Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Abstract

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth.

Figure 1.

Model of the TetR/Pip OFF system. In the absence of tetracycline (Tc), TetR binds to its operators turning off pip transcription, and allowing lacZ expression. In the presence of Tc, transcription of pip is allowed. As a result, Pip represses lacZ expression.

Figure 2.

Characterization of the TetR/Pip OFF system. (A) β-galactosidase assay in M. smegmatis in liquid media: MS82 and MS83 were grown in Middlebrook 7H9 medium with or without 50 ng/ml ATc. White bars: bacteria grown without ATc; grey bars: bacteria exposed to ATc for 6 h; black bars: bacteria exposed to ATc for 18 h. (B) β-galactosidase assay in M. tuberculosis in liquid media: TB38.1 and TB38.2 were grown in Middlebrook 7H9 medium with or without 200 ng/ml ATc. White bars: bacteria grown without ATc; black bars: bacteria exposed to ATc for 48 h. Results are expressed in Miller units.

Figure 3.

β-Galactosidase assay in liquid media. (A) M. smegmatis MS82 was pre-grown in Middlebrook 7H9 containing 50 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 24 h and for 3 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 24 h; gray bar: 48 h; white bars: 72 h. (B) TB38.2 was pre-grown in Middlebrook 7H9 containing 200 ng/ml ATc, and diluted into fresh media containing different ATc concentrations. Every 48 h and for 6 days, one aliquot of each culture was used to measure β-galactosidase activity and one aliquot was diluted into fresh medium containing the same amount of ATc as before. Black bars: 48 h; grey bar: 96 h; white bars: 144 h. Values are indicated as percentages of maximal activity.

Figure 4.

β-Galactosidase assay in liquid media. TB38.2 and TB29 were pregrown in 7H9 in the absence of ATc and diluted in fresh media with (TB38.2) or without ATc (TB38.2 and TB29). Every 48 h and for 6 days, one aliquot of each culture was diluted into fresh medium containing the same amount of ATc as before. β-Galactosidase activity was measured at 96 (grey bars) and 144 h (white bars).

Figure 5.

Characterization of the M. smegmatis ftsZ mutant MS98. (A) MS98 was plated on Middlebrook 7H10 medium with or without 50 ng/ml ATc. (B) MS98 and its parental wild-type strain were grown in Middlebrook 7H9 with or without ATc.

Figure 6.

Characterization of the M. tuberculosis fadD32 conditional mutant TB47. (A) Growth curve in the presence of different concentrations of ATc. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc (empty triangles), 100 ng/ml ATc (crosses), 50 ng/ml ATc (filled triangles) or No ATc (empty squares). (B) Viable counts variation during growth inhibition due to fadD32 depletion. TB47 was grown in Middlebrook 7H9 medium containing 200 ng/ml ATc. An aliquot of the bacterial culture was collected at different time points starting from 48 h after the beginning of the experiment, diluted and plated for cfu determination. Squares: cfu/ml; diamonds:optical density at 540 nm.

Figure 7.

Changes in the relative amounts of fadD32 mRNA measured by quantitative RT-PCR. The values are expressed as the ratio between the number of cDNA copies detected in a TB47 culture grown in the absence of ATc, and the number of cDNA copies detected in cultures grown with 200 ng/ml ATc. The values are normalized to sigA. Samples for RNA extraction were collected at 24 and 48 h after addition of ATc.

Figure 8.

Growth of M. tuberculosis conditional mutant TB47 in THP-1-derived human macrophages in the presence (triangles) or in the absence (squares) of 200 ng/ml ATc.

Figure 9.

Growth curve of the fadD32 conditional mutant TB47 in the presence of ATc and pristinamycin I. Bacteria were grown in Middlebrook 7H9 without ATc (diamonds) or with 200 ng/ml ATc (all the others). Forty-eight hours after the beginning of the experiment cultures were supplemented with 20 (filled triangles), 200 (circles) or 2000 ng/ml (open triangles) of pristinamycin I. As a control, one culture grown in 200 ng/ml ATc did not receive pristinamycin I (squares).