Gene Replacement in Mycobacterium intracellulare

Much of the present knowledge of the bacterial cell emanates from studies of spontaneous mutants and mutants obtained as a result of random or directed mutagenesis. Spontaneous mutants in a specific gene can generally be Isolated only if the mutation leads to a selectable phenotype, e.g., mutations in the katG gene that result in resistance to the antimycobacterial agent isoniazld. Random mutagenesis of mycobacteria has recently been reported using mycobacterlal transposons (1 ). However, the hpld-rich cell wall of mycobacterla makes them hydrophobic, which leads to cell clumping. This clumping makes it laborious to purify a large number of separate clones and thereby obtain representative mutant libraries. In addition, the expansion and screening of such clones, which 1s necessary unless the mutant phenotype can be selected for, will be very tlmeconsummg, especially If working with slow-growing mycobacteria.