Over expression of the constitutive expression refractory Mycobacterium tuberculosis Rv3488 protein with an...

As inducible protein expression plasmids available for mycobacterial species are limited, here we demonstrate the utility of an acid-inducible promoter driving gene and subsequent protein expression of a difficult to express protein. We wanted to assess the use of an acid inducible promoter expression system to produce proteins in a mycobacterial system, specifically proteins that when overexpressed interfere with mycobacterial cell growth. Overexpression of those types of proteins would require a tightly regulated promoter system. We employed the Mycobacterium tuberculosis lipF minimal acid-inducible promoter (mpr) which had previously been shown to be upregulated by acidic stress >100 X and to have a low basal level of expression in the absence of acidic stress. It is active from pH 4.3 up to pH 6.4 making this an acid range that is compatible with mycobacterial growth or survival and active at acidic ranges encountered in vivo within acidified phagosomes of macrophages. We therefore cloned the M. tuberculosis gene Rv3488, whose product had been unable to be expressed constitutively, into a plasmid downstream of the lipF mpr promoter and overexpressed this gene in the presence of acidic pH in Mycobacterium smegmatis. Sustained overexpression of the gene leads to inhibition of replication of mycobacterial cells as well as inhibition of carotenoid pigment synthesis, while short-term overexpression leads to appropriate protein expression.