EXPRESSION SYSTEMS FOR STUDY OF MYCOBACTERIAL GENE-REGULATION AND DEVELOPMENT OF RECOMBINANT BCG VACCINES

Successful genetic engineering of mycobacteria is crucial for developi ng new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of Mycobacterium tuberculosis. We have constructed a Mycobacterium-Escherichia coli shuttle expression vector pSD5. It carries a modular expression cassette which provides s ites for cloning of promoters, a ribosome binding site (RBS) with an a ppropriately placed initiation codon and multiple cloning sites for cl oning the genes of interest. We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycob acteria. This vector permits stable expression of genes in M.bovis BCC , M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter. These vectors enable the expression of a ge ne to be regulated by several hundred fold depending upon the strength of mycobacterial promoter. We propose that expression of protective a ntigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host. We have further employed the integratio n proficient expression system for comparing the efficiency and specif icity of transcriptional recognition in M.bovis BCG, M.tuberculosis, a nd M.smegmatis. We show that fast growing M.smegmatis and slow growing M.tuberculosis and M.bovis BCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates. ( C) 1998 Academic Press.