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, Raul G Barletta

Research output: Contribution to journalArticle

34 Citations (Scopus)

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

Fingerprint

Paratuberculosis
Mycobacterium avium
Mutagenesis
Mycobacteriophages
Mycobacterium avium subsp. paratuberculosis
Mycobacterium
Southern Blotting
Bacteriophages
Libraries
Sequence Analysis
Genome
Bacteria
DNA
Infection

Keywords

  • Mutant library
  • Paratuberculosis
  • Transposon mutagenesis

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology
  • Applied Microbiology and Biotechnology
  • Microbiology

Cite this

Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis. / Harris, N. Beth; Feng, Zhengyu; Liu, Xiaofei; Cirillo, Suat L G; Cirillo, Jeffrey D.; Barletta, Raul G.

In: FEMS Microbiology Letters, Vol. 175, No. 1, 01.06.1999, p. 21-26.

Research output: Contribution to journalArticle

Harris, N. Beth ; Feng, Zhengyu ; Liu, Xiaofei ; Cirillo, Suat L G ; Cirillo, Jeffrey D. ; Barletta, Raul G. / Development of a transposon mutagenesis system for Mycobacterium avium subsp. paratuberculosis. In: FEMS Microbiology Letters. 1999 ; Vol. 175, No. 1. pp. 21-26.
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