Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality.

UNLABELLED: SWTTING: Demonstrating that a gene is essential is always difficult, but this is particularly true for a slow-growing organism such as Mycobacterium tuberculosis. One method currently used is to show that homologous recombination leading to gene inactivation only occurs in the presence of a second copy of the gene, but obtaining statistically significant data can be prohibitively difficult. L5-based integrating plasmids have been widely used in the genetic analysis of mycobacteria. The L5 excisionase has been used in Mycobacterium smegmatis to excise and recover these plasmids from chromosome. OBJECTIVE: Our aims were to establish whether the L5 excisionase could function in M. tuberculosis to remove an L5-based integrated plasmid and, if so, to use this technology as the basis for an improved method for determining whether a gene is essential. DESIGN: We took two strains of M. tuberculosis carrying the essential gene glnE integrated into the chromosome on an L5-based plasmid, one of which lacked the functional chromosomal copy of the gene. We transformed these with vectors expressing the L5 excisionase and looked for loss of the integrated plasmid. RESULTS: We obtained efficient excision of an integrated vector from the wild-type strain. However, when the integrated vector carried the only functional copy of the essential gene glnE, the numbers of colonies recovered were reduced to background levels. CONCLUSION: The L5 excisionase does function in M. tuberculosis and can be used to confirm the essentiality of a gene. This technology also allows further analysis of essential genes that is difficult or impossible using current methods.