Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis.
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.
Carroll
Paul
P
Centre for Infectious Disease, Institute for Cell and Molecular Science, Barts and London, Turner Street, London E1 2AD, United Kingdom.
Muttucumaru
D G Niranjala
DG
Parish
Tanya
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D013752:Tetracycline / Q000494:pharmacology*; D014364:Tryptophan / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; D014364:Tryptophan; D013752:Tetracycline