In vitro restoration of nitrate reductase: Investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10⁻³ M Na₂MoO₄ was active in the restoration assay.Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract.The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 μg molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

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.     

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase

Summary A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrate reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such exeriments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

Nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary The wild-type line and 14 nitrate reductase-deficient mutant cell lines of Nicotiana tabacum were tested for the presence of nitrate reductase partial activities, and for nitrite reductase and xanthine dehydrogenase activity. Data characterizing the electron donor specificity of nitrate reductase (EC 1.6.6.1., NADH:nitrate oxidoreductase) and nitrite reductase (EC 1.7.7.1., ferredoxin:nitrite oxidoreductase) of the wild-type line are presented. Three lines (designated cnx) simultaneously lack NADH-, FADH₂-, red. benzyl viologen-nitrate reductase, and xanthine dehydrogenase activities, but retain the nitrate reductase-associated NADH-cytochrome c reductase activity. These mutants are, therefore, interpreted to be impaired in gene functions essential for the synthesis of an active molybdenum-containing cofactor. For cnx-68 and cnx-101, the sedimentation coefficient of the defective nitrate reductase molecules does not differ from that of the wild-type enzyme (7.6S). In 11 lines (designated nia) xanthine dehydrogenase activity is unaffected, and the loss of NADH-nitrate reductase is accompanied by a loss of all partial activities, including NADH-cytochrome c reductase. However, one line (nia-95) was found to possess a partially active nitrate reductase molecule, retaining its FADH₂- and red. benzyl viologen nitrate reductase activity. It is likely that nia-95 is a mutation in the structural gene for the apoprotein. Both, the nia and cnx mutant lines exhibit nitrite reductase activity, being either nitrate-inducible or constitutive. Evidence is presented that, in Nicotiana tabacum, nitrate, without being reduced to nitrite, is an inducer of the nitrate assimilation pathway.     

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover

Summary The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.     

Formation of NADPH-nitrate reductase activity in vitro from Aspergillus nidulans niaD and cnx mutants

Summary Mutants of A. nidulans at several loci lack detectable NADPH-nitrate reductase activity. These loci include niaD, the structural gene for the nitrate reductase polypeptide, and five other loci termed cnxABC, E, F, G and H which are presumed to be involved in the formation of a molybdenum-containing component (MCC) necessary for nitrate reductase activity. When frozen mycelia from A. nidulans deletion mutant niaD26 were homogenized in a Ten Broeck homogenizer together with frozen mycelia from either enzA6, cnxE29, cnxF12, enxG4 or cnxH3 strains grown on urea+nitrate as the nitrogen source, nitrate reductase activity was detectable in the extract. Similar results were obtained by co-homogenizing niaD mycelia with Neurospora crassa nit-1 mycelia induced on nitrate. Thus, all A. nidulans cnx mutants are similar to the N. crassa nit-1 strain in their capacity to yield NADPH-nitrate reductase in the presence of the presumed MCC. As judged by the amounts of nitrate reductase formed, niaD26 mycelia grown on urea±nitrate contained much more available MCC than ammonium-grown mycelia. No NADPH-nitrate reductase activity was found in extracts prepared by co-homogenizing mycelia from all five A. nidulans cnx strains. Wild-type A. nidulans NADPH-nitrate reductase acid dissociated by adjustment to pH 2.0–2.5 and re-adjusted to pH 7 could itself re-assemble to form active nitrate reductase and thus was not a sueful source of MCC for these experiments. These results are consistent with the conclusion that the active nitrate reductase complex is composed of polypeptide components which are the niaD gene product, plus the MCC which is formed through the combined action of the cnx gene products. Further, the production of MCC may be regulated in response to the nitrogen nutrition available to the organism.     

Regulation of the nitrate-reducing system enzymes in wild-type and mutant strains of Chlamydomonas reinhardii

Summary Six mutant strains (301, 102, 203, 104, 305, and 307) affected in their nitrate assimilation capability and their corresponding parental wild-type strains (6145c and 21gr) from Chlamydomonas reinhardii have been studied on different nitrogen sources with respect to NAD(P)H-nitrate reductase and its associated activities (NAD(P)H-cytochrome c reductase and reduced benzyl viologen-nitrate reductase) and to nitrite reductase activity. The mutant strains lack NAD(P)H-nitrate reductase activity in all the nitrogen sources. Mutants 301, 102, 104, and 307 have only NAD(P)H-cytochrome c reductase activity whereas mutant 305 solely has reduced benzyl viologen-nitrate reductase activity. Both activities are repressible by ammonia but, in contrast to the nitrate reductase complex of wild-type strains, require neither nitrate nor nitrite for their induction. Moreover, the enzyme from mutant 305 is always obtained in active form whereas nitrate reductase from wild-types needs to be reactivated previously with ferricyanide to be fully detected. Wild-type strains and mutants 301, 102, 104, and 307, when properly induced, exhibit an NAD(P)H-cytochrome c reductase distinguishable electrophoretically from contitutive diaphorases as a rapidly migrating band. Nitrite reductase from wild-type and mutant strains is also repressible by ammonia and does not require nitrate or nitrite for its synthesis. These facts are explained in terms of a regulation of nitrate reductase synthesis by the enzyme itself.     

Presence of the molybdenum-cofactor in nitrate reductase-deficient mutant cell lines of Nicotiana tabacum

Summary Nicotiana tabacum mutant cell cultures lacking nitrate reductase activity were assayed for the presence of the molybdenum-cofactor using its ability to restore NADPH-nitrate reductase activity in extracts of Neurospora crassa nit-1 mycelia. The molybdenum-cofactor of the tobacco wild-type line was shown to complement efficiently the N. crassa nit-1 mutant in vitro. The molybdenum-cofactor seems to exist in a bound form, as acid-treatment was required for release of cofactor activity. Molybdate (5–10 mM), ascorbic acid, and anaerobic conditions greatly increased the activity of the cofactor, demonstrating its high lability and sensitivity to oxygen. Similar results were obtained with two tobacco nia mutants, which are defective in the apoprotein of nitrate reductase. The four cnx mutants studied were shown to contain exclusively an inactive form of the molybdenum-cofactor. This inactive cofactor could be reactivated in vitro and in vivo by unphysiologically high concentrations of molybdate (1–10 mM), thereby converting the cnx cells into highly active cofactor sources in vitro, and restoring nitrate reductase and xanthine dehydrogenase in vivo to partial acitivity. Thus the defect of the cnx mutants resides in a lack of molybdenum as a catalytically active ligand metal for the cofactor, while the structural moiety of the cofactor seems not to be impaired by the mutation. The subunit assembly of the nitrate reductase was found to be independent of the molybdenum content of the cofactor.     

Ammonium repression of the nitrite-nitrate ( nasAB ) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Received: 4 December 1996 / Accepted: 27 March 1997     

A mutation in Aspergillus nidulans which affects the regulation of nitrite reductase and is tightly linked to its structural gene.

Summary The selection of nis-5, a mutation which is tightly linked to the structural genes for nitrate reductase (niaD) and nitrite reductase (niiA) but which only affects nitrite reductase activities, is described. nis-5 single mutants have only 40% of the wild type activity of nitrite reductase after induction by nitrate and, for this reason, grow poorly on nitrate and nitrite. Nitrate reductase activity is not affected, and nis-5 is shown to complement with a niaD^- mutation but not with a niiA^- mutation.When grown without inducer, nis-5 strains have higher than the non-induced wild type activity of nitrite reductase. This low, constitutive activity is insensitive to repression by ammonium. These facts explain why the nis-5 mutation weakly suppresses many nirA^- and areA^r mutations for utilization of nitrite.Three of the possible explanations of this unusual phenotype are considered. Studies of nitrite reductase in cell-free extracts provided no evidence for the already unlikely possibility that nis-5 is a structural gene mutation resulting in the observed phenotype because of alteration in the catalytic activity and/or stability of the nitrite reductase.A more plausible explanation is that it defines a receptor site for either the nirA gene product and/or the areA gene product. However, no evidence for this has yet been obtained from a study of double mutants carrying nis-5 and areA or nirA mutations.A third possibility is that nis-5 creates a new, but inefficient promoter or initiator, which is not subject to the normal control systems (and therefore causes constitutive, deprepressed synthesis) but whose physical presence reduces maximal enzyme synthesis. The presence of a translocation in nis-5 strains suggests a means by which niiA could come to be under the control of another promoter/initiator.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

GENETIC STUDIES OF NITRATE ASSIMILATION IN ASPERGILLUS NIDULANS

(1) In Aspergillus nidulans, at least 16 genes can mutate to affect the reduction of nitrate to ammonium, a process requiring two enzymes, nitrate reductase and nitrite reductase. (2) niaD is the only gene whose effects on enzyme structure are confined to nitrate reductase alone. It specifies a core polypeptide, one or more of which form the basic subunit of nitrate reductase, molecular weight 50000. (3) At least five cnx genes together specify a molybdenum co-factor, necessary for the activity of nitrate reductase, and of xanthine dehydrogenases I and II. The cnxH gene specifies a polypeptide component of this co-factor, and the cnxE and F gene products are involved in co-factor elaboration, The role of the remaining cnx genes is at present unknown. (4) Functional nitrate reductase has a molecular weight of 200000 and is likely to consist of four subunits, together with one or more molecules of the cnx-specified co-factor. (5) The co-factor plays a catalytic role in the aggregation of nitrate-reductase subunits. (6) The niiA gene is the structural gene for nitrite reductase. (7) Other genes affecting nitrate assimilation are either regulatory or bring about their effects indirectly. (8) Of the genes affecting nitrate assimilation, close linkage is found only between the niiA and niaD genes. (9) Nitrate and nitrite reductases are subject to control by nitrate induction and ammonium repression. (10) Nitrate induction is mediated by the nirA gene whose product must be active for the niiA and niaD genes to be expressed. Since most niaD mutants produce nitrite reductase constitutively, it is likely that the nirA gene product is normally inactivated by nitrate reductase, but only when the latter is not complexed with nitrate, (11) Ammonium repression is mediated by the areA gene, whose product must be active for the expression of the niiA and niaD genes, and which is inactive in the presence of ammonium. (12) The tamA gene may function similarly to the areA gene, both gene products being necessary for the expression of the niiA and niaD genes. (13) Although the niiA and niiD genes are probably contiguous, they are not likely to be organized into a structure equivalent to a bacterial operon. (14) Whereas the areA and nirA genes regulate the synthesis of nitrate and nitrite reductases, it is probable that at least nitrate reductase is also subject to post-translational control, the presence of active enzyme being correlated with high levels of NADPH. (15) The regulation of the pentose-phosphate pathway, of mannitol-I-phosphate dehydrogenase and of certain activities required for the catabolism of some nitrogen-containing compounds appears to be connected with that of nitrate assimilation. In all cases, it is probable that the nirA gene and nitrate reductase itself are involved.     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France.

Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c₅₅₂ biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c₅₅₂ cytochrome c₅₅₂     

Inheritance and expression of NAD(P)H nitrate reductase in barley

Summary NADH-specific and NAD(P)H bispecific nitrate reductases are present in barley (Hordeum vulgare L.). Wild-type leaves have only the NADH-specific enzyme while mutants with defects in the NADH nitrate reductase structural gene (nar1) have the NAD(P)H bispecific enzyme. A mutant deficient in the NAD(P)H nitrate reductase was isolated in a line (nar1a) deficient in the NADH nitrate reductase structural gene. The double mutant (nar1a;nar7w) lacks NAD(P)H nitrate reductase activity and has xanthine dehydrogenase and nitrite reductase activities similar to nar1a. NAD(P)H nitrate reductase activity in this mutant is controlled by a single codominant gene designated nar7. The nar7 locus appears to be the NAD(P)H nitrate reductase structural gene and is not closely linked to nar1. From segregating progeny of a cross between the wild type and nar1a;nar7w, a line was obtained which has the same NADH nitrate reductase activity as the wild type in both the roots and leaves but lacks NADPH nitrate reductase activity in the roots. This line is assumed to have the genotype Nar1Nar1nar7nar7. Roots of wild type seedlings have both nitrate reductases as shown by differential inactivation of the NADH and NAD(P)H nitrate reductases by a monospecific NADH-nitrate reductase antiserum. Thus, nar7 controls the NAD(P)H nitrate reductase in roots and in leaves of barley.Scientific Paper No. 7617, College of Agriculture Research Center and Home Economics, Washington State University, Pullman, WA, USA. Project Nos. 0233 and 0745     

Isolation and characterization of two new negative regulatory mutants for nitrate assimilation inChlamydomonas reinhardtii obtained by insertional mutagenesis

Plasmid DNA carrying either the nitrate reductase (NR) gene or the argininosuccinate lyase gene as selectable markers and the correspondingChlamydomonas reinhardtii mutants as recipient strains have been used to isolate regulatory mutants for nitrate assimilation by insertional mutagenesis. Identification of putative regulatory mutants was based on their chlorate sensitivity in the presence of ammonium. Among 8975 transformants, two mutants, N1 and T1, were obtained. Genetic characterization of these mutants indicated that they carry recessive mutations at two different loci, namedNrg1 andNrg2. The mutation in N1 was shown to be linked to the plasmid insertion. Two copies of the nitrate reductase plasmid, one of them truncated, were inserted in the N1 genome in inverse orientation. In addition to the chlorate sensitivity phenotype in the presence of ammonium, these mutants expressed NR, nitrite reductase and nitrate transport activities in ammonium-nitrate media. Kinetic constants for ammonium (¹⁴C-methylammonium) transport, as well as enzymatic activities related to the ammonium-regulated metabolic pathway for xanthine utilization, were not affected in these strains. The data strongly suggest thatNrg1 andNrg2 are regulatory genes which specifically mediate the negative control exerted by ammonium on the nitrate assimilation pathway inC. reinhardtii.