Querying Legionella Genomes Using Transposition-Sequencing.

Transposition-sequencing (Tn-seq) has recently emerged as a powerful technique to query bacterial genomes. Tn-seq can be used to query the bacterial genome with unprecedented resolution, allowing the identification of small genes (e.g., noncoding RNA) that may be missed in conventional screening approaches. Tn-seq can be used to predict genes essential for in vitro growth and to directly identify genetic requirements for survival under multiple conditions. For instance, Tn-seq can be applied to determine the genes, and cellular processes, required to resist an antibacterial treatment or to acquire new resistance genes, to adapt to intracellular life or to compete with other bacteria. Virtually any assay that involves a selection pressure can be used to identify the associated genetic determinants. So far, genome-wide Tn-seq has not been applied to Legionella species. Here, we provide a protocol covering all the different steps to conduct a Tn-seq analysis in L. pneumophila. This includes generating a high-density library of insertional mutants, setting up a selection screen, sequencing the libraries, mapping the insertion sites, and analyzing the data to obtain the list of genes involved in surviving the applied selection.