Production of recombinant multiheme cytochromes c in Wolinella succinogenes.

Respiratory nitrogen cycle processes like nitrification, nitrate reduction, denitrification, nitrite ammonification, or anammox involve a variety of dissimilatory enzymes and redox-active cofactors. In this context, an intriguing protein class are cytochromes c, that is, enzymes containing one or more covalently bound heme groups that are attached to heme c binding motifs (HBMs) of apo-cytochromes. The key enzyme of the corresponding maturation process is cytochrome c heme lyase (CCHL), an enzyme that catalyzes the formation of two thioether linkages between two vinyl side chains of a heme and two cysteine residues arranged in the HBM. In recent years, many multiheme cytochromes c involved in nitrogen cycle processes, such as hydroxylamine oxidoreductase and cytochrome c nitrite reductase, have attracted particular interest. Structurally, these enzymes exhibit conserved heme packing motifs despite displaying very different enzymic properties and largely unrelated primary structures. The functional and structural characterization of cytochromes c demands their purification in sufficient amounts as well as the feasibility to generate site-directed enzyme variants. For many interesting organisms, however, such systems are not available, mainly hampered by genetic inaccessibility, slow growth rates, insufficient cell yields, and/or a low capacity of cytochrome c formation. Efficient heterologous cytochrome c overproduction systems have been established using the unrelated proteobacterial species Escherichia coli and Wolinella succinogenes. In contrast to E. coli, W. succinogenes uses the cytochrome c biogenesis system II and contains a unique set of three specific CCHL isoenzymes that belong to the unusual CcsBA-type. Here, W. succinogenes is presented as host for cytochrome c overproduction focusing on a recently established gene expression system designed for large-scale production of multiheme cytochromes c.

The endophytic fungus Piriformospora indica stimulates the expression of nitrate reductase and the starch-degrading enzyme glucan-water dikinase in tobacco and Arabidopsis roots through a homeodomain transcription factor that binds to a conserved motif in their promoters.

Piriformospora indica, an endophytic fungus of the Sebacinaceae family, promotes growth of Arabidopsis and tobacco seedlings and stimulates nitrogen accumulation and the expression of the genes for nitrate reductase and the starch-degrading enzyme glucan-water dikinase (SEX1) in roots. Neither growth promotion nor stimulation of the two enzymes requires heterotrimeric G proteins. P. indica also stimulates the expression of the uidA gene under the control of the Arabidopsis nitrate reductase (Nia2) promoter in transgenic tobacco seedlings. At least two regions (-470/-439 and -103/-89) are important for Nia2 promoter activity in tobacco roots. One of the regions contains an element, ATGATAGATAAT, that binds to a homeodomain transcription factor in vitro. The message for this transcription factor is up-regulated by P. indica. The transcription factor also binds to a CTGATAGATCT segment in the SEX1 promoter in vitro. We propose that the growth-promoting effect initiated by P. indica is accompanied by a co-regulated stimulation of enzymes involved in nitrate and starch metabolisms.

Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation.

When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55-70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.

Diurnal changes in nitrogen assimilation of tobacco roots.

To gain an insight into the diurnal changes of nitrogen assimilation in roots the in vitro activities of cytosolic and plasma membrane-bound nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1) and cytosolic and plastidic glutamine synthetase (EC 6.3.1.2) were studied. Simultaneously, changes in the contents of total protein, nitrate, nitrite, and ammonium were followed. Roots of intact tobacco plants (Nicotiana tabacum cv. Samsun) were extracted every 3 h during a diurnal cycle. Nitrate reductase, nitrite reductase and glutamine synthetase were active throughout the day-night cycle. Two temporarily distinct peaks of nitrate reductase were detected: during the day a peak of soluble nitrate reductase in the cytosol, in the dark phase a peak of plasma membrane-bound nitrate reductase in the apoplast. The total activities of nitrate reduction were similar by day and night. High activities of nitrite reductase prevented the accumulation of toxic amounts of nitrite throughout the entire diurnal cycle. The resulting ammonium was assimilated by cytosolic glutamine synthetase whose two activity peaks, one in the light period and one in the dark, closely followed those of nitrate reductase. The contribution of plastidic glutamine synthetase was negligible. These results strongly indicate that nitrate assimilation in roots takes place at similar rates day and night and is thus differently regulated from that in leaves.

Maximal expression of membrane-bound nitrate reductase in Paracoccus is induced by nitrate via a third FNR-like regulator named NarR.

Respiratory reduction of nitrate to nitrite is the first key step in the denitrification process that leads to nitrate loss from soils. In Paracoccus pantotrophus, the enzyme system that catalyzes this reaction is encoded by the narKGHJI gene cluster. Expression of this cluster is maximal under anaerobic conditions in the presence of nitrate. Upstream from narK is narR, a gene encoding a member of the FNR family of transcriptional activators. narR is transcribed divergently from the other nar genes. Mutational analysis reveals that NarR is required for maximal expression of the membrane-bound nitrate reductase genes and narK but has no other regulatory function related to denitrification. NarR is shown to require nitrate and/or nitrite is order to activate gene expression. The N-terminal region of the protein lacks the cysteine residues that are required for formation of an oxygen-sensitive iron-sulfur cluster in some other members of the FNR family. Also, NarR lacks a crucial residue involved in interactions of this family of regulators with the sigma(70) subunit of RNA polymerase, indicating that a different mechanism is used to promote transcription. narR is also found in Paracoccus denitrificans, indicating that this species contains at least three FNR homologues.

Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate.

Ectomycorrhizal fungi contribute to the nitrogen nutrition of their host plants, but no information is available on the molecular control of their nitrogen metabolism. The cloning and pattern of transcriptional regulation of two nitrite reductase genes of the symbiotic basidiomycete Hebeloma cylindrosporum are presented. The genomic copy of one of these genes (nar1) was entirely sequenced; the coding region is interrupted by 12 introns. The nar1 gene, which is transcribed and codes for a putative 908-amino acid polypeptide complemented nitrate reductase-deficient mutants of H. cylindrosporum upon transformation, thus demonstrating that the gene is functional. The second gene (nar2), for which no mRNA transcripts were detected, is considered to be an ancestral, non-functional duplication of nar1. In a 462-nt partial sequence of nar2 two introns were identified at positions identical to those of introns 8 and 9 of nar1, although their respective nucleotide sequences were highly divergent; the exon sequences were much more conserved. In wild-type strains, transcription of nar1 is repressed in the presence of a high concentration of ammonium. High levels of transcription are observed in the presence of either very low nitrogen concentrations or high concentrations of nitrate or organic N sources such as urea, glycine or serine. This indicates that in H. cylindrosporum, in contrast to all nitrophilous organisms studied so far, an exogenous supply of nitrate is not required to induce transcription of a nitrate reductase gene. In contrast, repression by ammonium suggests the existence of a wide-domain regulatory gene, as already characterized in ascomycete species.

ZmcPKC70, a protein kinase C-type enzyme from maize. Biochemical characterization, regulation by phorbol 12-myristate 13-acetate and its possible involvement in nitrate reductase gene expression.

The crucial enzyme in diacylglycerol-mediated signaling is protein kinase C (PKC). In this paper we provide evidence for the existence and role of PKC in maize. A protein of an apparent molecular mass of 70 kDa was purified. The protein showed kinase activity that was stimulated by phosphatidylserine and oleyl acetyl glycerol (OAG) in the presence of Ca2+. Phorbol 12-myristate 13-acetate (PMA) replaced the requirement of OAG. [3H]PMA binding to the 70-kDa protein was competed by unlabeled PMA and OAG but not by 4alpha-PMA, an inactive analog. The kinase phosphorylates histone H1 at serine residue(s), and this activity was inhibited by H-7 and staurosporine. These properties suggest that the 70-kDa protein is a conventional serine/threonine protein kinase C (cPKC). Polyclonal antibodies raised against the polypeptide precipitate the enzyme activity and immunostained the protein on Western blots. The antibodies also cross-reacted with a protein of expected size from sorghum, rice, and tobacco. A rapid increase in the protein level was observed in maize following PMA treatments. In order to assign a possible role of PKC in gene regulation, the nitrate reductase transcript level was investigated. The transcript level increased by PMA, not by 4alpha-PMA treatments, and the increase was inhibited by H-7 but not by okadaic acid. The data show the existence and possible function of PKC in higher plants.

The narA locus of Synechococcus sp. strain PCC 7942 consists of a cluster of molybdopterin biosynthesis genes.

The narA locus required for nitrate reduction in Synechococcus sp. strain PCC 7942 is shown to consist of a cluster of genes, namely, moeA, moaC, moaD, moaE, and moaA, involved in molybdenum cofactor biosynthesis. The product of the moaC gene of strain PCC 7942 shows homology in its N-terminal half to MoaC from Escherichia coli and in its C-terminal half to MoaB or Mog. Overexpression of the Synechococcus moaC gene in E. coli resulted in the synthesis of a polypeptide of 36 kDa, a size that would conform to a protein resembling a fusion of the MoaC and MoaB or Mog polypeptides of E. coli. Insertional inactivation of the moeA, moaC, moaE, and moaA genes showed that the moeA-moa gene cluster is required for growth on nitrate and expression of nitrate reductase activity in strain PCC 7942. The moaCDEA genes constitute an operon which is transcribed divergently from the moeA gene. Expression of the moeA gene and the moa operon was little affected by the nitrogen source present in the culture medium.

Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes.

Cucumber mosaic cucumovirus (CMV) infection but not tomato black ring nepovirus infection counteracted post-transcriptional gene silencing (PTGS) of nitrate reductase (Nia) or beta-glucuronidase (uidA) transgenes in newly developing leaves of tobacco and Arabidopsis plants. PTGS did not affect meristems of noninfected silenced plants, indicating that the interfering effect of CMV is not likely to occur in the meristem. Models are proposed to explain how CMV (which has no sequence similarity to the Nia or uidA transgenes) can inhibit cellular factors involved in the RNA degradation step of PTGS and/or inhibit the systemic spread of the silencing signal to tissues emerging from the meristem.

Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase.

Although nitrate reductase (NR. EC 1.6.6.1) is thought to control the rate of nitrate assimilation, mutants with 40-45% of wildtype (WT) NR activity (NRA) grow as fast as the WT. We have investigated how tobacco (Nicotiana tabacum L. cv. Gatersleben) mutants with one or two instead of four functional nia genes compensate. (i) The nia transcript was higher in the leaves of the mutants. However, the diurnal rhythm was retained in the mutants, with a maximum at the end of the night and a strong decline during the photoperiod. (ii) Nitrate reductase protein and NRA rose to a maximum after 3-4 h light in WT leaves, and then decreased by 50-60% during the second part of the photoperiod and the first part of the night. Leaves of mutants contained 40-60% less NR protein and NRA after 3-4 h illumination, but NR did not decrease during the photoperiod. At the end of the photoperiod the WT and the mutants contained similar levels of NR protein and NRA. (iii) Darkening led to a rapid inactivation of NR in the WT and the mutants. However, in the mutants, this inactivation was reversed after 1-3 h darkness. Calyculin A prevented this reversal. When magnesium was included in the assay to distinguish between the active and inactive forms of NR, mutants contained 50% more activity than the WT during the night. Conversion of [15N]-nitrate to organic compounds in leaves in the first 6 h of the night was 60% faster in the mutants than in the WT. (iv) Growth of WT plants in enhanced carbon dioxide prevented the decline of NRA during the second part of the photoperiod, and led to reactivation of NR in the dark. (v) Increased stability of NR in the light and reversal of dark-inactivation correlated with decreased levels of glutamine in the leaves. When glutamine was supplied to detached leaves it accelerated the breakdown of NR, and led to inactivation of NR, even in the light. (vi) Diurnal changes were also investigated in roots. In the WT, the amount of nia transcript rose to a maximum after 4 h illumination and then gradually decreased. The amplitude of the changes in transcript amount was smaller in roots than in leaves, and there were no diurnal changes in NRA. In mutants, nia transcript levels were high through the photoperiod and the first part of the night. The NRA was 50% lower during the day but rose during the night to an activity almost as high as in the WT. The rate of [15N]-nitrate assimilation in the roots of the mutants resembled that in the WT during the first 6 h of the night. (vii) Diurnal changes were also compared in Nia30(145) transformants with very low NRA, and in nitrate-deficient WT plants. Both sets of plants had similar low growth rates. Nitrate reductase did not show a diurnal rhythm in leaves or roots of Nia30(145), the leaves contained very low glutamine, and NR did not inactivate in the dark. Nitrate-deficient WT plants were watered each day with 0.2 mM nitrate. After watering, there was a small peak of nia transcript NR protein and NRA and, slightly later, a transient increase of glutamine and other amino acids in the leaves. During the night glutamine was low, and NR did not inactivate. In the roots, there was a very marked increase of nitrate, nia transcript and NRA 2-3 h after the daily watering with 0.2 mM nitrate. (viii) It is concluded that WT plants have excess capacity for nitrate assimilation. They only utilise this potential capacity for a short time each day, and then down-regulate nitrate assimilation in response, depending on the conditions, to accumulation of the products of nitrate assimilation or exhaustion of external nitrate. Genotypes with a lower capacity for nitrate assimilation compensate by increasing expression of NR and weakening the feedback regulation, to allow assimilation to continue for a longer period each day.

Identification of cDNA clones corresponding to two inducible nitrate reductase genes in soybean: analysis in wild-type and nr1 mutant.

Among higher plants, soybean is unique in that biochemically it has been characterized as having two constitutive nitrate reductase (cNR) isoforms and one substrate-inducible nitrate reductase (iNR) isoform in leaves. All three NR isoforms are expressed in cv. Williams 82 while the nr1 mutant expresses only the iNR isoform. The genetic and molecular mechanisms for regulation of these isoforms have not been elucidated. We describe here the isolation, by reverse transcription-polymerase chain reaction (RT-PCR), of two cDNA clones encoding soybean NR. They were designated as iNR1 and iNR2, respectively, since both were inducible by nitrate. The iNR1 and iNR2 cDNAs cover total encoding regions of 2661 and 2673 nucleotides, respectively. The iNR1 clone shows a 12 bp deletion at the 5' end, relative to iNR2. They show overall similarity of 89% at the nucleotide level, and 87% at the amino acid level. Like all plant NRs cloned so far, deduced amino acid sequences between iNR1 and iNR2 show greatest variation at the N-terminal region while no difference was observed at the C-terminus. Soybean iNR mRNAs were found to be different from those of maize and tobacco in response to tungsten inhibitor treatment, since the inhibitor decreased the steady-state levels of mRNA for soybean iNR and for NiR. Using the same 5' regions of both cDNAs as the probes, Southern blot analysis of genomic DNA revealed differences in organization between iNR1 and iNR2. The genomic DNA from wild-type Williams 82 soybean was shown to have three Eco RI fragments while the nr1 mutant lacked an 8 kb fragment when probed with iNR1 cDNA. Likewise, the nr1 mutant lacked three Hae III restriction fragments when probed with iNR1 cDNA. When probed with iNR2, both wildtype and nr1 mutant showed one identical Eco RI band and two identical Hae III bands. In northern blots, the steady-state level of iNR1 mRNA was similar for the nr1 mutant and the wild-type parent after 20 to 48 h induction by nitrate. Based on the Eco RI and Hae III restriction enzyme digestion patterns observed in Southern blot analysis of soybean DNA, it is concluded that in soybean iNR1 is encoded by a small multiple gene family and iNR2 is a single gene.

Organization and expression of the Rhodobacter sphaeroides cycFG operon.

The Rhodobacter sphaeroides cycFG operon has been cloned, sequenced, and mapped to approximately coordinate 2500 of chromosome I. The cycF gene encodes cytochrome c554, a member of the class II family of soluble cytochrome c proteins. The cycF open reading frame includes a 20-amino acid extension at its N terminus which has not been detected in cytochrome c554. Antiserum against cytochrome c554 shows that this protein is localized to the periplasm of wild-type cells, which suggests that this N-terminal extension functions as a signal peptide. The predicted cycG gene product is a diheme cytochrome c with a subunit molecular mass of approximately 32 kDa. While a cytochrome with the properties predicted for CycG has not been reported for R. sphaeroides, we have tentatively identified this protein as a heme-staining polypeptide that is associated with membranes. CycG could have an overall structure similar to that of several other electron carriers, since the similarity between the predicted amino acid sequence of CycG and other multiheme cytochrome c proteins extends throughout the polypeptide. The cycFG transcript is approximately 1,500 nucleotides long and has a single 5' end 26 nucleotides upstream of the start of cycF translation. Expression of cycFG is regulated at the level of mRNA accumulation, since approximately fivefold-higher levels of both cycF-specific transcript and cytochrome c554 protein are detected in cell extracts from aerobic cultures in comparison with those from anaerobically grown cells. Although cytochrome c554 was detected under all growth conditions tested, the highest levels of this protein were found when cells generate energy via aerobic respiration.

Post-transcriptional regulation of nitrate reductase by light is abolished by an N-terminal deletion.

Higher plant nitrate reductases (NRs) carry an N-terminal domain whose sequence is not conserved in NRs from other organisms. A gene composed of a full-length tobacco NR cDNA with an internal deletion of 168 bp in the 5' end fused to the cauliflower mosaic virus 35S promoter and appropriate termination signals was constructed and designated as delta NR. An NR-deficient mutant of Nicotiana plumbaginifolia was transformed with this delta NR gene. In transgenic plants expressing this construct, NR activity was restored and normal growth resulted. Apart from a higher thermosensitivity, no appreciable modification of the kinetic parameters of the enzyme was detectable. The post-transcriptional regulation of NR by light was abolished in delta NR transformants. Consequently, deregulated production of glutamine and asparagine was detected in delta NR transformants. The absence of in vitro delta NR activity modulation by ATP suggests the impairment of delta NR phosphorylation and thereby suppression of delta NR post-translational regulation. These data imply that post-transcriptional control of NR expression is important for the flow of the nitrate assimilatory pathway.

The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch.

Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis.

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.

Integration of a cyanobacterial protein involved in nitrate reduction (narB) into isolated Synechococcus but not into pea thylakoid membranes.

Chimeric genes comprised of Rubisco small subunit transit peptide fused in frame with full-length and truncated sequences of a nitrate reductase (narB) structural gene of Synechococcus were constructed. Fusion proteins were synthesized in a rabbit reticulocyte system. In thylakoido integration of synthetic proteins resulted in the association of the full-length narB-coded protein to the Synechococcus photosynthetic membranes. The membrane-associated protein was sensitive to trypsin treatment but could not be removed by washing in the presence of NaBr. Trypsin pretreatment of thylakoids abolished the capability for association. The association of the narB-coded protein with thylakoids might require another membrane protein whose identity is not known. It is proposed that the Synechococcus narB polypeptide is a peripheral, membrane bound protein anchored to the thylakoids via a short hydrophobic domain while the major part of the protein resides on the outer side of the thylakoid membranes. The chimeric narB proteins were processed and imported by intact pea chloroplasts in vitro; however, the mature proteins were found localized in the stroma and not in the thylakoid membrane fraction. Similarly, the attempt to integrate the protein in vitro into isolated pea thylakoid membranes failed although these membranes incorporate early light-inducible proteins.

Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites.

Nitrate (NR) and nitrite reductase (NiR) catalyse the reduction of nitrate to ammonium. The regulation of NR and NiR gene expression by carbohydrates (C) and nitrogen (N) metabolites was studied using detached leaves. In the dark, glucose fructose and sucrose supplied to detached green leaves of dark-adapted Nicotiana plumbaginifolia plants resulted in NR mRNA and protein accumulation and the loss of circadian rhythmicity in the size of the transcript pool. The characterization of transgenic plants expressing either a NR cDNA controlled by the 35S CaMV promoter or a transcriptional fusion between the tobacco nia1 (NR structural gene) promoter and the beta-glucuronidase reporter gene, led us to conclude that C metabolite control is taking place at the transcriptional level. Under low light conditions (limiting photosynthetic conditions), the supply of glutamine or glutamate resulted in a drop in the level of NR mRNA. Exogenously supplied carbohydrates partially antagonized this inhibitory effect suggesting that the availability of N and C metabolites affects the expression of the NR gene. The effects of carbohydrates and glutamine on NiR expression were also studied. NiR mRNA levels in the dark were relatively insensitive to feeding with glucose. Glutamate and glutamine were less efficient at decreasing NiR mRNA than NR mRNA levels. In contrast to NR, NiR mRNA levels were significantly increased by light treatments, indicating that NiR display regulatory characteristics reminiscent of photosynthetic genes such as the small subunit of ribulose bisphosphate carboxylase than to NR.

The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics.

The nitrate reductase (NR) structural gene (nitA) of Volvox carteri has been cloned and characterized. There is a single copy of this gene in the genome, and RFLP (restriction-fragment length polymorphism) analysis assigns it to the previously defined nitA/chlR locus on linkage group IX, 20-30 cM from the two beta-tubulin-encoding loci. Determination of the 5871-nt sequence of the coding region of genomic clones, and comparisons to a cDNA sequence, revealed ten introns and eleven exons that encode a 864-aa polypeptide. Detailed comparisons with higher-plant and fungal NRs indicate that, whereas the aa sequence is strongly conserved within functional domains for the flavin adenine dinucleotide-, heme- and molybdenum-pterin cofactor-binding sites, substantial differences in the aa sequence occur in the N-terminal end and the two inter-domain regions. Two potential transcription start points 439 and 452 nt upstream from the start codon and a polyadenylation signal 355 nt downstream from the stop codon have been identified by primer-extension analysis and cDNA sequencing, respectively. Accumulation of the nitA transcript is both induced by nitrate and repressed by ammonium and urea: after the organism is transferred from ammonium to nitrate as the nitrogen source, a 3.6-kb NR transcript is readily detectable on Northern blots by 10 min, reaches maximum abundance by 30 min, and then rapidly declines to an intermediate level that is subsequently maintained. Substantial induction by nitrate is observed at the end of the dark portion of the daily light/dark cycle, but the inductive response peaks in the first hour of the light period.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli.

Two membrane-bound nitrate reductases, NRA and NRZ, exist in Escherichia coli. Both isoenzymes are composed of three structural subunits, alpha, beta, and gamma encoded by narG/narZ, narH/narY and narI/narV, respectively. The genes are in transcription units which also contain a fourth gene encoding a polypeptide, delta, which is not part of the final enzyme. A strain which is devoid of, or does not express, the nar genes, was used to investigate the role of the delta and gamma polypeptides in the formation and/or processing of the nitrate reductase. When only the alpha and beta polypeptides are produced, an (alpha beta) complex exists which is inactive and soluble. When the alpha, beta and delta polypeptides are produced, the (alpha beta) complex is active with artificial donors such as benzyl viologen but is soluble. When the alpha, beta and gamma polypeptides are produced, the (alpha beta) complex is inactive but partially binds the membrane. It was concluded that the gamma polypeptide is involved in the binding of the (alpha beta) complex to the membrane while the delta polypeptide is indispensable for the (alpha beta) nitrate reductase activity. The activation by the delta polypeptide does not seem to involve the insertion of the redox centres of the enzyme since the purified inactive (alpha beta) complex was shown to contain the four iron-sulphur centres and the molybdenum cofactor, which are normally present in the native purified enzyme. The extreme sensitivity of this inactive complex to thermal denaturation or tryptic treatment favours the idea that the delta polypeptide promotes the correct assembly of the alpha and beta subunits. Although this corresponds to the definition of a chaperone protein this possibility has been rejected. In this study we have also demonstrated that the delta or gamma polypeptide encoded by one nar operon can be substituted successfully for by its respective counterpart from the other nar operon to give an active membrane bound heterologous nitrate reductase enzyme.