title: Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis.
abstract: Mycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis 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 M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis 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 M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium 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.
authors.affiliation: Department of Veterinary and Biomedical Sciences, Center for Biotechnology, University of Nebraska, Lincoln, NE 68583-0905, USA.
authors.forename: Ellen M
authors.initials: EM
authors.lastname: Foley-Thomas
authors.affiliation: United States Department of Agriculture, National Animal Disease Center, Ames, IA 50010, USA.
authors.forename: Diana L
authors.initials: DL
authors.lastname: Whipple
authors.affiliation: Kuzell Institute for Arthritis and Infectious Diseases, California Pacific Medical Center Research Institute, San Francisco, CA 94115-1821, USA.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: Bermudez
authors.affiliation: Department of Veterinary and Biomedical Sciences, Center for Biotechnology, University of Nebraska, Lincoln, NE 68583-0905, USA.
authors.forename: Raul G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D017088:AIDS-Related Opportunistic Infections / Q000821:virology; D000583:Amikacin / Q000494:pharmacology; D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D006801:Humans; D007612:Kanamycin / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis; D008826:Microbial Sensitivity Tests; D015269:Mycobacterium avium Complex / Q000187:drug effects / Q000235:genetics* / Q000821:virology*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000187:drug effects / Q000235:genetics* / Q000821:virology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D013045:Species Specificity; D014162:Transfection; D014169:Transformation, Bacterial; D014376:Tuberculosis / Q000209:etiology / Q000821:virology
chemical_list: D011994:Recombinant Proteins; D007612:Kanamycin; D000583:Amikacin; D008156:Luciferases