GENETIC-TRANSFORMATION OF MYCOBACTERIA BY HOMOLOGOUS RECOMBINATION

Mycobacteria are highly potent adjuvants; therefore, expression of for eign genes in mycobacteria provides a delivery system to induce strong immune responses against foreign proteins. In this study we report tr ansformation of Mycobacterium smegmatis by homologous recombination us ing pUC19-based plasmid vectors with pyrF gene of M. smegmatis (pY6001 ) or pyrF gene disrupted by introducing the aminoglycoside phosphotran sferase (aph) gene (pY6002). Both of these plasmids were used to trans form the host cells by electroporation. The transformation and selecti on conditions were optimized with respect to cell number, stage of cel l growth, DNA concentration, postelectroporation incubation time, and kanamycin concentration, With the plasmid Y6002, the transformation wa s usually a result of single crossover (class I transformants) and onl y 5% transformants were generated by double crossover (class II transf ormants). The double crossover led to the replacement of wild-type pyr F gene with the aph-disrupted pyrF gene. The gene replacement could al so occur by resolution of the class I transformants into class II, but at a very low frequency. Further experiments were done to determine i f the wild-type genotype could be rescued by retransformation with pY6 001. Similar transformation efficiencies, as reported above, were obta ined, but the frequency of double crossover increased to 35%. This tra nsformation strategy provides a way by which the mycobacteria transfor med with foreign genes will not require drug selection, a trait prefer red to develop recombinant vaccines.