Scar-Free Genome Editing in Legionella pneumophila

Studying bacterial physiology and pathogenesis often requires isolation of targeted mutants. From the early days of bacterial genetics, many genetic tools have been developed to achieve this goal in a lot of bacteria species, and a major key is to be able to manipulate the targeted genome region with a minimum impact on the rest of the genome. Here, we described a two-step protocol relevant in Legionella pneumophila. This efficient two-step protocol uses the natural transformability of L. pneumophila and linear DNA fragments as substrates for recombination without the necessity of intermediate hosts to amplify targeted DNA. Based on a suicide cassette strategy, this genetic toolbox enables to generate clean scar-free deletions, single-nucleotide mutation, transcriptional or translational fusions, as well as insertion at any chosen place in L. pneumophila chromosome, therefore enabling multiple mutations with no need of multiple selection markers.