The three components of the pyruvate dehydrogenase complex of are encoded by three linked genes, (pyruvate dehydrogenase, E1), (dihydrolipoamide acetyltransferase, E2) and (lipoamide dehydrogenase, E3), situated close to the (quinolinate phosphoribosyltransferase) and (general aromatic amino acid permease) genes with the gene order: Several types of transducing phages, λ and λ, carrying the and genes were isolated from populations of artificially constructed transducing phages containing R.dIII or R.RI 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 operon by methods involving prophage insertion into the region and aberrant excision to yield λ and λ phages. These contained overlapping segments of bacterial DNA capable of expressing the and genes. A physical map of a 20 kilobase pairs (kb) segment of bacterial DNA encoding the entire region, bounded by R.dIII and R.RI targets and possessing several internal restriction targets, R.dIII (3) and R.RI (2), was constructed. Using a combination of nutritional and enzymological studies with dilysogens and genetic analysis with 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 gene was expressed in the complete absence of a functional operon and when the major λ promoters were repressed. This confirms that the gene can be independently transcribed from its own promoter.


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