Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b

Abstract

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)...

Regulation of nitrogen metabolism and gene expression in fungi.

“…Under conditions where GSase of wild-type strains was inactivated by MSX, only glutamine could repress nitrate reductase synthesis. Ammonia was transported and accumulated intracellularly but could not evoke nitrogen repression in wild-type cells lacking GSase activity (75).…”

“…Although this issue has not been settled, certain evidence suggests that in Neurospora glutamine is the effector metabolite. Premakumar et al (73 ,75) have examined the effect that various nitrogen metabolites have on the induced rate of appearance of nitrate reductase. Such studies are complicated, since many points exist at which a metabolite might have an effect on nitrate reduction expression.…”

Deletion of the Gibberella fujikuroi glutamine synthetase gene has significant impact on transcriptional control of primary and secondary metabolism

“…nidulans and N . crassa , respectively, have led to the suggestion that beside glutamine GS itself might have a regulatory role in nitrogen metabolite repression (Dunn-Coleman and Garrett, 1980;1981;Premakumar et al ., 1980;MacDonald, 1982;Cornwell and MacDonald, 1984). Recently, glnA oblation was achieved by gene replacement from A .…”

Regulation of Expression of Nitrate and Dinitrogen Assimilation by Anabaena Species

“…In cyanobacteria, exogenous ammonium per se does not appear to be the primary regulatory signal, at least for the synthesis of nitrogenase and differentiation of heterocysts (27). Rather, glutamine or a metabolic derivative of it has been implicated (10,24,30), as it also has been implicated in the control of nitrate metabolism in Neurospora species (22).…”