Assess the utility of the PIP-ON system in M. tb and overexpress or silence several mycobacterial genes

In this system the gene of interest is under transcriptional control of the Streptomyces Pip protein, the pristinamycin sensitive repressor. The pip gene of S. coelicolor has been cloned downstream of a constitutive mycobacterial promoter (the mutant pfurA102 of M. tb), and introduced in Mycobacterium smegmatis (see pMYS696 in Fig. 1). Furthermore, a Pip-ON system has been constructed, by cloning the S. pristinaespiralis ptr promoter upstream of a reporter gene (lacZ), in the above plasmid (see pMY718 in Fig. 1). These constructs have been introduced in M. smegmatis and beta-galactosidase activity measured: the activity expressed from pMY718 is very low (less than 10 Miller units) and inducible, in a dose dependent manner, by addition of pristinamycin.