Improving the stability of the TetR/Pip-OFF mycobacterial repressible promoter system

Abstract

Tightly regulated gene expression systems are powerful tools to study essential genes and characterize potential drug targets. In a past work we reported the construction of a very stringent and versatile repressible promoter system for Mycobacterium tuberculosis based on two different repressors (TetR/Pip-OFF system). This system, causing the repression of the target gene in response to anhydrotetracycline (ATc), has been successfully used in several laboratories to characterize essential genes in different mycobacterial species both in vitro and in vivo . One of the limits of this system was its instability, leading to the selection of mutants in which the expression of the target gene was no longer repressible. In this paper we demonstrated that the instability was mainly due either to the loss of the integrative plasmid carrying the genes encoding the two repressors, or to the selection of a frameshift mutation in the gene encoding the repressors Pip. To solve these problems, we (i) constructed a new integrative vector in which the gene encoding the integrase was deleted to increase its stability, and (ii) developed a new integrative vector carrying the gene encoding Pip to introduce a second copy of this gene in the chromosome. The use of these new tools was shown to reduce drastically the selection of escape mutants.

Construction and Use of Transposon MycoTetOP2 for Isolation of Conditional Mycobacteria Mutants

“…It is noted that the genes identified by Forti et al (2011) and those here have no overlaps. We suggest that it is advantageous to have different and new regulatory schemes (Schnappinger and Ehrt, 2014;Leotta et al, 2015;Boldrin et al, 2018Boldrin et al, , 2019 for the design of transposons for the investigation of mycobacterial biology in the future.…”

“…The significant road block here is that mutants in these genes are not readily available for follow-up studies. Rather, their investigation requires reverse genetics to construct conditional mutants using regulated promoters to deplete a gene product (Schnappinger and Ehrt, 2014;Leotta et al, 2015;Boldrin et al, 2018Boldrin et al, , 2019. A tetracycline regulatable system has been successfully adapted for use in mycobacteria for the construction of conditional mutations (Ehrt et al, 2005;Ehrt and Schnappinger, 2006).…”