RANDOM MUTAGENESIS OF LEGIONELLA-PNEUMOPHILA WITH MINI-TN10

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