Efficient homologous recombination in fast-growing and slow-growing mycobacteria

Although homologous recombination is a major mechanism for DNA rearrangement in most living organisms, it has been difficult to detect in slowly growing mycobacteria by a classical suicide vector approach. Among the possible reasons for this are the low levels of transformation efficiency, the relatively high levels of illegitimate recombination, and the peculiar nature of the recA gene in slowly growing mycobacteria. In this report, we present an efficient homologous recombination system for these organisms based on the use of replicative plasmids which facilitates the detection of rare recombination events, because the proportions of recombined molecules increase over time. Intraplasmid homologous recombination in Mycobacterium smegmatis and Mycobacterium bovis BCG was easily selected by the reconstitution of an interrupted kanamycin resistance gene. Chromosomal integration via homologous recombination was selected by the expression of the kanamycin resistance gene under the control of a chromosomal promoter that was not present in the plasmid before recombination. This technique was termed STORE (for selection technique of recombination events). All the clones selected by STORE had undergone homologous recombination, as evidenced by PCR analyses of the kanamycin-resistant clones. This technique should be applicable to all organisms for which homologous recombination has been difficult to achieve, provided the gene of interest is expressed.