Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K _m values for nitrate and nitrite were determined to be 32 and 10 μM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (–207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry. Received: 26 March 1999 / Accepted: 16 August 1999     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane.

Escherichia coli grown anaerobically on nitrate exhibited the same transport barrier to reduction of chlorate, relative to nitrate, as that exhibited by Paracoccus denitrificans. This establishes that the nitrate binding site of nitrate reductase (EC 1.7.99.4) in E. coli must also lie on the cell side of the nitrate transporter which is associated with the plasma membrane. Because nitrate reductase is membrane bound, the nitrate binding site is thus located on the inner aspect of the membrane. Nitrate pulse studies on E. coli in the absence of valinomycin showed a small transient alkalinization (leads to H+/NO3- congruent to --0.07) which did not occur with oxygen pulses. By analogy with P. denitrificans, the alkaline transient is interpreted to arise from proton-linked nitrate uptake which is closely followed by nitrite efflux. The result is consistent with internal reduction of nitrate, whereas external reduction would be expected to give leads to H+/NO3-ratios approaching --2.     

The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification

Both membrane-bound and periplasmic nitrate reductases have been found in denitrifying bacteria. Yet the role of periplasmic nitrate reductase in denitrification has not been clearly defined. To analyze the function of the periplasmic nitrate reductase in Pseudomonas sp. strain G-179, the nap gene cluster was identified and found to be linked to genes involved in reduction of nitrite and nitric oxide and anaerobic heme biosynthesis. Mutation in the nap region rendered the cells incapable of growing under anaerobic conditions with nitrate as the alternative electron acceptor. No nitrate reduction activity was detected in the Nap- mutant, but that activity could be restored by complementation with the nap region. Unlike the membrane-bound nitrate reductase, the nitrate reduction activity in strain G-179 was not inhibited by a low concentration of azide. Nor could it use NADH as the electron donor to reduce nitrate or use chlorate as the alternative substrate. These results suggest that the periplasmic nitrate reductase in this strain plays a primary role in dissimilatory nitrate reduction.     

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.     

Proton translocation and the respiratory nitrate reductase of Escherichia coli.

Stoicheometries and rates of proton translocation associated with respiratory reduction of NO3- have been measured for spheroplasts of Escherichia coli grown anaerobically in the presence of NO3-. Observed stoicheiometries [leads to H+/NO3- ratio; P. Mitchell (1966) Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin] were approx. 4 for L-malate oxidation and approx. 2 for succinate, D-lactate and glycerol oxidation. Measurements of the leads to H+/2e- ratio with formate as the reductant and oxygen or NO3- as the oxidant were complicated by pH changes associated with formate uptake and CO2 formation. Nevertheless, it was possible to conclude that the site of formate oxidation is on the inner aspect of the cytoplasmic membrane, that the leads to H+/O ratio for formate oxidation is approx. 4, and that the leads to H+/NO3- ratio is greater than 2. Measurements of the rate of NO3- penetration into osmotically sensitive spheroplasts demonstrated an electrogenic entry of NO3- anion. The permeability coefficient for nitrate entry at 30 degrees C was between 10(-9) and 10(-10) cm- s(-1). The calculated rate of nitrate entry at the concentration typically used for the assay of nitrate reductase (EC 1.7.99.4) activity was about 0.1% of that required to support the observed rate of nitrate reduction by reduced Benzyl Viologen. Measurements of the distribution of nitrate between the intracellular and extracellular spaces of a haem-less mutant, de-repressed for nitrate reductase but unable to reduce nitrate by the respiratory chain, showed that, irrespective of the presence or the absence of added glucose, nitrate was not concentrated intracellularly. Osmotic-swelling experiments showed that the rate of diffusion of azid anion across the cytoplasmic membrane is relatively low in comparison with the fast diffusion of hydrazoic acid. The inhibitory effect of azide on nitrate reductase was not altered by treatments that modify pH gradients across the cytoplasmic membrane. It is concluded that the nitrate-reducing azide-sensitive site of nitrate reductase is located on the outer aspect of the cytoplasmic membrane. The consequences of this location for mechanisms of proton translocation driven by nitrate reduction are discussed, and lead to the proposal that the nitrate reductase of the cytoplasmic membrane is vectorial, reducing nitrate on the outer aspect of the membrane with 2H+ and 2e- that have crossed from the inner aspect of the membrane.     

Interaction of Pseudomonas cytochrome cd1 with the cytoplasmic membrane.

Labelling with ferritin-conjugated antibody shows that Pseudomonas cytochrome cd1 is associated with the inner surface of the cytoplasmic membrane. Cytochrome cd1 is however, enriched to the soluble fraction obtained after destruction of Pseudomonas spheroplasts. Comparison of the respiratory nitrite reductase activities, due to this cytochrome, between different cellular fractions and the purified enzyme shows that while the kinetic pattern and the temperature dependence of the activity remain almost the same the molecular activity is enhanced when the enzyme is released from cells. A new assay of respiratory nitrite reductase was developed in this study. The method is based on determination of the stoichiometrical proton consumption accompanying nitrite reduction.     

The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli

Low concentrations (1-50mum) of ubiquinol(1) were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol(1) drove an outward translocation of protons with a corrected -->H(+)/2e(-) stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309-323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol(1) was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA(-)menA(-)), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol(1). Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ(+)) to the oxidized species (DQ(++)) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV(+)) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV(++). In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol(1) are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate-->H(+)/2e(-) stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol(1) (QH(2)), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed-->H(+)/2e(-) stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]     

The identification of a periplasmic nitrate reductase in Paracoccus denitrificans

Abstract A nitrate reductase activity has been identified in periplasmic extracts of Paracoccus denitrificans . The enzyme is relatively insensitive to azide and does not reduce chlorate, features which distinguish it from the well-characterised membrane-associated nitrate reductase. The specific activity of the enzyme was higher in intact cells grown with butyrate rather than succinate as the sole source of carbon.     

Nitrate Reductase and Chlorate Toxicity in Chlorella vulgaris Beijerinck

A study of the growth-inhibiting effect of chlorate on the Berlin strain of Chlorella vulgaris Beijerinck provided complete confirmation of the theory of chlorate toxicity first proposed by Åberg in 1947. Chlorate was toxic to the cells growing on nitrate, and relatively nontoxic to the cells growing on ammonium. The latter cells contained only 0.01 as much NADH-nitrate reductase as the nitrate-grown cells. Chlorate could substitute for nitrate as a substrate of the purified nitrate reductase with Km = 1.2 mm, and V_max = 0.9V_max for nitrate. Bromate, and to a much smaller extent, iodate, also served as alternate substrates. Nitrate is a reversible competitive inhibitor of chlorate reduction, which accounts for the partial reversal, by high nitrate concentrations, of the observed inhibition of cell growth by chlorate. During the reduction of chlorate by NADH in the presence of purified nitrate reductase, there was a progressive, irreversible inhibition of the enzyme activity, presumably brought about by the reduction product, chlorite. Both the NADH-nitrate reductase activity and the associated NADH-cytochrome c reductase activity were inactivated to the same extent by added chlorite. The spectral properties of the cytochrome b₅₅₇ associated with the purified enzyme were not affected by chlorite. The inactivation of the nitrate reductase by chlorite could account for the toxicity of chlorate to cells grown on nitrate, though the destruction of other cell components by chlorite or its decomposition products cannot be excluded.     

Nitrate reductase activity of plasma membranes from cultured carrot cells

Summary Cultured carrot cells (Daucus carota L.) reduced nitrate to nitrite at a slow rate (0.4 moles/g dry wt · h) without any additions to the reaction medium. This rate was doubled or tripled in presence of 100 M NADH. Ethanol and other alcohols stimulated the basal rate 8–10-fold. Isolated carrot plasma membranes also reduced nitrate to nitrite at a rate of 80 nmoles/mg protein · h. This plasma membrane-bound nitrate reductase activity was estimated to be 1.7% of the total activity. Nitrate reduction by carrot cells was inhibited 56% by sodium tungstate, 57% by potassium cyanide, and 87% by gold chloride. It was stimulated by plasma membrane electron transport inhibitors (retinoic acid and chloroquine) and ATPase inhibitors (diethylstilbestrol). From differential effects of some stimulators or inhibitors in the presence or absence of NADH, it can be implied that the nitrate reductase activity of cultured carrot cells was due to a transmembrane enzyme exhibiting an exogenous nitrate reductase activity when NADH was added.Abbreviation DMSO dimethyl sulfoxide - SHAM salicyl hydroxamic acid     

Effect of N oxyanions on anaerobic induction of nitrate reductase in subcellular fractions of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (Lupinus)

Anaerobic induction of nitrate reductase in subcellular fractions of Bradyrhizobium sp. strain USDA 3045 showed fivefold increase of the enzyme activity in spheroplasts, considered as the source of intact-membrane-bound nitrate reductase, within a 3 h time frame after nitrate addition. Such a dynamics was confirmed at the protein level, with antibodies specific to membrane-bound nitrate reductase. Nitrate reductase activity in the periplasm was one order of magnitude lower and significant only at initial 3 h of induction, within a narrow range of nitrate added. Nitrite induced the membrane-bound nitrate reductase at least 70% as effectively as nitrate, as judged from its activity pattern and Western blot analysis. The limited ability of Bradyrhizobium sp. to dissimilate ≥5 mM nitrate is not due to direct inhibition of respiratory nitrate reductase by accumulated nitrite. Moreover, a synergistic induction of membrane-bound nitrate reductase by nitrate and nitrite was indicated due to a twofold higher protein synthesis after simultaneous addition of these N oxyanions than when they were given separately.     

Enzyme localization and orientation of the active site of dissimilatory nitrite reductase from Bacillus firmus

The location of the dissimilatory nitrite reductase and orientation of its reducing site of the Grampositive denitrifier, Bacillus firmus NIAS 237 were examined. Approximately 90% of the total dissimilatory nitrite reductase activity with ascorbate-reduced phenazine methosulfate (PMS) as the electron donor was on the protoplast membrane. Nitrite induced with intact Bacillus cells an alkalinization in the external medium, followed by acidification. The electron transfer inhibitor, 2-heptyl-4-hydroxyquinoline-N-oxide, which blocked nitrite reduction with endogenous substrates, inhibited the acidification, but not the alkalinization. Alkalinization was not affected with ascorbate-reduced PMS as the artificial electron donor. This indicated that the alkalinization is not associated with proton consumption outside the cytoplasmic membrane by the extracellular nitrite reduction. The dissimilatory nitrite reductase of B. firmus NIAS 237 was located on the cytoplasmic membrane, and its reducing site is suggested to be on the inner side of this membrane.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - PMS phenazine methosulfate - H⁺/NO _inf2 ^sup- ratio number of consumed protons in the external medium per one ion of NO _inf2 ^sup- reduced     

Aerobic and anaerobic bacterial respiration monitored by electrodes.

A technique is described by which both oxygen and nitrate (or nitrate or chlorate) levels were continuously monitored during bacterial respiration. Paracoccus (Micrococcus) denitrificans and Escherichia coli oxidizing succinate rapidly ceased to reduce nitrate when oxygen was available, and equally rapidly commenced nitrate reduction when all the oxygen had been consumed. By contrast, membrane vesicles isolated from P. denitrificans reduced oxygen and nitrate simultaneously. The respiratory nitrate reductase in intact cells of P. denitrificans appeared to be inacessible to chlorate present in the reaction medium, and it is suggested that the nitrate reductase is orientated on the plasma membrane so that nitrate gains access from the inner (cytosolic) face.     

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.     

The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

(1) The apparent K_m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K_m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K_m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K_m (NO₃⁻) is obtained. It is suggested that the very low K_m (NO₃⁻) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN⁻ > NO₃⁻, NO₂⁻, pyruvate, acetate > CI⁻ > SO₄²⁻. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO₂ and CH₃COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO⁻) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.     

Physiology and enzymology involved in denitrification by Shewanella putrefaciens

Nitrate reduction to N2O was investigated in batch cultures of Shewanella putrefaciens MR-1, MR-4, and MR-7. All three strains reduced nitrate to nitrite to N2O, and this reduction was coupled to growth, whereas ammonium accumulation was very low (0 to 1 micromol liter-1). All S. putrefaciens isolates were also capable of reducing nitrate aerobically; under anaerobic conditions, nitrite levels were three- to sixfold higher than those found under oxic conditions. Nitrate reductase activities (31 to 60 micromol of nitrite min-1 mg of protein-1) detected in intact cells of S. putrefaciens were equal to or higher than those seen in Escherichia coli LE 392. Km values for nitrate reduction ranged from 12 mM for MR-1 to 1.3 mM for MR-4 with benzyl viologen as an artifical electron donor. Nitrate and nitrite reductase activities in cell-free preparations were demonstrated in native gels by using reduced benzyl viologen. Detergent treatment of crude and membrane extracts suggested that the nitrate reductases of MR-1 and MR-4 are membrane bound. When the nitrate reductase in MR-1 was partially purified, three subunits (90, 70, and 55 kDa) were detected in denaturing gels. The nitrite reductase of MR-1 is also membrane bound and appeared as a 60-kDa band in sodium dodecyl sulfate-polyacrylamide gels after partial purification.