Cloning of an EcoRI-generated fragment of the leucine operon of Salmonella typhimurium

Abstract

Recombinant plasmids carrying part of the leucine operon of Salmonella typhimurium were isolated following transformation of an Escherichia coli leucine auxotroph to prototrophy with a ligated mixture of EcoRI-treated Salmonella DNA and plasmid pSC101 DNA. Plasmids pCV11 and pCV13, containing a 3.4-10(6) dalton DNA fragment ligated to the vector, had the leu operon oriented in opposite directions. The orientation of the leu operon relative to plasmid genes was determined. The 3.4-10(6) dalton fragment was ligated in to the EcoRI site of plasmid pMB9 yielding plasmids pCV12 (orientation as in pCV11) and pCV14 (orientation as in pCV13). The results of enzyme assays and complementation tests indicated that these plasmids carry functional leuA, leuB, and leuC genes but not a functional leuD gene. Furthermore, the following results indicated that they have a functional leu control region and promoter. Expression of plasmid leu genes was markedly enhanced under conditions of leucine limitation whereas introduction of a leu promoter mutation into the operon oriented in either direction with respect to plasmid genes had a strong negative effect upon leu operon expression. Transcriptional readthrough from plasmid promoters, if it occurs at all, must be small in comparison with transcription initiated at the leu promoter. RNA was isolated from leucine auxotrophs grown under conditions of repression and derepression and from prototrophic strains derepressed for the leucine operon as a result of mutations in leuO, leuS, and flrB. The rate of synthesis of leu mRNA, measured by hybridization to plasmid pCV12 DNA, was proportional in each case to leu enzyme levels.