Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries

Mycobacterium paratuberculosis causes Johne's disease, a common wasting dis ease in ruminants. As a first step in studying virulence mechanisms, librar ies of random mutants were produced in two M. paratuberculosis strains by u sing the conditionally replicating shuttle phasmid phAE94 which contains th e transposon Tn5367. Two thousand mutants were screened for auxotrophy, car bon source preference, and altered cell wall. Genes interrupted by insertio n were identified for seven mutants isolated from the screening process. Tw o mutants had insertions in putative genes involved in synthesis of the cel l wall.