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Legionella pp 107-122 | Cite as

Querying Legionella Genomes Using Transposition-Sequencing

  • Léo Hardy
  • Xavier CharpentierEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1921)

Abstract

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.

Key words

Transposition Sequencing Gene Genome Gene function Legionella pneumophila 

Notes

Acknowledgments

We thank Jean-Marc Ghigo for providing the strain MFDpir. Clinical isolates tested for the construction of L. pneumophila mutant libraries have been collected from the French Legionella Reference Center (Lyon, France). This work was supported by the LABEX ECOFECT (ANR-11-LABX-0048) of Université de Lyon, within the program “Investissements d‘Avenir” (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.CIRI, Centre International de Recherche en Infectiologie, Team “Horizontal gene transfer in bacterial pathogens”, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, École Normale Supérieure de Lyon, Univ LyonVilleurbanneFrance

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