Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis

N. Beth Harris, Zhengyu Feng, Xiaofei Liu, Suat L.G. Cirillo, Jeffrey D. Cirillo, Raúl G. Barletta

Research output: Contribution to journalArticle

35 Scopus citations

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 1.0x10-6 transposition events per recipient cell. Auxotrophic mutants were observed in this library at a frequency of 0.3%. Copyright (C) 1999 Federation of European Microbiological Societies.

Original languageEnglish (US)
Pages (from-to)21-26
Number of pages6
JournalFEMS Microbiology Letters
Volume175
Issue number1
DOIs
StatePublished - Jun 1 1999

Keywords

  • Mutant library
  • Paratuberculosis
  • Transposon mutagenesis

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

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