Mycobacterium avium subsp. paratuberculosis (Map)is the causative pathogen of Johne's disease, a chronic inflammatory wasting disease in ruminants. The disease has been difficult to control because of the lack of an effective vaccine. To develop a live attenuated vaccine for Map, as well for the study of specific gene function in Map, an efficient method for generating targeted gene mutation is urgently needed. Here, we report an efficient allelic exchange mutagenesis system in Map using an in-vitro generated specialized transducing mycobacteriophage (phAE87). Three genes were selected for this study based on their known function in other mycobacteria: pknG and relA, genes known to be important virulence factors in pathogenic mycobacteria and lsr2, a gene regulating several important pathways related to lipid biosynthesis and multi-drug tolerance in mycobacteria. All three genes were successfully disrupted in a virulent strain, Map K10, as well as in a recombinant strain expressing the green fluorescent protein gene, gfp. The GFP tagged mutants will prove useful for intracellular studies as well as distinguishing vaccinated animals from naturally infected animals. With the optimized conditions we developed, we obtained allelic exchange frequencies of 78 - 100 % with a transduction frequency of 9.5 x 10^-8 - 1.6 x 10^-7. As predicted by its role in other mycobacteria, Δlsr2 showed strikingly different morphology on agar medium compared to wild type. In addition, it failed to form a pellicle in standing broth culture without shaking. To investigate whether the disrupted genes affect the capacity of Map to survive following phagocytosis, an in-vitro infection assay was conducted. Peripheral blood mononuclear cell derived macrophages were infected with each mutant or wild type at MOI of 10. Colony forming units (CFUs) were measured at each time point. On day 1 after infection, while CFU of K10 was increased about 38 % compared to baseline CFU (Time 0), CFU of three mutants was similar or slightly decreased compared to baseline CFU. On day 3, all mutants showed a significant decrease in survival compared to wild type (Percentage of CFU on day 3 compared to baseline CFU: K10, 84.4 %; Δlsr2, 38.9 %; ΔrelA, 37.8 %; ΔpknG, 49.5 %). Further studies on characterization of the mutants are now in progress. The improved method of selectively disrupting genes provides an opportunity to gain insight into specific gene function, mechanisms of pathogenesis and development of an effective vaccine for Map.