Open AccessRandom mutagenesis of Legionella pneumophila with mini‐Tn 10
Author(s) -
Dumais Pope C.,
Dhand Leena,
Cianciotto Nicholas P.
Publication year - 1994
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1994.tb07269.x
Subject(s) - transposon mutagenesis , legionella pneumophila , tn10 , biology , transposable element , mutagenesis , kanamycin , legionella , microbiology and biotechnology , insertional mutagenesis , virulence , infectivity , plasmid , transposase , genetics , mutant , gene , bacteria , virus
To more effectively study the genetic basis of Legionnaires' disease, we characterized a system for mini‐Tn 10 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‐Tn 10 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.