Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains.
METHODS
Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression.


OBJECTIVE
To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains.


METHODS
Plasmids were constructed using mycobacterial promoters (hsp60, 19-kDa antigen, 85A antigen--from the Mycobacterium tuberculosis complex--and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes.


RESULTS
The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product. Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters. BCG Moreau appeared to be more susceptible to deletions than other BCG strains.


CONCLUSIONS
The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product. Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation. With intact plasmid there was no marked difference in expression between BCG strains.
Al-Zarouni
Mansour
M
School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, UK.
Dale
Jeremy W
JW
D000818:Animals; D015153:Blotting, Western; D018834:Chaperonin 60 / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D001618:beta-Lactamases / Q000378:metabolism
D018834:Chaperonin 60; D001618:beta-Lactamases