Post-Translational Modifications of Nitrate Reductases Autoregulates Nitric Oxide Biosynthesis in Arabidopsis.

Nitric oxide (NO) is a regulator of growth, development, and stress responses in living organisms. Plant nitrate reductases (NR) catalyze the reduction of nitrate to nitrite or, alternatively, to NO. In plants, NO action and its targets remain incompletely understood, and the way NO regulates its own homeostasis remains to be elucidated. A significant transcriptome overlapping between NO-deficient mutant and NO-treated wild type plants suggests that NO could negatively regulate its biosynthesis. A significant increase in NO content was detected in transgenic plants overexpressing NR1 and NR2 proteins. In turn, NR protein and activity as well as NO content, decreased in wild-type plants exposed to a pulse of NO gas. Tag-aided immunopurification procedures followed by tandem mass spectrometry allowed identifying NO-triggered post-translational modifications (PTMs) and ubiquitylation sites in NRs. Nitration of tyrosine residues and S-nitrosation of cysteine residues affected key amino acids involved in binding the essential FAD and molybdenum cofactors. NO-related PTMs were accompanied by ubiquitylation of lysine residues flanking the nitration and S-nitrosation sites. NO-induced PTMs of NRs potentially inhibit their activities and promote their proteasome-mediated degradation. This auto-regulatory feedback loop may control nitrate assimilation to ammonium and nitrite-derived production of NO under complex environmental conditions.

Phylogenetic diversity of nitrogen-utilizing genes in hydrothermal chimneys from 3 middle ocean ridges.

Nitrogen-metabolizing genes, including nitrogenase (nifH), periplasmic nitrate reductase (napA), and cytochrome cd 1-type nitrite reductase (nirS), were collected from hydrothermal chimney sulfides on 3 middle ocean ridges and compared for the first time. There was a clear phylogenetic distinction of these nifH genes between different hydrothermal ecosystems, which supported the colonization and potential adaptation by different nitrogen fixing microbes in those sulfides. In particular, in sulfides from low-temperature hydrothermal vents of the Southwest Indian Ocean Ridge, the prevalence of nifH genes appears to be attributed to sulfate-reducing bacteria, suggesting their ecological significance. Phylogenetic analysis of nitrate/nitrite reductase genes indicated that nitrate was a critical electron acceptor for sulfur- or metal-oxidizing bacteria in these hydrothermal ecosystems. Our results provided information about the compositions and diversity of the 3 important genes involved in nitrogen fixation and nitrate/nitrite reduction processes in hydrothermal ecosystems and is the first comprehensive genetic repertoire of genes related to potential nitrogen fixation and denitrification processes in various hydrothermal environments.

High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

Effect of short-term salinity on the nitrate reductase activity in cucumber roots.

In short-term experiments, the effect of high salinity on cucumber (Cucumis sativus) nitrate reductase activity was studied. The 60-min exposure of cucumber roots to 200 mM NaCl resulted in significant increase of the actual NR activity (measured in the presence of Mg²+), whereas the total enzyme activity (measured with EDTA) was not affected. NaCl-induced stimulation of the actual NR activity was rapidly reversed upon transfer of roots to salt-free solution. The increase in actual activity was completely prevented by microcystin-LR and cantharidin, protein phosphatases inhibitors. In addition, a significant decrease in ATP level was also observed in roots incubated with NaCl. These data suggest that the reversible protein phosphorylation is involved in the induction of NR activity during the first hour of salt stress. The effect of short-term salinity on the expression of genes encoding for nitrate reductase in cucumber roots was also studied. 200 mM NaCl diminished the increase in CsNR1 expression observed in control roots. During the same time period, the expression of CsNR2 was not affected, whereas the expression of CsNR3 decreased significantly after 1h incubation of the excised roots in both, control and salt-containing nutrient solutions. Incubation of roots in the presence of iso-osmotic concentration of PEG had no effect on both, NR activity and expression. This indicates that only the ionic component of salt stress was involved in the salt-induced modifications of nitrate reductase activity.

Periplasmic nitrate reduction in Wolinella succinogenes: cytoplasmic NapF facilitates NapA maturation and requires the menaquinol dehydrogenase NapH for membrane attachment.

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periplasmic nitrate reductase NapA. The composition of the corresponding nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap loci predict accessory proteins such as the iron-sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium Wolinella succinogenes was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a napH deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron-sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CX(3)CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

Dissimilatory iron reduction in Escherichia coli: identification of CymA of Shewanella oneidensis and NapC of E. coli as ferric reductases.

Over geological time scales, microbial reduction of chelated Fe(III) or Fe(III) minerals has profoundly affected today's composition of our bio- and geosphere. However, the electron transfer reactions that are specific and defining for dissimilatory iron(III)-reducing (DIR) bacteria are not well understood. Using a synthetic biology approach involving the reconstruction of the putative electron transport chain of the DIR bacterium Shewanella oneidensis MR-1 in Escherichia coli, we showed that expression of cymA was necessary and sufficient to convert E. coli into a DIR bacterium. In intact cells, the Fe(III)-reducing activity was limited to Fe(III) NTA as electron acceptor. In vitro biochemical analysis indicated that CymA, which is a cytoplasmic membrane-associated tetrahaem c-type cytochrome, carries reductase activity towards Fe(III) NTA, Fe(III) citrate, as well as to AQDS, a humic acid analogue. The in vitro specific activities of Fe(III) citrate reductase and AQDS reductase of E. coli spheroplasts were 10x and 30x higher, respectively, relative to the specific rates observed in intact cells, suggesting that access of chelated and insoluble forms of Fe(III) and AQDS is restricted in whole cells. Interestingly, the E. coli CymA orthologue NapC also carried ferric reductase activity. Our data support the argument that the biochemical mechanism of Fe(III) reduction per se was not the key innovation leading to environmental relevant DIR bacteria. Rather, the evolution of an extension of the electron transfer pathway from the Fe(III) reductase CymA to the cell surface via a system of periplasmic and outer membrane cytochrome proteins enabled access to diffusion-impaired electron acceptors.

Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon.

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was -27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.

Nitrate reduction by Desulfovibrio desulfuricans: a periplasmic nitrate reductase system that lacks NapB, but includes a unique tetraheme c-type cytochrome, NapM.

Many sulphate reducing bacteria can also reduce nitrite, but relatively few isolates are known to reduce nitrate. Although nitrate reductase genes are absent from Desulfovibrio vulgaris strain Hildenborough, for which the complete genome sequence has been reported, a single subunit periplasmic nitrate reductase, NapA, was purified from Desulfovibrio desulfuricans strain 27774, and the structural gene was cloned and sequenced. Chromosome walking methods have now been used to determine the complete sequence of the nap gene cluster from this organism. The data confirm the absence of a napB homologue, but reveal a novel six-gene organisation, napC-napM-napA-napD-napG-napH. The NapC polypeptide is more similar to the NrfH subgroup of tetraheme cytochromes than to NapC from other bacteria. NapM is predicted to be a tetra-heme c-type cytochrome with similarity to the small tetraheme cytochromes from Shewanella oneidensis. The operon is located close to a gene encoding a lysyl-tRNA synthetase that is also found in D. vulgaris. We suggest that electrons might be transferred to NapA either from menaquinol via NapC, or from other electron donors such as formate or hydrogen via the small tetraheme cytochrome, NapM. We also suggest that, despite the absence of a twin-arginine targeting sequence, NapG might be located in the periplasm where it would provide an alternative direct electron donor to NapA.

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.

Partial sequences of nitrogen metabolism genes in hexaploid wheat.

Our objective was to partially sequence genes controlling nitrogen metabolism in wheat species in order to find sequence polymorphism that would enable their mapping. Primers were designed for nitrate reductase, nitrite reductase, glutamate dehydrogenase and glutamate synthase (GOGAT), and gene fragments were amplified on Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii. We obtained more than 8 kb of gene sequences, mainly as coding regions (60%). Polymorphism was quantified by comparing two-by-two the three genomes of the hexaploid cultivar Arche and genomes of diploid wheat species. On average, the polymorphism rate was higher for non-coding regions, where it ranged from 1/60 to 1/23, than for coding regions (range: 1/110-1/40) except when the hexaploid D genome was compared to that of T. tauschii (1/800 and 1/816, respectively). Genome-specific primers were devised for the ferredoxin-dependent (Fd)-GOGAT gene, and they enabled the mapping of this gene on homoeologous chromosomes of group 2 using Chinese Spring deletion lines. A single nucleotide polymorphism (SNP) detected between the two hexaploid wheat cultivars Arche and Recital was used to genetically map Fd-GOGAT on chromosome 2D using a population of dihaploid lines. Fd-GOGAT-specific primers were used to estimate the SNP rate on a set of 11 hexaploid and nine Durum wheat genotypes leading to the estimate of 1 SNP/515 bp. We demonstrate that polymorphism detection enables heterologous, homeologous and even paralogous copies to be assigned, even if the elaboration of specific primer pairs is time-consuming and expensive because of the sequencing.

Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris.

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

Expression of a deregulated tobacco nitrate reductase gene in potato increases biomass production and decreases nitrate concentration in all organs.

We investigated the physiological consequences for nitrogen metabolism and growth of the deregulated expression of an N-terminal-deleted tobacco nitrate reductase in two lines of potato (Solanum tuberosum L. cv Safrane). The transgenic plants showed a higher biomass accumulation, especially in tubers, but a constant nitrogen content per plant. This implies that the transformed lines had a reduced nitrogen concentration per unit of dry weight. A severe reduction in nitrate concentrations was also observed in all organs, but was more apparent in tubers where nitrate was almost undetectable in the transgenic lines. In leaves and roots, but not tubers, this nitrate decrease was accompanied by a statistically significant increase in the level of malate, which acts as a counter-anion for nitrate reduction. Apart from glutamine in tubers, no major changes in amino acid concentration were seen in leaves, roots or tubers. We conclude that enhancement of nitrate reduction rate leads to higher biomass production, probably by allowing a better allocation of N-resources to photosynthesis and C-metabolism.

Mechanism and importance of post-translational regulation of nitrate reductase.

In higher plants, nitrate reductase (NR) is inactivated by the phosphorylation of a conserved Ser residue and binding of 14-3-3 proteins in the presence of divalent cations or polyamines. A transgenic Nicotiana plumbaginifolia line (S521) has been constructed where the regulatory, conserved Ser 521 of tobacco NR (corresponding to Ser 534 in Arabidopsis) was mutated into Asp. This mutation resulted in the complete abolition of activation/inactivation in response to light/dark transitions or other treatments known to regulate the activation state of NR. Analysis of the transgenic plants showed that, under certain conditions, when whole plants or cut tissues are exposed to high nitrate supply, post-translational regulation is necessary to avoid nitrite accumulation. Abolition of the post-translational regulation of NR also results in an increased flux of nitric oxide from the leaves and roots. In view of the results obtained from examining the different transgenic N. plumbaginifolia lines, compartmentation of nitrate into an active metabolic pool and a large storage pool appears to be an important factor for regulating nitrate reduction. The complex regulation of nitrate reduction is likely to have evolved not only to optimize nitrogen assimilation, but also to prevent and control the formation of toxic, and possibly regulatory, products of NR activities. Phos phorylation of NR has previously been found to influence the degradation of NR in spinach leaves and Arabidopsis cell cultures. However, experiments with whole plants of N. plumbaginifolia, Arabidopsis, or squash are in favour of NR degradation being the same in light and darkness and independent of phosphorylation at the regulatory Ser.

A novel gene (narM) required for expression of nitrate reductase activity in the cyanobacterium Synechococcus elongatus strain PCC7942.

A new class of mutants deficient in nitrate assimilation was obtained from the cyanobacterium Synechococcus elongatus strain PCC7942 by means of random insertional mutagenesis. A 0.5-kb genomic region had been replaced by a kanamycin resistance gene cassette in the mutant, resulting in inactivation of two genes, one of which was homologous to the recently characterized cnaT gene of Anabaena sp. strain PCC7120 (J. E. Frías, A. Herrero, and E. Flores, J. Bacteriol. 185:5037-5044, 2003). While insertional mutation of the cnaT homolog did not affect expression of the nitrate assimilation operon or the activity of the nitrate assimilation enzymes in S. elongatus, inactivation of the other gene, designated narM, resulted in specific loss of the cellular nitrate reductase activity. The deduced NarM protein is a hydrophilic protein consisting of 161 amino acids. narM was expressed constitutively at a low level. The narM gene has its homolog only in the cyanobacterial strains that are capable of nitrate assimilation. In most of the cyanobacterial strains, narM is located downstream of narB, the structural gene of the cyanobacterial nitrate reductase, suggesting the functional link between the two genes. NarM is clearly not the structural component of the cyanobacterial nitrate reductase. The narM insertional mutant normally expressed narB, indicating that narM is not the transcriptional regulator of the structural gene of nitrate reductase. These results suggested that narM is required for either synthesis of the prosthetic group of nitrate reductase or assembly of the prosthetic groups to the NarB polypeptide to form functional nitrate reductase in cyanobacteria.

The role of nitrate reductase in the regulation of the nitrate assimilation pathway in the yeast Hansenula polymorpha.

The role of nitrate reductase (NR) in the regulation of the nitrate assimilation pathway was evaluated in the yeast Hansenula polymorpha. Posttranscriptional regulation of NR in response to reduced nitrogen sources and the effect of a heterologous NR on the transcriptional regulation of nitrate-assimilatory gene expression was examined. The strain bearing YNR1 (nitrate reductase gene) under the control of the methanol-induced MOX (methanol oxidase) promoter showed that NR is active in the presence of reduced nitrogen sources. In cells incubated with glutamine plus nitrate, rapamycin abolished nitrogen catabolite repression, NR activity being very similar to that in cells induced by nitrate alone. This reveals the involvement of the Tor-signalling pathway in the transcriptional regulation of H. polymorpha nitrate assimilation genes. To assess the role of NR in nitrate-assimilatory gene expression, different strains lacking YNR1, or both YNR1 and YNT1 (high-affinity nitrate transporter) genes, or expressing the tobacco NR under the YNR1 promoter, were used. Tobacco NR abolished the constitutive nitrate-assimilatory gene induction shown by an NR gene disruptant strain. Moreover, in strains lacking the high-affinity nitrate transporter and NR this deregulation disappeared. These facts discard the role of NR protein in the transcriptional induction of the nitrate-assimilatory genes and point out the involvement of the high-affinity nitrate transporter as a part of the nitrate-signalling pathway.

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation.

In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.

Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris.

In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed from most other higher-plant NRs by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099-1105]. In order to elucidate these deviating properties in more detail, the NR gene from R. communis was cloned, expressed heterologously and characterized. The deduced protein sequence showed that Ricinus NR has a serine phosphorylation site and a 14-3-3 binding motif, a common characteristic of NRs. Functional Ricinus NR protein was expressed in the yeast Pichia pastoris and compared with the features of Arabidopsis thaliana NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was not inactivated by incubation with MgATP. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3 proteins were prepared from 4-day-darkened (and therefore NR-free) leaves of Ricinus, and added to the assay of RcNR to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, its unusually high Mg2+-sensitivity was restored, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixture and ATP. Thus, insensitivity to ATP appears to be an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and appeared to interact specifically with recombinant Ricinus NR to provide the Mg2+-sensitivity of the authentic leaf enzyme.

Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18.

We present the 4.8-Mb complete genome sequence of Salmonella enterica serovar Typhi strain Ty2, a human-specific pathogen causing typhoid fever. A comparison with the genome sequence of recently isolated S. enterica serovar Typhi strain CT18 showed that 29 of the 4,646 predicted genes in Ty2 are unique to this strain, while 84 genes are unique to CT18. Both genomes contain more than 200 pseudogenes; 9 of these genes in CT18 are intact in Ty2, while 11 intact CT18 genes are pseudogenes in Ty2. A half-genome interreplichore inversion in Ty2 relative to CT18 was confirmed. The two strains exhibit differences in prophages, insertion sequences, and island structures. While CT18 carries two plasmids, one conferring multiple drug resistance, Ty2 has no plasmids and is sensitive to antibiotics.

The nitrate reductase and nitrite reductase operons and the narT gene of Staphylococcus carnosus are positively controlled by the novel two-component system NreBC.

In Staphylococcus carnosus, the nreABC (for nitrogen regulation) genes were identified and shown to link the nitrate reductase operon (narGHJI) and the putative nitrate transporter gene narT. An nreABC deletion mutant, m1, was dramatically affected in nitrate and nitrite reduction and growth. Transcription of narT, narGHJI, and the nitrite reductase (nir) operon was severely reduced even when cells were cultivated anaerobically without nitrate or nitrite. nreABC transcripts were detected when cells were grown aerobically or anaerobically with or without nitrate or nitrite. NreA is a GAF domain-containing protein of unknown function. In vivo and in vitro studies showed that NreC is phosphorylated by NreB and that phospho-NreC specifically binds to a GC-rich palindromic sequence to enhance transcription initiation. This binding motif was found at the narGHJI, nir, and narT promoters but not at the moeB promoter. NreB is a cytosolic protein with four N-terminal cysteine residues. The second cysteine residue was shown to be important for NreB function. In vitro autophosphorylation of NreB was not affected by nitrate, nitrite, or molybdate. The nir promoter activity was iron dependent. The data provide evidence for a global regulatory system important for aerobic and anaerobic metabolism, with NreB and NreC forming a classical two-component system and NreB acting as a sensor protein with oxygen as the effector molecule.

Tobacco Nia2 cDNA functionally complements a Hansenula polymorpha yeast mutant lacking nitrate reductase. A new expression system for the study of plant proteins involved in nitrate assimilation.

An integrative expression vector based on promoter and terminator transcriptional sequences from the Hansenula polymorpha nitrate reductase gene (YNR1) has been developed to express nitrate assimilation plant genes in the nitrate assimilatory yeast H. polymorpha. Using this vector a plant nitrate reductase cDNA (tobacco Nia2) was expressed for the first time in a nitrate assimilatory yeast. The heterologous nitrate reductase produced retained its biochemical and physiological properties such as its NADH-dependent nitrate reductase activity, and allowed growth in nitrate containing media in a strain lacking endogenous nitrate reductase activity. In the transgenic strain, maximum tobacco nitrate reductase activity was about 70% of that presented in the wild-type. On the other hand, the disappearance of nitrate reductase activity correlated with that of the enzyme protein in response to the addition of ammonium to the medium and took place more rapidly in the transgenic strain than in the wild-type. Nitrate reductase activity of the recombinant strain assayed in the presence of Mg2+ was about 30% of that observed when assayed with EDTA. This result, together with a decreased growth rate in nitrate, suggests that tobacco nitrate reductase could be partially inactivated in H. polymorpha by phosphorylation and binding of 14-3-3-like proteins. These results show that H. polymorpha is a useful yeast heterologous expression system for studying plant proteins involved in nitrate assimilation.