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. 1988 Dec;170(12):5452–5459. doi: 10.1128/jb.170.12.5452-5459.1988

Role of the Bradyrhizobium japonicum ntrC gene product in differential regulation of the glutamine synthetase II gene (glnII).

G B Martin 1, K A Chapman 1, B K Chelm 1
PMCID: PMC211637  PMID: 2903856

Abstract

We isolated the ntrC gene from Bradyrhizobium japonicum, the endosymbiont of soybean (Glycine max), and examined its role in regulating nitrogen assimilation. Two independent ntrC mutants were constructed by gene replacement techniques. One mutant was unable to produce NtrC protein, while the other constitutively produced a stable, truncated NtrC protein. Both ntrC mutants were unable to utilize potassium nitrate as a sole nitrogen source. In contrast to wild-type B. japonicum, the NtrC null mutant lacked glnII transcripts in aerobic, nitrogen-starved cultures. However, the truncated-NtrC mutant expressed glnII in both nitrogen-starved and nitrogen-excess cultures. Both mutants expressed glnII under oxygen-limited culture conditions and in symbiotic cells. These results suggest that nitrogen assimilation in B. japonicum is regulated in response to both nitrogen limitation and oxygen limitation and that separate regulatory networks exist in free-living and symbiotic cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams T. H., Chelm B. K. The nifH and nifDK promoter regions from Rhizobium japonicum share structural homologies with each other and with nitrogen-regulated promoters from other organisms. J Mol Appl Genet. 1984;2(4):392–405. [PubMed] [Google Scholar]
  2. Adams T. H., McClung C. R., Chelm B. K. Physical organization of the Bradyrhizobium japonicum nitrogenase gene region. J Bacteriol. 1984 Sep;159(3):857–862. doi: 10.1128/jb.159.3.857-862.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  4. Brown C. M., Dilworth M. J. Ammonia assimilation by rhizobium cultures and bacteroids. J Gen Microbiol. 1975 Jan;86(1):39–48. doi: 10.1099/00221287-86-1-39. [DOI] [PubMed] [Google Scholar]
  5. Carlson T. A., Guerinot M. L., Chelm B. K. Characterization of the gene encoding glutamine synthetase I (glnA) from Bradyrhizobium japonicum. J Bacteriol. 1985 May;162(2):698–703. doi: 10.1128/jb.162.2.698-703.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carlson T. A., Martin G. B., Chelm B. K. Differential transcription of the two glutamine synthetase genes of Bradyrhizobium japonicum. J Bacteriol. 1987 Dec;169(12):5861–5866. doi: 10.1128/jb.169.12.5861-5866.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chelm B. K., Hallick R. B. Changes in the expression of the chloroplast genome of Euglena gracilis during chloroplast development. Biochemistry. 1976 Feb 10;15(3):593–599. doi: 10.1021/bi00648a022. [DOI] [PubMed] [Google Scholar]
  8. Darr S. C., Somerville S. C., Arntzen C. J. Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II. J Cell Biol. 1986 Sep;103(3):733–740. doi: 10.1083/jcb.103.3.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Darrow R. A., Knotts R. R. Two forms of glutamine synthetase in free-living root-nodule bacteria. Biochem Biophys Res Commun. 1977 Sep 23;78(2):554–559. doi: 10.1016/0006-291x(77)90214-5. [DOI] [PubMed] [Google Scholar]
  10. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dixon R. A. The genetic complexity of nitrogen fixation. The ninth Fleming lecture. J Gen Microbiol. 1984 Nov;130(11):2745–2755. doi: 10.1099/00221287-130-11-2745. [DOI] [PubMed] [Google Scholar]
  12. Dixon R. Tandem promoters determine regulation of the Klebsiella pneumoniae glutamine synthetase (glnA) gene. Nucleic Acids Res. 1984 Oct 25;12(20):7811–7830. doi: 10.1093/nar/12.20.7811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drummond M., Whitty P., Wootton J. Sequence and domain relationships of ntrC and nifA from Klebsiella pneumoniae: homologies to other regulatory proteins. EMBO J. 1986 Feb;5(2):441–447. doi: 10.1002/j.1460-2075.1986.tb04230.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Elhai J., Wolk C. P. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene. 1988 Aug 15;68(1):119–138. doi: 10.1016/0378-1119(88)90605-1. [DOI] [PubMed] [Google Scholar]
  15. Ferro-Luzzi Ames G., Nikaido K. Nitrogen regulation in Salmonella typhimurium. Identification of an ntrC protein-binding site and definition of a consensus binding sequence. EMBO J. 1985 Feb;4(2):539–547. doi: 10.1002/j.1460-2075.1985.tb03662.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  17. Guerinot M. L., Chelm B. K. Bacterial delta-aminolevulinic acid synthase activity is not essential for leghemoglobin formation in the soybean/Bradyrhizobium japonicum symbiosis. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1837–1841. doi: 10.1073/pnas.83.6.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gussin G. N., Ronson C. W., Ausubel F. M. Regulation of nitrogen fixation genes. Annu Rev Genet. 1986;20:567–591. doi: 10.1146/annurev.ge.20.120186.003031. [DOI] [PubMed] [Google Scholar]
  19. Hawkes T., Merrick M., Dixon R. Interaction of purified NtrC protein with nitrogen regulated promoters from Klebsiella pneumoniae. Mol Gen Genet. 1985;201(3):492–498. doi: 10.1007/BF00331345. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Johnson G. V., Evans H. J., Ching T. Enzymes of the glyoxylate cycle in rhizobia and nodules of legumes. Plant Physiol. 1966 Oct;41(8):1330–1336. doi: 10.1104/pp.41.8.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Leary J. J., Brigati D. J., Ward D. C. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4045–4049. doi: 10.1073/pnas.80.13.4045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol. 1962 Jul;5:109–118. doi: 10.1016/s0022-2836(62)80066-7. [DOI] [PubMed] [Google Scholar]
  26. MacFarlane S. A., Merrick M. The nucleotide sequence of the nitrogen regulation gene ntrB and the glnA-ntrBC intergenic region of Klebsiella pneumoniae. Nucleic Acids Res. 1985 Nov 11;13(21):7591–7606. doi: 10.1093/nar/13.21.7591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Magasanik B. Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet. 1982;16:135–168. doi: 10.1146/annurev.ge.16.120182.001031. [DOI] [PubMed] [Google Scholar]
  28. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  29. Merrick M. J., Gibbins J. R. The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors. Nucleic Acids Res. 1985 Nov 11;13(21):7607–7620. doi: 10.1093/nar/13.21.7607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Nixon B. T., Ronson C. W., Ausubel F. M. Two-component regulatory systems responsive to environmental stimuli share strongly conserved domains with the nitrogen assimilation regulatory genes ntrB and ntrC. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7850–7854. doi: 10.1073/pnas.83.20.7850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. O'Gara F., Shanmugam K. T. Regulation of nitrogen fixation by Rhizobia. Export of fixed N2 as NH+4. Biochim Biophys Acta. 1976 Jul 21;437(2):313–321. doi: 10.1016/0304-4165(76)90001-5. [DOI] [PubMed] [Google Scholar]
  33. Ow D. W., Sundaresan V., Rothstein D. M., Brown S. E., Ausubel F. M. Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region. Proc Natl Acad Sci U S A. 1983 May;80(9):2524–2528. doi: 10.1073/pnas.80.9.2524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rao R. N., Rogers S. G. Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA segments. Gene. 1979 Sep;7(1):79–82. doi: 10.1016/0378-1119(79)90044-1. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Reitzer L. J., Magasanik B. Isolation of the nitrogen assimilation regulator NR(I), the product of the glnG gene of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5554–5558. doi: 10.1073/pnas.80.18.5554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reitzer L. J., Magasanik B. Transcription of glnA in E. coli is stimulated by activator bound to sites far from the promoter. Cell. 1986 Jun 20;45(6):785–792. doi: 10.1016/0092-8674(86)90553-2. [DOI] [PubMed] [Google Scholar]
  38. Rossbach S, Schell J, de Bruijn F J. The ntrC gene of Agrobacterium tumefaciens C58 controls glutamine synthetase (GSII) activity, growth on nitrate and chromosomal but not Ti-encoded arginine catabolism pathways. Mol Gen Genet. 1987 Oct;209(3):419–426. doi: 10.1007/BF00331144. [DOI] [PubMed] [Google Scholar]
  39. Rothstein D. M., Pahel G., Tyler B., Magasanik B. Regulation of expression from the glnA promoter of Escherichia coli in the absence of glutamine synthetase. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7372–7376. doi: 10.1073/pnas.77.12.7372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Szeto W. W., Nixon B. T., Ronson C. W., Ausubel F. M. Identification and characterization of the Rhizobium meliloti ntrC gene: R. meliloti has separate regulatory pathways for activation of nitrogen fixation genes in free-living and symbiotic cells. J Bacteriol. 1987 Apr;169(4):1423–1432. doi: 10.1128/jb.169.4.1423-1432.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Toukdarian A., Kennedy C. Regulation of nitrogen metabolism in Azotobacter vinelandii: isolation of ntr and glnA genes and construction of ntr mutants. EMBO J. 1986 Feb;5(2):399–407. doi: 10.1002/j.1460-2075.1986.tb04225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsang V. C., Peralta J. M., Simons A. R. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 1983;92:377–391. doi: 10.1016/0076-6879(83)92032-3. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Wolk C. P., Vonshak A., Kehoe P., Elhai J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1561–1565. doi: 10.1073/pnas.81.5.1561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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