Cloning and initial characterization of the metJ and metB genes from Salmonella typhimurium LT2

Abstract

The metJ and metB genes of Salmonella typhimurium have been cloned into Escherichia coli K-12 on a 19-kb EcoRI fragment in the plasmid vector pACYC184. The presence of a functional metB+ gene on this plasmid, designated pGS89, was demonstrated by its ability to complement a metB- E. coli mutant. The presence of a functional metJ+ gene on this plasmid was demonstrated by its ability to repress metC+ gene expression in a metJ- mutant transformed with this plasmid. The metJ gene product was identified in a minicell system as a polypeptide of Mr 12000. This polypeptide was not produced when the metJ gene was inactivated by insertion of a Tn5 element. Transformation of an E. coli metB- mutant with plasmid pGS89 (metB+, metJ+) results in transformants that grow slowly on glucose-minimal medium or glucose-minimal medium supplemented with homocysteine. Methionine addition, however, restores normal growth. This phenotype requires the relA- mutation in the host strain and at least two other plasmid loci, one of which is the metJ+ gene. Transformation of an E. coli metJ- mutant with metJ- derivatives of plasmid pGS89 results in transformants that are unable to grow on either glucose-minimal medium or glucose-minimal medium supplemented with methionine. This phenotype requires the presence of a functional metB+ gene on the plasmid, and is unrelated to the status of the relA gene.