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.     

Nitrogen regulation of amino acid catabolism in Neurospora crassa

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.This work was supported by Grant R01 GM-23367 from the National Institutes of Health. T. J. F. was supported by an NIH Predoctoral Traineeship in Developmental Biology; G. A. M. is supported by NIH Career Development Award GM-00052.     

Nitrogen regulation of acid phosphatase in Neurospora crassa.

Neurospora crassa possesses a repressible acid phosphatase with phosphodiesterase activity which appears to permit it to utilize ribonucleic acid as a phosphorus and as a nitrogen source. This acid phosphatase, which is specified by the pho-3 locus, is derepressed approximately eightfold during nitrogen limitation and to an even greater extent during phosphorus limitation, but is unaffected by sulfur limitation. Derepression of the enzyme did not occur when adenosine 5'-monophosphate was the sole phosphorus or nitrogen source. Synthesis of the acid phosphatase is not under the control of the nit-2 locus, which regulates the expression of a large number of other nitrogen catabolic enzymes. The structural gene of the acid phosphatase appears to be a member of both the phosphorus and nitrogen regulatory circuits.     

Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa

A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed.     

A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain.

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.     

The regulatory gene nit-2 of Neurospora crassa complements a nnu mutant of Gibberella zeae (Fusarium graminearum).

Summary The nnu mutant of Gibberella zeae (= Fusarium graminearum) is unable to catabolize many of the nitrogen sources utilized by its wild-type parent, and may have suffered a mutation in the major nitrogen regulatory locus. Transformation of this mutant with the major nitrogen regulatory gene from Neurospora crassa, nit-2, restored the wild-type phenotype, thus confirming that the nnu mutation is in the major nitrogen regulatory locus of G. zeae. Our results are consistent with the premise of conservation of the structure of regulatory factors and suggest the possibility that functional DNA homologues of this regulatory element occur across a broad range of ascomycetous fungi.     

Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

Site-directed mutagenesis of the ''zinc finger'' DNA-binding domain of the nitrogen-regulatory protein NIT2 of Neurospora

The major nitrogen-regulatory gene nit-2 of Neurospora crassa activates the expression of numerous unlinked structural genes which specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein of 1036 amino acid residues with a single 'zinc finger' and a downstream basic region, which together may constitute a DNA-binding domain. The zinc finger domain of the NIT2 protein was synthesized in vitro and also expressed as a fusion protein in Escherichia coli to examine its DNA-binding activity. The wild-type NIT2 finger domain protein binds to the promoter region of nit-3, the nitrate reductase structural gene. A series of NIT2 mutant proteins obtained by site-directed mutagenesis was expressed and tested for functional activity. The results demonstrate that both the single zinc-finger motif and the downstream basic region of NIT2 are critical for its trans-activating function in vivo and specific DNA-binding in vitro.     

Molecular cloning of nit-2, a regulatory gene required for nitrogen metabolite repression in Neurospora crassa

We used an efficient sib-selection procedure to isolate a cosmid clone that complemented a mutated nit-2 gene of Neurospora crassa. Restriction fragment length polymorphism mapping indicated that the cosmid DNA insert was derived from linkage group IL, between 5S rDNA locus 12 and mt, the region of the N. crassa genome that contains nit-2. We conclude that the cosmid carries the nit-2 gene.     

Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa

The nitrogen regulatory circuit of Neurospora crassa contains structural genes that encode nitrogen catabolic enzymes which are subject to complex genetic and metabolic regulation. This set of genes is controlled by nitrogen limitation, by specific induction, and by the action of nit-2, a major positive-acting regulatory gene, and nmr, a negative-acting control gene. The complete nucleotide sequence of alc, the gene that encodes allantoicase, a purine catabolic enzyme, is presented. The alc gene contains a single intron, is transcribed from two initiation sites situated approximately 50 nb upstream of the translation start site, and encodes a protein comprised of 354 amino acids. Mobility shift and DNA footprint experiments identified a single binding site for the NIT2 regulatory protein in the alc promoter region. The binding site contains a 10 nucleotide base pair symmetrical sequence which is flanked by two possible core binding sequences, TATCT and TATCG. Mutant NIT2/beta-gal fusion proteins with amino acid substitutions in a putative zinc-finger motif were shown to be completely deficient in the ability to bind to the alc promoter DNA fragment.     

The global nitrogen regulator, FNR1, regulates fungal nutrition-genes and fitness during Fusarium oxysporum pathogenesis

Fusarium oxysporum is a soil-borne pathogen that infects plants through the roots and uses the vascular system for host ingress. Specialized for this route of infection, F. oxysporum is able to adapt to the scarce nutrient environment in the xylem vessels. Here we report the cloning of the F. oxysporum global nitrogen regulator, Fnr1 , and show that it is one of the determinants for fungal fitness during in planta growth. The Fnr1 gene has a single conserved GATA-type zinc finger domain and is 96% and 48% identical to AREA-GF from Gibberella fujikuroi , and NIT2 from Neurospora crassa , respectively. Fnr1 cDNA, expressed under a constitutive promoter, was able to complement functionally an N. crassa nit-2 ^RIP mutant, restoring the ability of the mutant to utilize nitrate. Fnr1 disruption mutants showed high tolerance to chlorate and reduced ability to utilize several secondary nitrogen sources such as amino acids, hypoxanthine and uric acid, whereas growth on favourable nitrogen sources was not affected. Fnr1 disruption also abolished in vitro expression of nutrition genes , normally induced during the early phase of infection. In an infection assay on tomato seedlings, infection rate of disruption mutants was significantly delayed in comparison with the parental strain. Our results indicate that FNR1 mediates adaptation to nitrogen-poor conditions in planta through the regulation of secondary nitrogen acquisition, and as such acts as a determinant for fungal fitness during infection.     

Sequence-specific DNA binding by NIT4, the pathway-specific regulatory protein that mediates nitrate induction in Neurospora

The expression of the structural genes nit-3 and nit-6, which encode the nitrate assimilatory enzymes nitrate reductase and nitrite reductase, respectively, is highly regulated by the global-acting NIT2 regulatory protein. These structural genes are also controlled by nitrogen catabolite repression and by specific induction via nitrate. A pathway-specific regulatory protein, NIT4, appears to mediate nitrate induction of nit-3 and of nit-6. The NIT4 protein, composed of 1090 amino acids, contains a putative GAL4-like Cys-6 zinc cluster DNA-binding motif, which is joined by a short segment to a stretch of amino acids that appear to constitute a coiled-coil dimerization domain. Chemical crosslinking studies demonstrated that a truncated form of NIT4 forms homodimers. Mobility-shift and DNA-footprinting experiments have identified two NIT4-binding sites of significantly different strengths in the promoter region of the nit-3 gene. The stronger binding site contains a symmetrical octameric sequence, TCCGCGGA, whereas the weaker site has a related sequence. Sequences related to this palindromic element can be found upstream of the nit-6 gene.