p lead to the identification of the genes

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Last updated: September 24, 2019

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ctl { font-family: “FreeSans”; font-size: 12pt; }a:link { }Pathogenscan adopt specific strategies to colonize and infect a host, or toadapt and resist therapeutic and prophylactic interventions.Deciphering these strategies requires determining their geneticbasis. For a given pathogen this is typically done by analyzing thephenotype of a given gene mutant in a model ofinfection/adaptation/resistance. Alternatively, random mutagenesiscan generate libraries of thousands of mutants that can beindividually tested in a phenotyping model. This can lead to theidentification of the genes required for a specific process (i.e.

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,virulence, resistance). Yet, these strategies are tedious and alimited number of infection model or conditions can be tested for agiven pathogen. Transposition-sequencing (Tn-seq, HITS, Tradis,Inseq) has recently emerged as a way to drastically increase thethroughput of such approaches (Gawronski et al., 2009; Goodman etal., 2009; Langridge et al., 2009; van Opijnen et al., 2009).Transposition-sequencing (Tn-seq)combinestransposition mutagenesis and deep-sequencing mapping and allows theidentification of mutants from an insertional library that have losta given function (negative selection) without the need to testindividual mutants.

By monitoring a large library of singletransposon insertion mutants with high throughput sequencing, thismethod can rapidly identify genomic regions that contribute toorganismal fitness under any condition thatcan be assayedin the laboratory with high resolution (Figure1)(van Opijnen and Camilli, 2013). For instance, a recent study defineda “fine scale phenotype-genotype virulence map” of the humanpathogen Streptococcuspneumoniaeby screening 17 invitroand 2 invivo(carriage and infection) conditions(van Opijnen and Camilli, 2012).Moreover, Tn-seqcan be used to query the bacterial genome with unprecedentedresolution, allowing the identification of small genes (non-codingRNA) that may be missed in conventional screening approaches(Barquist et al., 2013). Once a Tn-seqlibrary has been established for a pathogen, it can be screened inmultiple assays with near endless possibilities, from predictinggenes essential for in vitro growth to directly assayingrequirements for survival under infective conditions in vivo.Tn-seq can be applied to determine the genes, and cellular processes,required to resist an antibacterial treatment or to acquire newresistance genes, to adapt to intracellular life or to compete withother bacteria.

Virtually any assay that applies a selection pressurecan be used to identify the genetic determinants involved in aselection process.We here provide a protocol to conduct a Tn-seqanalysis in a Legionella pneumophila isolate. First, wedescribe the procedure to generate a collection of insertion mutantsthat will be suitable for a Tn-seq analysis. Several hundredthousands mutants can be obtained with this protocol. Thetransposition mutant library can then be used in any selection-basedscreen.

Second, we provide a detailed protocol to identify thetransposition insertion sites with the TdT method. Finally, weprovide an example of data analysis to identify the genes requiredfor the function tested in the selection-based screen.

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