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. 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.

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Selected References

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