Genetic analysis of the proBA genes of Salmonella typhimurium: physical and genetic analyses of the cloned proB+ A+ genes of Escherichia coli and of a mutant allele that confers proline overproduction and enhanced osmotolerance

Abstract

Because of the fact that proline overproduction relieves the inhibitory effects of high external osmotic strength in a number of procaryotes, we wished to clone a mutant allele, pro-74, that confers proline overproduction and enhanced osmotolerance on Salmonella typhimurium and Escherichia coli. Therefore, the pro-74 allele, originally located on an E. coli episome F'128, was cloned into pBR322. In a parallel experiment, the wild type proB+ A+ genes of E. coli were also cloned from F'128 into pBR322. Both the pro-74 and the proB+ A+ alleles were obtained on a 10.4-kilobase-pair fragment that also contained the unrelated phoE gene. Strains carrying either the wild-type proB+ A+ or the pro-74 alleles on pBR322 grew more slowly, both in minimal medium and media of elevated osmotic strength, than strains carrying the same alleles on the low-copy plasmid, F'128, indicating that some gene in the cloned region is deleterious in high copy. We constructed Tn5 insertion mutations in the proB and the proA genes of E. coli, carried on F'128 in S. typhimurium. Using P22 transduction in S. typhimurium, we transferred these proB and proA::Tn5 insertions from F'128 into the cloned proBA genes on pBR322. From the restriction maps of the plasmids thus generated, we determined the approximate locations of the proB and the proA genes. We also performed complementation tests of S. typhimurium and E. coli proB and proA mutants by using the F'128 proB and proA::Tn5 insertions. These tests revealed that the proBA genes of S. typhimurium form an operon, whose direction of transcription is from proB to proA. They also indicated that in S. typhimurium, as in E. coli, the proB+ gene encodes gamma-glutamyl kinase, and the proA+ gene encodes gamma-glutamyl phosphate reductase. Complementation tests also indicated that the pro-74 mutation is either in the proB structural gene, or its promoter-operator.