The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory to the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

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

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The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-lb, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.Nitrate reductase from Neurospora crassa, the first enzyme in the assimilation of nitrate, is induced by nitrate (12,16,25) and repressed by ammonium (12,25,29) by what seems to be a form of nitrogen metabolite repression (1,20). The mechanism of "ammonium repression" of nitrate reductase in Neurospora has been the subject of several recent investigations (6,9,19). Dantzig et al. (6) found that in a N. crassa mutant lacking NADP-glutamate dehydrogenase, ammonium was unable to repress the formation of nitrate reductase, although certain amino acids could still repress the enzyme. This observation led the authors to suggest that ammonium repression was, in fact, mediated by a nitrogen metabolite(s) other than ammonium ions, an idea that is not new (3,5,8). Studies in our laboratory with a glutamine auxotroph of N. crassa, identified glutamine (or a close metabolite of glutamine) as a corepressor of nitrate reductase (19). Dunn-Coleman et al. (9) confirmed our results by a similar approach. They hypothesized that octameric glutamine synthetase, the predominant form of the enzyme under conditions of nitrogen sufficiency (9, 18), is the putative repressor.In the present study, the situation obtained in the glutamine auxotroph, namely the inability to convert ammonia to glutamine, is simulated in the wild-type strain by the use of L-methionine-DL-sulfoximine (MSX). This drug, long known to be an inhibitor ofglutamine synthetase (17), is phosphorylated on the enzyme to form methionine sulfoximine phosphate, which binds tightly to the active site (13,(21)(22)(23). The data indica,te that MSX inhibits the synthetase by acting as an analog of the tetrahedral intermediate or the transition state (15,23,30). In this study we attempt to demonstrate that when glutamine synthetase is inactivated in the above fashion, ammonium ions do not repress nitrate reductase in Neurospora, whereas glutamine still does repress the reductase. We also propose to demonstrate that in glutamine auxotropic Neurospora strain gln-lb glutamine represses the reductase.MATERIALS AND METHODS Neurospora strains. All strains except glutamine auxotroph gln-lb came from the fungal genetics stock center at Humboldt State University Foundation, Arcata, Calif. Strain 3-la (FGSC 935) was used as the wild-type strain. The mutant strain used was kindly provided by R. H. Garrett, University of Virginia, Charlottesville, Va. This mutant recently has been shown to have an impaired glutamine synthetase (7)...