Random mutagenesis of Legionella pneumophila with mini-Tn10

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.

The TolC Protein of Legionella pneumophila Plays a Major Role in Multi-Drug Resistance and the Early Steps of Host Invasion

“…A derivative plasmid of pCDP05 was constructed in the laboratory. pCDP05 plasmid is a suicide vector which was used in a previous study to obtain random insertions of a kanamycine resistance cassette on the chromosome of Legionella pneumophila [39]. This plasmid bears the sacB gene of Bacillus subtilis which expression in the presence of sucrose leads to the accumulation of levans at noxious concentrations to Gram-negative bacterial cells when accumulated in periplasmic space.…”

Counterselectable Markers: Untapped Tools for Bacterial Genetics and Pathogenesis

“…Transposon mutagenesis. Despite the similarities between allelic exchange and transposon mutagenesis, counterselectable markers have rarely been used for transposon mutagenesis (12,25,35). Clearly, all the above-described double-selection strategies can be used for transposon mutagenesis if the vector used to deliver the transposon is lost, or in other words, when the mobile element transposes in a conservative fashion, or when the cointegrate is resolved by a specific resolvase or by homologous recombination.…”

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

“…Legionella is amenable for gene deletion by homologous recombination (Merriam et al, 1997 ; Bryan et al, 2011 ; O'Connor et al, 2011 ) and mutagenesis with transposons (Ott, 1994 ; Pope et al, 1994 ; Edelstein et al, 1999 ; O'Connor et al, 2011 ). Assays recording intracellular growth by colony counting or continuously, using fluorescent or bioluminescent strains, are established (Coers et al, 2007 ; Tiaden et al, 2013 ; Schroeder et al, 2015 ).…”