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

A positive, genetic selection against the activity of the nitrogen regulatory (NTR) system was used to isolate insertion mutations affecting nitrogen regulation in Klebsiella 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.     

Cloning of the Klebsiella aerogenes nac gene, which encodes a factor required for nitrogen regulation of the histidine utilization (hut) operons in Salmonella typhimurium.

The nac (nitrogen assimilation control) gene from Klebsiella aerogenes, cloned in a low-copy-number cloning vector, restored the ability of K. aerogenes nac mutants to activate histidase and repress glutamate dehydrogenase formation in response to nitrogen limitation and to limit the maximum expression of the nac promoter. When present in Salmonella typhimurium, the K. aerogenes nac gene allowed the hut genes to be activated during nitrogen-limited growth. Thus, the nac gene encodes a cytoplasmic factor required for activation of hut expression in S. typhimurium during nitrogen-limited growth.     

Nitrogen regulation system of Klebsiella aerogenes: the nac gene.

In Klebsiella aerogenes, the product of a his-linked gene, nac, appears to play a crucial role in tying the synthesis of enzymes activated or repressed by ammonia deprivation, such as histidase and glutamate dehydrogenase, to the known regulators of nitrogen assimilation, the products of glnG and glnF.     

A nonsense mutation in the structural gene for glutamine synthetase leading to loss of nitrogen regulation in Klebsiella aerogenes

Summary An amber mutation (glnA3711), the first nonsense mutation isolated in Klebsiella aerogenes, is described. When amber suppressors were present, the mutant made active glutamine synthetase which was more thermolabile than wild type, showing that glnA3711 lies in the structural gene for glutamine synthetase. Strains carrying the glnA3711 allele were unable to express nitrogen regulation of genes coding for histidase, asparaginase, and glutamate dehydrogenase unless amber suppressors were also present. These results support a model that expression of gene(s) from the glnA promoter is required for nitrogen regulation in K. aerogenes.     

Nitrogen control in Pseudomonas aeruginosa: A role for glutamine in the regulation of the synthesis of NADP-dependent glutamate dehydrogenase,urease and histidase

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.     

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from gamma-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.     

Sequence analysis of the hutH gene encoding histidine ammonia-lyase in Pseudomonas putida.

The complete nucleotide sequence of the hutH gene, encoding histidine ammonia-lyase (histidase), in Pseudomonas putida ATCC 12633 has been determined from the appropriate portions of the hut region that had been cloned into Escherichia coli. The resulting DNA sequence revealed an open reading frame of 1,530 base pairs, corresponding to a protein subunit of approximate molecular weight 53,600, in the location previously identified for the histidase gene by Tn1000 mutagenesis. Translation began at a GTG codon, but direct protein sequencing revealed that the initiating amino acid was removed posttranslationally to provide an N-terminal threonine; 11 additional residues completely agreed with the predicted amino acid sequence. This sequence excluded the possibility that a dehydroalanine unit, the postulated coenzyme for histidase, is attached at the N terminus of histidase subunits. Comparison of the P. putida histidase gene sequence with that of a Bacillus subtilis region encoding histidase revealed 42% identity at the protein level. Although the hutU (urocanase) and hutH (histidase) genes are induced by urocanate and normally are transcribed as a unit beginning with hutU, analysis of the region immediately upstream of the histidase gene revealed a potential weak promoter that may possibly be used to maintain a basal level of histidase for the generation of inducer (urocanate) when histidine levels are elevated.     

Regulation of Klebsiella pneumoniae hut operons by oxygen.

We investigated the regulation of genes concerned with nitrogen metabolism by oxygen in the facultative anaerobe Klebsiella pneumoniae. We found oxygen to be required for the expression of the hut operons; the effect of O2 on the glutamine synthetase and urease was less pronounced than on the hut operons. Glutamine synthetase was transiently repressed during the transition from an aerobic to an anaerobic environment. Regulation of hut by O2 suppressed the effect of nitrogen limitation on the expression of these genes.