Molecular cloning and characterization of supQ/newD, a gene substitution system for the leuD gene of Salmonella typhimurium

Abstract

The isopropylmalate isomerase of Salmonella typhimurium and Escherichia coli is a complex of the leuC and leuD gene products. The supQ/new D gene substitution system in S. typhimurium restores leucine prototrophy to leuD mutants of S. typhimurium. Previous genetic evidence supports a model that indicates the replacement of the missing LeuD polypeptide by the newD gene product. This model proposed that this gene substitution is possible when a mutation at the supQ locus (near newD) liberates unaltered newD polypeptide from its normal complex with the supQ protein product. In this study, recombinant plasmids carrying newD, supQ, or both were transformed into E. coli and S. typhimurium strains deleted for the leuD and supQ genes to test the supQ/newD gene substitution model for suppression of leucine auxotrophy. It was determined that the newD gene encodes a 22-kilodalton polypeptide which can restore leucine prototrophy to leuD deletion strains and that a functional supQ gene prevents this suppression. It was also determined that the supQ and newD genes are separated by a gene encoding a 50-kilodalton protein, pB. While there is extensive DNA sequence homology between the leucine operons of S. typhimurium and E. coli, DNA hybridization experiments did not indicate substantial homology between the newD and leuD genes. These data, taken together with previously obtained genetic data, eliminate the possibility that supQ and newD are recently translocated segments of the leucine operon.