Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq

ABSTRACT We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle met...

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Autores principales:	Larry A. Gallagher, Jay Shendure, Colin Manoil
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2011
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/83c770bd0f9f4f3e95b196ea55633f16
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spelling	oai:doaj.org-article:83c770bd0f9f4f3e95b196ea55633f162021-11-15T15:38:46ZGenome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq10.1128/mBio.00315-102150-7511https://doaj.org/article/83c770bd0f9f4f3e95b196ea55633f162011-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00315-10https://doaj.org/toc/2150-7511ABSTRACT We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle method), a method which can be used with virtually any transposon. The procedure reliably identified more than 100,000 transposon insertions in a single experiment, providing near-saturation coverage of the genome. To test the effectiveness of the procedure for mutant identification, we screened for mutations reducing intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic tobramycin resistance had been previously analyzed at genome scale using mutant-by-mutant screening and thus provided a benchmark for evaluating the new method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the majority of which were then verified with individual mutants. The group of genes with the strongest mutant phenotypes included nearly all (13 of 14) of those with strong mutant phenotypes identified in the previous screening, as well as a nearly equal number of new genes. The results thus show the effectiveness of the Tn-seq method in defining the genetic basis of a complex resistance trait of P. aeruginosa and indicate that it can be used to analyze a variety of growth-related processes. IMPORTANCE Research progress in microbiology is technology limited in the sense that the analytical methods available dictate how questions are experimentally addressed and, to some extent, what questions are asked. This report describes a new transposon tracking procedure for defining the genetic basis of growth-related processes in Pseudomonas aeruginosa, an important bacterial pathogen. The method employs next-generation sequencing to monitor the makeup of mutant populations (Tn-seq) and has several potential advantages over other Tn-seq methodologies. The new method was validated through the analysis of a clinically relevant antibiotic resistance trait.Larry A. GallagherJay ShendureColin ManoilAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 2, Iss 1 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Larry A. Gallagher Jay Shendure Colin Manoil Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
description	ABSTRACT We describe a deep-sequencing procedure for tracking large numbers of transposon mutants of Pseudomonas aeruginosa. The procedure employs a new Tn-seq methodology based on the generation and amplification of single-strand circles carrying transposon junction sequences (the Tn-seq circle method), a method which can be used with virtually any transposon. The procedure reliably identified more than 100,000 transposon insertions in a single experiment, providing near-saturation coverage of the genome. To test the effectiveness of the procedure for mutant identification, we screened for mutations reducing intrinsic resistance to the aminoglycoside antibiotic tobramycin. Intrinsic tobramycin resistance had been previously analyzed at genome scale using mutant-by-mutant screening and thus provided a benchmark for evaluating the new method. The new Tn-seq procedure identified 117 tobramycin resistance genes, the majority of which were then verified with individual mutants. The group of genes with the strongest mutant phenotypes included nearly all (13 of 14) of those with strong mutant phenotypes identified in the previous screening, as well as a nearly equal number of new genes. The results thus show the effectiveness of the Tn-seq method in defining the genetic basis of a complex resistance trait of P. aeruginosa and indicate that it can be used to analyze a variety of growth-related processes. IMPORTANCE Research progress in microbiology is technology limited in the sense that the analytical methods available dictate how questions are experimentally addressed and, to some extent, what questions are asked. This report describes a new transposon tracking procedure for defining the genetic basis of growth-related processes in Pseudomonas aeruginosa, an important bacterial pathogen. The method employs next-generation sequencing to monitor the makeup of mutant populations (Tn-seq) and has several potential advantages over other Tn-seq methodologies. The new method was validated through the analysis of a clinically relevant antibiotic resistance trait.
format	article
author	Larry A. Gallagher Jay Shendure Colin Manoil
author_facet	Larry A. Gallagher Jay Shendure Colin Manoil
author_sort	Larry A. Gallagher
title	Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
title_short	Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
title_full	Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
title_fullStr	Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
title_full_unstemmed	Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq
title_sort	genome-scale identification of resistance functions in <named-content content-type="genus-species">pseudomonas aeruginosa</named-content> using tn-seq
publisher	American Society for Microbiology
publishDate	2011
url	https://doaj.org/article/83c770bd0f9f4f3e95b196ea55633f16
work_keys_str_mv	AT larryagallagher genomescaleidentificationofresistancefunctionsinnamedcontentcontenttypegenusspeciespseudomonasaeruginosanamedcontentusingtnseq AT jayshendure genomescaleidentificationofresistancefunctionsinnamedcontentcontenttypegenusspeciespseudomonasaeruginosanamedcontentusingtnseq AT colinmanoil genomescaleidentificationofresistancefunctionsinnamedcontentcontenttypegenusspeciespseudomonasaeruginosanamedcontentusingtnseq
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Genome-Scale Identification of Resistance Functions in <named-content content-type="genus-species">Pseudomonas aeruginosa</named-content> Using Tn-seq

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