An Inducible System for the Identification of Target Genes for a Regulator in Mycobacteria

Abstract

We described principle and application of an inducible system for identification of target genes of a given mycobacterial protein with a regulatory function. This vector system, a promoterprobe vector, carries (i) promoterless lacZ as the reporter gene and (ii) the mycobacterial gene encoding the regulatory protein under the transcriptional control of the promoter of highly inducible mycobacterial gene encoding acetamidase. A promoter library of M.tuberculosis is constructed in this vector upstream of the lacZ gene. It is then possible to screen for those promoters that are responsive to the presence of the regulatory protein by inducing the expression of the regulatory gene. The presence of lacZ permits the screening of the promoters based on simple blue-white selection. This system is specifically designed for those regulatory genes of M.tuberculosis which are associated with virulence and thus are absent from M.smegmatis (the non-pathogenic, saprophytic species of mycobacteria), although in principle, modifications can be incorporated in the selection scheme to make it applicable for the identification of target promoter(s) of any regulatory gene of mycobacterial origin. This strategy will be helpful in quick identification of targets for the development of antitubercular drugs and in alleviating some of the stumbling blocks faced by the investigators working in the area of molecular genetics of M.tuberculosis.