Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis.

ABSTRACT

The Escherichia coli-mycobacterium shuttle vector pJAM2 has been used to inducibly express genes in mycobacteria. The vector carries the promoter region from the highly inducible acetamidase gene of Mycobacterium smegmatis which is used to drive expression of heterologous genes. We used pJAM2 to over-express the Mycobacterium tuberculosis gene Rv2868c, a homologue of gcpE. In M. smegmatis the plasmid was stable, but the promoter region was readily deleted when the parental vector or recombinant plasmids were transformed into M. tuberculosis. We mapped the deletion by sequencing and found that it encompassed the entire acetamidase promoter and adjacent sequence totalling approximately 7.3 kb and occurred very soon after introduction into M. tuberculosis. This is the first report of instability of a vector carrying the acetamidase promoter in M. tuberculosis.