Physiological characterization of a Neurospora crassa mutant with impaired regulation of nitrate reductase

Abstract

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation. Nitrate reductase from Neurospora crassa, the first enzyme in the assimilation of nitrate, is induced by nitrate (15, 18, 24) and repressed by ammonium (15, 18, 31), by what seems to be a form of nitrogen metabolite regulation (1, 22, 23, 33). There is evidence that the nitrogen metabolite preventing the induction of nitrate reductase is glutamine (8, 20). The isolation and characterization of mutants insensitive to the above effect of glutamine (ammonium) in these systems should contribute to our understanding of the mechanism of repression. In this report, we describe the isolation and physiological characterization of one such mutant. MATERIALS AND METHODS N. cras8a strains. The strains of N. crassa came either from the Fungal Genetics stock center at Humboldt State University, Arcata, Calif., or from our own collection. Strain 3.la (FGSC no. 935) was used as the wild-type strain. Strain gin-lb, a glutamine-requiring mutant, was obtained from R. Garrett (Department of Biology, University of Virginia, Charlottesville). The other mutant strains used were nit-3A (FGSC no. 3009), nit-4A (FGSC no. 2993), nit-5A (FGSC no. 985), and NR37.6A (FGSC no. 983), which is allelic to nit-2 (28). There is no induced formation of nitrate or nitrite reductase in mutants nit-4A, nit-5A, and NR37.6A; hence, these are postulated to be regulatory mutants. Mutant nit-3A, which is thought to be the structural gene for nitrate reductase, produces an altered nitrate reductase possessing only a partial enzymatic activity, namely, reduced benzyl viologen-nitrate reductase. Culture conditions. The basic medium without a

Two distinct protein–protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

“…Mutation analysis has demonstrated that the NMR protein is required to establish nitrogen repression, as nmr mutants are largely insensitive to sufficient glutamine to exert full repression of nitrate reductase synthesis in nmr þ (Premakumar et al, 1980;Tomsett et al, 1981). One intriguing possibility is that the 12-amino-acid carboxy-terminal tail shown above to be essential for nitrogen repression represents a motif that mediates specific NIT2-NMR proteinprotein binding.…”

“…When the cells have an adequate supply of a primary nitrogen source, ammonium ion, glutamate or glutamine, NIT2 does not activate transcription of nit-3 or other genes encoding nitrogen catabolic enzymes, a consequence of nitrogen metabolite repression (Fu and Marzluf, 1987). Mutations in another gene, nmr (for 'nitrogen metabolite repression'), result in the loss of nitrogen repression; nitrate reductase and other related enzymes are expressed in nmr mutants when the cells have sufficient glutamine or ammonia to inhibit their synthesis in the wild type completely (Premakumar et al, 1980;Tomsett et al, 1981). These observations imply that the nmr gene product functions in some aspect of repression.…”

Characterization of the Aspergillus nidulans nmrA Gene Involved in Nitrogen Metabolite Repression

“…In N. crassa, the gene nmr-1 has been found to be important for the response to nitrogen metabolite repression. Recessive mutations in this gene result in derepression of some nitrogencontrolled activities, suggesting a negative role for the gene (16,31,32,37). Cloning and characterization of this gene have enabled studies of its role in nitrogen metabolite repression (19,23,24,40).…”

Regulation of amino acid utilization in Neurospora crassa: effect of nmr-1 and ms-5 mutations

“…The nmr-I strain of N. crassa has been characterized by its ability to produce nitrate reductase in the presence of the nitrogen metabolite repressor glutamine, which normally prevents synthesis of enzymes involved with the utilization of secondary nitrogen sources such as nitrate (13,25). The ms-S strain has a similar phenotype except that glutamine repression of nitrite reductase, histidase, acetamidase, and L-amino acid oxidase is additionally eliminated (4,21). One possible explanation for the failure of glutamine to repress is that these mutations interfere with glutamine entry into cells.…”

“…The nmr-l locus has been defined through its release of nitrate reductase from glutamine-mediated nitrogen metabolite repression (13,25). The ms-5 locus similarly relieves glutamine-mediated repression of nitrate reductase production but additionally allows nitrite reductase, histidase, acetamidase, and L-amino acid oxidase production in the presence of glutamine (4,21).…”