ColE1 hybrid plasmids containing Escherichia coli genes involved in the biosynthesis of glutamate and glutamine

Abstract

The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA + allele. GS synthesis by pACR1 glnA + glnA20 heterozygous merodiploids was subjected to repression by growth on 15 m m NH 4 + and had a twofold high derepressed level than wild-type ( glnA +) haploid cells when grown on 0.5 m m NH 4 + or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the glnA + glnA20 merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA + haploid cells.