Genetic transformation of mycobacteria by homologous recombination

Mycobacteria are highly potent adjuvants; therefore, expression of foreign genes in mycobacteria provides a delivery system to induce strong immune responses against foreign proteins. In this study we report transformation of Mycobacterium smegmatis by homologous recombination using pUC19-based plasmid vectors with pyrF gene of M. smegmatis (pY6001) or pyrF gene disrupted by introducing the aminoglycoside phosphotransferase (aph) gene (pY6002). Both of these plasmids were used to transform the host cells by electroporation. The transformation and selection conditions were optimized with respect to cell number, stage of cell growth, DNA concentration, postelectroporation incubation time, and kanamycin concentration. With the plasmid Y6002, the transformation was usually a result of single crossover (class I transformants) and only 5% transformants were generated by double crossover (class II transformants). The double crossover led to the replacement of wild-type pyrF gene with the aph-disrupted pyrF gene. The gene replacement could also occur by resolution of the class I transformants into class II, but at a very low frequency. Further experiments were done to determine if the wild-type genotype could be rescued by retransformation with pY6001. Similar transformation efficiencies, as reported above, were obtained, but the frequency of double crossover increased to 35%. This transformation strategy provides a way by which the mycobacteria transformed with foreign genes will not require drug selection, a trait preferred to develop recombinant vaccines.