Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase.

Rothstein, David M.; Pahel, G; Tyler, Bonnie; Magasanik, Boris

doi:10.1073/pnas.77.12.7372

Cited by 65 publications

(57 citation statements)

References 18 publications

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“…The salts for the minimal medium have been described previously (15). In addition, minimal medium contained 0.4% glucose, 0.02% thiamine, and 0.1% of each nitrogen source unless otherwise noted.…”

Section: Methodsmentioning

confidence: 99%

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

2000

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Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential 54 -dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the 54 -dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced 14 CO 2 from minimal medium containing [ 14 C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated 14 CO 2 production from [ 14 C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.

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Section: Methodsmentioning

confidence: 99%

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

2000

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“…Cell Growth and Enzyme Assays-The minimal growth medium contained W salts, 0.4% of the carbon source, 0.2% of each nitrogen source, and 0.02% thiamine (34). Assays for glutamine synthetase and ␤-galactosidase were performed as described (34,35).…”

Section: Methodsmentioning

confidence: 99%

“…Assays for glutamine synthetase and ␤-galactosidase were performed as described (34,35). Units are nmol of product min Ϫ1 mg protein Ϫ1 , unless otherwise specified.…”

Section: Methodsmentioning

confidence: 99%

Phosphorylation-independent Dimer-Dimer Interactions by the Enhancer-binding Activator NtrC of Escherichia coli

Yang

Schneider³

et al. 2004

Journal of Biological Chemistry

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The response regulator NtrC transcriptionally activates genes of the nitrogen-regulated (Ntr) response. Phosphorylation of its N-terminal receiver domain stimulates an essential oligomerization of the central domain. Deletion of the central domain reduces, but does not eliminate, intermolecular interactions as assessed by cooperative binding to DNA. To analyze the structural determinants and function of this central domain-independent as well as phosphorylation-independent oligomerization, we randomly mutagenized DNA coding for an NtrC without its central domain and isolated strains containing NtrC with defective phosphorylation-independent cooperative binding. The alterations were primarily localized to helix B of the C-terminal domain. Site-specific mutagenesis that altered surface residues of helix B confirmed this localization. The purified NtrC variants, with or without the central domain, were specifically defective in phosphorylation-independent cooperative DNA binding and had little defect, if any, on other functions, such as noncooperative DNA binding. We propose that this region forms an oligomerization interface. Full-length NtrC variants did not efficiently repress the glnA-ntrBC operon when NtrC was not phosphorylated, which suggests that phosphorylation-independent cooperative binding sets the basal level for glutamine synthetase and the regulators of the Ntr response. The NtrC variants in these cells generally, but not always, supported wild-type growth in nitrogen-limited media and wild-type activation of a variety of Ntr genes. We discuss the differences and similarities between the NtrC C-terminal domain and the homologous Fis, which is also capable of intermolecular interactions.

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“…The assay for glutamine synthetase activity has been described previously (30). The values for glutamine synthetase activity are the average of two to six determinations, each determination using an independent transformant.…”

Section: Methodsmentioning

confidence: 99%

“…Growth and harvesting of cells and all standard techniques involving nucleic acids have been described elsewhere (17,30). The strains were derivatives of the E. coli K-12 strain YMC10 (thi-1 endAl hsdRJ7 lacU169 hutCk) (2), which is considered wild type.…”

Section: Methodsmentioning

confidence: 99%

Activation of glnA transcription by nitrogen regulator I (NRI)-phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase

1989

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Growth of cels of Eschericha coli in nitrogen-limited medium induces the formato of glutmine synthetase, product of the ginA gene, and of other proteins that facilitate the assimilation of compounds. Transcription from the gInAp2 promoter of the glnALG operon Among nitrogen sources, ammonia supports the fastest growth of cells of Escherichia coli and Salmonella typhimurium. Growth on other sources of metabolizable nitrogen, such as histidine or glutamine, is nitrogen limited and induces the synthesis of glutamine synthetase, the product of the glnA gene, and of other proteins that accelerate transport or degradation of nitrogen-containing compounds (reviewed in reference 28). The first operon activated during the transition to nitrogen-limited growth is the glnALG operon. Transcription from glnAp2, the major promoter of the glnALG operon, requires the phosphorylated form of NRI (nitrogen regulator I), product of the glnG (or ntrC) gene, and ar"4, product of the rpoN (or ntrA) gene (9,10,14,19,26,28). Nitrogen regulator II, the product of the glnL (or ntrB) gene, phosphorylates NRI during nitrogen-limited growth and dephosphorylates NRI-phosphate when ammonia is in the growth medium (12, 19).Core RNA polymerase complexed with oF54 transcribes many nitrogen-regulated genes and recognizes promoters with a consensus sequence CTGGYAYR-N4-TTGCA instead of promoters with the sequence of the canonical promoter of enteric bacteria, TTGACA-N,7-TATAAT (1, 9, 10). Core RNA polymerase complexed to oS4 protects DNA in the vicinity of glnAp2 against DNaseI digestion from 45 bases upstream to 10 bases downstream of the start of transcription (9, 20, 31). The binding of r54-RNA polymerase to glnAp2 DNA does not require and is not facilitated by NRI or NRI-phosphate (9,20,31 . Small increases in the distance between the binding sites for an activator and RNA polymerase diminish transcription of other positively regulated procaryotic genes (4,15,16). For these and other positively controlled procaryotic genes, the activator has been postulated to stimulate transcription by contacting RNA polymerase (4,11,23,29

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Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase.

Cited by 65 publications

References 18 publications

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage

Phosphorylation-independent Dimer-Dimer Interactions by the Enhancer-binding Activator NtrC of Escherichia coli

Activation of glnA transcription by nitrogen regulator I (NRI)-phosphate in Escherichia coli: evidence for a long-range physical interaction between NRI-phosphate and RNA polymerase

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