Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1986 Sep;52(3):413–419. doi: 10.1128/aem.52.3.413-419.1986

Cloning, Expression, and Purification of Glutamine Synthetase from Clostridium acetobutylicum

Karen P Usdin 1, Harold Zappe 1, David T Jones 1, David R Woods 1,*
PMCID: PMC203548  PMID: 16347143

Abstract

A glutamine synthetase (GS) gene, glnA, from the gram-positive obligate anaerobe Clostridium acetobutylicum was cloned on recombinant plasmid pHZ200 and enabled Escherichia coli glnA deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. The cloned C. acetobutylicum gene was expressed from a regulatory region contained within the cloned DNA fragment. glnA expression was subject to nitrogen regulation in E. coli. This cloned glnA DNA did not enable an E. coli glnA ntrB ntrC deletion mutant to utilize arginine or low levels of glutamine as sole nitrogen sources, and failed to activate histidase activity in this strain which contained the Klebsiella aerogenes hut operon. The GS produced by pHZ200 was purified and had an apparent subunit molecular weight of approximately 59,000. There was no DNA or protein homology between the cloned C. acetobutylicum glnA gene and GS and the corresponding gene and GS from E. coli. The C. acetobutylicum GS was inhibited by Mg2+ in the γ-glutamyl transferase assay, but there was no evidence that the GS was adenylylated.

Full text

PDF
414

Images in this article

414Image
on p.414
416Image
on p.416
416Image
on p.416
417Image
on p.417
417Image
on p.417

Selected References

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

  1. Allcock E. R., Reid S. J., Jones D. T., Woods D. R. Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum. Appl Environ Microbiol. 1981 Dec;42(6):929–935. doi: 10.1128/aem.42.6.929-935.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backman K., Chen Y. M., Magasanik B. Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3743–3747. doi: 10.1073/pnas.78.6.3743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bender R. A., Janssen K. A., Resnick A. D., Blumenberg M., Foor F., Magasanik B. Biochemical parameters of glutamine synthetase from Klebsiella aerogenes. J Bacteriol. 1977 Feb;129(2):1001–1009. doi: 10.1128/jb.129.2.1001-1009.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bodasing S. J., Brandt P. W., Robb F. T., Woods D. R. Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain. Arch Microbiol. 1985 Jan;140(4):369–374. doi: 10.1007/BF00446980. [DOI] [PubMed] [Google Scholar]
  5. Dean D. R., Hoch J. A., Aronson A. I. Alteration of the Bacillus subtilis glutamine synthetase results in overproduction of the enzyme. J Bacteriol. 1977 Sep;131(3):981–987. doi: 10.1128/jb.131.3.981-987.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  7. Fisher R., Tuli R., Haselkorn R. A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3393–3397. doi: 10.1073/pnas.78.6.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fisher S. H., Rosenkrantz M. S., Sonenshein A. L. Glutamine synthetase gene of Bacillus subtilis. Gene. 1984 Dec;32(3):427–438. doi: 10.1016/0378-1119(84)90018-0. [DOI] [PubMed] [Google Scholar]
  9. Frey T. G., Eisenberg D., Eiserling F. A. Glutamine synthetase forms three- and seven-stranded helical cables. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3402–3406. doi: 10.1073/pnas.72.9.3402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garcia E., Bancroft S., Rhee S. G., Kustu S. The product of a newly identified gene, gInF, is required for synthesis of glutamine synthetase in Salmonella. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1662–1666. doi: 10.1073/pnas.74.4.1662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gardner A. L., Aronson A. I. Expression of the Bacillus subtilis glutamine synthetase gene in Escherichia coli. J Bacteriol. 1984 Jun;158(3):967–971. doi: 10.1128/jb.158.3.967-971.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ish-Horowicz D., Burke J. F. Rapid and efficient cosmid cloning. Nucleic Acids Res. 1981 Jul 10;9(13):2989–2998. doi: 10.1093/nar/9.13.2989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones D. T., van der Westhuizen A., Long S., Allcock E. R., Reid S. J., Woods D. R. Solvent Production and Morphological Changes in Clostridium acetobutylicum. Appl Environ Microbiol. 1982 Jun;43(6):1434–1439. doi: 10.1128/aem.43.6.1434-1439.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kustu S., Burton D., Garcia E., McCarter L., McFarland N. Nitrogen control in Salmonella: regulation by the glnR and glnF gene products. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4576–4580. doi: 10.1073/pnas.76.9.4576. [DOI] [PMC free article] [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. 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]
  17. 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]
  18. McFarland N., McCarter L., Artz S., Kustu S. Nitrogen regulatory locus "glnR" of enteric bacteria is composed of cistrons ntrB and ntrC: identification of their protein products. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2135–2139. doi: 10.1073/pnas.78.4.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Brien R. W., Morris J. G. Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol. 1971 Nov;68(3):307–318. doi: 10.1099/00221287-68-3-307. [DOI] [PubMed] [Google Scholar]
  20. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  23. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  24. Schreier H. J., Fisher S. H., Sonenshein A. L. Regulation of expression from the glnA promoter of Bacillus subtilis requires the glnA gene product. Proc Natl Acad Sci U S A. 1985 May;82(10):3375–3379. doi: 10.1073/pnas.82.10.3375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith G. E., Summers M. D. The bidirectional transfer of DNA and RNA to nitrocellulose or diazobenzyloxymethyl-paper. Anal Biochem. 1980 Nov 15;109(1):123–129. doi: 10.1016/0003-2697(80)90019-6. [DOI] [PubMed] [Google Scholar]
  26. Smith G. R., Halpern Y. S., Magasanik B. Genetic and metabolic control of enzymes responsible for histidine degradation in Salmonella typhimurium. 4-imidazolone-5-propionate amidohydrolase and N-formimino-L-glutamate formiminohydrolase. J Biol Chem. 1971 May 25;246(10):3320–3329. [PubMed] [Google Scholar]
  27. Streicher S. L., Tyler B. Purification of glutamine synthetase from a variety of bacteria. J Bacteriol. 1980 Apr;142(1):69–78. doi: 10.1128/jb.142.1.69-78.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sykes R. B., Nordström K. Microiodometric determination of beta-lactamase activity. Antimicrob Agents Chemother. 1972 Feb;1(2):94–99. doi: 10.1128/aac.1.2.94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tuli R., Fisher R., Haselkorn R. The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae. Gene. 1982 Jul-Aug;19(1):109–116. doi: 10.1016/0378-1119(82)90195-0. [DOI] [PubMed] [Google Scholar]
  30. Tyler B. Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem. 1978;47:1127–1162. doi: 10.1146/annurev.bi.47.070178.005403. [DOI] [PubMed] [Google Scholar]
  31. Wedler F. C., Shreve D. S., Kenney R. M., Ashour A. E., Carfi J., Rhee S. G. Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile. Isolation, physicochemical and kinetic properties. J Biol Chem. 1980 Oct 10;255(19):9507–9516. [PubMed] [Google Scholar]
  32. Zabeau M., Stanley K. K. Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda. EMBO J. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zappe H., Jones D. T., Woods D. R. Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli. J Gen Microbiol. 1986 May;132(5):1367–1372. doi: 10.1099/00221287-132-5-1367. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES