Essentiality testing with inducible promoters

Objectives: Assess the utility of the PIP-ON system in M.tb. Progress: The PIP-ON system was transferred in M. tb where we could show a very efficient repression of Pip on the ptr promoter. In response to pristinamicin we could see a dose dependent induction of the promoter obtaining a maximum induction of about 100 fold with 150 ng/ml of pristinamicin. The construction of plasmids where the expression of pknB, glnA1 and fad32 genes and of their antisense RNA is under the ptr promoter control and thus responding to pristinamicin is under way. An alternative approach using a TET/PIP repressible system was developed. The reporter gene is represented by the same one used for the PIP ON system (lacZ under ptr promoter transcriptional control). In this case the PIP repressor is provided in trans under the control of a TET dependant promoter. In the absence of inducer PIP should not be produced and the ptr promoter should be expressed constitutively. Addition of tetracycline should induce the expression of the PIP repressor structural gene causing the repression of transcription of the ptr promoter. Removal of tetracycline and/or addition of pristinamycin should restore its transcription. The system, introduced in M. smegmatis did not give the expected results probably due to the high strength of the TET-dependant promoter used in these constructs. We decided to place PIP under the control of a weaker promoter. We chose a mutated furA promoter previously shown to express PIP at functional level. The promoter was mutagenized to introduce two tet-operators, one immediately upstream the -35 region and the other between the -35 and the -10, placed upstream the PIP conding region and finally cloned into an integrative vector containing a lacZ gene under ptr- promoter transcriptional control. This plasmid has been sequenced and introduced in a M smegmatis strain expressing the TET repressor from a replicative plasmid and is actually under analysis