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Adam P Arkin - One of the best experts on this subject based on the ideXlab platform.
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the genetic basis of energy conservation in the sulfate reducing bacterium desulfovibrio alaskensis g20
Frontiers in Microbiology, 2014Co-Authors: Morgan N Price, Kelly M Wetmore, Jennifer V Kuehl, Stefan Bauer, Adam M Deutschbauer, Adam P ArkinAbstract:Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the Ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde_1207:13) is sometimes important alongside Rnf and may perform an electron bifurcation to generate more Reduced Ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde_1250:1) is important and may generate Reduced Ferredoxin to allow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce Ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions, although we cannot rule out genetic redundancy.
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the genetic basis of energy conservation in the sulfate reducing bacterium desulfovibrio alaskensis g20
bioRxiv, 2014Co-Authors: Morgan N Price, Kelly M Wetmore, Jennifer V Kuehl, Stefan Bauer, Adam M Deutschbauer, Adam P ArkinAbstract:Sulfate-reducing bacteria play major roles in the global carbon and sulfur cycles, but it remains unclear how reducing sulfate yields energy. To determine the genetic basis of energy conservation, we measured the fitness of thousands of pooled mutants of Desulfovibrio alaskensis G20 during growth in 12 different combinations of electron donors and acceptors. We show that ion pumping by the Ferredoxin:NADH oxidoreductase Rnf is required whenever substrate-level phosphorylation is not possible. The uncharacterized complex Hdr/flox-1 (Dde 1207:13) is sometimes important alongside Rnf and may perform a n electron bifurcation to generate more Reduced Ferredoxin from NADH to allow further ion pumping. Similarly, during the oxidation of malate or fumarate, the electron-bifurcating transhydrogenase NfnAB-2 (Dde 1250:1) is important and may generate Reduced Ferredoxin to a llow additional ion pumping by Rnf. During formate oxidation, the periplasmic [NiFeSe] hydrogenase HysAB is required, which suggests that hydrogen forms in the periplasm, diffuses to the cytoplasm, and is used to reduce Ferredoxin, thus providing a substrate for Rnf. During hydrogen utilization, the transmembrane electron transport complex Tmc is important and may move electrons from the periplasm into the cytoplasmic sulfite reduction pathway. Finally, mutants of many other putative electron carriers have no clear phenotype, which suggests that they are not important under our growth conditions.
Volker Muller - One of the best experts on this subject based on the ideXlab platform.
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The Rnf complex from the acetogenic bacterium Acetobacterium woodii: Purification and characterization of RnfC and RnfB.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2020Co-Authors: Martin Kuhns, Verena Schuchmann, Silke Schmidt, Thorsten Friedrich, Anja Wiechmann, Volker MullerAbstract:Abstract rnf genes are widespread in anaerobic bacteria and hypothesized to encode a respiratory enzyme that couples exergonic reduction of NAD with Reduced Ferredoxin as a reductant to vectorial ion (Na+, H+) translocation across the cytoplasmic membrane. However, despite its importance for the physiology of these bacteria, little is known about the subunit composition and the function of subunits. Here, we have purified the entire Rnf complex from the acetogen Acetobacterium woodii or after its production in Escherichia coli. These studies revealed covalently bound flavin in RnfB and RnfD. Unfortunately, the complex did not catalyze electron transfer from Reduced Ferredoxin to NAD. We, therefore, concentrated on the two cytosolic subunits RnfC and RnfB. RnfC was produced in E. coli, purified and shown to have 8.3 mol iron and 8.6 mol sulfur per mol of the subunit, consistent with the presence of two [4Fe-4S] centers, which were verified by EPR analysis. Flavins could not be detected, but RnfC catalyzed NADH-dependent FMN reduction. These data confirm RnfC as NADH-binding subunit and FMN as an intermediate in the electron transport chain. RnfB could only be produced as a fusion to the maltose-binding protein. It contained 25 mol iron and 26 mol sulfur, consistent with the predicted six [4Fe 4S] centers. The FeS centers in RnfB were Reduced with Reduced Ferredoxin as reductant. These data are consistent with RnfB as the Ferredoxin-binding subunit of the complex.
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the rnf complex is an energy coupled transhydrogenase essential to reversibly link cellular nadh and Ferredoxin pools in the acetogen acetobacterium woodii
Journal of Bacteriology, 2018Co-Authors: Lars Westphal, Anja Wiechmann, Jonathan Baker, Nigel P Minton, Volker MullerAbstract:ABSTRACT The Rnf complex is a respiratory enzyme that catalyzes the oxidation of Reduced Ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and Ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of Ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of Reduced Ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces Ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of Ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates. IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of Ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of Ferredoxin and NAD+. When Ferredoxin is greater than NADH, exergonic electron flow from Ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than Ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide Reduced Ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.
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The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii.
Journal of Bacteriology, 2018Co-Authors: Lars Westphal, Anja Wiechmann, Jonathan Baker, Nigel P Minton, Volker MullerAbstract:The Rnf complex is a respiratory enzyme that catalyzes the oxidation of Reduced Ferredoxin to the reduction of NAD+, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H2 plus CO2, nor did it produce acetate or ATP from H2 plus CO2, and Ferredoxin:NAD+ oxidoreductase activity and Na+ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of Ferredoxin but only of NAD+, and we speculated that the growth phenotype is caused by a loss of Reduced Ferredoxin, indispensable for biosynthesis and CO2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces Ferredoxin, and indeed, the addition of H2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of Ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD+ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of Ferredoxin is lower than that of the NADH/NAD+ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of Ferredoxin and NAD+ When Ferredoxin is greater than NADH, exergonic electron flow from Ferredoxin to NAD+ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than Ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide Reduced Ferredoxin, indispensable for biosynthesis and CO2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.
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the Ferredoxin nad oxidoreductase rnf from the acetogen acetobacterium woodii requires na and is reversibly coupled to the membrane potential
Journal of Biological Chemistry, 2013Co-Authors: Verena Hess, Kai Schuchmann, Volker MullerAbstract:Abstract The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na+-translocating electron transport chain that couples electron transfer from Reduced Ferredoxin to NAD+ with the generation of a primary electrochemical Na+ potential across its cytoplasmic membrane. In previous assays in which Ti3+ was used to reduce Ferredoxin, Na+ transport was observed, but not a Na+ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for Ferredoxin in which Ferredoxin is Reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-Reduced Ferredoxin, NAD+ reduction was highly specific and strictly dependent on Ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na+ dependence of this electron transfer reaction. The Km was 0.2 mm. Na+ could be partly substituted by Li+. Na+ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na+ as a coupling ion. Electron transport from Reduced Ferredoxin to NAD+ was coupled to electrogenic Na+ transport, indicating the generation of ΔμNa+. Vice versa, endergonic Ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔμNa+, and was accompanied by Na+ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes Ferredoxin reduction at the expense of an electrochemical Na+ gradient. The physiological significance of this finding is discussed.
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The Ferredoxin:NAD+ oxidoreductase (Rnf) from the acetogen Acetobacterium woodii requires Na+ and is reversibly coupled to the membrane potential.
Journal of Biological Chemistry, 2013Co-Authors: Verena Hess, Kai Schuchmann, Volker MullerAbstract:Abstract The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na+-translocating electron transport chain that couples electron transfer from Reduced Ferredoxin to NAD+ with the generation of a primary electrochemical Na+ potential across its cytoplasmic membrane. In previous assays in which Ti3+ was used to reduce Ferredoxin, Na+ transport was observed, but not a Na+ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for Ferredoxin in which Ferredoxin is Reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-Reduced Ferredoxin, NAD+ reduction was highly specific and strictly dependent on Ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na+ dependence of this electron transfer reaction. The Km was 0.2 mm. Na+ could be partly substituted by Li+. Na+ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na+ as a coupling ion. Electron transport from Reduced Ferredoxin to NAD+ was coupled to electrogenic Na+ transport, indicating the generation of ΔμNa+. Vice versa, endergonic Ferredoxin reduction with NADH as reductant was possible, but only in the presence of ΔμNa+, and was accompanied by Na+ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes Ferredoxin reduction at the expense of an electrochemical Na+ gradient. The physiological significance of this finding is discussed.
Francisco J. Florencio - One of the best experts on this subject based on the ideXlab platform.
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A loop unique to Ferredoxin-dependent glutamate synthases is not absolutely essential for Ferredoxin-dependent catalytic activity
Photosynthesis Research, 2015Co-Authors: Jatindra N. Tripathy, Masakazu Hirasawa, Anurag P. Srivastava, R. Bryan Sutton, Afia Dasgupta, Nanditha Vaidyanathan, Masoud Zabet-moghaddam, Francisco J. Florencio, David B. KnaffAbstract:It had been proposed that a loop, typically containing 26 or 27 amino acids, which is only present in monomeric, Ferredoxin-dependent, “plant-type” glutamate synthases and is absent from the catalytic α-subunits of both NADPH-dependent, heterodimeric glutamate synthases found in non-photosynthetic bacteria and NADH-dependent heterodimeric cyanobacterial glutamate synthases, plays a key role in productive binding of Ferredoxin to the plant-type enzymes. Site-directed mutagenesis has been used to delete the entire 27 amino acid-long loop in the Ferredoxin-dependent glutamate synthase from the cyanobacterium Synechocystis sp. PCC 6803. The specific activity of the resulting loopless variant of this glutamate synthase, when Reduced Ferredoxin serves as the electron donor, is actually higher than that of the wild-type enzyme, suggesting that this loop is not absolutely essential for efficient electron transfer from Reduced Ferredoxin to the enzyme. These results are consistent with the results of an in-silico study that suggests that the loop is unlikely to interact directly with Ferredoxin in the energetically most favorable model of a 1:1 complex of Ferredoxin with the wild-type enzyme.
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Effect of Glucose Utilization on Nitrite Excretion by the Unicellular Cyanobacterium Synechocystis sp. Strain PCC 6803.
Applied and Environmental Microbiology, 1993Co-Authors: José C. Reyes, Sebastián Chávez, M. I. Muro-pastor, Pedro Candau, Francisco J. FlorencioAbstract:Up to 1 mM nitrite was excreted by Synechocystis strain 6803 cells growing under mixotrophic or photoheterotrophic conditions. This excretion is not due to a lower ratio of nitrite and nitrate reductase activities in the presence of glucose but seems to be related to a shortage of Reduced Ferredoxin, their electron donor, as a result of a decrease in noncyclic photosynthetic flow observed under these circumstances. Because about 60% of the Reduced nitrate is excreted, the potential utilization of cyanobacteria for removal of nitrate from contaminated waters containing high concentrations of organic compounds is questioned.
Michael W W Adams - One of the best experts on this subject based on the ideXlab platform.
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engineering redox balanced ethanol production in the cellulolytic and extremely thermophilic bacterium caldicellulosiruptor bescii
Metabolic Engineering Communications, 2018Co-Authors: Amanda M Williamsrhaesa, Gabriel M Rubinstein, Israel M Scott, Gina L Lipscomb, Farris L Poole, Robert M Kelly, Michael W W AdamsAbstract:Abstract Caldicellulosiruptor bescii is an extremely thermophilic cellulolytic bacterium with great potential for consolidated bioprocessing of renewable plant biomass. Since it does not natively produce ethanol, metabolic engineering is required to create strains with this capability. Previous efforts involved the heterologous expression of the gene encoding a bifunctional alcohol dehydrogenase, AdhE, which uses NADH as the electron donor to reduce acetyl-CoA to ethanol. Acetyl-CoA produced from sugar oxidation also generates Reduced Ferredoxin but there is no known pathway for the transfer of electrons from Reduced Ferredoxin to NAD in C. bescii. Herein, we engineered a strain of C. bescii using a more stable genetic background than previously reported and heterologously-expressed adhE from Clostridium thermocellum (which grows optimally (Topt) at 60 °C) with and without co-expression of the membrane-bound Rnf complex from Thermoanaerobacter sp. X514 (Topt 60 °C). Rnf is an energy-conserving, Reduced Ferredoxin NAD oxidoreductase encoded by six genes (rnfCDGEAB). It was produced in a catalytically active form in C. bescii that utilized the largest DNA construct to be expressed in this organism. The new genetic lineage containing AdhE resulted in increased ethanol production compared to previous reports. Ethanol production was further enhanced by the presence of Rnf, which also resulted in decreased production of pyruvate, acetoin and an uncharacterized compound as unwanted side-products. Using crystalline cellulose as the growth substrate for the Rnf-containing strain, 75 mM (3.5 g/L) ethanol was produced at 60 °C, which is 5-fold higher than that reported previously. This underlines the importance of redox balancing and paves the way for achieving even higher ethanol titers in C. bescii.
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Engineering the respiratory membrane-bound hydrogenase of the hyperthermophilic archaeon Pyrococcus furiosus and characterization of the catalytically active cytoplasmic subcomplex.
Protein Engineering Design and Selection, 2014Co-Authors: Patrick M. Mcternan, Sanjeev K. Chandrayan, Brian J. Vaccaro, W. Andrew Lancaster, Michael W W AdamsAbstract:The archaeon Pyrococcus furiosus grows optimally at 100°C by converting carbohydrates to acetate, carbon dioxide and hydrogen gas (H2), obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of Reduced Ferredoxin with generation of a sodium ion gradient. MBH is classified as a Group 4 hydrogenase and is encoded by a 14-gene operon that contains hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged 4-subunit cytoplasmic subcomplex of MBH (C-MBH) was engineered and expressed in P. furiosus by differential transcription of the MBH operon. It was purified under anaerobic conditions by affinity chromatography without detergent. Purified C-MBH had a Fe : Ni ratio of 14 : 1, similar to the predicted value of 13 : 1. The O2 sensitivities of C-MBH and the 14-subunit membrane-bound version were similar (half-lives of ∼15 h in air), but C-MBH was more thermolabile (half-lives at 90°C of 8 and 25 h, respectively). C-MBH evolved H2 with the physiological electron donor, Reduced Ferredoxin, optimally at 60°C. This is the first report of the engineering and characterization of a soluble catalytically active subcomplex of a membrane-bound respiratory hydrogenase.
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the iron hydrogenase of thermotoga maritima utilizes Ferredoxin and nadh synergistically a new perspective on anaerobic hydrogen production
Journal of Bacteriology, 2009Co-Authors: Gerrit J Schut, Michael W W AdamsAbstract:The hyperthermophilic and anaerobic bacterium Thermotoga maritima ferments a wide variety of carbohydrates, producing acetate, CO2, and H2. Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and Reduced Ferredoxin are generated. The oxidation of these electron carriers must be coupled to H2 production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from T. maritima does not use either Reduced Ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H2 production. The enzyme oxidizes NADH and Ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H2. It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of Ferredoxin (midpoint potential, −453 mV) is used to drive the unfavorable oxidation of NADH (E0′ = −320 mV) to produce H2 (E0′ = −420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric Ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H2-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler Ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H2 production and of anaerobic metabolism in general.
Ed W J Van Niel - One of the best experts on this subject based on the ideXlab platform.
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hydrogenomics of the extremely thermophilic bacterium caldicellulosiruptor saccharolyticus
Applied and Environmental Microbiology, 2008Co-Authors: Harmen J G Van De Werken, Marcel R A Verhaart, Amy L Vanfossen, Karin Willquist, Derrick L Lewis, Jason D Nichols, Heleen P Goorissen, Emmanuel F Mongodin, Karen E Nelson, Ed W J Van NielAbstract:Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO2, and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and Reduced Ferredoxin. NADH and Reduced Ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium.