Molecular Cloning of the Pyruvate Dehydrogenase Complex Genes of Escherichia coli

Abstract

The 3 components of the pyruvate dehydrogenase complex of E. coli are encoded by 3 linked genes, aceE(pyruvate dehydrogenase, E1), aceF(dihydrolipoamideacetyltransferase, E2) and lpd (lipoamide dehydrogenase, E3), situated close to the nadC quinolinate phosphoribosyltransferase) and aroP (general aromatic amino acid permease) genes with the gene order: nadC-aroP-aceE-aceF-lpd. Several types of transducing phages, .lambda.nadC and .lambda.lpd, carrying the nadC and lpd genes were isolated from populations of artificially constructed transducing phages containing hindIII or EcoRI fragments of bacterial DNA, by selecting for their ability to complement the metabolic lesions of the corresponding mutants. The cloned fragments were extended to include a functional ace operon by in vivo methods involving prophage insertion into the nadC-lpd region and aberrant excision to yield .lambda.nadC-lpd and .lambda.lpd-ace phages. These contained overlapping segments of bacterial DNA capable of expressing the aceE, aceF and lpd genes. A physical map of a 20 kilobase pairs (kb) segment of bacterial DNA encoding the entire nadC-lpd region, bound by HindIII and EcoRI targets and possessing 3 internal HindIII cleavage sites and 2 internal EcoRI cleavage sites was constructed. Using a combination of nutritional and enzymological studies with dilysogens and genetic analysis with ace mutants the approximate positions of the genes specifying the pyruvate dehydrogenase complex were traced to a 9.5 kb segment of the restriction map. The cloned lpd gene was expressed in the complete absence of a functional ace operon and when the major .lambda. promoters were repressed. The lpd gene can be independently transcribed from its own promoter.