ATP formation associated with fumarate and nitrate reduction in growing cultures of Veillonella alcalescens

Molar growth yields, fermentation balances and enzyme activities were measured in Veillonella alcalescens grown anaerobically with different substrates in the absence or presence of fumarate or nitrate. The molar growth yields on malate (14.3 g dry wt bacteria/mole substrate) and citrate (19.3) were higher than that on lactate (8.6). The molar growth yield on lactate was increased to 15.5 or 19.8 by the addition of fumarate or nitrate, respectively, to the growth medium, and the molar growth yield on citrate was increased to 25.3 by addition of nitrate. Active growth yield was 25.5. From fermentation balances and fermentation systems similar Y_ATP values (g dry wt bacteria/mole ATP) were calculated for all substrates or mixtures of substrates assuming that one mole of ATP is generated at the electron transport from pyruvate, NADH and NADPH to nitrate or fumarate whereas ATP is not produced in the electron transport from lactate to fumarate or nitrate, and, therefore, this assumption was considered to reflect the actual situation. The mean Y_ATP value at a doubling time of 1 h was 16.5 g dry wt bacteria/mole ATP for growth without an added hydrogen acceptor, 14.4 for growth with fumarate, and 14.2 for growth with nitrate.     

Influence of nar (nitrate reductase) genes on nitrate inhibition of formate-hydrogen lyase and fumarate reductase gene expression in Escherichia coli K-12.

In Escherichia coli, aerobiosis inhibits the synthesis of enzymes for anaerobic respiration (e.g., nitrate reductase and fumarate reductase) and for fermentation (e.g., formate-hydrogen lyase). Anaerobically, nitrate induces nitrate reductase synthesis and inhibits the formation of both fumarate reductase and formate-hydrogen lyase. Previous work has shown that narL+ is required for the effects of nitrate on synthesis of both nitrate reductase and fumarate reductase. Another gene, narK (whose function is unknown), has no observable effect on formation of these enzymes. We report here our studies on the role of nar genes in fumarate reductase and formate-hydrogen lyase gene expression. We observed that insertions in narX (also of unknown function) significantly relieved nitrate inhibition of fumarate reductase gene expression. This phenotype was distinct from that of narL insertions, which abolished this nitrate effect under certain growth conditions. In contrast, insertion mutations in narK and narGHJI (the structural genes for the nitrate reductase enzyme complex) significantly relieved nitrate inhibition of formate-hydrogen lyase gene expression. Insertions in narL had a lesser effect, and insertions in narX had no effect. We conclude that nitrate affects formate-hydrogen lyase synthesis by a pathway distinct from that for nitrate reductase and fumarate reductase.     

Comparative growth analysis of the facultative anaerobes Bacillus subtilis,Bacillus licheniformis,and Escherichia coli

Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli. In glycerol defined medium, B. subtilis grew with nitrate, but not nitrite or fumarate, while B. licheniformis grew with nitrate or fumarate, but not nitrite. Growth of E. coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate. In order to grow by fermentation, B. subtilis required both glucose and pyruvate, while B. licheniformis and E. coli were capable of using either glucose or pyruvate.     

Anaerobic growth of Escherichia coli on formate by reduction of nitrate, fumarate, and trimethylamine N-oxide.

Anaerobic growth of E. coli, strain K-10, depending on formate oxidation by nitrate, fumarate, and trimethylamine N-oxide was followed in a medium containing peptone. The presence of formate and peptone was indispensable for growth with fumarate and trimethylamine N-oxide reduction. While there was no growth in the absence of acceptor, growth was observed in the absence of formate by nitrate reduction though not as much as under aerobic conditions. Per mole consumed formate equimolar succinate or trimethylamine was formed, but 1.2 mole of nitrate was produced, probably depending partly on peptone oxidation. The molar growth yield on formate was found to be 6.5, 7.6, and 7.0 g cells/mole depending on the reduction of nitrate, fumarate, and trimethylamine N-oxide, respectively, suggesting the formation of one mole ATP coupled to the anaerobic electron transfers from formate.     

Metabolic characterization of lactic acid bacterium Lactococcus garvieae sk11, capable of reducing ferric iron, nitrate, and fumarate

A lactic acid bacterium capable of anaerobic respiration was isolated from soil with ferric iron-containing glucose basal medium and identified as L. garvieae by using 16S rDNA sequence homology. The isolate reduced ferric iron, nitrate, and fumarate to ferrous iron, nitrite, and succinate, respectively, under anaerobic N2 atmosphere. Growth of the isolate was increased about 30-39% in glucose basal medium containing nitrate and fumarate, but not in the medium containing ferric iron. Specifically, metabolic reduction of nitrate and fumarate is thought to be controlled by the specific genes fnr, encoding FNR-like protein, and nir, regulating fumarate-nitrate reductase. Reduction activity of ferric iron by the isolate was estimated physiologically, enzymologically, and electrochemically. The results obtained led us to propose that the isolate metabolized nitrate and fumarate as an electron acceptor and has specific enzymes capable of reducing ferric iron in coupling with anaerobic respiration.     

Respiration-linked proton translocation coupled to anaerobic reduction of manganese(IV) and iron(III) in Shewanella putrefaciens MR-1.

An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked proton translocation in the manganese- and iron-reducing bacterium Shewanella putrefaciens MR-1. Cells grown anaerobically with fumarate or nitrate as the electron acceptor translocated protons in response to manganese (IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response to iron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocated protons only in response to oxygen. Proton translocation with all electron acceptors was abolished in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 microM) and was partially to completely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 microM).     

Enzymatic catalysis on conducting graphite particles

A new concept for enzyme-catalyzed redox transformations features pairs of electron donor and acceptor enzymes attached to conducting particles. Electrons furnished by oxidation at one enzyme are used at the other. Graphite microparticles modified with hydrogenase and nitrate reductase or fumarate reductase catalyze reductions of nitrate or fumarate by H2.     

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.     

Transport of C(4)-dicarboxylates in Wolinella succinogenes

C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.     

Anaerobic L- -glycerophosphate dehydrogenase of Escherichia coli: its genetic locus and its physiological role

In mutant cells of Escherichia coli missing the particulate l-alpha-glycerophosphate (l-alpha-GP) dehydrogenase necessary for aerobic growth on glycerol or l-alphaGP, a soluble, flavine-dependent l-alpha-GP dehydrogenase supports normal anaerobic growth rates on either of the two substrates with fumarate or nitrate as exogenous hydrogen acceptor. In an experiment in which glycerol served as the carbon source and nitrate as the acceptor, the growth of such a mutant was arrested upon the admission of air, whereas the growth of wild-type cells continued smoothly. Mutant cells lacking the soluble l-alpha-GP dehydrogenase, but possessing the particulate enzyme, can grow at normal rates aerobically on glycerol and l-alpha-GP or anaerobically on these compounds with nitrate, but not fumarate, as the hydrogen acceptor. Double mutants lacking both of the dehydrogenases fail to show significant growth on either glycerol or l-alpha-GP under any condition. Mutations affecting the anaerobic dehydrogenase (glpA locus) are situated at about minute 43 of the Taylor map, just clockwise beyond glpT, and show cotransduction with purF (1.5%), glpT (91%), and nalA (50%). The anaerobic dehydrogenase is a member of the glp regulon as judged by its inducibility by l-alpha-GP and by its constitutive formation in strains of glpR(c) genotype. The level of the anaerobic dehydrogenase is about the same in cells grown either aerobically or anaerobically with nitrate serving as a terminal hydrogen acceptor. With fumarate as terminal acceptor, the level is elevated several fold.     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Regulation of anaerobic respiratory pathways in Wolinella succinogenes by the presence of electron acceptors

In Wolinella succinogenes ATP synthesis and consequently bacterial growth can be driven by the reduction of either nitrate (E₀=+0.42 V), nitrite (E₀=+0.36 V), fumarate (E₀=+0.03 V) or sulphur (E₀=-0.27 V) with formate as the electron donor. Bacteria growing in the presence of nitrate and fumarate were found to reduce both acceptors simultaneously, while the reduction of both nitrate and fumarate is blocked during growth with sulphur. These observations were paralleled by the presence and absence of the corresponding bacterial reductase activities. Using a specific antiserum, fumarate reductase was shown to be present in bacteria grown with fumarate and nitrate, and to be nearly absent from bacteria grown in the presence of sulphur. The contents of polysulphide reductase, too, corresponded to the enzyme activities found in the bacteria. This suggests that the activities of anaerobic respiration are regulated at the biosynthetic level in W. succinogenes. Thus nitrate and fumarate reduction are repressed by the most electronegative acceptor of anacrobic respiration, sulphur. By contrast, in Escherichia coli a similar effect is exerted by the most electropositive acceptor, O₂. W. succinogenes also differs from E. coli in that fumarate reductase is not repressed by nitrate.Abbreviations BV benzyl viologen - DMN 2,3-dimethyl-1,4-naphthoquinone - DMSO dimethylsulfoxide - TMAO trimethylamine-N-oxide