Organization and expression of the pyruvate dehydrogenase complex genes of Escherichia coli K12

The organization and expression of the pyruvate dehydrogenase complex genes, aceE (E1), aceF (E2) and lpd (E3), were investigated using a series of λ transducing phages carrying different segments of the nadC-aroP-aceE-aceF-lpd region of the chromosome of Escherichia coli. The polypeptides synthesized following the infection of u.v.-irradiated lysogenic and non-lysogenic hosts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. The aceE and lpd gene products were readily identified by their sizes, M r 100000 (pyruvate dehydrogenase. E1 component) and 56500 (lipoamide dehydrogenase. E3 component), but considerable heterogeneity was detected for the aceF gene product (acetyltransferase, E2 component). The main E2 polypeptides, M r 80000 and 83000, were accompanied by a family of minor polypeptides. M r 86000, 89000 and 91000, but no precursor-product relationships were apparent. A very large polypeptide, M r 190000, was also detected and found to contain the E1 component but in uncertain combination. In addition, the product of a gene in the nadC-aroP region was detected as a polypeptide with M r 36500. The existence of a single promoter for the aceE and aceF genes and a separate promoter for the lpd gene was confirmed. The lpd gene was shown to be transcribed with the same polarity as the ace genes (clockwise with respect to the E. coli linkage map). The relative rates of expression of the three genes from the bacterial promoters were estimated as 0.94:1.0:1.4-2.3 (E1:E2:E3) on a molar basis. The excess production of lipoamide dehydrogenase components (E3) indicates that the lpd promoter functions independently, at least in supplying components for the 2-oxoglutarate dehydrogenase complex. When expressed from PL, the powerful phage promoter, the ace and lpd gene products accounted for the greater part of the newly synthesized protein.