Transposon mutagenesis in mycobacteria using conditionally replicating mycobacteriophages

Genetic analyses of pathogenic mycobacteria such as Mycobacterium tubeculosis and Mycobacterium bovis required improvement of existing methodologies for the generation of large representative libraries of mutants. Two basic methodologies have been used to generate mutant libraries in both fast- and slow-growing mycobacteria: chemical mutagenesis and transposon mutagenesis. Chemical mutagenesis has successfully been used to produce different auxotrophic mutants in the fast growing mycobacteria Mycobacterium phlei (1,2) and Mycobacterium smegmatis (3,4). A detailed chemical mutagenesis protocol for the generation of mutant libraries in the fast-growing mycobacteria can be found in the previous volume of this manual (5). Chemical mutagenesis is not the ideal method for producing large representative mutant libraries for the slow-growing mycobacteria because: (1) the mutation frequency is relatively low,(2) multiple mutations may occur in the same cells,(3) clumping of the mycobacteria makes the identification and purification of the mutant clones very difficult,and (4) no generalized transducing phage has been described for the slow-growing mycobacteria to allow transfer of the point mutations and construction of isogenic strains.