title: Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis.
abstract: Mycobacterium avium subspecies paratuberculosis, a slow-growing Mycobacterium, is the causative agent of Johne's disease. Although M. paratuberculosis is difficult to manipulate genetically, our laboratory has recently demonstrated the ability to introduce DNA into these bacteria by transformation and phage infection. In the current study we develop the first transposon mutagenesis system for M. paratuberculosis using the conditionally replicating 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. paratuberculosis genome. We constructed a comprehensive bank of 5620 insertion mutants using this transposon. The transposition frequency obtained using this delivery system was 1.0 x 10(-6) transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%.
authors.affiliation: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, USA.
authors.forename: N B
authors.initials: NB
authors.lastname: Harris
authors.forename: Z
authors.initials: Z
authors.lastname: Feng
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: S L
authors.initials: SL
authors.lastname: Cirillo
authors.forename: J D
authors.initials: JD
authors.lastname: Cirillo
authors.forename: R G
authors.initials: RG
authors.lastname: Barletta
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D004251:DNA Transposable Elements; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009160:Mycobacteriophages / Q000235:genetics; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000254:growth & development / Q000821:virology; D017422:Sequence Analysis, DNA
chemical_list: D004251:DNA Transposable Elements