Abstract
The signal-transducing kinase/phosphatase nitrogen regulator II (NRII or NtrB) is required for the efficient positive and negative regulation of glnA, encoding glutamine synthetase, and the Ntr regulon in response to the availability of ammonia. Alteration of highly conserved residues within the kinase/phosphatase domain of NRII revealed that the positive and negative regulatory functions of NRII could be genetically separated and that negative regulation by NRII did not require the highly conserved His-139, Glu-140, Asn-248, Asp-287, Gly-289, Gly-291, Gly-313, or Gly-315 residue. These mutations affected the positive regulatory function of NRII to various extents. Certain substitutions at codons 139 and 140 resulted in mutant NRII proteins that were transdominant negative regulators of glnA and the Ntr regulon even in the absence of nitrogen limitation. In addition, we examined three small deletions near the 3' end of the gene encoding NRII; these resulted in altered proteins that retained the negative regulatory function but were defective to various extents in the positive regulatory function. A truncated NRII protein missing the C-terminal 59 codons because of a nonsense mutation at codon 291 lacked entirely the positive regulatory function but was a negative regulator of glnA even in the absence of nitrogen limitation. Thus, we have identified both point and deletion mutations that convert NRII into a negative regulator of glnA and the Ntr regulon irrespective of the nitrogen status of the cell.
Full text
PDF







Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adler S. P., Purich D., Stadtman E. R. Cascade control of Escherichia coli glutamine synthetase. Properties of the PII regulatory protein and the uridylyltransferase-uridylyl-removing enzyme. J Biol Chem. 1975 Aug 25;250(16):6264–6272. [PubMed] [Google Scholar]
- Atkinson M. R., Ninfa A. J. Characterization of Escherichia coli glnL mutations affecting nitrogen regulation. J Bacteriol. 1992 Jul;174(14):4538–4548. doi: 10.1128/jb.174.14.4538-4548.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bloom F. R., Levin M. S., Foor F., Tyler B. Regulation of glutamine synthetase formation in Escherichia coli: characterization of mutants lacking the uridylyltransferase. J Bacteriol. 1978 May;134(2):569–577. doi: 10.1128/jb.134.2.569-577.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown M. S., Segal A., Stadtman E. R. Modulation of glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the P II -regulatory protein. Proc Natl Acad Sci U S A. 1971 Dec;68(12):2949–2953. doi: 10.1073/pnas.68.12.2949. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bueno R., Pahel G., Magasanik B. Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J Bacteriol. 1985 Nov;164(2):816–822. doi: 10.1128/jb.164.2.816-822.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen Y. M., Backman K., Magasanik B. Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli. J Bacteriol. 1982 Apr;150(1):214–220. doi: 10.1128/jb.150.1.214-220.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engleman E. G., Francis S. H. Cascade control of E. coli glutamine synthetase. II. Metabolite regulation of the enzymes in the cascade. Arch Biochem Biophys. 1978 Dec;191(2):602–612. doi: 10.1016/0003-9861(78)90398-3. [DOI] [PubMed] [Google Scholar]
- Feng J., Atkinson M. R., McCleary W., Stock J. B., Wanner B. L., Ninfa A. J. Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J Bacteriol. 1992 Oct;174(19):6061–6070. doi: 10.1128/jb.174.19.6061-6070.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foor F., Cedergren R. J., Streicher S. L., Rhee S. G., Magasanik B. Glutamine synthetase of Klebsiella aerogenes: properties of glnD mutants lacking uridylyltransferase. J Bacteriol. 1978 May;134(2):562–568. doi: 10.1128/jb.134.2.562-568.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Foor F., Reuveny Z., Magasanik B. Regulation of the synthesis of glutamine synthetase by the PII protein in Klebsiella aerogenes. Proc Natl Acad Sci U S A. 1980 May;77(5):2636–2640. doi: 10.1073/pnas.77.5.2636. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Francis S. H., Engleman E. G. Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli. Arch Biochem Biophys. 1978 Dec;191(2):590–601. doi: 10.1016/0003-9861(78)90397-1. [DOI] [PubMed] [Google Scholar]
- Garcia E., Rhee S. G. Cascade control of Escherichia coli glutamine synthetase. Purification and properties of PII uridylyltransferase and uridylyl-removing enzyme. J Biol Chem. 1983 Feb 25;258(4):2246–2253. [PubMed] [Google Scholar]
- Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
- Hess J. F., Bourret R. B., Simon M. I. Histidine phosphorylation and phosphoryl group transfer in bacterial chemotaxis. Nature. 1988 Nov 10;336(6195):139–143. doi: 10.1038/336139a0. [DOI] [PubMed] [Google Scholar]
- Hirschman J., Wong P. K., Sei K., Keener J., Kustu S. Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a sigma factor. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7525–7529. doi: 10.1073/pnas.82.22.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hunt T. P., Magasanik B. Transcription of glnA by purified Escherichia coli components: core RNA polymerase and the products of glnF, glnG, and glnL. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8453–8457. doi: 10.1073/pnas.82.24.8453. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Keener J., Kustu S. Protein kinase and phosphoprotein phosphatase activities of nitrogen regulatory proteins NTRB and NTRC of enteric bacteria: roles of the conserved amino-terminal domain of NTRC. Proc Natl Acad Sci U S A. 1988 Jul;85(14):4976–4980. doi: 10.1073/pnas.85.14.4976. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- MacNeil T., MacNeil D., Tyler B. Fine-structure deletion map and complementation analysis of the glnA-glnL-glnG region in Escherichia coli. J Bacteriol. 1982 Jun;150(3):1302–1313. doi: 10.1128/jb.150.3.1302-1313.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacNeil T., Roberts G. P., MacNeil D., Tyler B. The products of glnL and glnG are bifunctional regulatory proteins. Mol Gen Genet. 1982;188(2):325–333. doi: 10.1007/BF00332696. [DOI] [PubMed] [Google Scholar]
- Ninfa A. J., Bennett R. L. Identification of the site of autophosphorylation of the bacterial protein kinase/phosphatase NRII. J Biol Chem. 1991 Apr 15;266(11):6888–6893. [PubMed] [Google Scholar]
- Ninfa A. J., Magasanik B. Covalent modification of the glnG product, NRI, by the glnL product, NRII, regulates the transcription of the glnALG operon in Escherichia coli. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5909–5913. doi: 10.1073/pnas.83.16.5909. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ninfa A. J. Protein phosphorylation and the regulation of cellular processes by the homologous two-component regulatory systems of bacteria. Genet Eng (N Y) 1991;13:39–72. doi: 10.1007/978-1-4615-3760-1_2. [DOI] [PubMed] [Google Scholar]
- Ninfa E. G., Atkinson M. R., Kamberov E. S., Ninfa A. J. Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits. J Bacteriol. 1993 Nov;175(21):7024–7032. doi: 10.1128/jb.175.21.7024-7032.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pahel G., Rothstein D. M., Magasanik B. Complex glnA-glnL-glnG operon of Escherichia coli. J Bacteriol. 1982 Apr;150(1):202–213. doi: 10.1128/jb.150.1.202-213.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parkinson J. S., Kofoid E. C. Communication modules in bacterial signaling proteins. Annu Rev Genet. 1992;26:71–112. doi: 10.1146/annurev.ge.26.120192.000443. [DOI] [PubMed] [Google Scholar]
- Reitzer L. J., Magasanik B. Expression of glnA in Escherichia coli is regulated at tandem promoters. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1979–1983. doi: 10.1073/pnas.82.7.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reitzer L. J., Movsas B., Magasanik B. 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. J Bacteriol. 1989 Oct;171(10):5512–5522. doi: 10.1128/jb.171.10.5512-5522.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rhee S. G., Park S. C., Koo J. H. The role of adenylyltransferase and uridylyltransferase in the regulation of glutamine synthetase in Escherichia coli. Curr Top Cell Regul. 1985;27:221–232. doi: 10.1016/b978-0-12-152827-0.50026-8. [DOI] [PubMed] [Google Scholar]
- Smith R. A., Parkinson J. S. Overlapping genes at the cheA locus of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5370–5374. doi: 10.1073/pnas.77.9.5370. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ueno-Nishio S., Mango S., Reitzer L. J., Magasanik B. Identification and regulation of the glnL operator-promoter of the complex glnALG operon of Escherichia coli. J Bacteriol. 1984 Oct;160(1):379–384. doi: 10.1128/jb.160.1.379-384.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss V., Claverie-Martin F., Magasanik B. Phosphorylation of nitrogen regulator I of Escherichia coli induces strong cooperative binding to DNA essential for activation of transcription. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5088–5092. doi: 10.1073/pnas.89.11.5088. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weiss V., Magasanik B. Phosphorylation of nitrogen regulator I (NRI) of Escherichia coli. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8919–8923. doi: 10.1073/pnas.85.23.8919. [DOI] [PMC free article] [PubMed] [Google Scholar]