Escherichia coli cytochrome c nitrite reductase NrfA.

The periplasmic cytochrome c nitrite reductase (Nrf) system of Escherichia coli utilizes nitrite as a respiratory electron acceptor by reducing it to ammonium. Nitric oxide (NO) is a proposed intermediate in this six-electron reduction and NrfA can use exogenous NO as a substrate. This chapter describes the method used to assay Nrf-catalyzed NO reduction in whole cells of E. coli and the procedures for preparing highly purified NrfA suitable for use in kinetic, spectroscopic, voltammetric, and crystallization studies.

Physiological and gene expression analysis of inhibition of Desulfovibrio vulgaris hildenborough by nitrite.

A Desulfovibrio vulgaris Hildenborough mutant lacking the nrfA gene for the catalytic subunit of periplasmic cytochrome c nitrite reductase (NrfHA) was constructed. In mid-log phase, growth of the wild type in medium containing lactate and sulfate was inhibited by 10 mM nitrite, whereas 0.6 mM nitrite inhibited the nrfA mutant. Lower concentrations (0.04 mM) inhibited the growth of both mutant and wild-type cells on plates. Macroarray hybridization indicated that nitrite upregulates the nrfHA genes and downregulates genes for sulfate reduction enzymes catalyzing steps preceding the reduction of sulfite to sulfide by dissimilatory sulfite reductase (DsrAB), for two membrane-bound electron transport complexes (qmoABC and dsrMKJOP) and for ATP synthase (atp). DsrAB is known to bind and slowly reduce nitrite. The data support a model in which nitrite inhibits DsrAB (apparent dissociation constant K(m) for nitrite = 0.03 mM), and in which NrfHA (K(m) for nitrite = 1.4 mM) limits nitrite entry by reducing it to ammonia when nitrite concentrations are at millimolar levels. The gene expression data and consideration of relative gene locations suggest that QmoABC and DsrMKJOP donate electrons to adenosine phosphosulfate reductase and DsrAB, respectively. Downregulation of atp genes, as well as the recorded cell death following addition of inhibitory nitrite concentrations, suggests that the proton gradient collapses when electrons are diverted from cytoplasmic sulfate to periplasmic nitrite reduction.

NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing.

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC ). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+ H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with beta-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron-sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone-ubiquinol couple via NapC and NapB to NapA.

A new role for an old enzyme: nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in Arabidopsis thaliana.

The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in Arabidopsis guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type Arabidopsis generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant, nia1, nia2 that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI) abi1-1 and abi2-1 mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in Arabidopsis guard cells.

Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli.

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated. Nitric-oxide reductase activity could not be detected in Nrf(-) strains. Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains. A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain. The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes. The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments.

Evolution of nitrate reductase: molecular and structural variations on a common function.

The biological transformation of nitrogen oxyanions is widespread in nature and gives rise to a robust biogeochemical cycle. The first step in nitrate reduction is carried out by the enzyme nitrate reductase (NR). Although NR always catalyzes the same chemical reaction (conversion of nitrate into nitrite), its location in the cell, structure, and function are organism-dependent. We use protein sequence data to determine phylogenetic relationships and to examine similarities in structure and function. Three distinct clades of NR are apparent: the eukaryotic assimilatory NR (Euk-NR) clade, the membrane-associated prokaryotic NR (Nar) clade, and a clade that includes both the periplasmic NR (Nap) and prokaryotic assimilatory NR (Nas). The high degree of sequence similarity and a phylogenetic distribution that follows taxonomic classification suggest a monophyletic origin for the Euk-NR early on in the evolution of eukaryotic cells. In contrast, sequence conservation, phylogenetic analysis, and physiology suggest that both Nar and Nap were acquired by horizontal gene transfer. Nap and Nas share a lesser degree of similarity, with Nap a subclade of Nas. Nap from strict anaerobic bacteria such as Desulfovibrio desulfuricans is ancestral to facultative species and may provide an evolutionary link between Nap and Nas. We observed conserved binding sites for molybdenum and pterin cofactors in all four proteins. In pathways involving Euk-NR, Nas, and Nar, for which ammonia is the end product, nitrite is reduced to ammonia by a siroheme nitrite reductase. Nap, however, is coupled to a pentaheme nitrite reductase. In denitrification, whether Nar or Nap is involved, nitrite is reduced to nitric oxide by either a cytochrome cd1 or a copper-containing nitrite reductase. This complexity underscores the importance of nitrate reduction as a key biological process.

Dependence of Mycobacterium bovis BCG on anaerobic nitrate reductase for persistence is tissue specific.

Mycobacterium bovis BCG, the only presently available vaccine against tuberculosis, was obtained from virulent M. bovis after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immune-deficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that M. bovis BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of narG, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of M. bovis BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.

Survival of bacteria during oxygen limitation.

Regulatory mechanisms that enable bacteria associated with food, drinks and the human body to adapt to changes in the availability of oxygen are reviewed. Excess oxygen induces two adaptive responses to oxidative stress. Five or more control circuits enable enteric bacteria to generate energy and grow well in anaerobic environments. Two sets of enzymes catalyse both nitrate and nitrite reduction, and dual two-component regulatory systems sense and respond to the available nitrate and nitrite in the environment. The periplasmic nitrate reductase enables bacteria to scavenge low concentrations of nitrate: similar systems are found in food-borne and other pathogens.

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli.

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.

Site-directed mutagenesis of nitrate reductase from Aspergillus nidulans. Identification of some essential and some nonessential amino acids among conserved residues.

Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.

The role of ntrA in the anaerobic metabolism of Salmonella typhimurium.

The possible involvement of NtrA in the expression of several anaerobically induced genes in Salmonella typhimurium was investigated. Unlike Escherichia coli, where hydrogenase 3 is ntrA dependent, the introduction of a mutation in ntrA had virtually no effect on the hydrogenase activity, thought to be hydrogenase 3, of S. typhimurium LT7. Fumarate reductase and alcohol dehydrogenase activities were found to be diminished in ntrA mutant strains, but this may very well be indirect since fdhF mutant strains showed the same effect. These results suggest that in S. typhimurium NtrA is highly specific for the anaerobic expression of fdhF.

Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings.

Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana.

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrate uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.

Latent nitrate reductase activity is associated with the plasma membrane of corn roots.

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

Nitrate assimilation in Neurospora crassa: enzymatic and immunoblot analysis of wild-type and nit mutant protein products in nitrate-induced and glutamine-repressed cultures.

The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are alpha 2 type homodimers. Enzyme-bound prosthetic groups mediate the electron transfer reactions which reduce inorganic nitrate to an organically utilizable form, ammonium. One, a molybdenum-containing cofactor, is required by nitrate reductase for both enzyme activity and holoenzyme assembly. Three modes of regulation are imposed on the expression of nitrate assimilation, namely: nitrogen metabolite repression, nitrate induction and autogenous regulation by nitrate reductase. In this study, nitrocellulose blots of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) resolved proteins from crude extracts of the wild type and specific nitrate-nonutilizing (nit) mutants were examined for material cross-reactive with antibodies against nitrate reductase and nitrite reductase. The polyclonal antibody preparations used were rendered monospecific by reverse affinity chromatography. Growth conditions which alter the regulatory response of the organism were selected such that new insight could be made into the complex nature of the regulation imposed on this pathway. The results indicate that although nitrate reductase and nitrite reductase are coordinately expressed under specific nutritional conditions, the enzymes are differentially responsive to the regulatory signals.