IDENTIFICATION OF MYCOBACTERIUM-TUBERCULOSIS DNA-SEQUENCES ENCODING EXPORTED PROTEINS BY USING PHOA GENE FUSIONS

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11 ) allowing fusions between phoA and genes encoding exported proteins w as constructed to study protein export in mycobacteria. Introduction o f the Mycobacterium fortuitum beta-lactamase gene (blaF) into this ve ctor led to the production in M. smegmatis of protein fusions with Pho A activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Seq uences of the M. tuberculosis inserts directing the production of prot ein fusions in these PhoA-positive clones were determined. They includ e part of the already-known exported 19-kDa lipoprotein, a sequence wi th similarities to the exported 28-kDa antigen from M. leprae, a seque nce encoding a protein sharing conserved amino acid motifs with stearo yl-acyl-carrier-protein desaturases, and unknown sequences. This appro ach thus appears to identify sequences directing protein export, and w e expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.