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.     

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.     

Diverse effects of formate on the assimilatory metabolism of nitrate and nitrite inRhizobium

Two strains ofRhizobium, cowpeaRhizobium 32H1 andRhizobium japonicum CB 1809, showed a marked stimulation in growth on addition of formate to the minimal medium containing nitrate as the sole source of nitrogen. The amount of accumulated nitrite and specific nitrate reductase activity was much higher in cultures supplemented with formate than in the control medium. In contrast, growth, consumption of nitrite and specific nitrite reductase activity in minimal medium + nitrite was greatly reduced by the addition of formate. A chlorate resistant mutant (Chl-16) was isolated spontaneously which contained a nitrite reductase which was not inhibited by formate. The results suggest that formate serves as an electron donor for nitrate reductase and inhibits nitrite assimilation inRhizobium     

Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana

Summary Chlorate resistant mutants of Arabidopsis thaliana were isolated, of which 10 exhibited a lowered nitrate reductase activity and 51 were chlorate-resistant because of an impaired uptake of chlorate. The 51 mutants of this type are all affected in the same gene. The mutants with a lowered nitrate reductase activity fall into 7 different complementation groups. Three of these mutants grow poorly on media with nitrate as the sole nitrogen source, while the others apparently can reduce sufficient nitrate to bring about growth. In all cases a low nitrate reductase activity coincides with an enhanced nitrite reductase activity. After sucrose gradient centrifugation of wildtype extracts nitrate reductase is found at the 8S position, whereas cytochrome-c reductase is found both at 4 and 8S positions. It is suggested that the functional nitrate reductase is a complex consisting of 4S subunits showing cytochrome-c reductase activity and a Mo-bearing cofactor. All mutants except B25 are capable of assembling the 4S subunits into complexes which for most mutants have a lower S value and exhibit a lower nitrate reductase activity than the wildtype complexes. Since the mutants B25 and B73 exhibit a low xanthine dehydrogenase activity, the Mo-bearing cofactor is probably less available in these mutants than in the wildtype. B73 appears to be the only mutant which is partly repaired by excessive Mo. The possible role of several genes is discussed.     

The correlation between the protein composition of cytoplasmic membranes and the formation of nitrate reductase A,chlorate reductase C and tetrathionate reductase in Proteus mirabilis wild type and some chlorate resistant mutants

Three genotypically different chlorate resistant mutants, chl I, chl II and chl III, appeared to lack completely nitrate reductase A, chlorate reductase C and tetrathionate reductase activity. Fumarate reductase is only partially affected in chl I and chl III and unaffected in chl II. Formate dehydrogenase is only partially diminished in chl II, hydrogenase is diminished in chl I and chl II and completely absent in chl III.Subunits of nitrate reductase A, chlorate reductase C and tetrathionate reductase have been identified in protein profiles of purified cytoplasmic membranes from the wild type and the three mutant strains, grown under various conditions. Only the presence and absence of the largest subunits of these enzymes appeared to be correlated with their repression and derepression in the wild type membranes. On the cytoplasmic membranes of the chl I and chl III mutants these subunits lack for the greater part. In the chl II mutant, however, these subunits are inserted in the membrane all together after anaerobic growth with or without nitrate.A model for the repression/derepression mechanism for the reductases has been proposed. It includes repression by cytochrome b components, whereas the redox-state of the nitrate reductase A molecule itself is also involved in its derepression under anaerobic conditions.     

Physiological and biochemical characterization of chlorate-resistant mutants ofAnabaena doliolum

Chlorate-resistant mutants of the filamentous cyanobacterium,Anabaena doliolum, were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG)¹ mutagenesis. Three classes of mutants were obtained that were altered either in the nitrate uptake activity or nitrate reductase enzyme activity or both. These results suggest that the genetic determinant of the uptake system was distinct from that of the reductase system.Uptake studies of nitrite and ammonium and rate of nitrite reductase activity in the mutants revealed that the nitrite and ammonium metabolisms were not affected by this mutation.Both nitrate and chlorate acted like a pair of antagonists, with nitrate protecting the growth against chlorate with increase in its concentration; similarly, increasing chlorate concentrations counteracted the growth-protective action of nitrate.     

Toxicity of and mutagenesis by chlorate are independent of nitrate reductase activity in Chlamydomonas reinhardtii

Summary Spontaneous chlorate-resistant (CR) mutants have been isolated from Chlamydomonas reinhardtii wildtype strains. Most of them, 244, were able to grow on nitrate minimal medium, but 23 were not. Genetic and in vivo complementation analyses of this latter group of mutants indicated that they were defective either at the regulatory locus nit-2, or at the nitrate reductase (NR) locus nit-1, or at very closely linked loci. Some of these nit-1 or nit-2 mutants were also defective in pathways not directly related to nitrate assimilation, such as those of amino acids and purines. Chlorate treatment of wild-type cells resulted in both a decrease in cell survival and an increase in mutant cells resistant to a number of different chemicals (chlorate, methylammonium, sulphanilamide, arsenate, and streptomycin). The toxic and mutagenic effects of chlorate in minimal medium were not found when cells were grown either in darkness or in the presence of ammonium, conditions under which nitrate uptake is drastically inhibited. Chlorate was also able to induce reversion of nit ^– mutants of C. reinhardtii, but failed to produce His ⁺ revertants or Ara^r mutants in the BA-13 strain of Salmonella typhimurium. In contrast, chlorate treatment induced mutagenesis in strain E1F1 of the phototrophic bacterium Rhodobacter capsulatus. Genetic analyses of nitrate reductase-deficient CR mutants of C. reinhardtii revealed two types of CR, to low (1.5 mM) and high (15 mM) chlorate concentrations. These two traits were recessive in heterozygous diploids and segregated in genetic crosses independently of each other and of the nit-1 and nit-2 loci. Three her loci and four lcr loci mediating resistance to high (HC) and low (LC) concentrations of chlorate were identified. Mutations at the nit-2 locus, and deletions of a putative locus for nitrate transport were always epistatic to mutations responsible for resistance to either LC or HC. In both nit ⁺ and nit ^– chlorate-sensitive (CS) strains, nitrate and nitrite gave protection from the toxic effect of chlorate. Our data indicate that in C. reinhardtii chlorate toxicity is primarily dependent on the nitrate transport system and independent of the existence of an active NR enzyme. At least seven loci unrelated to the nitrate assimilation pathway and mediating CR are thought to control indirectly the efficiency of the nitrate transporter for chlorate transport. In addition, chlorate appears to be a mutagen capable of inducing a wide range of mutations unrelated to the nitrate assimilation pathway.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.     

Isolation and characterization of chlorate resistant mutants from nitrate-nonutilizing fungusPhycomyces blakesleeanus

Chlorate resistant mutants, which were first isolated in the zygomycetous fungusPhycomyces blakesleeanus, were found to be resistant up to a concentration of at least 300 mM of potassium chlorate. The dose-response relationship showed that although the mutants could be divided into two groups based on chlorate resistance in the mycelial elongation assay on the solid minimal medium, this was not observed in the assay using liquid culture. Genetic analysis of heterokaryons revealed the mutant alleles to be dominant. Enzymatic activities of three nitrate reductases and chlorate reductase were deficient in both the parent strain and the mutants. Intracellular incorporation of chlorate ion varied from strain to strain; however, the variation could not explain the mechanism of chlorate resistance. One unexpected characteristic of the mutants was that the intracellular sulfate ion concentration was 3.5 to 5.5 times higher than in the parent strain. We designated this mutant genotypecrw, chlorate resistant mutant from nitrate-nonutilizing wild type.     

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     

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     

Nitrate and nitrite reductase negative mutants of N2-fixingAzospirillum spp.

Chlorate resistant spontaneous mutants ofAzospirillum spp. (syn.Spirillum lipoferum) were selected in oxygen limited, deep agar tubes with chlorate. Among 20 mutants fromA. brasilense and 13 fromA. lipoferum all retained their functional nitrogenase and 11 from each species were nitrate reductase negative (nr^–). Most of the mutants were also nitrite reductase negative (nir^–), only 3 remaining nir⁺. Two mutants from nr⁺ nir⁺ parent strains lost only nir and became like the nr⁺ nir^– parent strain ofA. brasilense. No parent strain or nr⁺ mutant showed any nitrogenase activity with 10 mM NO ₃ ^– . In all nr^– mutants, nitrogenase was unaffected by 10 mM NO ₃ ^– . Nitrite inhibited nitrogenase activity of all parent strains and mutants including those which were nir^–. It seems therefore, that inhibition of nitrogenase by nitrate is dependent on nitrate reduction. Under aerobic conditions, where nitrogenase activity is inhibited by oxygen, nitrate could be used as sole nitrogen source for growth of the parent strains and one mutant (nr^– nir^–) and nitritite of the parent strains and 10 mutants (all types). This indicates the loss of both assimilatory and dissimilatory nitrate reduction but only dissimilatory nitrite reduction in the mutants selected with chlorate.     

Evidence for a Second Nitrate Reductase Activity That Is Distinct from the Respiratory Enzyme in Salmonella typhimurium

Significant nitrate reductase activity was detected in mutants of Salmonella typhimurium which mapped at or near chlC and which were incapable of growth with nitrate as electron acceptor. The same mutants were sensitive to chlorate and performed sufficient nitrate reduction to permit anaerobic growth with nitrate as the sole nitrogen source in media containing glucose. The mutant nitrate-reducing protein did not migrate with the wild-type nitrate reductase in polyacrylamide electrophoretic gels. Studies of the electrophoretic mobility in gels of different polyacrylamide concentration revealed that the wild-type and mutant nitrate reductases differed significantly in both size and charge. The second enzyme also differed from the wild-type major enzyme in its response to repression by low pH and its lack of response to repression by glucose. The same mutants were found to be derepressed for nitrite reductase and for a cytochrome with a maximal reduced absorbance at 555 nm at 25°C. This cytochrome was not detected in preparations of the wild type grown under the same conditions. Extracts of these mutants contained normal amounts of the b-type cytochromes which, in the wild type, were associated with nitrate reductase and formate dehydrogenase, respectively, although they could not mediate the oxidation of these cytochromes with nitrate. They were capable of oxidizing the derepressed 555-nm peak cytochrome with nitrate. It is suggested that these mutants synthesize a nitrate-reducing enzyme which is distinct from the chlC gene product and which is repressed in the wild type during anaerobic growth with nitrate.     

Chemolithotrophic growth ofDesulfovibrio desulfuricans with hydrogen coupled to ammonification of nitrate or nitrite

Two of nine sulfate reducing bacteria tested,Desulfobulbus propionicus andDesulfovibrio desulfuricans (strain Essex 6), were able to grow with nitrate as terminal electron acceptor, which was reduced to ammonia. Desulfovibrio desulfuricans was grown in chemostat culture with hydrogen plus limiting concentrations of nitrate, nitrite or sulfate as sole energy source. Growth yields up to 13.1, 8.8 or 9.7 g cell dry mass were obtained per mol nitrate, nitrite or sulfate reduced, respectively. The apparent half saturation constants (K _s) were below the detection limits of 200, 3 or 100 mol/l for nitrate, nitrite of sulfate, respectively. The maximum growth rates {ie63-1} raised from 0.124 h^-1 with sulfate and 0.150 h^-1 with nitrate to 0.193 h^-1 with nitrite as electron acceptor. Regardless of the electron acceptor in the culture medium, cell extracts exhibited absorption maxima corresponding to cytochromec and desulfoviridin. Nitrate reductase was found to be inducible by nitrate or nitrite, whereas nitrite reductase was synthesized constitutively. The activities of nitrate and nitrite reductases with hydrogen as electron donor were 0.2 and 0.3 mol/min·mg protein, respectively. If limiting amounts of hydrogen were added to culture bottles with nitrate as electron acceptor, part of the nitrate was only reduced to the level of nitrite. In media containing nitrate plus sulfate or nitrite plus sulfate, sulfate reduction was suppressed.The results demonstrate that the ammonification of nitrate or nitrite can function as sole energy conserving process in some sulfate-reducing bacteria.     

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O₂ pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids. Received: 17 April 1996 / Accepted: 28 May 1996     

Regulation of reductase formation in Proteus mirabilis

Summary Prteus mirabilis can form four reductases after anaerobic growth: nitrate reductase A, chlorate reductase C, thiosulfate reductase and tetrathionate reductase. The last three enzymes are formed constitutively. Nitrate reductase is formed only after growth in the presence of nitrate, which causes repression of the formation of thiosulfate reductase, chlorate reductase C, tetrathionate reductase and hydrogenase. Formic dehydrogenase assayed with methylene blue as hydrogen acceptor is formed under all conditions.Two groups of chlorate resistant mutants were obtained. One group does not form the reductases and formic dehydrogenase. The second group does not form nitrate reductase, chlorate reductase and hydrogenase, but forms formic dehydrogenase and small amounts of formic hydrogenlyase after growth without hydrogen acceptor or after growth in the presence of thiosulfate or tetrathionate. Nitrate prevents the formation of formic dehydrogenase, thiosulfate reductase and tetrathionate reductase in this group of mutants. Only after growth with thiosulfate or tetrathionate the reductases for these compounds are formed. Anaerobic growth of the wild type in complex medium without a fermentable carbon source is strongly stimulated by the presence of nitrate. Tetrathionate and thiosulfate have no effect at all or only a small effect. The results show that in the presence of tetrathionate or thiosulfate the bacterial metabolism is fully anaerobic, as these cells also contain formic hydrogenlyase.     

Structural and mechanistic insights on nitrate reductases

Nitrate reductases (NR) belong to the DMSO reductase family of Mo‐containing enzymes and perform key roles in the metabolism of the nitrogen cycle, reducing nitrate to nitrite. Due to variable cell location, structure and function, they have been divided into periplasmic (Nap), cytoplasmic, and membrane‐bound (Nar) nitrate reductases. The first crystal structure obtained for a NR was that of the monomeric NapA from Desulfovibrio desulfuricans in 1999. Since then several new crystal structures were solved providing novel insights that led to the revision of the commonly accepted reaction mechanism for periplasmic nitrate reductases. The two crystal structures available for the NarGHI protein are from the same organism (Escherichia coli) and the combination with electrochemical and spectroscopic studies also lead to the proposal of a reaction mechanism for this group of enzymes. Here we present an overview on the current advances in structural and functional aspects of bacterial nitrate reductases, focusing on the mechanistic implications drawn from the crystallographic data.     

Chlorate toxicity in Aspergillus nidulans. Studies of mutants altered in nitrate assimilation.

Summary It had previously been held that chlorate is not itself toxic, but is rendered toxic as a result of nitrate reductase-catalysed conversion to chlorite. This however cannot be the explanation of chlorate toxicity in Aspergillus nidulans, even though nitrate reductase is known to have chlorate reductase activity. Among other evidence against the classical theory for the mechanism of chlorate toxicity, is the finding that not all mutants lacking nitrate reductase are clorate resistant. Both chlorate-sensitive and resistant mutants lacking nitrate reductase, also lack chlorate reductase. Data is presented which implicates not only nitrate reductase but also the product of the nirA gene, a positive regulator gene for nitrate assimilation, in the mediation of chlorate toxicity. Alternative mechanisms for chlorate toxicity are considered. It is unlikely that chlorate toxicity results from the involvement of nitrate reductase and the nirA gene product in the regulation either of nitrite reductase, or of the pentose phosphate pathway. Although low pH has an effect similar to chlorate, chlorate is not likely to be toxic because it lowers the pH; low pH and chlorate may instead have similar effects. A possible explanation for chlorate toxicity is that it mimics nitrate in mediating, via nitrate reductase and the nirA gene product, a shut-down of nitrogen catabolism. As chlorate cannot act as a nitrogen source, nitrogen starvation ensures.     

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.