Assimilatory reduction of nitrate in Rhizobium meliloti

Nitrate and nitrite reductases in the crude extract of aerobically grown Rhizobium meliloti were determined with methylviologen as electron donor at pH 7. Nitrate reductase was detected in the cells grown in the medium that did not contain nitrate, and in the presence of nitrate the specific activity increased about 2-fold. Nitrite reductase was induced by nitrate and produced ammonia from nitrite. In nitrate reducing cells, two kinds of O₂ labile nitrate reductase were found. One enzyme had optimal pH at 7 and was stabilized to O₂ by treating with DEAE-Toyopearl 650M. The other had optimal pH at 9 and was stabilized by the addition of dithiothreitol and EDTA. Nitrate reductase stabilized by DEAE-Toyopearl 650M treatment was purified 3,360-fold from crude extract. The purified enzyme showed a single protein band in polyacrylamide gel electrophoresis, and there was no absorption peak in the visible region. It had a molecular weight of 64,000 in SDS PAGE and 58,000 on Sephadex G-100 gel filtration. K_m for nitrate was 0.9 mM. It was inhibited by p-chloromercuribenzoate, cyanide, and α,α'-dipyridyl.     

Constitutive nitrate- and nitrite reductase activities of Rhizobium in relation to denitrification

Nitrate reductase (NR) activity of R. meliloti SU 47 grown in yeast extract mannitol (YM) medium not containing any nitrate was highest during early stationary phase of growth. The strain did not express nitrite reductase (NiR) activity under this condition. In this medium stationary phase cells of some strains of rhizobia expressed nitrite reductase activity and produced gas from nitrate or nitrite in Durham tubes under anaerobic condition in 72 hrs. The strain SU 47, although, did not produce any gas in 72 hrs, did so and expressed NiR activity when grown for 8 days under anaerobic condition. The data indicated a positive correlation between gas production and NiR activity.     

The expression profile of the <Emphasis Type="Italic">Tuber borchii</Emphasis> nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.     

Isolation and characterization of a methylammonium resistant mutant of Neurospora crassa

Summary A mutant of Neurospora crassa has been isolated which is resistant to methylammonium, a structural analog of ammonium. In contrast to wild type, this mutant, mea-1, has derepressed nitrate reductase and nitrite reductase activities in the presence of ammonium. However, glutamine still represses these nitrate assimilation enzymes in mea-1. The nit-2 mutant was epistatic to mea-1 since the mea-1; nit-2 double mutant has the nit-2 mutant phenotype. In addition, mea-1; nit-2 double mutants cannot utilize ammonium as a nitrogen source. We suggest therefore that nit-2 and mea-1 loci play a role in ammonia/methylamine uptake.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

Assimilatory nitrate reductase in a chlorate-resistant mutant of Escherichia coli

Nitrate reductase was investigated in extracts from cells of a chlorate-resistant mutant strain of E. coli which grew anaerobically on nitrate as the sole source of nitrogen. The nitrate reductase was of particulate nature and reduced chlorate like the nitrate reductase from the wild strain, but in contrast was inhibited only weakly by azide or cyanide. Nitrate reductase activity was found in extracts from the mutant cells grown on nitrate as the sole source of nitrogen, but not in extracts from cells grown in complex nutrient medium. Addition of ammonia also caused a decrease in activity. Accordingly, the nitrate reductase in the chlorateresistant mutant is of the assimilatory type.     

The Relationship between Gastric Juice Nitrate/Nitrite Concentrations and Gastric Mucosal Surface pH

Purpose

To investigate gastric juice nitrate/nitrite concentration according to mucosal surface pH extent (area) of gastric corpus intimately contacting the gastric juice.

Materials and Methods

We included ninety-nine patients with dyspepsia. To evaluate gastric mucosal surface pH and its extent, gastric chromosocpy was performed by spraying phenol red dye on the corpus mucosa and estimating the extent of area with color changed. Nitrate/nitrite concentrations and pH of gastric juice were measured by ELISA and pH meter, respectively. Silver staining was done to histologically confirm the presence of Helicobacter pylori.

Results

Intragastric nitrate/nitrite concentrations in patients, showing phenol red staining mucosa were higher than those of unstaining mucosa (p=0.001): the more extensive in the area of phenol red staining area of corpus, the higher gastric juice pH found (r=0.692, p<0.001). Furthermore, the intragastric nitrate/nitrite concentrations correlated positively with gastric juice pH (r=0.481, p<0.001).

Conclusion

The changes of mucosal surface pH and its extent in gastric corpus might affect either pH or nitrate/nitrite level of gastric juice.     

Effect of nitrate reduction on metabolic products and growth of Propionibacterium acidi-propionici

From a culture of P. acidi-propionici (formerly P. arabinosum) in a medium containing lactate and nitrate, a strain with high activity of nitrate reduction was isolated and used in this study. The growth rates on lactate and glucose were not significantly affected by nitrate under anaerobic and aerobic conditions. Addition of nitrate resulted in a decrease of propionate and an increase of acetate and pyruvate produced from lactate and glucose with accumulation of nitrite. In the reaction of cell suspensions in the absence of added nitrogeneous nutrient, a similar result was obtained. Accumulation of large amounts of polysaccharide observed in the presence of glucose under these conditions was not affected by the addition of nitrate. The molar growth yield on lactate but not on glucose in a defined medium was increased in the presence of nitrate. In cell suspensions glycerol, glucose, lactate, and propionate were utilized as electron donors for nitrate reduction to nitrite. Nitrate reduction was also found in cells grown in the absence of nitrate. Cytochrome b, found in the organism, was reduced by lactate and oxidized by nitrate or by fumarate in cell suspensions and in cell-free extracts.     

Cu2+ efflux and its regulation in Cu-resistant (Cur) cyanobacterium Nostoc calcicola BRÉB

Cu²⁺-tolerant/resistant (Cu^r) strain of the cyanobacterium Nostoc calcicola BRÉB ., developed in the laboratory, showed remarkable growth in ambient medium containing 40 μM Cu²⁺ (5 μM Cu²⁺ being lethal for wild type strain). It showed 22% less Cu²⁺ uptake compared to Cu^s (Cu-sensitive) cells. Cu^r cyanobacterial cells were characterized by a time-dependent Cu²⁺ efflux in contrast to no cation efflux by Cu^s cells. Cu²⁺ efflux pattern in Cu^r cells consisted of two distinct phases: First rapid efflux (10 min) followed by slower second one at least up to 1 h. Cu²⁺ efflux depended to a major extent on light generated ATP¹), as the dark exposed Cu^r cells showed 87% reduction in Cu²⁺ efflux. The lowering in Cu²⁺ efflux in presence of various metabolic inhibitors/uncouplers like carbonyl cyanide-m-chloro phenyl hydrazone (CCCP)¹), N,N'-dicyclohexycarbodiimide (DCCD), azide (NaN₃) and p-chloromercuribenzoate (pCMB) indicated the involvement of -SH group, membrane potential and ATP hydrolysis in the regulation of this process. Cu²⁺ efflux was also initiated in such a medium containing 100-fold lower Cu²⁺ concentration (0.4 μM) compared to 40 μM Cu²⁺ (saturing concentration for metal uptake) and not before. It is suggested that Cu²⁺ efflux in N. calcicola cells may be the predominant mechanism of Cu²⁺ resistance/tolerance in this organism and are regulated at different levels by different modifying factors.     

Co-ordination of the nitrate and nitrite assimilation,the glutathione and free radical metabolisms,and the pentose phosphate pathway in Penicillium chrysogenum

The specific glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD) activities increased significantly in Penicillium chrysogenum grown on glucose in the presence of either nitrate or nitrite. These changes kept the intracellular peroxide levels low and prevented the onset of any glutathione/glutathione disulphide redox imbalances. On the other hand, the specific activity of several other glutathione metabolic enzymes including glutathione peroxidase, glutathione S-transferase, γ-glutamyltranspeptidase and the glutathione producing activity was not effected by NO₃^— and NO₂^—. When P. chrysogenum mycelia were challenged with oxidative stress caused by high concen-trations of H₂O₂, tert-butyl hydroperoxide, menadione, diamide or phenoxyacetic acid the intracellular peroxide concentrations increased significantly, and the nitrate reductase and nitrite reductase activities were eliminated. When glucose was replaced with lactose the GR and G6PD activities were not influenced by the nitrogen sources but in this case the intracellular GSH concentration was twice higher than that observed with glucose.     

Aerobic inhibition of nitrate assimilation in Escherichia coli

Growth of Escherichia coli, strain K-10, in synthetic medium with nitrate as a sole source of nitrogen was not found under aerobic conditions, but found in the medium under anaerobic conditions as well as in synthetic medium with ammonium or nitrite under both aerobic and anaerobic conditions. The presence of nitrate reductase activity in cells indicates that the growth inhibition is due to the inhibition of the enzyme by oxygen and not due to its absence. Conditions of preculture, inoculation size, pH, and composition of the nitratesynthetic medium did not affect the inhibition. All the 37 strains of E. coli tested were grown in the nitrate medium only anaerobically, while 18 strains of Klebsiella-Aerobacter among 22 strains tested were grown in the nitrate medium both aerobically and anaerobically.     

Production of pollution-tolerant strains of unicellular algae through mutagenic treatment. I. Ethyl methane sulphonate

The unicellular algae Anacystis nidulans and Chlorella vulgaris were collected from effluents of the Indian Oil Refinery Barauni and the Sindri Fertilizer Factory (India), respectively, and isolated in unialgal and bacteria-free culture. The algae were treated with the mutagen ethyl methane sulphonate and such mutagenically treated algae were compared with untreated controls with respect to growth characteristics and the capacity to absorb the two important components of polluted habitats, viz., phosphate and nitrate. Treated A. nidulans grew faster and showed a consistently higher uptake of phosphate than untreated. Treated C. vulgaris cells grew more slowly than untreated cells and showed lesser phosphate uptake during early phases of growth but greater uptake during the later phases. Nitrate uptake capacity was increased in both algae following treatment.     

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster

 The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus. Received: 17 November 1995/16 January 1996     

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47

Effect of cyanate on nitrate reduction by Rhizobium meliloti SU47 was studied. In the presence of cyanate assimilatory nitrate reduction activity appeared in the culture after a long lag and a conspicuously low level of nitrite was accumulated. Dissimilatory nitrate reduction activity was low in the intact cells precultured in KCNO of KNO₃ as compared to that in the control. Dissimilatory nitrate reductase activity in the extracts of cells precultured in KCNO was also compared to be low. In the extracts, dissimilatory nitrate reductase activity assayed with increasing concentrations of KCNO confirmed that KCNO acts as an inhibitor of the enzyme.     

The regulatory protein NIT4 that mediates nitrate induction inNeurospora crassa contains a complex tripartite activation domain with a novel leucine-rich,acidic motif

Expression ofnit-3 andnit-6, the structural genes which encode nitrate reductase and nitrite reductase inNeurospora crassa, requires the global-acting NIT2 and the pathway specific NIT4 regulatory proteins. NIT4, which consists of 1090 amino-acid residues, possesses a Cys₆/Zn₂ zinc cluster DNA-binding-domain. NIT4 was dissected to localize transactivation domains by fusion of various segments of NIT4 to the DNA-binding domain of GAL4 for in vivo analysis in yeast. Three separate activation subdomains, and one negative-acting region, which function in yeast were located in the carboxyl-terminal region of NIT4. The C-terminal tail of 28 amino-acid residues was identified as a minimal activation domain and consists of a novel leucine-rich, acidic region. Most deletions which removed even small segments of the NIT4 protein were found to lead to the loss of NIT4 function in vivo inN. crassa, implying that the central region of the protein which lies between the DNA-binding and activation domains is essential for function. The yeast two-hybrid system was employed to identify regions of NIT4 responsible for dimer formation. A short isoleucine-rich segment downstream from the zinc cluster, predicted to form a coiled coil, allowed dimerization in vivo; this same isoleucine-rich region also showed dimerization in vitro when examined via chemical cross linking. The enzyme nitrate reductase has been postulated to exert autogenous regulation by directly interacting with the NIT4 protein. This possible nitrate reductase-NIT4 interaction was investigated with the yeast two-hybrid system and by direct in vitro binding assays; both assays failed to identify such a protein-protein interaction.     

Relationship between systemic nitric oxide metabolites and cyclic GMP in healthy male volunteers

Aim: Nitric oxide (NO) is an endogenous mediator of many physiological processes, many of which are mediated by cyclic guanosine 3′,5′‐monophosphate (cGMP). Much effort has been made to validate clinical markers of NO production or bioavailability. While the measurement of plasma nitrate, nitrite, and cGMP concentrations have been suggested to reflect endogenous production of NO, there is no study showing whether there is correlation between these three markers. In the present study, we investigate whether there is correlation between the plasma concentrations of nitrate, nitrite, and cGMP in a relatively homogeneous group of 141 healthy subjects. Methods: Venous blood samples were collected from healthy male subjects and plasma aliquots were then immediately removed and stored at ?70 °C until analysed in duplicate for their nitrite and nitrate content using ozone‐based chemiluminescence assays. Plasma cGMP levels were determined by using a commercial enzyme immunoassay. Results: While we found no significant correlation between plasma nitrite and nitrate concentrations (P = 0.747), or between plasma nitrate and cGMP concentrations (P = 0.221), a significant positive correlation was found between plasma cGMP and nitrite concentrations (P = 0.017, r_s = 0.270). Conclusions: The significant correlation we found between plasma nitrite and cGMP concentrations is consistent with the notion that nitrite or cGMP concentrations in plasma may be useful clinical markers of NO formation in healthy subjects.     

Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number,growth rate and enzyme activity

Summary nit-4 is a pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa, and appears to mediate nitrate induction of nitrate and nitrite reductase. The NIT4 protein consists of 1090 amino-acid residues and possesses a single GAL4-like putative DNA-binding domain plus acidic, glutaminerich, and polyglutamine regions. Several mutants with amino-acid substitutions in the putative DNA-binding domain and a nit-4 deletion mutant, which encodes a truncated NIT4 protein lacking the polyglutamine region, are functional, i.e., they are capable of transforming a nit-4 mutant strain. However, transformants obtained with most of these nit-4 mutant genes possess a markedly reduced level of nitrate reductase and grow only slowly on nitrate, emphasizing the need to examine quantitatively the affects of in vitro-manipulated genes. The possibility that some mutant genes could yield transformants only if multiple copies were integrated was examined. The presence of multiple copies of wild-type or mutant nit-4 genes did not generally lead to increased enzyme activity or growth rate, but instead frequently appeared to be detrimental to nit-4 function. A hybrid nit-4-nirA gene transforms nit-4 mutants but only allows slow growth on nitrate and has a very low level of nitrate reductase.     

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea. Received: 19 February / 8 April 1997     

Biochemical characterization of the molybdenum cofactor mutants of Neurospora crassa: in vivo and in vitro reconstitution of NADPH-nitrate reductase activity

Summary Molybdenum cofactor (MoCo) mutants of Neurospora crassa lack both NADPH-nitrate reductase and xanthine dehydrogenase activity. In vivo and in vitro studies to further characterize these mutants are now reported. The MoCo mutants nit-9A and nit-9B are capable of growing, albeit poorly, with nitrate as the sole nitrogen source, provided high levels of molybdate are present. The MoCo mutants nit-9A, nit-9B and nit-9C, but not nit-1, nit-7 or nit-8, have significant levels of NADPH-nitrate reductase when grown in nitrate medium containing 30 mM molybdate. In vitro reconstitution experiments using cell free extracts of the N. crassa MoCo mutants and E. coli HB101 as a source of wild-type MoCo were performed. MoCo from E. coli was capable of reconstituting NADPH-nitrate reductase activity to nit-1, nit-7 and nit-8. Molybdate is required for the in vitro reconstitution of NADPH-nitrate reductase activity. It was not possible to in vitro reconstitute NADPH-nitrate reductase activity in the MoCo mutants nit-9A, nit-9B or nit-9C.     

Characterisation of the nitrite reductase gene (NII1) and the nitrate-assimilation gene cluster of Stagonospora (Septoria) nodorum

By sequencing downstream of the cloned nitrate reductase gene (NIA1) in the phytopathogenic fungus Stagonospora (Septoria) nodorum, a second open reading frame was found. Further analysis revealed this to be the nitrite reductase gene (NII1). Both genes are transcribed in the same direction, and are separated by an intergenic region of 829-bp. The coding sequence of NII1 is interrupted by three small introns and corresponds to a predicted protein of 1141 amino acids in length. Consensus binding sites for regulatory proteins are present in the promoter region of NII1. There is no indication, however, from hybridisation or sequence analysis that the nitrate transporter gene is closely associated with the NIA1-NII1 cluster, as has been found for a number of fungi. Received: 23 November 1998 / 26 April 1999