The positive-acting major nitrogen regulatory gene, nit-2, and the pathway-specific regulatory gene, nit-4, are each presumed to encode a regulatory protein which is required for the expression of nit-3 (encoding NR) and nit-6 (encoding NiR).

Two models for operation of the nitrogen control circuit are shown below. (a) The nit-2 gene is postulated to be expressed constitutively to yield a regulatory product active for turning on nit-3 and nit-6 unless it is inhibited by the metabolite repressor molecule, glutamine. (b) The nmr gene is visualized as producing a repressor protein which is activated by glutamine and precludes nit-2 gene expression. In the absence of glutamine, nit-2 is expressed and in turn activates nit-3 and nit-6 (Marzluf and Fu, 1989).

Nitrate reductase is expressed in a constitutive fashion in nmr mutant strains, which appear to be largely insensitive to nitrogen catabolite repression (Fu et al, 1988). nmr mutants are sensitive to chlorate in the presence of ammonia or glutamine, whereas the wild type is chlorate resistant under these conditions (Fu et al, 1988).

Ammonium repression of NR in blue-green algae and Neurospora is no longer evident in cells without an active glutamine synthetase (GS). Glutamine has been suggested to be the putative repressor of NR in Neurospora. Glutamine prevents the synthesis of the short half-life NR mRNA in Neurospora. Specific inhibitors of ammonium assimilation also block the ammonium inhibition of nitrate uptake in cyanobacteria (Guerrero et al, 1981). Two genes (glnA and glnN) encode glutamine synthetase (GS) in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 (Reyes and Florencio,1994). In a glnA-disrupted Synechocystis mutant in which the only GS activity is that corresponding to the glnN product [and is unable to grow in ammonium-containing medium because of its very low levels of GS activity], nitrate and nitrite reductases are no longer repressed by ammonium, and short-term ammonium-promoted inhibition of nitrate uptake is impaired (Reyes and Florencio, 1994). Thus, metabolic signals that control the nitrate assimilation system in Synechocystis require ammonium metabolism through GS (Reyes and Florencio, 1994).

In the cyanobacterium Synechococcus sp. strain PCC 7942, the phosphorylation states of the signal transducer PII protein (GlnB) can change rapidly depending on the nitrogen and carbon supply and is implicated in ammonium-dependent inhibition of the nitrate and nitrite uptake (Lee et al, 1998; 2000). Both unphosphorylated and phosphorylated-like forms of PII are able to inhibit nitrate uptake in the presence of ammonium, but the unphosphorylated form also has a negative effect in the absence of this nitrogen source (Lee et al, 2000). An additional effector (possibly 2-oxoglutarate) is required for the PII protein to relieve inhibition of nitrate uptake in the absence of ammonium (Lee et al, 2000).

Evidence has been obtained for NIT2-NIT4 protein-protein interactions in the regulation of expresion of nit-3 and nit-6 (Chiang and Marzluf, 1995). Expression of nit-3 is dependent upon a synergistic interaction of NIT2 with the pathway-specific control protein, NIT4. The NIT2 and NIT4 proteins both bind at specific recognition elements in the nit-3 promoter. A direct protein-protein interaction between NIT2 and NIT4 is essential for optimal expression of nit-3 (Feng and Marzluf, 1998). NIT4 consists of 1090 amino-acid residues, possesses a Cys6/Zn2 zinc cluster DNA-binding-domain, and is a member of the GAL4 family of fungal regulatory factors (Yuan et al, 1991; Yuan and Marzluf, 1992; Feng and Marzluf, 1996; 1998).

The NIT2 protein is also required to activate various other nitrogen-related structural genes. For example, the NIT2 regulatory protein binds to the alc promoter region: alc encodes allantoicase, a purine catabolic enzyme (Lee et al, 1990). The negative-acting nmr gene is involved in control of these activities and specifies a protein which interacts directly with the NIT2 protein. Thus, the negative-acting NMR protein interacts via specific protein-protein binding with two distinct regions of the NIT2 protein; a short alpha-helical motif within the NIT2 DNA-binding domain, and a second motif at its carboxy terminus (Pan et al, 1997).

Chiang TY, Marzluf GA 1995 Binding affinity and functional significance of NIT2 and NIT4 binding sites in the promoter of the highly regulated nit-3 gene, which encodes nitrate reductase in Neurospora crassa. J. Bacteriol. 177: 6093-6099.

Feng B, Marzluf GA 1996 The regulatory protein NIT4 that mediates nitrate induction in Neurospora crassa contains a complex tripartite activation domain with a novel leucine-rich, acidic motif. Curr. Genet. 29: 537-548.

Feng B, Marzluf GA 1998 Interaction between major nitrogen regulatory protein NIT2 and pathway-specific regulatory factor NIT4 is required for their synergistic activation of gene expression in Neurospora crassa. Mol. Cell. Biol. 18: 3983-3990.

Fu YH, Young JL, Marzluf GA 1988 Molecular cloning and characterization of a negative-acting nitrogen regulatory gene of Neurospora crassa. Mol. Gen. Genet. 214: 74-79.

Guerrero MG, Vega JM, Losada M 1981 The assimilatory nitrate-reducing system and its regulation. Annu. Rev. Plant Physiol. 32: 169-204.

Lee H, Fu YH, Marzluf GA 1990 Nucleotide sequence and DNA recognition elements of alc, the structural gene which encodes allantoicase, a purine catabolic enzyme of Neurospora crassa. Biochemistry 29: 8779-8787.

Lee HM, Flores E, Forchhammer K, Herrero A, Tandeau De Marsac N 2000 Phosphorylation of the signal transducer PII protein and an additional effector are required for the PII-mediated regulation of nitrate and nitrite uptake in the cyanobacterium Synechococcus sp. PCC 7942. Eur. J. Biochem. 267: 591-600.

Lee HM, Flores E, Herrero A, Houmard J, Tandeau de Marsac N 1998 A role for the signal transduction protein PII in the control of nitrate/nitrite uptake in a cyanobacterium. FEBS Lett. 427: 291-295.

Marzluf GA, Fu Y-H 1989 Genetics, regulation, and molecular studies of nitrate assimilation in Neurospora crassa. In (JL Wray, JR Kinghorn eds) "Molecular and Genetic Aspects of Nitrate Assimilation", Oxford Science Publications, Oxford, pp 315-327.

Pan H, Feng B, Marzluf GA 1997 Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa. Mol. Microbiol. 26: 721-729.

Reyes JC, Florencio FJ 1994 A mutant lacking the glutamine synthetase gene (glnA) is impaired in the regulation of the nitrate assimilation system in the cyanobacterium Synechocystis sp. strain PCC 6803. J. Bacteriol. 176: 7516-7523.

Yuan GF, Fu YH, Marzluf GA 1991 nit-4, a pathway-specific regulatory gene of Neurospora crassa, encodes a protein with a putative binuclear zinc DNA-binding domain. Mol. Cell Biol. 11: 5735-5745.

Yuan GF, Marzluf GA 1992 Transformants of Neurospora crassa with the nit-4 nitrogen regulatory gene: copy number, growth rate and enzyme activity. Curr. Genet. 22: 205-211.