complex strains and are closely related intracellular pathogens affecting humans and animals. complex infections are a leading cause of morbidity and mortality in AIDS patients, and is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in complex and . The mycobacteriophage TM4 propagated on or plaqued at the same efficiency on these two mycobacterial hosts. Screening of complex and clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of with TM4 DNA and applied to isolate kanamycin-resistant transformants of complex and with shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species.


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