EFFICIENT TRANSPOSITION IN MYCOBACTERIA - CONSTRUCTION OF MYCOBACTERIUM-SMEGMATIS INSERTIONAL MUTANT LIBRARIES

The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons. The resulting plasmid , pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis. These are the first mycobacterial insertio nal mutant libraries to be constructed by transposition directly into a mycobacterium. No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the in sertion mutants. Auxotrophic mutants with various phenotypes were isol ated at a frequency ranging from 0.1 to 0.4%, suggesting that the libr aries are representative. The pCG79 system thus seems to be a useful t ool for the study of M. smegmatis genetics and may be applicable to ot her mycobacteria, such as the M. tuberculosis complex.