CONSTRUCTION OF SHUTTLE VECTORS FOR GENETIC MANIPULATION AND MOLECULAR ANALYSIS OF MYCOBACTERIA

Two novel shuttle vectors for mycobacteria are described which have be en derived from the expression system pSD5 developed in our laboratory . Plasmid pSD5B is a promoter-selection vector containing a promoterle ss lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal. Mor eover, the chronological order of appearance of blue colonies and inte nsity of colour provide a qualitative index of transcriptional strengt hs of the cloned promoters. Plasmid pSD5C has been designed to constru ct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria. Libra ries 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 Escherich ia coil and mycobacterial mutants. These vectors combine the ease of w orking in E. coli with the advantage of directly propagating them in m ycobacteria without further manipulations. Finally, we demonstrate tha t these vectors function efficiently both in fast growing Mycobacteriu m smegmatis and slow growing mycobacteria including Mycobacterium tube rculosis and Mycobacterium bovis BCG.