Molecular cloning and expression of the ilvGEDAY genes from Salmonella typhimurium

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Abstract
The ilvGEDAY genes of S. typhimurium, which codes for proteins and enzymes involved in L-isoleucine and L-valine synthesis, 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 [valinomycin resistant] Ampr [ampicillin resistant] transformants of strain SK1592. pDU1 contained 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 occupied 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 ilvY between ilvA and ilvC. The presence of a rRNA 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 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 2 internal promoter sequences, one located proximal to ilvE and the other located proximal to ilvD.