsdA from the Epsilonproteobacterium Wolinella succinogenes ( Ws TsdA). The situation in W. succinogenes is unique since TsdA is closely associated with the unprecedented lipoprotein TsdC encoded immediately downstream of tsdA in the same direction of transcription. Ws TsdA purified from Escherichia coli catalyzed both thiosulfate oxidation and tetrathionate reduction. After co-production of TsdC and Ws TsdA in E. coli , TsdC was found to mediate membrane attachment of TsdA and to ensure its full catalytic activity. This effect was much stronger in the tetrathionate-reducing than in the thiosulfate-oxidizing direction. It is concluded that the TsdAC complex predominantly acts as a tetrathionate reductase in vivo .
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three transcription regulators of the nss family mediate the adaptive response induced by nitrate nitric oxide or nitrous oxide in Wolinella succinogenes
Environmental Microbiology, 2016Co-Authors: Melanie Kern, Jörg SimonAbstract:Summary Sensing potential nitrogen-containing respiratory substrates such as nitrate, nitrite, hydroxylamine, nitric oxide (NO) or nitrous oxide (N2O) in the environment and subsequent upregulation of corresponding catabolic enzymes is essential for many microbial cells. The molecular mechanisms of such adaptive responses are, however, highly diverse in different species. Here, induction of periplasmic nitrate reductase (Nap), cytochrome c nitrite reductase (Nrf) and cytochrome c N2O reductase (cNos) was investigated in cells of the Epsilonproteobacterium Wolinella succinogenes grown either by fumarate, nitrate or N2O respiration. Furthermore, fumarate respiration in the presence of various nitrogen compounds or NO-releasing chemicals was examined. Upregulation of each of the Nap, Nrf and cNos enzyme systems was found in response to the presence of nitrate, NO-releasers or N2O, and the cells were shown to employ three transcription regulators of the Crp-Fnr superfamily (homologues of Campylobacter jejuni NssR), designated NssA, NssB and NssC, to mediate the upregulation of Nap, Nrf and cNos. Analysis of single nss mutants revealed that NssA controls production of the Nap and Nrf systems in fumarate-grown cells, while NssB was required to induce the Nap, Nrf and cNos systems specifically in response to NO-generators. NssC was indispensable for cNos production under any tested condition. The data indicate dedicated signal transduction routes responsive to nitrate, NO and N2O and imply the presence of an N2O-sensing mechanism.
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production and consumption of nitrous oxide in nitrate ammonifying Wolinella succinogenes cells
Microbiology, 2014Co-Authors: Monique Luckmann, Melanie Kern, Daniel Mania, Asa Frostegard, Lars R Bakken, Jörg SimonAbstract:Global warming is moving more and more into the public consciousness. Besides the commonly mentioned carbon dioxide and methane, nitrous oxide (N2O) is a powerful greenhouse gas in addition to its contribution to depletion of stratospheric ozone. The increasing concern about N2O emission has focused interest on underlying microbial energy-converting processes and organisms harbouring N2O reductase (NosZ), such as denitrifiers and ammonifiers of nitrate and nitrite. Here, the epsilonproteobacterial model organism Wolinella succinogenes is investigated with regard to its capacity to produce and consume N2O during growth by anaerobic nitrate ammonification. This organism synthesizes an unconventional cytochrome c nitrous oxide reductase (cNosZ), which is encoded by the first gene of an atypical nos gene cluster. However, W. succinogenes lacks a nitric oxide (NO)-producing nitrite reductase of the NirS- or NirK-type as well as an NO reductase of the Nor-type. Using a robotized incubation system, the wild-type strain and suitable mutants of W. succinogenes that either produced or lacked cNosZ were analysed as to their production of NO, N2O and N2 in both nitrate-sufficient and nitrate-limited growth medium using formate as electron donor. It was found that cells growing in nitrate-sufficient medium produced small amounts of N2O, which derived from nitrite and, most likely, from the presence of NO. Furthermore, cells employing cNosZ were able to reduce N2O to N2. This reaction, which was fully inhibited by acetylene, was also observed after adding N2O to the culture headspace. The results indicate that W. succinogenes cells are competent in N2O and N2 production despite being correctly grouped as respiratory nitrate ammonifiers. N2O production is assumed to result from NO detoxification and nitrosative stress defence, while N2O serves as a terminal electron acceptor in anaerobic respiration. The ecological implications of these findings are discussed.
Achim Kroger - One of the best experts on this subject based on the ideXlab platform.
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solution structure of the 30 kda polysulfide sulfur transferase homodimer from Wolinella succinogenes
Biochemistry, 2004Co-Authors: Felician Dancea, Oliver Klimmek, Stefania Pfeiffermarek, Hans Wienk, Achim Kroger, Frank Löhr, Michael Nilges, Heinz RüterjansAbstract:The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel ‚-sheet enclosing a hydrophobic core, a two-stranded antiparallel ‚-sheet, and six R-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.
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multifrequency cw epr investigation of the catalytic molybdenum cofactor of polysulfide reductase from Wolinella succinogenes
Journal of Biological Inorganic Chemistry, 2003Co-Authors: Thomas F Prisner, Sevdalina Lyubenova, Yener Atabay, Fraser Macmillan, Achim Kroger, Oliver KlimmekAbstract:Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different MoV states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the MoV states, based on the experimentally observed shift of the g av value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the MoV G-tensor supports this assignment.
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psrr a member of the arac family of transcriptional regulators is required for the synthesis of Wolinella succinogenes polysulfide reductase
Archives of Microbiology, 2002Co-Authors: Stephan Braatsch, Jörg Simon, Oliver Klimmek, Roland Gross, Torsten Krafft, Achim KrogerAbstract:Wolinella succinogenes grows by polysulfide respiration with formate or hydrogen as electron donor. Polysulfide reduction is catalyzed by the membrane-bound polysulfide reductase complex encoded by the psrABC operon. An open reading frame, designated psrR, was found in close proximity upstream of the psr operon, but oriented in the opposite direction. The deduced amino acid sequence of PsrR is similar to those of transcriptional regulators of the AraC family and includes all typical features. Polysulfide reductase is not detectable in a Delta psrR deletion mutant of W. succinogenes. Mutant cells grown with fumarate as terminal electron acceptor did not catalyze polysulfide reduction with formate or hydrogen, in contrast to the wild-type strain. The phenotype of W. succinogenes wild-type cells was restored by genomic complementation of W. succinogenes Delta psrR. The results suggest that the gene product of psrR is involved in the regulation of polysulfide reductase synthesis.
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psrr a member of the arac family of transcriptional regulators is required for the synthesis of Wolinella succinogenes polysulfide reductase
Archives of Microbiology, 2002Co-Authors: Stephan Braatsch, Jörg Simon, Oliver Klimmek, Roland Gross, Torsten Krafft, Achim KrogerAbstract:Wolinella succinogenes grows by polysulfide respiration with formate or hydrogen as electron donor. Polysulfide reduction is catalyzed by the membrane-bound polysulfide reductase complex encoded by the psrABC operon. An open reading frame, designated psrR, was found in close proximity upstream of the psr operon, but oriented in the opposite direction. The deduced amino acid sequence of PsrR is similar to those of transcriptional regulators of the AraC family and includes all typical features. Polysulfide reductase is not detectable in a ΔpsrR deletion mutant of W. succinogenes. Mutant cells grown with fumarate as terminal electron acceptor did not catalyze polysulfide reduction with formate or hydrogen, in contrast to the wild-type strain. The phenotype of W. succinogenes wild-type cells was restored by genomic complementation of W. succinogenes ΔpsrR. The results suggest that the gene product of psrR is involved in the regulation of polysulfide reductase synthesis.
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reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes
FEBS Journal, 2002Co-Authors: Simone Biel, Jörg Simon, Roland Gross, Teresa Ruiz, Maarten Ruitenberg, Achim KrogerAbstract:Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.
Roy C D Lancaster - One of the best experts on this subject based on the ideXlab platform.
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an unconventional anaerobic membrane protein production system based on Wolinella succinogenes
Methods in Enzymology, 2015Co-Authors: Michael Lafontaine, Roy C D LancasterAbstract:Abstract In cases where membrane protein production attempts in more conventional Escherichia coli -based systems have failed, a solution is to resort to a system based on the nonpathogenic epsilon-proteobacterium Wolinella succinogenes . This approach has been demonstrated to be successful for structural and mechanistic analyses not only for homologous production of W . succinogenes membrane proteins but also for the heterologous production of membrane protein complexes from the human pathogens Helicobacter pylori and Campylobacter jejuni . The procedure to establish a system for the production of native and variant enzymes in W . succinogenes is presented in detail for the examples of the quinol:fumarate reductase and the SdhABE complexes of W . succinogenes . Subsequently, further projects using W . succinogenes as expression host are covered.
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design synthesis and biological testing of novel naphthoquinones as substrate based inhibitors of the quinol fumarate reductase from Wolinella succinogenes
Journal of Medicinal Chemistry, 2013Co-Authors: Hamid R Nasiri, Roy C D Lancaster, Gregor M Madej, Robin Panisch, Jan W. Bats, Michael Lafontaine, Harald SchwalbeAbstract:Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure–activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b–k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a–c) and 2-methoxy-1,4-naphthoquinone (5a–b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in lo...
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production characterization and determination of the real catalytic properties of the putative succinate dehydrogenase from Wolinella succinogenes
Molecular Microbiology, 2009Co-Authors: Hanno D Juhnke, Harald Schwalbe, Heiko Hiltscher, Hamid R Nasiri, Roy C D LancasterAbstract:Both the genomes of the epsilonproteobacteria Wolinella succinogenes and Campylobacter jejuni contain operons (sdhABE) that encode for so far uncharacterized enzyme complexes annotated as ‘non-classical’ succinate:quinone reductases (SQRs). However, the role of such an enzyme ostensibly involved in aerobic respiration in an anaerobic organism such as W. succinogenes has hitherto been unknown. We have established the first genetic system for the manipulation and production of a member of the non-classical succinate:quinone oxidoreductase family. Biochemical characterization of the W. succinogenes enzyme reveals that the putative SQR is in fact a novel methylmenaquinol:fumarate reductase (MFR) with no detectable succinate oxidation activity, clearly indicative of its involvement in anaerobic metabolism. We demonstrate that the hydrophilic subunits of the MFR complex are, in contrast to all other previously characterized members of the superfamily, exported into the periplasm via the twin-arginine translocation (tat)-pathway. Furthermore we show that a single amino acid exchange (Ala86→His) in the flavoprotein of that enzyme complex is the only additional requirement for the covalent binding of the otherwise non-covalently bound FAD. Our results provide an explanation for the previously published puzzling observation that the C. jejuni sdhABE operon is upregulated in an oxygen-limited environment as compared with microaerophilic laboratory conditions.
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heterologous production in Wolinella succinogenes and characterization of the quinol fumarate reductase enzymes from helicobacter pylori and campylobacter jejuni
Biochemical Journal, 2006Co-Authors: Mauro Mileni, Jörg Simon, Fraser Macmillan, Christos Tziatzios, Klaus Zwicker, Alexander H Haas, Werner Mantele, Roy C D LancasterAbstract:The ϵ-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic ϵ-proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation–reduction (‘redox’) midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.
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heterologous production in Wolinella succinogenes and characterization of the quinol fumarate reductase enzymes from helicobacter pylori and campylobacter jejuni
Biochemical Journal, 2006Co-Authors: Mauro Mileni, Jörg Simon, Fraser Macmillan, Christos Tziatzios, Klaus Zwicker, Alexander H Haas, Werner Mantele, Roy C D LancasterAbstract:The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.
Roland Gross - One of the best experts on this subject based on the ideXlab platform.
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characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes nife hydrogenase
Journal of Biological Chemistry, 2004Co-Authors: Roland Gross, Roy C D Lancaster, Rene Pisa, Monica Sanger, Jörg SimonAbstract:Abstract The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
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the hyde gene is essential for the formation of Wolinella succinogenes nife hydrogenase
Fems Microbiology Letters, 2003Co-Authors: Roland Gross, Ja Rg SimonAbstract:Wolinella succinogenes grows by anaerobic respiration using hydrogen gas as electron donor. The hydE gene is located on the genome downstream of the structural genes encoding the membrane-bound NiFe-hydrogenase complex (HydABC) and a putative protease (HydD) possibly involved in hydrogenase maturation. Homologs of hydE are found in the vicinity of NiFe-hydrogenase-encoding genes on the genomes of several other proteobacteria. A hydE deletion mutant of W. succinogenes does not catalyze hydrogen oxidation with various electron acceptors. The hydrogenase iron–sulfur subunit HydA is absent in mutant cells whereas the apparently processed NiFe subunit (HydB) is located exclusively in the soluble cell fraction. It is suggested that HydE is involved in the maturation and/or stability of HydA or the HydAB complex in some, but not all bacteria containing NiFe-hydrogenases.
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complete genome sequence and analysis of Wolinella succinogenes
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Claudia Baar, Jörg Simon, Oliver Klimmek, Roland Gross, Mark Eppinger, Andrea Rosinus, Christa Lanz, Ramkumar Nandakumar, Guenter Raddatz, Heike KellerAbstract:To understand the origin and emergence of pathogenic bacteria, knowledge of the genetic inventory from their nonpathogenic relatives is a prerequisite. Therefore, the 2.11-megabase genome sequence of Wolinella succinogenes, which is closely related to the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, was determined. Despite being considered nonpathogenic to its bovine host, W. succinogenes holds an extensive repertoire of genes homologous to known bacterial virulence factors. Many of these genes have been acquired by lateral gene transfer, because part of the virulence plasmid pVir and an N-linked glycosylation gene cluster were found to be syntenic between C. jejuni and genomic islands of W. succinogenes. In contrast to other host-adapted bacteria, W. succinogenes does harbor the highest density of bacterial sensor kinases found in any bacterial genome to date, together with an elaborate signaling circuitry of the GGDEF family of proteins. Because the analysis of the W. succinogenes genome also revealed genes related to soil- and plant-associated bacteria such as the nif genes, W. succinogenes may represent a member of the epsilon proteobacteria with a life cycle outside its host.
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electron transport to periplasmic nitrate reductase napa of Wolinella succinogenes is independent of a napc protein
Molecular Microbiology, 2003Co-Authors: Jörg Simon, Stephan C Schuster, Monica Sanger, Roland GrossAbstract:The rumen bacterium Wolinella succinogenes grows by respiratory nitrate ammonification with formate as electron donor. Whereas the enzymology and coupling mechanism of nitrite respiration is well known, nitrate reduction to nitrite has not yet been examined. We report here that intact cells and cell fractions catalyse nitrate and chlorate reduction by reduced viologen dyes with high specific activities. A gene cluster encoding components of a putative periplasmic nitrate reductase system (napA, G, H, B, F, L, D) was sequenced. The napA gene was inactivated by inserting a kanamycin resistance gene cassette. The resulting mutant did not grow by nitrate respiration and did not reduce nitrate during growth by fumarate respiration, in contrast to the wild type. An antigen was detected in wild-type cells using an antiserum raised against the periplasmic nitrate reductase (NapA) from Paracoccus pantotrophus. This antigen was absent in the W. succinogenes napA mutant. It is concluded that the periplasmic nitrate reductase NapA is the only respiratory nitrate reductase in W. succinogenes, although a second nitrate-reducing enzyme is apparently induced in the napA mutant. The nap cluster of W. succinogenes lacks a napC gene whose product is thought to function in quinol oxidation and electron transfer to NapA in other bacteria. The W. succinogenes genome encodes two members of the NapC/NirT family, NrfH and FccC. Characterization of corresponding deletion mutants indicates that neither of these two proteins is required for nitrate respiration. A mutant lacking the genes encoding respiratory nitrite reductase (nrfHA) had wild-type properties with respect to nitrate respiration. A model of the electron transport chain of nitrate respiration is proposed in which one or more of the napF, G, H and L gene products mediate electron transport from menaquinol to the periplasmic NapAB complex. Inspection of the W. succinogenes genome sequence suggests that ammonia formation from nitrate is catalysed exclusively by periplasmic respiratory enzymes.
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modification of heme c binding motifs in the small subunit nrfh of the Wolinella succinogenes cytochrome c nitrite reductase complex
FEBS Letters, 2002Co-Authors: Jörg Simon, Robert Eichler, Simone Biel, Rene Pisa, Roland GrossAbstract:The two multiheme c-type cytochromes NrfH and NrfA form a membrane-bound complex that catalyzes menaquinol oxidation by nitrite during respiratory nitrite ammonification of Wolinella succinogenes. Each cysteine residue of the four NrfH heme c binding motifs was individually replaced by serine. Of the resulting eight W. succinogenes mutants, only one is able to grow by nitrite respiration although its electron transport activity from formate to nitrite is decreased. NrfH from this mutant was shown by matrix-assisted laser desorption/ionization mass spectrometry to carry four covalently bound heme groups like wild-type NrfH indicating that the cytochrome c biogenesis system II organism W. succinogenes is able to attach heme to an SXXCH motif.
Oliver Klimmek - One of the best experts on this subject based on the ideXlab platform.
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biochemical studies of klebsiella pneumoniae nifl reduction using reconstituted partial anaerobic respiratory chains of Wolinella succinogenes
Journal of Biological Chemistry, 2007Co-Authors: Robert Thummer, Oliver Klimmek, Ruth A SchmitzAbstract:Abstract In the diazotroph Klebsiella pneumoniae the flavoprotein NifL inhibits the activity of the nif-specific transcriptional activator NifA in response to molecular oxygen and combined nitrogen. Sequestration of reduced NifL to the cytoplasmic membrane under anaerobic and nitrogen-limited conditions impairs inhibition of cytoplasmic NifA by NifL. To analyze whether NifL is reduced by electrons directly derived from the reduced menaquinone pool, we studied NifL reduction using artificial membrane systems containing purified components of the anaerobic respiratory chain of Wolinella succinogenes. In this in vitro assay using proteoliposomes containing purified formate dehydrogenase and purified menaquinone (MK6) or 8-methylmenaquinone (MMK6) from W. succinogenes, reduction of purified NifL was achieved by formate oxidation. Furthermore, the respective reduction rates, which were determined using equal amounts of NifL, have been shown to be directly dependent on the concentration of both formate dehydrogenase and menaquinones incorporated into the proteoliposomes, demonstrating a direct electron transfer from menaquinone to NifL. When purified hydrogenase and MK6 from W. succinogenes were inserted into the proteoliposomes, NifL was reduced with nearly the same rate by hydrogen oxidation. In both cases reduced NifL was found to be highly associated to the proteoliposomes, which is in accordance with our previous findings in vivo. On the bases of these experiments, we propose that the redox state of the menaquinone pool is the redox signal for nif regulation in K. pneumoniae by directly transferring electrons onto NifL under anaerobic conditions.
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solution structure of the 30 kda polysulfide sulfur transferase homodimer from Wolinella succinogenes
Biochemistry, 2004Co-Authors: Felician Dancea, Oliver Klimmek, Stefania Pfeiffermarek, Hans Wienk, Achim Kroger, Frank Löhr, Michael Nilges, Heinz RüterjansAbstract:The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel ‚-sheet enclosing a hydrophobic core, a two-stranded antiparallel ‚-sheet, and six R-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.
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complete genome sequence and analysis of Wolinella succinogenes
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Claudia Baar, Jörg Simon, Oliver Klimmek, Roland Gross, Mark Eppinger, Andrea Rosinus, Christa Lanz, Ramkumar Nandakumar, Guenter Raddatz, Heike KellerAbstract:To understand the origin and emergence of pathogenic bacteria, knowledge of the genetic inventory from their nonpathogenic relatives is a prerequisite. Therefore, the 2.11-megabase genome sequence of Wolinella succinogenes, which is closely related to the pathogenic bacteria Helicobacter pylori and Campylobacter jejuni, was determined. Despite being considered nonpathogenic to its bovine host, W. succinogenes holds an extensive repertoire of genes homologous to known bacterial virulence factors. Many of these genes have been acquired by lateral gene transfer, because part of the virulence plasmid pVir and an N-linked glycosylation gene cluster were found to be syntenic between C. jejuni and genomic islands of W. succinogenes. In contrast to other host-adapted bacteria, W. succinogenes does harbor the highest density of bacterial sensor kinases found in any bacterial genome to date, together with an elaborate signaling circuitry of the GGDEF family of proteins. Because the analysis of the W. succinogenes genome also revealed genes related to soil- and plant-associated bacteria such as the nif genes, W. succinogenes may represent a member of the epsilon proteobacteria with a life cycle outside its host.
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multifrequency cw epr investigation of the catalytic molybdenum cofactor of polysulfide reductase from Wolinella succinogenes
Journal of Biological Inorganic Chemistry, 2003Co-Authors: Thomas F Prisner, Sevdalina Lyubenova, Yener Atabay, Fraser Macmillan, Achim Kroger, Oliver KlimmekAbstract:Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different MoV states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the MoV states, based on the experimentally observed shift of the g av value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the MoV G-tensor supports this assignment.
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psrr a member of the arac family of transcriptional regulators is required for the synthesis of Wolinella succinogenes polysulfide reductase
Archives of Microbiology, 2002Co-Authors: Stephan Braatsch, Jörg Simon, Oliver Klimmek, Roland Gross, Torsten Krafft, Achim KrogerAbstract:Wolinella succinogenes grows by polysulfide respiration with formate or hydrogen as electron donor. Polysulfide reduction is catalyzed by the membrane-bound polysulfide reductase complex encoded by the psrABC operon. An open reading frame, designated psrR, was found in close proximity upstream of the psr operon, but oriented in the opposite direction. The deduced amino acid sequence of PsrR is similar to those of transcriptional regulators of the AraC family and includes all typical features. Polysulfide reductase is not detectable in a Delta psrR deletion mutant of W. succinogenes. Mutant cells grown with fumarate as terminal electron acceptor did not catalyze polysulfide reduction with formate or hydrogen, in contrast to the wild-type strain. The phenotype of W. succinogenes wild-type cells was restored by genomic complementation of W. succinogenes Delta psrR. The results suggest that the gene product of psrR is involved in the regulation of polysulfide reductase synthesis.
