Lacking Nitrate Reductase Activity Research Articles

In praise of erroneous hypotheses

In the sixties Cove and Pateman discovered that mutants of Aspergillus nidulans lacking nitrate reductase activity were constitutive for the expression of genes induced by nitrate and dependent on the transcription factor NirA. They proposed that the nitrate protein acted as a repressor, preventing the transcription factor activity of NirA. Nitrate-mediated regulation behaved similarly in other organisms. This “autogenous regulation hypothesis” has recently shown to be erroneous, in the very organism for which it was first proposed. Nevertheless this erroneous hypothesis have led to a thorough dissection of the process of regulation of nitrate assimilation and more importantly to a hypothesis bearing on the origin of metabolite-responsive transcription factors. In this article I discuss the heuristic value and evolutionary importance of autogenous regulation.

Regulation and Symbiotic Role of nirK and norC Expression in Rhizobium etli

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.

Effect of calcium ion on uptake of amino acids by symbiotic Chlorella F36-ZK isolated from Japanese Paramecium bursaria

Symbiotic Chlorella F36-ZK isolated from Paramecium bursaria F36 lacks nitrate reductase activity and has constitutive amino acid transport systems. Divalent cations generally accelerate amino acid uptake and Ca 2+ affects membrane permeability and H +-ATPase activity. However, Ser uptake by F36-ZK was decreased by divalent cations, especially Ca 2+, and the cation had no effect on membrane permeability or H +-ATPase activity. Extracellular Ca 2+ contributed to inhibit Ser uptake. The cation selectively inhibited uptake of Ser, Ala and Gln, which are transported via the same system, but not Arg. Based on kinetic analysis of Ser transport, inhibition was noncompetitive, i.e., Ca 2+ affected only the V max of Ser uptake. Calcium ions were found in symbiotic Chlorella cells associated with their host by transmission electron microscopy using an oxalate-pyroantimonate method, suggesting the transfer of Ca 2+ during symbiosis. From these results obtained in this paper, we discuss the role of divalent cations on amino acid transport in the Paramecium symbiosis.

Functional characterization of the Bradyrhizobium japonicum modA and modB genes involved in molybdenum transport

A modABC gene cluster that encodes an ABC-type, high-affinity molybdate transporter from Bradyrhizobium japonicum has been isolated and characterized. B. japonicum modA and modB mutant strains were unable to grow aerobically or anaerobically with nitrate as nitrogen source or as respiratory substrate, respectively, and lacked nitrate reductase activity. The nitrogen-fixing ability of the mod mutants in symbiotic association with soybean plants grown in a Mo-deficient mineral solution was severely impaired. Addition of molybdate to the bacterial growth medium or to the plant mineral solution fully restored the wild-type phenotype. Because the amount of molybdate required for suppression of the mutant phenotype either under free-living or under symbiotic conditions was dependent on sulphate concentration, it is likely that a sulphate transporter is also involved in Mo uptake in B. japonicum. The promoter region of the modABC genes has been characterized by primer extension. Reverse transcription and expression of a transcriptional fusion, P(modA)-lacZ, was detected only in a B. japonicum modA mutant grown in a medium without molybdate supplementation. These findings indicate that transcription of the B. japonicum modABC genes is repressed by molybdate.

Molybdate-dependent expression of the periplasmic nitrate reductase in Bradyrhizobium japonicum

The napEDABC genes of Bradyrhizobium japonicum encode the periplasmic nitrate reductase, an Mo-containing enzyme which catalyses the reduction of nitrate to nitrite when oxygen concentrations are limiting. In this bacterium, another set of genes, modABC, code for a high affinity ABC-type Mo transport system. A B. japonicum modA mutant has been obtained that is not capable of growing anaerobically with nitrate and lacks nitrate reductase activity. Under nitrate respiring conditions, when Mo concentrations are limiting, the B. japonicum modA mutant lacked both the 90 kDa protein corresponding to the NapA component of the periplasmic nitrate reductase, and the membrane-bound 25 kDa c-type cytochrome NapC. Regulatory studies using a napE-lacZ fusion indicated that napE expression was highly reduced in the modA mutant background when the cells were incubated anaerobically with nitrate under Mo-deficient conditions.

The Bradyrhizobium japonicum napEDABC genes encoding the periplasmic nitrate reductase are essential for nitrate respiration

The napEDABC gene cluster that encodes the periplasmic nitrate reductase from Bradyrhizobium japonicum USDA110 has been isolated and characterized. napA encodes the catalytic subunit, and the napB and napC gene products are predicted to be a soluble dihaem c and a membrane-anchored tetrahaem c-type cytochrome, respectively. napE encodes a transmembrane protein of unknown function, and the napD gene product is a soluble protein which is assumed to play a role in the maturation of NapA. Western blots of the periplasmic fraction from wild-type cells grown anaerobically with nitrate revealed the presence of a protein band with a molecular size of about 90 kDa corresponding to NapA. A B. japonicum mutant carrying an insertion in the napA gene was unable to grow under nitrate-respiring conditions, lacked nitrate reductase activity, and did not show the 90 kDa protein band. Complementation of the mutant with a plasmid bearing the napEDABC genes restored both nitrate-dependent anaerobic growth of the cells and nitrate reductase activity. A membrane-bound and a periplasmic c-type cytochrome, with molecular masses of 25 kDa and 15 kDa, respectively, were not detected in the napA mutant strain incubated anaerobically with nitrate, which identifies those proteins as the NapC and the NapB components of the B. japonicum periplasmic nitrate reductase enzyme. These results suggest that the periplasmic nitrate reductase is the enzyme responsible for anaerobic growth of B. japonicum under nitrate-respiring conditions. The promoter region of the napEDABC genes has been characterized by primer extension. A major transcript initiates 66.5 bp downstream of the centre of a putative FNR-like binding site.

The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii.

The expression of nitrite uptake activity and the induction of transcripts from several nitrate assimilation genes (Nii1, Nrt2;1, Nrt2;3, and Nar1) have been analysed in Chlamydomonas reinhardtii Dang. strains bearing several sets of genes encoding nitrate transport systems. Different nitrate concentrations resulted in a differential induction pattern of nitrite uptake activity depending on which particular nitrate transport system was present in the cells, and that was directly related to their relative efficiency for nitrate transport. The presence of the high-affinity nitrate transport system I (NRT2;1, NAR2) made cells able to sense the very low concentrations of nitrate present in culture medium with no added nitrate and to express optimally Nii1, Nrt2;1, Nrt2;3, and Nar1 genes involved in nitrate/nitrite assimilation. In addition, strains lacking nitrate reductase activity overexpressed these gene transcripts as a result of continuous signalling by nitrate, but only those bearing an active system I. This study supports the hypothesis that signalling of nitrate assimilation genes occurs intracellularly in a process dependent on the nitrate uptake activity. The bispecific nitrate/nitrite transport system I with the highest affinity for nitrate is the most efficient one for signalling the expression of nitrate/nitrite assimilation genes in C. reinhardtii.

Isolation of periplasmic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases

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

The terminal reductases for selenate and nitrate respiration in Thauera selenatis are two distinct enzymes.

A number of approaches have been used to show that a recently isolated selenate-respiring bacterium, Thauera selenatis, is able to synthesize both a selenate reductase (SR) and a nitrate reductase (NR). (i) The pH optimum of the SR was found to be 6.0; that of the NR was 7.0. (ii) The presence of nitrate did not inhibit selenate reduction in selenate-grown cells. (iii) In cell extracts, the highest SR or NR activity was observed in cells grown with the respective electron acceptor. (iv) Mutants that were unable to grow with nitrate as the terminal electron acceptor and lacked NR activity were isolated; these mutants grew normally with selenate and synthesized SR. (v) The SR was found in the periplasmic space of the cell, whereas the NR was present in the cytoplasmic membrane. A hypothetical electron transport system involving the SR is described.

Nitrate transport in the cyanobacterium Anacystis nidulans R2. Kinetic and energetic aspects.

Nitrate transport has been studied in the cyanobacterium Anacystis nidulans R2 by monitoring intracellular nitrate accumulation in intact cells of the mutant strain FM6, which lacks nitrate reductase activity and is therefore unable to reduce the transported nitrate. Kinetic analysis of nitrate transport as a function of external nitrate concentration revealed apparent substrate inhibition, with a peak velocity at 20-25 microM-nitrate. A Ks (NO3-) of 1 microM was calculated. Nitrate transport exhibited a stringent requirement for Na+. Neither Li+ nor K+ could substitute for Na+. Monensin depressed nitrate transport in a concentration-dependent manner, inhibition being more than 60% at 2 microM, indicating that the Na(+)-dependence of active nitrate transport relies on the maintenance of a Na+ electrochemical gradient. The operation of an Na+/NO3- symport system is suggested. Nitrite behaved as an effective competitive inhibitor of nitrate transport, with a Ki (NO2-) of 3 microM. The time course of nitrite inhibition of nitrate transport was consistent with competitive inhibition by mixed alternative substrates. Nitrate and nitrite might be transported by the same carrier.

Specimen cross-contamination by a strain of Mycobacterium tuberculosis lacking nitrate reductase activity

An unusual strain of Mycobacterium tuberculosis lacking nitrate reductase activity was isolated from specimens from five patients over a 2-week period. Two patients, a father (patient 1) and his son (patient 5), had pulmonary tuberculosis. Specimens from three other patients (patients 2, 3, and 4) processed on the same 2 days as those from patient 1 yielded only sparse growth (approximately five colonies each) of M. tuberculosis. These and other data strongly suggest that specimens from the latter three patients were contaminated during processing with the strain of M. tuberculosis from patient 1.

No Correlation between Plasmid Content and Ability to Reduce Nitrate in Wild-Type Strains of Rhodobacter capsulatus

Patterns of endogenous plasmids and nitrate reductase activities were analyzed in the phototrophic bacterium Rhodobacter (Rb.) capsulatus. From 10 strains investigated (including a UV-induced plasmidless nit- mutant), 4 were unable to grow photosynthetically with nitrate as N-source and lacked nitrate reductase activity (nit strains). Irrespective of the nit phenotype, all wildtype strains contained at least one large plasmid with a size ranging from 93 to 134 kb. Thus, other than in plasmid- cured mutants (J. C. Willison, FEMS Microbiol. Lett. 66, 23-28[1990]), in wild-type strains of Rb. capsulatus the nit- character was not related to lack of endogenous plasmids.

Anaerobic Inductions of Active Forms of Superoxide Dismutases inEscherzchia Coli

Escherichia coli growing anaerobically respond to NO3- with a approximately 3-fold induction of active FeSOD and a approximately 5.5-fold induction of an inactive, but activatable form of MnSOD (pro-MnSOD). Paraquat, which mediates anaerobic electron flow to NO3-, increased the induction of pro-MnSOD to approximately 2.5-fold. Strains with defects in the SOD genes or which lacked nitrate reductase activity failed to accumulate active or pro-forms of SODs in response to NO3- +/- PQ++. Diamide caused anaerobic induction of active MnSOD and this effect was also observed in a glutathione-negative strain. These inductions required de novo synthesis of protein, even when cell content of pro-MnSOD had been elevated by exposure to NO3- +/- PQ++ prior to addition of diamide. These results indicate that oxidation of a cell component increases biosynthesis of the SOD gene product and this postulated oxidation can be caused by terminal electron acceptors, such as dioxygen or NO3-. In addition, it appears that insertion of the correct metal can be rate-limiting, leading to competition by other metals and to the accumulation of inactive, incorrectly substituted pro-forms. Metal insertion may be dependent upon the valence of the metal, which may be influenced, in turn, by the redox status of the cells. Diamide and redox active agents such as ferricyanide may thus allow anaerobic production of active MnSOD by favoring the production of a complexed form of Mn(III) which can compete favorably with other metal cations for the active site of nascent MnSOD.

Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.

We have developed a nuclear transformation system for Chlamydomonas reinhardtii, using micro-projectile bombardment to introduce the gene encoding nitrate reductase into a nit1 mutant strain which lacks nitrate reductase activity. By using either supercoiled or linear plasmid DNA, transformants were recovered consistently at a low efficiency, on the order of 15 transformants per microgram of plasmid DNA. In all cases the transforming DNA was integrated into the nuclear genome, usually in multiple copies. Most of the introduced copies were genetically linked to each other, and they were unlinked to the original nit1 locus. The transforming DNA and nit+ phenotype were stable through mitosis and meiosis, even in the absence of selection. nit1 transcripts of various sizes were expressed at levels equal to or greater than those in wild-type nit+ strains. In most transformants, nitrate reductase enzyme activity was expressed at approximately wild-type levels. In all transformants, nit1 mRNA and nitrate reductase enzyme activity were repressed in cells grown on ammonium medium, showing that expression of the integrated nit1 genes was regulated normally. When a second plasmid with a nonselectable gene was bombarded into the cells along with the nit1 gene, transformants carrying DNA from both plasmids were recovered. In some cases, expression of the unselected gene could be detected. With the advent of nuclear transformation in Chlamydomonas, it becomes the first photosynthetic organism in which both the nuclear and chloroplast compartments can be transformed.

Selection of a Nicotiana plumbaginifolia universal hybridizer and its use in intergeneric somatic hybrid formation

A Nicotiana plumbaginifolia cell strain carrying a positive (dominant) trait, resistance to azetidine-2-carboxylate (A2C), was selected in strain NX1 which lacked nitrate reductase activity (a negative or recessive trait). This universal hybridizer strain, denoted NXAr, was fused with dextran to a Daucus carota strain, PR, which carried glyphosate (GLP) resistance. A large number of hybrids were selected in a medium with NO 3 - as the sole nitrogen source and A2C as inhibitor, conditions which prevent the growth of both parents. When the selected colonies were then tested for GLP resistance, 93% carried this trait. In addition the hybrid nature was indicated by additive chromosome numbers, both A2C and GLP resistance in suspension cultures, intermediate nitrate reductase activity and the presence of banding patterns for three isozymes which match those of the parents. Southern hybridization analysis using an enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) probe, pMON 6145, also showed the presence of the gene from both parents in the hybrid strains based on restriction length polymorphisms. The PR strain contains increased levels of EPSPS which confers GLPr due to gene amplification. Since the universal hybridizer can be used as a fusion partner with any wild-type line many protoplast fusion studies can be carried out easily.

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

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.

Nitrate reductase activity in isolated heterocysts of the cyanobacterium Nostoc muscorum

The nitrate reductase, nitrate reductase apoprotein and nitrate reductase Mo-cofactor activities were measured in the cell-free extracts of isolated heterocysts and whole filaments of the cyanobacterium Nostoc muscorum. The nitrate reductase activity of N 2- and NO − 3-grown whole filaments was 3.25 and 7.14 nmol NO − 2 formed·min −1·mg protein −1, respectively. No nitrate reducatase activity was found in the extracts of isolated heterocysts. However, when the heterocyst extract was supplemented with nitrate reductase apoprotein from the whole filaments, a reconstituted nitrate reductase activity of 6.4 nmol NO 2 − 2 formed·min −1·mg protein −1 was detectable. Therefore, it was concluded that nitrate reductase in Nostoc muscorum was localized in the vegetative cells, that the heterocysts lacked nitrate reductase activity due to the lack of apoprotein, and that the Mo-cofactor was present in heterocysts.

In vitro reconstitution of NADH: nitrate reductase in nitrate reductase-deficient mutants of barley

In vitro complementation of the nitrate reductase-deficient barley mutant nar2a extracts with molybdenum cofactor from commercial xanthine oxidase resulted in reactivation of NADH: nitrate reductase activity. Maximum reactivation was achieved with 7.5 μg/ml xanthine oxidase (final concentration), 10 mM glutathione (final concentration) and incubation for 30 min at room temperature (ca. 25°C). This in vitro complementation assay was used to determine the presence of functional apoprotein and molybdenum cofactor in 12 nitrate reductase-deficient barley mutants. Extracts of all nar1 alleles contained functional molybdenum cofactor (complemented with nar2a) but they lacked functional apoprotein (did not complement with molybdenum cofactor from xanthine oxidase). The nar2a, nar3a and nar3b extracts were able to donate functional apoprotein, but were poor sources of functional molybdenum cofactor. These data are in agreement with our previous assignment of nar1 to the barley NADH: nitrate reductase structural locus and nar2 and nar3 to molybdenum cofactor functions. Wild type cv. Steptoe barley seedlings grown in the absence of nitrate and lacking nitrate reductase activity contained low levels of molybdenum cofactor. Nitrate induction resulted in a several-fold increase in the measurable molybdenum cofactor levels that was correlated with the increase in nitrate reductase activity.

Isolation and characterisation of nitrate reductase mutants and regulation of nitrate reductase and nitrogenase in the cyanobacterium Nostoc muscorum

Chlorate resistant mutants of the cyanobacterium Nostoc muscorum isolated after N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenesis were found to be defective/blocked in nitrate reductase (NR). The parent strain possessed active NR in the presence of nitrogen as nitrate and only basal levels of activity in ammonia and N-free grown cultures. Addition of ammonia suppressed the NR activity in the parent strain whereas addition of L-methionine DL-sulphoximine (MSX) restored NR activity. A similar repression by ammonia, glutamine and derepression with MSX were also observed for nitrogenase synthesis. One class of mutants lacked NR activity (nar -) whereas the specific activity of NR was low in another class of mutants (nar def). Unlike the parent, the mutants synthesized nitrogenase and differentiated heterocysts in the presence of nitrate nitrogen. Uptake studies of nitrite and ammonia in mutants revealed that they possessed both nitrite reductase and glutamine synthetases (GS) at low levels, and the same level respectively in comparison with the parent.

Complementation analysis of nitrate reductase deficient mutants of Nicotiana tabacum by somatic hybridization

Mutant cell lines lacking nitrate reductase activity were analyzed genetically. Protoplasts from one apoprotein defective (nia) and four cofactor defective (cnx) mutants were fused in all possible pairwise combinations with the aid of polyethylene glycol. Complementing hybrids were detected by their ability to grow with nitrate as sole nitrogen source and confirmed by measuring their nitrate reductase activity. Strong complementation was observed in all types of nia+cnx hybrids, whereas the cnx mutants failed to complement each other. From the results it can be concluded that the mutants studied are recessive and that the four cnx mutants are alleles of the same pair of duplicate loci (cnxA1, cnxA2).