Molecular cloning of the phosphoenolpyruvate carboxylase gene of Anabaena variabilis

Purified Anabaena variabilis chromosomal DNA was partially digested with restriction endonuclease Sau3A and ligated into the BamHI site of plasmid pBR322. Escherichia coli 342-167, a mutant with a decreased level of phosphoenolpyruvate carboxylase (PEPCase) activity was transformed with plasmids from the A. variabilis genomic library. A transformant that grew on minimal media in the absence of glutamate was isolated and its plasmid, pTRH1, was shown to encode the A. variabilis PEPCase. E. coli HB101 cells transformed with plasmid pTRH1 have approx. 50 times the normal amount of PEPCase activity and also overproduce a protein with the apparent Mr (99,000) of the A. variabilis PEPCase.