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. 1988 Feb;170(2):821–827. doi: 10.1128/jb.170.2.821-827.1988

gltBDF operon of Escherichia coli.

I Castaño 1, F Bastarrachea 1, A A Covarrubias 1
PMCID: PMC210728  PMID: 2448295

Abstract

A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.

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Selected References

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  1. ADELBERG E. A., BURNS S. N. Genetic variation in the sex factor of Escherichia coli. J Bacteriol. 1960 Mar;79:321–330. doi: 10.1128/jb.79.3.321-330.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
  3. Berberich M. A. A glutamate-dependent phenotype in E. coli K12: the result of two mutations. Biochem Biophys Res Commun. 1972 Jun 28;47(6):1498–1503. doi: 10.1016/0006-291x(72)90242-2. [DOI] [PubMed] [Google Scholar]
  4. Betlach M., Hershfield V., Chow L., Brown W., Goodman H., Boyer H. W. A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA. Fed Proc. 1976 Jul;35(9):2037–2043. [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
  6. Brenchley J. E., Baker C. A., Patil L. G. Regulation of the ammonia assimilatory enzymes in Salmonella typhimurium. J Bacteriol. 1975 Oct;124(1):182–189. doi: 10.1128/jb.124.1.182-189.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brenchley J. E., Prival M. J., Magasanik B. Regulation of the synthesis of enzymes responsible for glutamate formation in Klebsiella aerogenes. J Biol Chem. 1973 Sep 10;248(17):6122–6128. [PubMed] [Google Scholar]
  8. Castaño I., Bastarrachea F. glnF-lacZ fusions in Escherichia coli: studies on glnF expression and its chromosomal orientation. Mol Gen Genet. 1984;195(1-2):228–233. doi: 10.1007/BF00332751. [DOI] [PubMed] [Google Scholar]
  9. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Covarrubias A. A., Sánchez-Pescador R., Osorio A., Bolivar F., Bastarrachea F. ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine. Plasmid. 1980 Mar;3(2):150–164. doi: 10.1016/0147-619x(80)90106-7. [DOI] [PubMed] [Google Scholar]
  11. Garciarrubio A., Lozoya E., Covarrubias A., Bolivar F. Structural organization of the genes that encode two glutamate synthase subunits of Escherichia coli. Gene. 1983 Dec;26(2-3):165–170. doi: 10.1016/0378-1119(83)90186-5. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Kingsbury D. T., Helinski D. R. DNA polymerase as a requirement for the maintenance of the bacterial plasmid colicinogenic factor E1. Biochem Biophys Res Commun. 1970 Dec 24;41(6):1538–1544. doi: 10.1016/0006-291x(70)90562-0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Lozoya E., Sanchez-Pescador R., Covarrubias A., Vichido I., Bolivar F. Tight linkage of genes that encode the two glutamate synthase subunits of Escherichia coli K-12. J Bacteriol. 1980 Nov;144(2):616–621. doi: 10.1128/jb.144.2.616-621.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Madonna M. J., Fuchs R. L., Brenchley J. E. Fine structure analysis of Salmonella typhimurium glutamate synthase genes. J Bacteriol. 1985 Jan;161(1):353–360. doi: 10.1128/jb.161.1.353-360.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Mantsala P., Zalkin H. Active subunits of Escherichia coli glutamate synthase. J Bacteriol. 1976 Apr;126(1):539–541. doi: 10.1128/jb.126.1.539-541.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meagher R. B., Tait R. C., Betlach M., Boyer H. W. Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977 Mar;10(3):521–536. doi: 10.1016/0092-8674(77)90039-3. [DOI] [PubMed] [Google Scholar]
  21. Meers J. L., Tempest D. W., Brown C. M. 'Glutamine(amide):2-oxoglutarate amino transferase oxido-reductase (NADP); an enzyme involved in the synthesis of glutamate by some bacteria. J Gen Microbiol. 1970 Dec;64(2):187–194. doi: 10.1099/00221287-64-2-187. [DOI] [PubMed] [Google Scholar]
  22. Miller R. E., Stadtman E. R. Glutamate synthase from Escherichia coli. An iron-sulfide flavoprotein. J Biol Chem. 1972 Nov 25;247(22):7407–7419. [PubMed] [Google Scholar]
  23. 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]
  24. Pahel G., Tyler B. A new glnA-linked regulatory gene for glutamine synthetase in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4544–4548. doi: 10.1073/pnas.76.9.4544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pahel G., Zelenetz A. D., Tyler B. M. gltB gene and regulation of nitrogen metabolism by glutamine synthetase in Escherichia coli. J Bacteriol. 1978 Jan;133(1):139–148. doi: 10.1128/jb.133.1.139-148.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Senior P. J. Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous-culture technique. J Bacteriol. 1975 Aug;123(2):407–418. doi: 10.1128/jb.123.2.407-418.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  30. Stüber D., Bujard H. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci U S A. 1981 Jan;78(1):167–171. doi: 10.1073/pnas.78.1.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]

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