Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

Abstract

The ilvGEDAY genes of Salmonella typhimurium were cloned in Escherichia coli K-12 by in vitro recombination techniques. A single species of recombinant plasmid, designated pDU1, was obtained by selecting for Valr Ampr transformants of strain SK1592. pDU1 was shown to contain a 14-kilobase EcoRI partial digestion product of the S. typhimurium chromosome inserted into the EcoRI site of the pVH2124 cloning vector. The ilvGEDAY genes were found to occupy a maximum length of 7.5 kilobases. Restriction endonuclease analysis of the S. typhimurium ilv gene cluster provided another demonstration of the gene order as well as established the location of ilv Y between ilvA and ilvC. The presence of a ribosomal ribonucleic acid operon on the pDU1 insert, about 3 kilobases from the 5' end of ilvG, was shown by Southern hybridization. The expression of the ilvGEDA operon from pDU1 was found to be elevated, reflecting the increased gene dosage of the multicopy plasmid. A polarity was observed with respect to ilvEDA expression which is discussed in terms of the possible translational effects of the two internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.