Hybrid Plasmids Containing The Pyruvate Dehydrogenase Complex Genes and Gene-Dna Relationships In The 2 To 3 Minute Region Of The Escherichia Coli Chromosome

Abstract

A sample of colonies from the Clarke-Carbon ColE1-E. coli DNA plasmid gene bank was screened by conjugation for complementation of the lipoamide dehydrogenase lesion of a deletion strain lacking all components of the pyruvate dehydrogenase complex, .DELTA.(aroP aceE aceF lpd). Two ColE1-lpd+ hybrid plasmids were identified: pGS2 (ColE1-ace lpd+; 24 kb [kilobase]) and pGS5 (ColE1-lpd+; 14 kb). Enzymological studies confirmed that pGS2 expressed all the activities of the pyruvate dehydrogenase complex; pGS5 expressed the lipoamide dehydrogenase and acetyltransferase activities (the latter from a ColE1 promoter). These and other plasmids were used to construct a 47-site (15 enzymes) restriction map for a 24.2 kb segment of bacterial DNA in the nadC-lpd region. A further 13 sites (6 enzymes) were defined in a 5.4 kb sub-segment containing the lpd gene. .lambda. Phage derivatives containing specific fragments were constructed and used in transduction studies which located the ace and lpd genes in a 7.78 kb sub-segment flanked by AccI and NruI sites.