Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines.

Successful genetic engineering of mycobacteria is crucial for developing 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 sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning 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 mycobacteria. This vector permits stable expression of genes in M.bovis BCG, M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter. These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter. We propose that expression of protective antigens 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 integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition in M.bovis BCG, M.tuberculosis, and 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.