Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis

Abstract

A number of essential genes have been identified in mycobacteria, but methods to study these genes have not been developed, leaving us unable to determine the function or biology of the genes. We investigated the use of a tetracycline-inducible expression system in Mycobacterium tuberculosis and Mycobacterium smegmatis. Using a reporter gene which encodes an unstable variant of GFP, we showed that tetracycline-inducible expression occurred in M. smegmatis and that expression levels were titratable to some extent by varying the concentration of tetracycline. The removal of tetracycline led to cessation of GFP expression, and we showed that this was a controllable on/off switch for fluorescence upon addition and removal of the antibiotic inducer. The system also functioned in M. tuberculosis, giving inducible expression of the reporter gene. We used homologous recombination to construct a strain of M. tuberculosis that expressed the only copy of the tryptophan biosynthetic enzyme, TrpD, from the tetracycline-inducible promoter. This strain was conditionally auxotrophic, showing auxotrophy only in the absence of tetracycline, confirming that trpD was tightly controlled by the foreign promoter. This is the first demonstration of the use of an inducible promoter to generate a conditional auxotroph of M. tuberculosis. The ability to tightly regulate genes now gives us the possibility to define the functions of essential genes by switching them off under defined conditions and paves the way for in vivo studies.

FIG. 1.

tetRPO system. The two operator sites at which the TetR repressor binds in the absence of tetracycline (Op) overlap two divergent promoters (P_rep and P_tet). One promoter directs expression of the tetR gene (P_rep), and the other directs expression of the inserted gene (P_tet). (Top) Simple tetRPO system. (Bottom) P_Ag85A-tetRPO system. TetR expression is driven by the antigen 85A promoter from M. tuberculosis.

FIG. 2.

Controlled expression of GFP using the tetracycline-inducible systems. M. smegmatis transformants were assayed for gfp expression after growth in various concentrations of tetracycline. (A) pFLAME3-derivedvectors. (B) pFLAME4-derived vectors. The experiment was repeated twice with independent transformants, and the results of a representative experiment are shown. The standard deviations in experiments were within the range from 5 to 15%.

FIG. 3.

Temporal control of expression in M. smegmatis. M. smegmatis transformants carrying different plasmids were subcultured on a daily basis in medium containing no tetracycline (OFF) and medium containing tetracycline (ON), and fluorescence was measured. For pFLAME-ace vectors acetamide was used for the ON media rather than tetracycline. (Top) pFLAME3-derived vectors. (Bottom) pFLAME4-derived vectors. The results are the averages of three independent transformants. The experiment was repeated twice with independent cultures, and the same results were obtained.

FIG. 4.

Inducible expression of gfp in M. tuberculosis. M. tuberculosis transformants carrying different plasmids were grown for 2 weeks, and gfp expression was assayed by measuring fluorescence in cell extracts. Tetracycline (200 ng/ml) was added to most ON cultures; the only exception was pFLAME3-ace, which was cultured in the presence of acetamide. The bars indicate the averages for three independent transformant cultures, and the error bars indicate standard deviations.

FIG. 5.

Inducible expression of trpD using the tetRPO system in M. tuberculosis: growth of transformants carrying pTACT1 and pTACT3. Wild-type (WT) and auxotrophic strains carrying both pTACT3 (P_tet-trpD) and pTACT1 (P_tet empty control) were grown in various media, as indicated; growth was read after 9 days. The results are the averages ± standard deviations for three independent transformants.

FIG. 6.

Construction of conditional auxotrophs. (A) The recombination vector pTACT21 was constructed by cloning the 5′ region of trpD downstream of tetRPO into a vector which does not replicate in mycobacteria. Single-crossover recombinants were generated by homologous recombination. The resulting strains had one complete functional copy of trpD downstream of P_tet, as well as the 5′ end of trpD only. EcoRI sites are indicated by numbers in parentheses. WT, wild type; kan, kanamycin resistance; hyg, hygromycin resistance. (B) Southern analysis of the recombinants. DNA was digested with EcoRI and probed with the complete trpD gene indicated in panel A. The two SCO bands and the resulting loss of the wild-type band (wt) expected in the Southern analysis are also shown as black lines in panel A. Lanes 1 to 5 contained five independent mutants, and lane 6 contained wild-type strain H37Rv.

FIG. 7.

Growth phenotypes of conditional auxotrophs. (A to C) Three independent recombinant strains were grown in liquid media containing hygromycin. (D) Control strain growth. The control strain carried both hygromycin and kanamycin resistance genes integrated into the chromosome via homologous recombination. Symbols: ♦, Middlebrook 7H9 medium; ▪, medium containing tetracycline (200 ng/ml); ▴, medium containing tryptophan (50 μg/ml); ×, medium containing tetracycline and tryptophan.

FIG. 8.

Titration of trpD expression. One representative recombinant strain (Tact 21) and the wild type were grown in the presence of various tetracycline concentrations for 12 days. Growth is expressed as a percentage of the growth of the strain with no tetracycline. Symbols: ♦, wild type; ▪, Tact 21.