Molecular cloning of the phosphoenolpyruvate carboxylase gene, ppc, of Escherichia coli

Abstract

The ColE1 hybrid plasmid, pLC20-10, carrying the ppc gene and the argECBH gene cluster of Escherichia coli K-12, was characterized. The ppc gene coding for phosphoenolpyruvate carboxylase (EC 4.1.1.31), was subcloned into the plasmid pBR322 to give the plasmids pS2 and pS3. These plasmids carried a 4.4-kb SalI segment containing the ppc gene, in both orientations. The specific activity of the enzyme was increased approx. 20-fold by these plasmids. Experiments with maxicells harboring pS2 showed that the 90-kDal enzyme subunit was encoded by the plasmid. The location of the ppc gene in pS2 and the direction of transcription of the gene were determined. In DNA-DNA hybridization experiments using pS2 as a probe, significant hybridizations were observed with DNAs from E. coli strains K-12 and W, and from Salmonella typhimurium, but not with those from Chlorella regularis, Anacystis nidulans, Rhodospirillum rubrum, and Pseudomonas AM-1.