Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria

Abstract

Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory. Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters. Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants. These vectors combine the ease of working in E. coli with the advantage of directly propagating them in mycobacteria without further manipulations. Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG.