The Use of Temperature-Sensitive Plasmids in Mycobacteria

The construction of allelic exchange mutants is one of the major strategies to decipher the function of a defined gene. In this chapter, protocols are described to perform allelic exchange in the mycobacterial species Mycobacterium smegmatis and Mycobacterium tuberculosis using thermosensitive counterselectable vectors. The antibiotic resistance cassette used makes it possible to efficiently rescue the marker, leaving an unmarked mutation on the chromosome. In addition, a method is provided to construct conditional M. smegmatis mutants and to test whether or not a gene is essential.