Development of a transposon mutagenesis system for Mycobacterium avium subsp paratuberculosis

Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacteri um, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demons trated the ability to introduce DNA into these bacteria by transformation a nd phage infection. In the current study we develop the first transposon mu tagenesis system for M. paratuberculosis using the conditionally replicatin g mycobacteriophage phAE94 to introduce the mycobacterial transposon Tn5367 . Southern blotting and sequence analysis demonstrated that the transposon insertion sites are distributed relatively randomly throughout the M. parat uberculosis genome. We constructed a comprehensive bank of 5620 insertion m utants using this transposon. The transposition frequency obtained using th is delivery system was 1.0x10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%. ( C) 1999 Federation of European Microbiological Societies. Published by Else vier Science B.V. All rights reserved.