Querying Legionella Genomes Using Transposition-Sequencing

Hardy, L.; Charpentier, Xavier

doi:10.1007/978-1-4939-9048-1_7

Cited by 2 publications

(4 citation statements)

References 15 publications

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“…Transposon-based random mutagenesis was performed as previously described (82) by conjugative delivery of the Himar1-based transposon suicide vector pBT20 from the donor strain E. coli MFDpir to the recipient strain of L. pneumophila to be mutagenized. To do so, both bacteria were cultivated overnight at 37°C with shaking in their corresponding liquid media : 7.5 mL LB broth containing 100 µg/mL ampicillin and 1% DAP for E. coli and 15 mL standard AYE medium for L. pneumophila.…”

Section: Generation Of Transposon Insertion Mutants Library Of Legionmentioning

confidence: 99%

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Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

et al. 2021

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Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdown horizontal gene transfer events. Transposon-insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of difficulty to obtain a high-saturation transposon insertion library. Indeed, we found that isolates of sequence type 1 (ST1), to which belong the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we therefore used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species. IMPORTANCE Legionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. The environmental life traits of L. pneumophila are essential to its ability to accidentally infect humans. A comprehensive identification of their genetic basis could be obtained through the use of transposon-insertion sequencing. Yet, this powerful approach, had not been fully implemented in L. pneumophila. Here we described the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step towards identifying the genetic basis of the many life traits of this environmental and pathogenic species.

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Section: Generation Of Transposon Insertion Mutants Library Of Legionmentioning

confidence: 99%

“…Libraries were prepared as previously described (82). Mutant libraries from the -80°C frozen stock were thawed and centrifuged at maximum speed to pellet them.…”

Section: Dna Library Preparation and Sequencingmentioning

confidence: 99%

Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

et al. 2021

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show abstract

“…Transposon-based random mutagenesis was performed as previously described (79) by conjugative delivery of the Himar1-based transposon suicide vector pBT20 from the donor strain E. coli MFDpir to the recipient strain of L. pneumophila to be mutagenized. To do so, both bacteria were cultivated overnight at 37°C with shaking in their corresponding liquid media : 7.5 mL LB broth containing 100 µg/mL ampicillin and 1% DAP for E. coli and 15 mL standard AYE medium for L. pneumophila.…”

Section: Generation Of Transposon Insertion Mutants Library Of Legionmentioning

confidence: 99%

“…Libraries were prepared as previously described (79). Mutant libraries from the -80°C frozen stock were thawed and centrifuged at maximum speed to pellet them.…”

Section: Dna Library Preparation and Sequencingmentioning

confidence: 99%

Transposon-insertion sequencing in a clinical isolate ofLegionella pneumophilaidentifies essential genes and determinants of natural transformation

Hardy

Juan

Coupat-Goutaland

et al. 2020

Preprint

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Legionella pneumophila is a Gram-negative bacterium ubiquitous in freshwater environments which, if inhaled, can cause a severe pneumonia in humans. The emergence of L. pneumophila is linked to several traits selected in the environment, the acquisition of some of which involved intra- and interkingdown horizontal gene transfer events. Transposon-insertion sequencing (TIS) is a powerful method to identify the genetic basis of selectable traits as well as to identify fitness determinants and essential genes, possible antibiotic targets. TIS has not yet been used to its full power in L. pneumophila, possibly because of difficulty to obtain a high-saturation transposon insertion library. Indeed, we found that ST1 isolates, to which belong the commonly used laboratory strains, are poorly permissive to saturating mutagenesis by conjugation-mediated transposon delivery. In contrast, we obtained high-saturation libraries in non-ST1 clinical isolates, offering the prospect of using TIS on unaltered L. pneumophila strains. Focusing on one of them, we therefore used TIS to identify essential genes in L. pneumophila. We also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identify all the genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome by TIS and the identification of the genetic basis of other life traits of this species.ImportanceLegionella pneumophila is the etiologic agent of a severe form of nosocomial and community-acquired pneumonia in humans. L. pneumophila is found in man-made and freshwater environments which are the causing source of the infection. The environmental life traits of L. pneumophila, such as its abilities to form biofilms, resist biocides and unicellular predators, are essential to its ability to accidentally infect humans. A comprehensive identification of the genetic basis of these life traits could be obtained through the use of transposon-insertion sequencing. Yet, this powerful approach, had not been fully implemented in L. pneumophila. Here we described the successful implementation of the transposon-sequencing approach in a clinical isolate of L. pneumophila. We identify essential genes, potential drug targets, and genes required for horizontal gene transfer by natural transformation. This work represents an important step towards identifying the genetic basis of the many life traits of this environmental and pathogenic species.

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Querying Legionella Genomes Using Transposition-Sequencing

Cited by 2 publications

References 15 publications

Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

Transposon Insertion Sequencing in a Clinical Isolate of Legionella pneumophila Identifies Essential Genes and Determinants of Natural Transformation

Transposon-insertion sequencing in a clinical isolate ofLegionella pneumophilaidentifies essential genes and determinants of natural transformation

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