Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona

Abstract

The leuB gene of Leptospira interrogans serovar pomona strain kenniwicki has been cloned on a 9.5 kb plasmid, pWVL1, by complementation of Escherichia coli leuB mutants. Subcloning and Tn5 mutagenesis showed that the region required for complementation was approximately 1.2 kb in length. Enzyme assays showed that the product of the cloned gene was a beta-isopropylmalate dehydrogenase. Defects in the leuA, leuC and leuD genes of E. coli were not complemented by pWVL1. The nucleotide sequence of the leuB-complementing region and surrounding DNA has been determined. Three open reading frames were found which encode proteins of 40.9, 38.8 and 15 kDa. Analysis of subclones containing nucleotide deletions of varying sizes showed that only the 38.8 kDa protein was necessary to obtain complementation of E. coli leuB mutations. The PIR data base was searched and the enzyme 3-isopropylmalate dehydrogenase from six different micro-organisms was found to share significant amino acid sequence similarity (43-57%) with the 38.8 kDa L. interrogans leuB gene product. The organization of the leucine biosynthetic genes in L. interrogans differs from that found in E. coli, Salmonella typhimurium and Bacillus subtilis.