Hypersensitive sites in the 5' promoter region of nit-3, a highly regulated structural gene of Neurospora crassa.

The nit-3 gene of the filamentous fungus Neurospora crassa encodes nitrate reductase, the enzyme which catalyzes the first step in nitrate assimilation. The nit-3 gene is subject to a high degree of regulation by metabolic inducers and repressors, and its expression requires two distinct trans-acting regulatory proteins. Hypersensitive sites in the 5' DNA sequence upstream of the nit-3 gene were mapped with the use of three different nucleases as molecular probes. Six hypersensitive sites, three of which are very strong, were detected at essentially identical positions by all three nucleases. The hypersensitive sites appear to develop in a constitutive fashion and are present under conditions in which the nit-3 structural gene is expressed but also when this gene is inactive, although these sites are considerably less prominent in cells subjected to nitrogen catabolite repression. The presence of the hypersensitive sites appears to depend upon both the positively acting NIT2 and the positively acting NIT4 regulatory proteins, which might play a role in positioning of chromatin protein.     

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.     

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.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

The Isolation and Characterization of Mutants Defective in Nitrate Assimilation in NEUROSPORA CRASSA

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth tests that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing co-factor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-7, nit-8 and nit-9), all of which are genetically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar to the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one locus (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.     

Ecological Physiology of Synechococcus sp. Strain SH-94-5, a Naturally Occurring Cyanobacterium Deficient in Nitrate Assimilation

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.     

Ecological physiology of Synechococcus sp. strain SH-94-5, a naturally occurring cyanobacterium deficient in nitrate assimilation.

Synechococcus sp. strain SH-94-5 is a nitrate assimilation-deficient cyanobacterium which was isolated from an ammonium-replete hot spring in central Oregon. While this clone could grow on ammonium and some forms of organic nitrogen as sole nitrogen sources, it could not grow on either nitrate or nitrite, even under conditions favoring passive diffusion. It was determined that this clone does not express functional nitrate reductase or nitrite reductase and that the lack of activity of either enzyme is not due to inactivation of the cyanobacterial nitrogen control protein NtcA. A few other naturally occurring cyanobacterial strains are also nitrate assimilation deficient, and phylogenetic analyses indicated that the ability to utilize nitrate has been independently lost at least four times during the evolutionary history of the cyanobacteria. This phenotype is associated with the presence of environmental ammonium, a negative regulator of nitrate assimilation gene expression, which may indicate that natural selection to maintain functional copies of nitrate assimilation genes has been relaxed in these habitats. These results suggest how the evolutionary fates of conditionally expressed genes might differ between environments and thereby effect ecological divergence and biogeographical structure in the microbial world.     

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.     

A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi

In higher plants, the expression of the nitrate assimilation pathway is highly regulated. Although the molecular mechanisms involved in this regulation are currently being elucidated, very little is known about the trans-acting factors that allow expression of the nitrate and nitrite reductase genes which code for the first enzymes in the pathway. In the fungus Neurospora crassa, nit-2, the major nitrogen regulatory gene, activates the expression of unlinked structural genes that specify nitrogen-catabolic enzymes during conditions of nitrogen limitation. The nit-2 gene encodes a regulatory protein containing a single zinc finger motif defined by the C-X₂-CX₁₇-C-X₂-C sequence. This DNA-binding domain recognizes the promoter region of N. crassa nitrogen-related genes and fragments derived from the tomato nia gene promoter. The observed specificity of the binding suggests the existence of a NIT2-like homolog in higher plants. PCR and cross-hybridization techniques were used to isolate, respectively, a partial cDNA from Nicotiana plumbaginifolia and a full-length cDNA from Nicotiana tabacum. These clones encode a NIT2-like protein (named NTL1 for nit-2-like), characterized by a single zinc finger domain, defined by the C-X₂-C-X₁₈-C-X₂-C amino acids, and associated with a basic region. The amino acid sequence of NTL1 is 60% homologous to the NIT2 sequence in the zinc finger domain. The Ntl1 gene is present as a unique copy in the diploid N. plumbaginifolia species. The characteristics of Ntl1 gene expression are compatible with those of a regulator of the nitrate assimilation pathway, namely weak nitrate inducibility and regulation by light.     

Regulation of nitrate reductase of Neurospora at the level of transcription and translation

The presence of nitrate is required for the induced synthesis of NADPH-nitrate reductase and its related partial activity Benzyl Viologen-nitrate reductase in a wild-type strain of Neurospora. In nit-3, a mutant lacking complete NADPH-nitrate reductase activity but retaining the partial activity Benzyl Viologen-nitrate reductase, the presence of nitrate ions is not required for the de-repressed synthesis of the latter enzyme. The accumulation of the capacity to synthesize nitrate reductase, and the related Benzyl Viologen-nitrate reductase, in the absence of protein synthesis does not require nitrate in the normal strain or in strain nit-3. Ammonia antagonizes the accumulation of this capacity in both strains. Nitrate is required for the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain. Nitrate is not required for the comparable function in strain nit-3. Ammonia appears to stop the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain and in strain nit-3. The effects of nitrate, or ammonia and of no nitrogen source on the induced synthesis of nitrate reductase cannot be explained on the basis of the effects of the different nitrogen sources on general synthesis of RNA or of protein.     

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.