Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp.

Abstract The effect of the nitrogen source on the cellular activity level of assimilatory nitrate reductase in the cyanobacteria Anabaena variabilis (ATCC29413) and Synechocystis sp. (PCC6714) has been examined. In the filamentous N₂-fixing A. variabilis , nitrate behaved as a nutritional inducer of nitrate reductase, with ammonium acting (via products of its assimilation) as an antagonist with regard to nitrate. Ammonium-promoted repression of nitrate reductase was also evident in the unicellular non-nitrogen fixer Synechocystis , but in this strain nitrate was not required as an obligatory inducer.     

The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants

1. Possible mechanisms regulating the activities of three enzymes involved in nitrate assimilation, nitrate reductase, nitrite reductase and glutamate dehydrogenase, were studied in radish cotyledons. 2. Nitrate-reductase and nitrite-reductase activities are low in nitrogen-deficient cotyledons, and are induced by their substrates. 3. Glutamate dehydrogenase is present regardless of the nitrogen status, and the enzyme can be increased only slightly by long-term growth on ammonia. 4. Although nitrate is the best inducer of nitrate reductase, lower levels of induction are also obtained with nitrite and ammonia. The experiments did not distinguish between direct or indirect induction by these two molecules. 5. Nitrite reductase is induced by nitrite and only indirectly by nitrate. 6. The induction of both nitrate reductase and nitrite reductase is prevented by the inhibitors actinomycin D, puromycin and cycloheximide, indicating a requirement for the synthesis of RNA and protein. 7. The decay of nitrate reductase, determined after inhibition of protein synthesis, is slower than the synthesis of the enzyme. Nitrite reductase is much more stable than nitrate reductase. 8. The synthesis of nitrate reductase is not repressed by ammonia, but is repressed by growth on a nitrite medium. 9. There is no inhibition of nitrate reductase, nitrite reductase or glutamate dehydrogenase by the normal end products of assimilation, but cyanate is a fairly specific inhibitor of nitrate reductase.     

The Regulation of Nitrite Reductase Level in Lemna minor L.

The regulation of nitrite reductase in Lemna minor has beenstudied. The evidence indicates that in nitrate-fed plants nitrateitself is the inducer of nitrite reductase. The enzyme is subjectto end-product repression by ammonia and various amino acids.Nitrate reductase is also repressed by a similar range of compounds.Most of the repressors tested are more effective when nitraterather than nitrite is supplied as the inducer. The effectsof cyclo-heximide, D-threo-chloramphenicol and lincomycin onthe induction by nitrate and nitrite suggest that both enzymesare synthesized on cytoplasmic ribosomes. The mechanism of repressionby ammonia and amino acids is discussed.     

Nitrite metabolism in the cyanobacterium Anabaena cycadeae: Regulation of nitrite uptake and nitrite reductase by ammonia

Abstract Nitrogen regulation of nitrite uptake and nitrite reductase was studied in the cyanobacterium Anabaena cycadeae and its glutamine-auxotrophic mutant. The development of the nitrite-uptake system preceded, and was independent of, the development of nitrate reductase. The levels of both of the systems were higher in the glutamine auxotroph lacking glutamine synthetase (GS) than in the wild-type strain having normal GS activity. The nitrite-uptake system was found to be constitutive and ammonia-repressible whereas the nitrite-reductase system was ammonia-repressible and nitrite-inducible. Ammonia did not inhibit the nitrite-uptake and nitrite reductase activities in the glutamine auxotroph whereas glutamine did so, suggesting that repression of nitrite-uptake and nitrite reductase systems by ammonia requires the operation of GS and probably involves the participation of some organic nitrogen metabolites like glutamine.     

Bacterial nitrate assimilation: gene distribution and regulation

In the context of the global nitrogen cycle, the importance of inorganic nitrate for the nutrition and growth of marine and freshwater autotrophic phytoplankton has long been recognized. In contrast, the utilization of nitrate by heterotrophic bacteria has historically received less attention because the primary role of these organisms has classically been considered to be the decomposition and mineralization of dissolved and particulate organic nitrogen. In the pre-genome sequence era, it was known that some, but not all, heterotrophic bacteria were capable of growth on nitrate as a sole nitrogen source. However, examination of currently available prokaryotic genome sequences suggests that assimilatory nitrate reductase (Nas) systems are widespread phylogenetically in bacterial and archaeal heterotrophs. Until now, regulation of nitrate assimilation has been mainly studied in cyanobacteria. In contrast, in heterotrophic bacterial strains, the study of nitrate assimilation regulation has been limited to Rhodobacter capsulatus, Klebsiella oxytoca, Azotobacter vinelandii and Bacillus subtilis. In Gram-negative bacteria, the nas genes are subjected to dual control: ammonia repression by the general nitrogen regulatory (Ntr) system and specific nitrate or nitrite induction. The Ntr system is widely distributed in bacteria, whereas the nitrate/nitrite-specific control is variable depending on the organism.     

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

Summary The nitrate assimilatory pathway in Neurospora crassa is composed of two enzymes, nitrate reductase and nitrite reductase. Both are ₂type homodimers. Enzymebound 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.     

Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119, and Nostoc sp. strain 6719.

The effect of the nitrogen source on the cellular activity of ferredoxin-nitrate reductase in different cyanobacteria was examined. In the unicellular species Anacystis nidulans, nitrate reductase was repressed in the presence of ammonium but de novo enzyme synthesis took place in media containing either nitrate or not nitrogen source, indicating that nitrate was not required as an obligate inducer. Nitrate reductase in A. nidulans was freed from ammonium repression by L-methionine-D,L-sulfoximine, an irreversible inhibitor of glutamine synthetase. Ammonium-promoted repression appears therefore to be indirect; ammonium has to be metabolized through glutamine synthetase to be effective in the repression of nitrate reductase. Unlike the situation in A. nidulans, nitrate appeared to play an active role in nitrate reductase synthesis in the filamentous nitrogen-fixing strains Anabaena sp. strain 7119 and Nostoc sp. strain 6719, with ammonium acting as an antagonist with regard to nitrate.     

Reduced nicotinamide–adenine dinucleotide–nitrite reductase from Azotobacter chroococcum

1. The assimilatory nitrite reductase of the N(2)-fixing bacterium Azotobacter chroococcum was prepared in a soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties have been studied. 2. The enzyme is a FAD-dependent metalloprotein (mol.wt. about 67000), which stoicheiometrically catalyses the direct reduction of nitrite to NH(3) with NADH as the electron donor. 3. NADH-nitrite reductase can exist in two either active or inactive interconvertible forms. Inactivation in vitro can be achieved by preincubation with NADH. Nitrite can specifically protect the enzyme against this inactivation and reverse the process once it has occurred. 4. A. chroococcum nitrite reductase is an adaptive enzyme whose formation depends on the presence of either nitrate or nitrite in the nutrient solution. 5. Tungstate inhibits growth of the microorganism very efficiently, by competition with molybdate, when nitrate is the nitrogen source, but does not interfere when nitrite or NH(3) is substituted for nitrate. The addition of tungstate to the culture media results in the loss of nitrate reductase activity but does not affect nitrite reductase.     

Physiology and interaction of nitrate and nitrite reduction in Staphylococcus carnosus.

Staphylococcus carnosus reduces nitrate to ammonia in two steps. (i) Nitrate was taken up and reduced to nitrite, and nitrite was subsequently excreted. (ii) After depletion of nitrate, the accumulated nitrite was imported and reduced to ammonia, which again accumulated in the medium. The localization, energy gain, and induction of the nitrate and nitrite reductases in S. carnosus were characterized. Nitrate reductase seems to be a membrane-bound enzyme involved in respiratory energy conservation, whereas nitrite reductase seems to be a cytosolic enzyme involved in NADH reoxidation. Syntheses of both enzymes are inhibited by oxygen and induced to greater or lesser degrees by nitrate or nitrite, respectively. In whole cells, nitrite reduction is inhibited by nitrate and also by high concentrations of nitrite (> or = 10 mM). Nitrite did not influence nitrate reduction. Two possible mechanisms for the inhibition of nitrite reduction by nitrate that are not mutually exclusive are discussed. (i) Competition for NADH nitrate reductase is expected to oxidize the bulk of the NADH because of its higher specific activity. (ii) The high rate of nitrate reduction could lead to an internal accumulation of nitrite, possibly the result of a less efficient nitrite reduction or export. So far, we have no evidence for the presence of other dissimilatory or assimilatory nitrate or nitrite reductases in S. carnosus.     

nasST, two genes involved in the induction of the assimilatory nitrite—nitrate reductase operon (nasAB) of Azotobacter vinelandii

An operon including two new genes ( nasS and nasT ) has been defined, cloned and sequenced. The deduced NASS protein is homologous to NRTA from Synechococcus sp. and to NASF from Klebsiella pneumoniae , two proteins involved in nitrate uptake. The predicted NAST polypeptide is homologous to the regulator proteins of the two-component regulatory systems. NASS plays a negative regulatory role in the synthesis of the nitrate and nitrite reductase. NAST is required for the expression of the nitrite—nitrate reductase operon ( nasAB ). Expression of the nasST operon is not under the control of the NTR system and is not regulated by the nitrogen source. A Φ( nasA—lacZ ) fusion has been used to analyse expression of the nasAB operon in three different genetic backgrounds with altered nitrate reductase activity. Beta-galactosidase activity in two of them was independent of nitrate but in a mutant unable to reduce nitrate, nas-4 , it was normally induced by nitrate.     

INORGANIC NITROGEN ASSIMILATION OF DITYLUM BRIGHTWELLII,A MARINE PLANKTON DIATOM1,2

Apparent K_m values for nitrite reductase, glutamic dehydrogenase, and nitrate reductase are of the order 10^?4 molar for nitrite, ammonia, and nitrate, respectively while half-saturation constants for the corresponding uptake mechanisms approximate 10^?6 molar. Ammonium and nitrate are accumulated in the vacuolated cells of the diatom (about 10 and 40 mmoles/liter cell volume, respectively) and these intracellular pools serve as substrate for the assimilatory enzymes. Nitrite is either not accumulated or is concentrated, in a very small cellular compartment. Ammonium and nitrate in the external medium exert modifying effects on uptake and assimilatory mechanisms which can be distinguished from effects of the ions accumulated within the cells. Several of these effects are described and fitted into a general scheme of nitrogen assimilation by D. brightwellii.     

Mutants of Azotobacter vinelandii altered in the regulation of nitrate assimilation

The two enzymes involved in the assimilatory pathway of nitrate in Azotobacter vinelandii are corregulated. Nitrate reductase and nitrite reductase are inducible by nitrate and nitrite. Ammonium represses induction by nitrate of both reductases. Repression by ammonium is higher in media containing 2-oxo-glutarate as carbon source than in media containing sucrose. Mutants in the gene ntrC lost nitrate and nitrite reductase simultaneously. Ten chlorate-resistant mutants with a new phenotype were isolated. In media without ammonium they had a normal phenotype, being sensitive to the toxic effect of chlorate. In media containing low ammonium concentrations they were resistant to chlorate. These mutants seem to be affected in the repression of nitrate and nitrite reductases by ammonium.     

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.     

Identification and Characterization of a Nitrate Transporter Gene in Neurospora crassa

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.     

Nitrogen Metabolism of Lemna minor. II. Enzymes of Nitrate Assimilation and Some Aspects of Their Regulation

In L. minor grown in sterile culture, the primary enzymes of nitrate assimilation, nitrate reductase (NR), nitrite reductase (NiR) and glutamate dehydrogenase (GDH) change in response to nitrogen source. NR and NiR levels are low when grown on amino acids (hydrolyzed casein) or ammonia; both enzymes are rapidly induced on addition of nitrate, while addition of nitrite induces NiR only. Ammonia represses the nitrate induced synthesis of both NR and NiR.NADH dependent GDH activity is low when grown on amino acids and high when grown on nitrate or ammonia, but the activities of NADPH dependent GDH and Alanine dehydro-genase (AIDH) are much less affected by nitrogen source. NADH-GDH and AIDH are induced by ammonia, and it is suggested that these enzymes are involved in primary nitrogen assimilation.     

Utilization of nitrate byBeggiatoa alba

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

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.     

A haloarchaeal ferredoxin electron donor that plays an essential role in nitrate assimilation

In the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcanii DS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependent assimilatory NO??/NO?? reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential role in the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmic NarB- and NirA-type reductases respectively. UV-visible absorbance and EPR spectroscopic characterization of purified Hv-Fd demonstrate that this protein binds a single [2Fe-2S] cluster, and potentiometric titration reveals that the cluster shares similar redox properties with those present in plant-type ferredoxins.     

Three nitrate reductase activities in Alcaligenes eutrophus

Three nitrate reductase activities were detected in Alcaligenes eutrophus strain H16 by physiological and mutant analysis. The first (NAS) was subject to repression by ammonia and not affected by oxygen indicating a nitrate assimilatory function. The second (NAR) membrane-bound activity was only formed in the absence of oxygen and was insensitive to ammonia repression indicating a nitrate respiratory function. The third (NAP) activity of potential respiratory function occurred in the soluble fraction of cells grown to the stationary phase of growth. In contrast to NAR and NAS, expression of NAP did not require nitrate for induction and was independent of the rpoN gene product. Genes for the three reductases map at different loci. NAR and NAS are chromosomally encoded whereas NAP is a megaplasmid-borne activity in A. eutrophus.