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     

Members of the genus Arthrobacter grow anaerobically using nitrate ammonification and fermentative processes: anaerobic adaptation of aerobic bacteria abundant in soil

Members of the genus Arthrobacter are usually regarded as obligate aerobic bacteria. The anaerobic growth and energy metabolism of two Arthrobacter species were investigated. Arthrobacter globiformis utilized both nitrate ammonification and lactate, acetate and ethanol producing fermentation processes for anaerobic growth. Only nitrate supported anaerobic growth of Arthrobacter nicotianae. Anaerobically induced respiratory nitrate reductase activity was detected in both strains. Neither of the tested strains used the alternative electron acceptors fumarate, dimethylsulfoxide or trimethylamine-N-oxide.     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

DMSO respiration by the anaerobic rumen bacterium Wolinella succinogenes

The anaerobic rumen bacterium Wolinella succinogenes was able to grow by respiration with dimethylsulphoxide (DMSO) as electron acceptor and formate or H₂ as electron donors. The growth yield amounted to 6.7 g and 6.4 g dry cells/mol DMSO with formate or H₂ as the donors, respectively. This suggested an ATP yield of about 0.7 mol ATP/mol DMSO. Cell homogenates and the membrane fraction contained DMSO reductase activity with a high K _m (43 mM) for DMSO. The electron transport from H₂ to DMSO in the membranes was inhibited by 2-(heptyl)-4-hydroxyquinoline N-oxide, indicating the participation of menaquinone. Formation of DMSO reductase activity occurred only during growth on DMSO, presence of other electron acceptors (fumarate, nitrate, nitrite, N₂O, and sulphur) repressed the DMSO reductase activity. DMSO can therefore be used by W. succinogenes as an acceptor for phosphorylative electron transport, but other electron acceptors are used preferentially.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - DMS Dimethylsulphide (CH₃)₂S - DMSO Dimethylsulphoxide (CH₃)₂SO - HQNO 2-(Heptyl)-4-hydroxyquinoline-N-oxide - TMAO Trimethylamine-N-oxide - Y _s Growth yield for substrate S     

Utilization of nitrate byBeggiatoa alba

Beggiatoa alba B18LD utilizes both nitrate and nitrite as sole nitrogen sources, although nitrite was toxic above 1 mM.B. alba coupledin vivo acetate oxidation, but not sulfide oxidation, with nitrate and nitrite reduction.B. alba could not, however, grow anaerobically with nitrate as the sole electron acceptor. Furthermore, the incorporation of acetate into macromolecules under anaerobic conditions with nitrate as the sole electron acceptor was less 10% of the incorporation with oxygen as the electron acceptor. The product of nitrate reduction byB. alba was ammonia; N₂ or N₂O were not produced. The nitrate reductase activity inB. alba was soluble and it utilized reduced flavins or methyl viologen and dithionite as electron donors. Pyrimidine nucleotides were not used as in vitro electron donors, either alone or with flavins in coupled assays. TheB. alba nitrate reductase activity was competitively inhibited with chlorate and was only mildly inhibited by azide and cyanide. Nitrate was not required for induction of theB. alba nitrate reductase, and neither oxygen nor ammonia repressed its activity. Thus,B. alba nitrate reductase appears to be an assimilatory nitrate reductase with unusual regulatory properties.Non-standard abbreviations MV Methyl viologen - DT dithionite - GS glutamine synthetase - GOGAT glutamine 2-oxoglutarate aminotransferase - PPO 2-diphenyloxazole - POPOP 1,4-(bis)-[2-(5-phenyloxazolyl)] benzene - TCA trichloroacetic acid - CCCP carbonylcyanidem-chlorophenylhydrazone - FCCP carbonylcyanidep-trifluoromethoxyphenylhydrazone - TTFA thenoyltrifluoroacetone - PHEN 1,10-phenanthroline - HOQNO 2-heptyl 4-hydroxyquinoline-n-oxide - 8HQ 8-hydroxyquinoline     

The periplasmic nitrite reductase of Thauera selenatis may catalyze the reduction of selenite to elemental selenium

Thauera selenatis grows anaerobically with selenate, nitrate or nitrite as the terminal electron acceptor; use of selenite as an electron acceptor does not support growth. When grown with selenate, the product was selenite; very little of the selenite was further reduced to elemental selenium. When grown in the presence of both selenate and nitrate both electron acceptors were reduced concomitantly; selenite formed during selenate respiration was further reduced to elemental selenium. Mutants lacking the periplasmic nitrite reductase activity were unable to reduce either nitrite or selenite. Mutants possessing higher activity of nitrite reductase than the wild-type, reduced nitrite and selenite more rapidly than the wild-type. Apparently, the nitrite reductase (or a component of the nitrite respiratory system) is involved in catalyzing the reduction of selenite to elemental selenium while also reducing nitrite. While periplasmic cytochrome C ₅₅₁ may be a component of the nitrite respiratory system, the level of this cytochrome was essentially the same in mutant and wild-type cells grown under two different growth conditions (i.e. with either selenate or selenate plus nitrate as the terminal electron acceptors). The ability of certain other denitrifying and nitrate respiring bacteria to reduce selenite will also be described.     

An Escherichia coli mutant containing only demethylmenaquinone,but no menaquinone: effects on fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate respiration

The mutant strain AN70 (ubiE) of Escherichia coli which is known to lack ubiquinone (Young IG et al. 1971), was analyzed for menaquinone (MK) and demethylmenaquinone (DMK) contents. In contrast to the wild-type, strain AN70 contained only DMK, but no MK. The mutant strain was able to grow with fumarate, trimethylamine N-oxide (TMAO) and dimethylsulfoxide (DMSO), but not with nitrate as electron acceptor. The membranes catalyzed anaerobic respiration with fumarate and TMAO at 69 and 74% of wild-type rates. DMSO respiration was reduced to 38% of wild-type activities and nitrate respiration was missing (8% of wild-type), although the respective enzymes were present in wild-type rates. The results complement earlier findings which demonstrated a role for DMK only in TMAO respiration (Wissenbach et al. 1990). It is concluded, that DMK (in addition to MK) can serve as a redox mediator in fumarate, TMAO and to some extent in DMSO respiration, but not in nitrate respiration. In strain AN70 (ubiE) the lack of ubiquinone (Q) is due to a defect in a specific methylation step of Q biosynthesis. Synthesis of MK from DMK appears to depend on the same gene (ubiE).Abbreviations DMSO = dimethylsulfoxide - DMS = dimethylsulfide - TMAO = trimethylamine N-oxide - TMA = trimethylamine - BV = benzylviologen - BV_red = reduced benzylyiologen - Q = ubiquinone - MK = menaquinone - DMK = demethylmenaquinone - NQ = naphthoquinone     

A membrane-bound nitrate reductase encoded by the narGHJI operon is responsible for anaerobic respiration in Halomonas maura

The halophilic bacterium Halomonas maura is capable of anaerobic respiration on nitrates. By insertional mutagenesis with the minitransposon Tn-5 we obtained the mutant Tc62, which was incapable of anaerobic respiration on nitrates. An analysis of the regions adjacent to the transposon allowed us to characterize the membrane-bound anaerobic-respiratory nitrate reductase narGHJI gene cluster in H. maura. We identified consensus sequences for fumarate and nitrate reductase regulator (FNR)-like protein-binding sites in the promoter regions of the nar genes and consensus sequences corresponding to the NarL binding sites upstream of the nar genes. RT-PCR analysis showed that the narGHJI operon was expressed in response to anaerobic conditions when nitrate was available as electron acceptor. This membrane-bound nitrate reductase is the only enzyme responsible for anaerobic respiration on nitrate in H. maura. In this article we discuss the possible relationship between this enzyme and a dissimilatory nitrate-reduction-to-ammonia process (DNRA) in H. maura and its role in the colonization of the rhizosphere.     

Kinetic studies on autohydrogenotrophic growth of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> with nitrate as terminal electron acceptor

Autohydrogenotrophic batch growth of Ralstonia eutropha H16 was studied in a stirred-tank reactor with nitrate and nitrite as terminal electron acceptors and the sole limiting substrates. Assuming product inhibition by nitrite, saturation kinetics with the two limiting substrates and a simple switching function, which allows growth on nitrite only at low nitrate concentrations, resulted in a kinetic growth model with nine model parameters. The data of two batch experiments were used to identify the kinetic model. The kinetic model was validated with two additional batch experiments. The model predictions are in very good agreement with the experimental data. The maximum nitrite concentration was estimated to be 30.7 mM (total inhibition of growth). After complete reduction of nitrate, the growth rate decreases almost to zero before it increases again because of the following nitrite respiration. The maximum autohydrogenotrophic growth rate of Ralstonia eutropha with nitrate as a final electron acceptor (0.509 d⁻¹) was found to be reduced by 90–95% compared to the so far reported autohydrogenotrophic growth rates with oxygen.     

Transformations of inorganic nitrogen by Azospirillum spp.

Azospirillum spp. participate in all steps of the nitrogen cycle except nitrification. They can fix molecular nitrogen and perform assimilatory nitrate reduction and nitrate respiration. Culture conditions have been defined under which nitrate is used both as terminal respiratory electron acceptor and as nitrogen source for growth. Nitrate and, possibly to a very limited extent, nitrite, but not sulfate, iron or fumarate support anaerobic respiration. Under anaerobic conditions, nitrate can also supply energy for nitrogen fixation but without supporting growth. Nitrate-dependent nitrogenase activity lasts only for 3–4 h until the enzymes of assimilatory nitrate reduction are synthesized. Nitrite accumulates during this period and inhibits nitrogenase activity at concentrations of about 1 mM.     

Assimilatory nitrate reductase from Acinetobacter calcoaceticus

A soluble nitrate reductase from the bacterium Acinetobacter calcoaceticus grown on nitrate has been characterized. The reduction of nitrate to nitrite is mediated by an enzyme of 96000 molecular weight that can use as electron donors either viologen dyes chemically reduced with dithionite or enzymatically reduced with NAD(P)H, through specific diaphorases which utilize viologens as electron acceptors. Nitrate reductase activity is molybdenum-dependent as shown by tungstate antagonistic experiments and is sensitive to -SH reagents and metal chelators such as KCN.The enzyme synthesis is repressed by ammonia. Moreover, nitrate reductase activity undergoes a quick inactivation either by dithionite and temperature or by dithionite in the presence of small amounts of nitrate. Cyanate prevents this inactivating process and can restore the activity once the inactivation had occurred, thus suggesting that an interconversion mechanism may participate in the regulation of Acinetobacter nitrate reductase.Abbreviations EDTA ethylenediaminetetraacetate - BV benzyl viologen - MV methyl viologen - MW molecular weight - NEM N-ethylmaleimide - p-HMB p-hydroxymercuribenzoate - DCPIP 2,6-dichlorophenol-indophenol - FMN flavin mononucleotide - FAD flavin adenine dinucleotide - KCNO potassium cyanate     

Anaerobic growth of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.     

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.     

Cell yield (YM) of Thauera selenatis grown anaerobically with acetate plus selenate or nitrate

Thauera selenatis was grown anaerobically in minimal medium with either selenate or nitrate as the terminal electron acceptor and acetate as the carbon source and electron donor. The molar cell protein yields, Y_M-protein (selenate) and Y_M-protein (nitrate), were found to be 7.8 g cell protein/mol selenite formed and 7.5 g cell protein/mol nitrite formed, respectively. These values represent Y_M values of 57 and 55 g (dry weight)/mol acetate when selenate or nitrate was the electron acceptor, respectively. Based upon a calculated Y_ATP value of 10.0 g (dry weight) cells/mol ATP, for growth on acetate in inorganic salts, growth with selenate as the terminal electron acceptor theoretically yielded 5.7 ATP/acetate oxidized, and 5.5 ATP when nitrate was the terminal electron acceptor. The results support the conclusion that energy is conserved via electron transport phosphorylation when selenate or nitrate reduction are the terminal electron acceptors during anaerobic growth with acetate.     

Growth yields and energy generation by Campylobacter sputorum subspecies bubulus during growth in continuous culture with different hydrogen acceptors

Campylobacter sputorum subspecies bubulus was grown in continuous culture with excess of l-lactate or formate, and growth-limiting amounts of oxygen, fumarate, nitrate or nitrite. l-Lactate was oxidized to acetate, fumarate was reduced to succinate, and nitrate and nitrite were reduced to ammonia. The Y _lactate values (g dry weight bacteria/g mol lactate) for the respective hydrogen acceptors were much higher than the Y _formate values. Steady state cultures on formate and nitrite could only be obtained at a low dilution rate and low nitrite concentrations in the growth medium. In H⁺/2e measurements with lactate-grown cells proton ejections were observed with lactate or pyruvate as a hydrogen donor, and oxygen or hydrogen peroxide as a hydrogen acceptor. Proton ejection was also observed with pyruvate and nitrate. Proton ejection did not occur with lactate and nitrate, neither with lactate or pyruvate and fumarate or nitrite. With formate as a hydrogen donor acidification occurred with all hydrogen acceptors mentioned. It has been concluded that during growth on lactate and fumarate or nitrite substrate level phosphorylation at acetate formation is the sole ATP-generating system. Growth on formate and fumarate or nitrite is explained by a proton gradient generated as a result of oxidation of formate at the periplasmic side of the cytoplasmic membrane. With oxygen and nitrate additional ATP is formed by electron transport-linked phosphorylation. The low molar growth yields with formate are explained by the observation that formate-grown cells had a great permeability to protons.Abbreviations H⁺/2e value number of protons ejected per electron pair transported in the respiratory system - P/2e value mol of ATP formed per electron pair transported in the respiratory system - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

The expression of redox proteins of denitrification inThiosphaera pantotropha grown with oxygen,nitrate, and nitrous oxide as electron acceptors

The redox proteins and enzymes involved in denitrification inThiosphaera pantotropha exhibited a differential expression in response to oxygen. Pseudoazurin was completely repressed during batch or continuous culture under oxic conditions. Cytochromecd ₁ nitrite reductase was also heavily repressed after aerobic growth. Nitrite, nitric oxide, and nitrous oxide reductase activities were detected in intact cells under some conditions of aerobic growth, indicating that aerobic denitrification might occur in some circumstances. However, the rates of denitrification were much lower after aerobic growth than after anaerobic growth. Growth with nitrous oxide as sole electron acceptor mimicked aerobic growth in some respects, implying that expression of parts of the denitrification apparatus might be controlled by the redox state of a component of the electron transport chain rather than by oxygen itself. Nevertheless, the regulation of expression of nitrous oxide reductase was linked to the oxygen concentration.