Identification and Characterization of a Nitrate Transporter Gene in Neurospora crassa

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Nitrogen Utilization in Lemna: I. Relations between Net Nitrate Flux, Nitrate Reduction, and in Vitro Activity and Stability of Nitrate Reductase

Cultures of Lemna gibba L. G3 were maintained at a constant, N-limited growth rate by adding nitrate daily in amounts calculated to sustain a rate of culture N increment of 0.20 day⁻¹. Nitrate added to the culture was consumed within 8 to 10 hours and the partitioning to reduction and accumulation during this phase corresponded to, on the average, 75 and 25% of net uptake, respectively. The calculated rate of nitrate reduction was stimulated by onset of net uptake without delay and decreased when net uptake ceased. NADH-nitrate reductase (NR) activity measured in vitro without inclusion of antiproteolytic agents more than doubled during the first hour after nitrate addition and then gradually fell to its original level over the rest of the 24 hour interval. In the presence of the proteinase inhibitor leupeptin during extraction, however, NR activity was in general much higher and without any apparent cycles. The relative stabilizing effect of leupeptin was greatest on NADH-NR and reduced flavin adenine mononucleotide-NR activities whereas the effect was less on NADH-cytochrome c reductase activity (diaphorase) and reduced methylviologen-NR activity. The constant nitrate reductase activity measured in the presence of proteinase inhibitors is assumed to reflect the physiological situation. It thus appeares that short-term changes in nitrate assimilation by N-limited Lemna is related to the flux of nitrate to the reducing site and not to changes in nitrate reductase activity.     

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

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

Molecular Components of Nitrate and Nitrite Efflux in Yeast

Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.     

Evidence for multiple nitrate uptake systems in the yeast Hansenula polymorpha

Hansenula polymorpha mutants disrupted in the high-affinity nitrate transporter gene (YNT1) are still able to grow in nitrate. To detect the nitrate transporter(s) responsible for this growth a strain containing disruption of the nitrate assimilation gene cluster and expressing nitrate reductase gene (YNR1) under the control of H. polymorpha MOX1 (methanol oxidase) promoter was used (FM31 strain). In this strain nitrate taken up is transformed into nitrite by nitrate reductase and excreted to the medium where it is easily detected. Nitrate uptake which is neither induced by nitrate nor repressed by reduced nitrogen sources was detected in the FM31 strain. Likewise, nitrate uptake detected in the strain FM31 is independent of both Ynt1p and Yna1p and is not affected by ammonium, glutamine or chlorate. The inhibition of nitrite extrusion by extracellular nitrite suggests that the nitrate uptake system shown in the FM31 strain could also be involved in nitrite uptake.     

Identification and characterization of narQ, a second nitrate sensor for nitrate-dependent gene regulation in Escherichia coli

In response to nitrate availability, Escherichia coli regulates the synthesis of a number of enzymes involved in anaerobic respiration and fermentation. When nitrate is present, nitrate reductase (narGHJI) gene expression is induced, while expression of the DMSO/TMAO reductase (dmsABC), fumarate reductase (frdABCD) and fermentation related genes are repressed. The narL and narX gene products are required for this nitrate-dependent control, and apparently function as members of a two-component regulatory system. NarX is a presumed sensor-transmitter for nitrate and possibly molybdenum detection. The presumed response-regulator, NarL, when activated by NarX then binds at the regulatory DNA sites of genes to modulate their expression. In this study a third nitrate regulatory gene, narQ, was identified that also participates in nitrate-dependent gene regulation. Strains defective in either narQ or narX alone exhibited no nitrate-dependent phenotype whereas mutants defective in both narQ and narX were fully inactive for nitrate-dependent repression or activation. In all conditions tested, this regulation required a functional narL gene product. These findings suggest that the narX and narQ products have complementary sensor-transmitter functions for nitrate detection, and can work independently to activate NarL, for eliciting nitrate-dependent regulation of anaerobic electron transport and fermentation functions. The narQ gene was cloned, sequenced, and compared with the narX gene. Both gene products are similar in size, hydrophobicity, and sequence, and contain a highly conserved histidine residue common to sensor-transmitter proteins.     

Nitrogen Utilization in Lemna: II. Studies of Nitrate Uptake Using NO(3)

¹³N-labeled nitrate was used to trace short-term nitrate influx into Lemna gibba L. G3 in experiments where disappearance of both radioactivity and total nitrate from the incubation medium was measured continuously and simultaneously. In plants performing net nitrate uptake from an initial nitrate concentration of 40 to 60 micromolar, there was no discrepancy between net uptake and influx, irrespective of the N status of the plants, indicating that concomitant nitrate efflux was low or nil. Plants treated with tungstate to inactivate nitrate reductase were able to take up nitrate following induction of the uptake system by exposure to a low amount of nitrate. Also, in this case, net uptake was equivalent to influx. In tungstate-treated plants preloaded with nitrate, both net uptake and influx were nil. In contrast to these observations, a clear discrepancy between net uptake and influx was observed when the plants were incubated at an initial nitrate concentration of approximately 5 micromolar, where net uptake is low and eventually ceases. It is concluded that plasmalemma nitrate transport is essentially unidirectional in plants performing net uptake at a concentration of 40 to 60 micromolar, and that transport is nil when internal nitrate sinks (vacuole, metabolism) are eliminated. The efflux component becomes increasingly important when the external concentration approaches the threshold value for net nitrate uptake (the nitrate compensation point) where considerable exchange between internal and external nitrate occurs.     

Microarray analysis of the nitrate response in Arabidopsis roots and shoots reveals over 1,000 rapidly responding genes and new linkages to glucose,trehalose-6-phosphate,iron, and sulfate metabolism

The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 microm nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism.     

Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.     

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

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

Genetic evidence that NarL function is not required for nitrate regulation of nitrate assimilation in Klebsiella pneumoniae M5al.

We cloned the narL gene, required for nitrate induction of respiratory nitrate reductase synthesis, from Klebsiella pneumoniae. The E. coli narL gene product shares sequence similarity with the response regulator proteins of two-component regulatory systems. We found that narL(+)-containing plasmids restored nitrate regulation of anaerobic respiratory gene expression in appropriate Escherichia coli hosts. The K. pneumoniae narL region encoded a protein whose migration in Laemmli gels was indistinguishable from that of the narL product of E. coli. We constructed a narL::Km mutant of K. pneumoniae. This mutation abolished nitrate induction of respiratory nitrate reductase synthesis but had no effect on nitrate induction of assimilatory nitrate and nitrite reductase synthesis. We conclude that K. pneumoniae has distinct nitrate-responsive regulators for controlling respiratory and assimilatory gene expression.     

Cystine reductase in the dimorphic fungus Histoplasma capsulatum.

Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.     

Interaction between electron transport at the plasma membrane and nitrate uptake by maize (Zea mays L.) roots

Summary In the present study nitrate uptake by maize (Zea mays L.) roots was investigated in the presence or absence of ferricyanide (hexacyanoferrate III) or dicumarol. Nitrate uptake caused an alkalization of the medium. Nitrate uptake of intact maize seedlings was inhibited by ferricyanide while the effect of dicumarol was not very pronounced. Nitrite was not detected in the incubation medium, neither with dicumarol-treated nor with control plants after application of 100 M nitrate to the incubation solution. In a second set of experiments interactions between nitrate and ferricyanide were investigated in vivo and in vitro. Nitrate (1 or 3 mM) did neither influence ferricyanide reductase activity of intact maize roots nor NADH-ferricyanide oxidoreductase activity of isolated plasma membranes. Nitrate reductase activity of plasma-membrane-enriched fractions was slightly stimulated by 25 M dicumarol but was not altered by 100 M dicumarol, while NADH-ferricyanide oxidoreductase activity was inhibited in the presence of dicumarol. These data suggest that plasma-membrane-bound standard-ferricyanide reductase and nitrate reductase activities of maize roots may be different. A possible regulation of nitrate uptake by plasmalemma redox activity, as proposed by other groups, is discussed.Abbreviations ADH alcohol dehydrogenase - HCF III hexacyanoferrate III (ferricyanide) - ME NADP-dependent malic enzyme - NR nitrate reductase - PM plasma membrane - PM NR nitrate reductase copurifying with plasma membranes     

Nitrate uptake,nitrate reductase distribution and their relation to proton release in five nodulated grain legumes

Nitrate uptake, nitrate reductase activity (NRA) and net proton release were compared in five grain legumes grown at 0.2 and 2 mM nitrate in nutrient solution. Nitrate treatments, imposed on 22-d-old, fully nodulated plants, lasted for 21 d. Increasing nitrate supply did not significantly influence the growth of any of the species during the treatment, but yellow lupin (Lupinus luteus) had a higher growth rate than the other species examined. At 0.2 mM nitrate supply, nitrate uptake rates ranged from 0.6 to 1.5 mg N g(-1) d(-1) in the order: yellow lupin > field pea (Pisum sativum) > chickpea (Cicer arietinum) > narrow-leafed lupin (L angustifolius) > white lupin (L albus). At 2 mM nitrate supply, nitrate uptake ranged from 1.7 to 8.2 mg N g(-1) d(-1) in the order: field pea > chickpea > white lupin > yellow lupin > narrow-leafed lupin. Nitrate reductase activity increased with increased nitrate supply, with the majority of NRA being present in shoots. Field pea and chickpea had much higher shoot NRA than the three lupin species. When 0.2 mM nitrate was supplied, narrow-leafed lupinreleased the most H+ per unit root biomass per day, followed by yellow lupin, white lupin, field pea and chickpea. At 2 mM nitrate, narrow-leafed lupin and yellow lupin showed net proton release, whereas the other species, especially field pea, showed net OH- release. Irrespective of legume species and nitrate supply, proton release was negatively correlated with nitrate uptake and NRA in shoots, but not with NRA in roots.