Nitrate reduction associated with respiration in <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> 2011 is performed by a membrane-bound molybdoenzyme

The purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR) is reported together with the optimal conditions for cell growth and enzyme production. The best biomass yield was obtained under aerobic conditions in a fed-batch system using Luria–Bertani medium with glucose as carbon source. The highest level of Sm NR production was achieved using microaerobic conditions with the medium supplemented with both nitrate and nitrite. Sm NR is a mononuclear Mo-protein belonging to the DMSO reductase family isolated as a heterodimeric enzyme containing two subunits of 118 and 45 kDa. Protein characterization by mass spectrometry showed homology with respiratory nitrate reductases. UV–Vis spectra of as-isolated and dithionite reduced Sm NR showed characteristic absorption bands of iron-sulfur and heme centers. Kinetic studies indicate that Sm NR follows a Michaelis–Menten mechanism (K _m = 97 ± 11 μM, V = 9.4 ± 0.5 μM min⁻¹, and k _cat = 12.1 ± 0.6 s⁻¹) and is inhibited by azide, chlorate, and cyanide with mixed inhibition patterns. Physiological and kinetic studies indicate that molybdenum is essential for NR activity and that replacement of this metal for tungsten inhibits the enzyme. Although no narGHI gene cluster has been annotated in the genome of rhizobia, the biochemical characterization indicates that Sm NR is a Mo-containing NR enzyme with molecular organization similar to NarGHI.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

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 nitrite 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.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Purification and Characterization of Nitrate Reductase from the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

Summary Factors affecting the activity of nitrate reductase (E.C.1.7.7.2) from the halotolerant cyanobacterium Aphanothece halophytica were investigated. Cells grown in nitrate-containing medium exhibited higher nitrate reductase activity than cells grown in medium in which nitrate was replaced by glutamine. When ammonium was present in the medium instead of nitrate, the activity of nitrate reductase was virtually non-detectable, albeit with normal cell growth. The enzyme was localized mainly in the cytoplasm. The enzyme was purified 406-fold with a specific activity of 40.6 μmol/min/mg protein. SDS-PAGE revealed a subunit molecular mass of 58 kDa. Gel filtration experiments revealed a native molecular mass of 61 kDa. The K _m value for nitrate was 0.46 mM. Both methyl viologen and ferredoxin could serve as electron donor with K _m values of 4.3 mM and 5.2 μM, respectively. The enzyme was strongly inhibited by sulfhydryl-reactive agents and cyanide. Nitrite, the product of the enzyme reaction, showed little inhibition. Chlorate, the substrate analog, could moderately inhibit the enzyme activity. NaCl up to 200 mM stimulated the activity of the enzyme whereas enzyme inhibition was observed at ≥300 mM NaCl.     

The identification of nitrate reductase fromEscherichia coli as the antigen for a monoclonal antibody of unknown specificity

The immunoaffinity chromatography of total membrane proteins fromEscherichia coli helped determine the specificity of the monoclonal antibody 3A6 that was obtained upon immunization of mice with nicotinamide nucleotide transhydrogenase preparations and reacted with an unknownE. coli antigen. Proteins with apparent molecular masses of 150, 45, and 20 kDa were isolated and identified byN-terminal sequencing as the subunits of nitrate reductase. This conclusion was confirmed by immunoblotting with the 3A6 antibody of the proteins from theE. coli cells grown upon induction of nitrate reductase. It was shown that the 3A6 antibody specifically recognizes the α subunit of nitrate reductase, and the formation of the enzyme-antibody complex does not result in a loss of the enzyme catalytic activity.     

The 4784V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermoiability in initiation of Escherichia coli DNA replication

The prototrophic bacterium Rhodobacter sphaeroides DSM 158 has a periplasmic nitrate reductase which is induced by nitrate and it is not repressed by ammonium or oxygen. In a Tn5 mutant lacking nitrate reductase activity, transposon insertion is localized in a 1.2 kb EcoRI fragment. A 0.6 kb BamHI-EcoRI segment of this region was used as a probe to isolate, from the wild-type strain, a 6.8 kb Pstl fragment carrying the putative genes coding for the periplasmic nitrate reductase. In vivo protein expression and DNA sequence analysis reveal the presence in this region of three genes, napABC, probably organized in an operon. These genes are required for nitrate reduction, as deduced by mutational and complementation studies. The napA gene codes for a protein with a high homology to the periplasmic nitrate reductase from Alcali-genes eutrophus and, to a lesser extent, to other prokaryotic nitrate reductases and molybdenum-containing enzymes. The napB gene product has two haem c-binding sites and shows a high homology with the cytochrome c-type subunit of the periplasmic nitrate reductase from A. eutrophus. NAPA and NAPB proteins appear to be translated with signal peptides of 29 and 24 residues, respectively, indicating that mature proteins are located in the periplasm. The napC gene codes for a 25 kDa protein with a transmembrane sequence of 17 hydrophobic residues. NAPC has four haem c-binding sites and is homologous to the membrane-bound c-type cytochromes encoded by Pseudomonas stutzeri nirT and Escherichia coli torC genes. The phenotypes of defined insertion mutants constructed for each gene also indicate that periplasmic nitrate reductase from R. sphaeroides DSM 158 is a dimeric complex of a 90kDa catalytic subunit (NAPA) and a 15kDa cytochrome c (NAPB), which receives electrons from a membrane-anchored tetrahaem protein (NAPC), thus allowing electron flow between membrane and periplasm. This nitrate-reducing system differs from the assimilatory and respiratory bacterial nitrate reductases at the level of cellular localization, regulatory properties, biochemical characteristics and gene organization.     

Isolation and preliminary characterization of a respiratory nitrate reductase from hydrocarbon-degrading bacterium <Emphasis Type="Italic">Gordonia alkanivorans</Emphasis> S7

Gordonia alkanivorans S7 is an efficient degrader of fuel oil hydrocarbons that can simultaneously utilize oxygen and nitrate as electron acceptors. The respiratory nitrate reductase (Nar) from this organism has been isolated using ion exchange chromatography and gel filtration, and then preliminarily characterized. PAGE, SDS-PAGE and gel filtration chromatography revealed that Nar consisted of three subunits of 103, 53 and 25 kDa. The enzyme was optimally active at pH 7.9 and 40°C. K _m values for NO₃ ⁻ (110 μM) and for ClO₃ ⁻ (138 μM) were determined for a reduced viologen as an electron donor. The purified Nar did not use NADH as the electron donor to reduce nitrate or chlorate. Azide was a strong inhibitor of its activity. Our results imply that enzyme isolated from G. alkanivorans S7 is a respiratory membrane-bound nitrate reductase. This is the first report of purification of a nitrate reductase from Gordonia species.     

Anaerobic energy metabolism of the sulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitrate reduction to ammonia

In a batch culture experiment the microaerophilic Campylobacter-like bacterium “Spirillum” 5175 derived its energy for growth from the reduction of nitrate to nitrite and nitrite to ammonia. Hereby, formate served as electron donor, acetate as carbon source, and l-cysteine as sulfur source. Nitrite was quantitatively accumulated in the medium during the reduction of nitrate; reduction of nitrite began only after nitrate was exhausted from the medium. The molar growth yield per mol formate consumed, Y_m, was 2.4g/mol for the reduction of nitrate to nitrite and 2.0 g/mol for the conversion of nitrite to ammonia. The gain of ATP per mol of oxidized formate was 20% higher for the reduction of nitrate to nitrite, compared to the reduction of nitrite to ammonia. With succinate as carbon source and nitrite as electron acceptor, Y_m was 3.2g/mol formate, i.e. 60% higher than with acetate as carbon source. No significant amount of nitrous oxide or dinitrogen was produced during growth with nitrate or nitrite both in the presence or absence of acetylene. No growth on nitrous oxide was found. The hexaheme c nitrite reductase of “Spirillum” 5175 was an inducible enzyme. It was present in cells cultivated with nitrate or nitrite as electron acceptor. It was absent in cells grown with fumarate, but appeared in high concentration in “Spirillum” 5175 grown on elemental sulfur. Furthermore, the dissimilatory enzymes nitrate reductase and hexaheme c nitrite reductase were localized in the periplasmic part of the cytoplasmic membrane.     

Rapid and slow modulation of nitrate reductase activity in the red macroalga <Emphasis Type="Italic">Gracilaria chilensis</Emphasis> (Gracilariales,Rhodophyta): influence of different nitrogen sources

Nitrate is one of the most important stimuli in nitrate reductase (NR) induction, while ammonium is usually an inhibitor. We evaluated the influence of nitrate, ammonium or urea as nitrogen sources on NR activity of the agarophyte Gracilaria chilensis. The addition of nitrate rapidly (2 min) induced NR activity, suggesting a fast post-translational regulation. In contrast, nitrate addition to starved algae stimulated rapid nitrate uptake without a concomitant induction of NR activity. These results show that in the absence of nitrate, NR activity is negatively affected, while the nitrate uptake system is active and ready to operate as soon as nitrate is available in the external medium, indicating that nitrate uptake and assimilation are differentially regulated. The addition of ammonium or urea as nitrogen sources stimulated NR activity after 24 h, different from that observed for other algae. However, a decrease in NR activity was observed after the third day under ammonium or urea. During the dark phase, G. chilensis NR activity was low when compared to the light phase. A light pulse of 15 min during the dark phase induced NR activity 1.5-fold suggesting also fast post-translational regulation. Nitrate reductase regulation by phosphorylation and dephosphorylation, and by protein synthesis and degradation, were evaluated using inhibitors. The results obtained for G. chilensis show a post-translational regulation as a rapid response mechanism by phosphorylation and dephosphorylation, and a slower mechanism by regulation of RNA synthesis coupled to de novo NR protein synthesis.     

<Emphasis Type="Italic">Haloarcula marismortui</Emphasis> cytochrome <Emphasis Type="Italic">b-</Emphasis>561 is encoded by the <Emphasis Type="Italic">narC</Emphasis> gene in the dissimilatory nitrate reductase operon

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was −27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.     

Purification of Two Nitrate Reductases from Xanthomonas maltophilia Grown in Aerobic Cultures

Xanthomonas maltophilia ATCC 17666 is an obligate aerobe that accumulates nitrite when grown on nitrate. Spectra of membranes from nitrate-grown cells exhibited b-type cytochrome peaks and A_615-630 indicative of d-type cytochrome but no absorption peaks corresponding to c-type cytochromes. The nitrate reductase (NR) activity was located in the membrane fraction. Triton X-100-extracted reduced methyl viologen-NRs were purified on DE-52, hydroxylapatite, and Sephacryl S-300 columns to specific activities of 52 to 67 μmol of nitrite formed per min per mg of protein. The cytochrome-containing NR_I separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis into a 135-kDa α-subunit, a 64-kDa β-subunit, and a 23-kDa γ-subunit with relative band intensities indicative of a 1:1:1 α/β/γ subunit ratio and a M_r of 222,000. The electronic spectrum of dithionite-reduced purified NR displayed peaks at 425, 528, and 558 nm, indicative of the presence of a cytochrome b, an interpretation consistent with the pyridine hemochrome spectrum formed. The cytochrome b of the NR was reduced under anaerobic conditions by menadiol and oxidized by nitrate with the production of nitrite. This NR contained 0.96 Mo, 12.5 nonheme iron, and 1 heme per 222 kDa: molybdopterin was detected with the Neurospora crassa nit-1 assay. A smaller reduced methyl viologen-NR (169 kDa), present in various concentrations in the Triton X-100 preparations, lacked a cytochrome spectrum and did not oxidize menadiol. The characteristics of the NRs and the absence of c-type cytochromes provide insights into why X. maltophilia accumulates nitrite.     

The nitrate reductase promoter of birch directs differential reporter gene expression in tissues of transgenic tobacco

A 1535 bp promoter of the nitrate reductase gene (nia) from birch (Betula pendula) and a series of 5′ deletions were fused to the β-glucuronidase (GUS) gene and introduced into Nicotiana plumbaginifolia. In transgenic plants the NR promoter sequences directed strong GUS expression in the root epidermal hair cells, and in phloem cells of leaf and stem vascular tissue. The NR promoter confers also a significant stimulation of the GUS gene expression by nitrate. These findings might indicate that nitrate flow is one of the signals involved into tissue and cell specific expression of the NR promoter GUS fusions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of phytohormones and tungsten on light-induced and nitrate-induced nitrate reductase activity of mustard cotyledons

Mustard (Brassica juncea Coss cv. T-59 ‘Varuna’) seedlings pretreated with gibberellic acid (GA) and kinetin (KiN) were grown in light. In vivo nitrate reductase (NR) activity was estimated and effect of tungsten on light-induced and NO ₃ ^su− -induced NR activity was investigated. Different concentrations of GA did not show any effect on induction of light-induced NR; addition of nitrate promoted in vivo NR activity but no concentration effect of GA was evident. Light-induced NR was promoted by KiN and like in GA treatment, addition of nitrate increased NR activity. Addition of Na-tungstate inhibited NO ₃ ⁻ induced NR while light-induced NR was not much affected in both GA and KiN treated seedlings. The two forms of NRs were further characterized by studying the decay kinetics using Na-tungstate. In light-induced NR, tungstate did not affect NR activity up to 11 h, while at later periods, a slight decay was observed. On the other hand, NO ₃ ⁻ -induced NR activity increased up to 4 h and subsequently a rapid fall was observed. It was therefore apparent that light-induced NR had a very low turnover rate as compared to NO ₃ ⁻ -induced NR. These results further support the earlier conclusion that in mustard seedlings two distinct types of NR enzyme exist and that nitrate requirement for NR induction is not absolute.     

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     

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Growth of <Emphasis Type="Italic">Pseudomonas chloritidismutans</Emphasis> AW-1<Superscript>T</Superscript> on <Emphasis Type="Italic">n</Emphasis>-alkanes with chlorate as electron acceptor

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1^T grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1^T also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 ± 0.1 and 0.4 ± 0.02 day⁻¹, respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.     

Constitutive and nitrate-induced,membrane-bound nitrate reductase fromBradyrhizobium japonicum

Nitrate reductase (NR) activity was detected in membranes from cells ofBradyrhizobium japonicum cultured in defined medium either with glutamate or nitrate as the only nitrogen source. With gel filtration, the relative molecular mass (Mr) of the NR in cells growth with glutamate was estimated to be about 78 kDa. The enzyme from cells grown aerobically with nitrate had an Mr of 236 kDa, the same as that of the NR from microaerobically nitrate-grown cells. When cells that had been grown with glutamate were incubated microaerobically in both the absence and the presence of nitrate, the enzyme from each source resembled that of nitrate-grown cells in having an Mr of 236 kDa. In glutamate-grown cells that were further incubated, both microaerobiosis and nitrate were required for fully expression of the activity of the enzyme.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996