Genetic and biochemical analysis of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium

Abstract

Pantothenate (pan) auxotrophs of E. coli K-12 and S. typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ('panA') mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase incidates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of α-ketoisovalerate rather than the synthesis of α-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. It was also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or β-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.