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.

Figure 1

Ratio between escape mutants able to growth in ATc (50 ng/ml) and total cells population after 24 h of growth of different M. smegmatis mutants. The reported values derive from three independent experiments. *P < 0.05; **P < 0.01 versus Ms98 (Student’s t-test).

Figure 2

Changes in ftsZ mRNA levels upon exposure of exponentially growing cultures to 50 ng/ml ATc for 7 hours in the conditional ftsZ mutants MS98 and MS165 and in their parental wt strain mc²155. Values are expressed as the ratio between the number of cDNA copies detected in samples obtained from bacteria exposed to ATc and the number of cDNA copies detected in samples from parallel cultures not exposed to ATc. The values were normalized to the level of mysA cDNA, which represented the internal invariant control. The reported values derive from two indipendent experiments.

Figure 3

Ratio between escape mutants able to growth in ATc (500 ng/ml) and total cells population after 5 days of incubation of different M. tuberculosis mutants. The reported values derive from two independent experiments. *P < 0.05; **P < 0.01 versus TB259 (Student’s t-test).

Figure 4

Growth curve of Ms98 and Ms165 ATc 50 ng/ml. ATc was added at T0, T24 and T48 to provide continue ftsZ repression (arrows). (A) Effects of ftsZ gene repression after 24 hours of incubation in the presence of ATc: clumping of both mutants is visible on the bottom of the tubes; (B) Effects of ftsZ gene repression after 48 hours of incubation in the presence of ATc: growth resumption in MS98 culture, but not in MS165 culture is visible. Data represent the average of two independent experiments. Circles: MS98; triangles: MS165.