Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes

Abstract

A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Kiebsiella aerogenes. Two classes of mutation were obtained: those affecting the NTR system itself and leading to the loss of almost all nitrogen regulation, and those affecting the nac locus and leading to a loss of nitrogen regulation of a family of nitrogen-regulated enzymes. The set of these nac-dependent enzymes included histidase, glutamate dehydrogenase, glutamate synthase, proline oxidase, and urease. The enzymes shown to be nac independent included glutamine synthetase, asparaginase, tryptophan permease, nitrate reductase, the product of the nifLA operon, and perhaps nitrite reductase. The expression of the nac gene was itself highly nitrogen regulated, and this regulation was mediated by the NTR system. The loss of nitrogen regulation was found in each of the four insertion mutants studied, showing that loss of nitrogen regulation resulted from the absence of nac function rather than from an altered form of the nac gene product. Thus we propose two classes of nitrogen-regulated operons: in class I, the NTR system directly activates expression of the operon; in class II, the NTR system activates nac expression and the product(s) of the nac locus activates expression of the operon.The enteric bacterium Klebsiella aerogenes is capable of using a large number of compounds as its sole source of nitrogen. The formation of the enzymes needed to metabolize these compounds is in most, if not all, cases regulated by the availability of the preferred nitrogen source, ammonium (22). The formation of the enzymes needed for the conversion of poor nitrogen sources to either ammonia or glutamate is increased when ammonia is absent. Conversely, the formation of the two enzymes ultimately responsible for assimilating ammonia into glutamate (glutamate dehydrogenase and glutamate synthase) is repressed when ammonia is absent. As early as 1973, Prival et al. (33) recognized a role for the glnA locus in the genetic control of nitrogen-regulation in K. aerogenes. After a false start suggesting that the product of the glnA gene (glutamine synthetase) was itself the activator of transcription for nitrogen-regulated genes (23), it eventually became clear that the glutamine synthetase encoded by the glnA locus was physiologically important for nitrogen regulation because it synthesized the internal signal of nitrogen excess (glutamine or a product thereof) from ammonia, but that glnA was not directly involved in the genetic control of nitrogen regulation. The work from several groups (for reviews, see references 19,22 can cause the conversion of promoter-bound RNA polymerase containing the novel sigma subunit sigma-54, encoded by rpoN (instead of the normal sigma-70), from a closed complex to an open complex-at least in the case of the glnA promoter (32). Thus NTRC can activate transcription directly.This transcriptional activation was directly demonstrated in vitro ...

The Alternative Sigma Factor RpoN Is Required for hrp Activity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

“…54 , which is encoded by rpoN, works in conjunction with members of the NtrC superfamily of transcriptional activators (1, 50). Among the different enzymatic pathways under 54 control are those responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (8,39,47,79).In the case of some phytopathogenic bacterial species, rpoN has been implicated indirectly as a regulator of a large cluster of pathogenicity-related genes known as the hrp gene cluster (17,27). For example, Pseudomonas syringae pv.…”

“…54 , which is encoded by rpoN, works in conjunction with members of the NtrC superfamily of transcriptional activators (1, 50). Among the different enzymatic pathways under 54 control are those responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (8,39,47,79).…”

Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

“…For example, genes involved in nitrogen, hydrogen, and catabolite utilization are frequently regulated by 54 (6,35,48,95). In the case of pathogenic bacteria, rpoN mediates expression of virulence-related factors such as pilin in Pseudomonas aeruginosa and flagellin in Vibrio anguillarum (24,61,86).…”

Activation of the Escherichia coli lacZ promoter by the Klebsiella aerogenes nitrogen assimilation control protein (NAC), a LysR family transcription factor

“…Essentially, nitrogen starvation results in the activation of NtrC, a DNA-binding protein that enhances transcription from 54 -dependent promoters. Although the Ntr system is necessary for the modulation of all nitrogen-regulated operons, transcriptional activity of a subset of these operons is also dependent on the nitrogen assimilation control protein (NAC) (4,5,15). NAC is a member of the LysR family of DNA binding transcriptional factors (23,24).…”

“…NAC is a member of the LysR family of DNA binding transcriptional factors (23,24). The transcription of the nac gene is 54 dependent and under positive control by the Ntr system (8,15). The apparent paradox posed by a second, positively regulated transcription factor in the Ntr cascade is explained by the functions NAC adds to the Ntr repertoire: NAC regulates 70 -dependent promoters and can either activate or repress transcription (see reference 2 for a review).…”