Random mutagenesis of Legionella pneumophila with mini-Tn10

C. Dumais Pope, Leena Dhand, Nicholas P. Cianciotto*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

To more effectively study the genetic basis of Legionnaires' disease, we characterized a system for mini-Tn10 mutagenesis in Legionella pneumophila. The mini-transposons were first electroporated into Legionella on counterselectable vectors expressing altered target site transposases. Then, by simultaneously selecting for the kanamycin-resistance gene within the transposon and counterselecting against the maintenance of the plasmid, we directly and readily isolated strains bearing single chromosomal insertions. Southern hybridization analysis further demonstrated that the insertions were randomly distributed throughout the Legionella genome. The mini-Tn10 insertions were stable during extracellular and intracellular growth, and did not alter the infectivity of L. pneumophila. Thus, this mutagenesis system offers an easy, one-step approach toward isolating large populations of random mutants which can be screened for defects in virulence.

Original languageEnglish (US)
Pages (from-to)107-111
Number of pages5
JournalFEMS Microbiology Letters
Volume124
Issue number1
DOIs
StatePublished - Nov 15 1994

Keywords

  • Counterselectable plasmids
  • Legionella pneumophila
  • Legionnaires's disease
  • Mini-Tn10
  • Random mutagenesis
  • U937 cells

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Genetics

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