Physical and genetic analysis of the phosphoenolpyruvate carboxykinase (pckA) locus from Escherichia coli K12

Abstract

An 8 kb BamHI fragment of the Escherichia coli K12 chromosome has been cloned which complemented the pheotype of CRM+ pckA mutants with inactive phosphoenolpyruvate (PEP) carboxykinase. The pckA+ clones expressed levels of enzyme activity elevated up to 30-fold and produced a Mr 55,000 product in maxicells, which co-electrophoresed with purified PEP carboxykinase. The cloned fragment expressed the pckA, ompR and envZ gene products in maxicells. The order of genes on the chromosome inferred from restriction mapping, was (74 min)...pckA envZ ompR...(75 min). Transcription of the pckA gene cloned on multicopy plasmids increased in stationary phase and was also regulated by catabolite repression. The transcriptional control region has been located by genetic fusions to the chloramphenicol acetyltransferase (cat) gene and pckA was transcribed in the direction of envZ (clockwise direction on the chromosome).