Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the develop...

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Autores principales:	Madeleine G. Moule, Claudia M. Hemsley, Qihui Seet, José Afonso Guerra-Assunção, Jiali Lim, Mitali Sarkar-Tyson, Taane G. Clark, Patrick B. O. Tan, Richard W. Titball, Jon Cuccui, Brendan W. Wren
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/944e443574194337893472bae4c81c02
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spelling	oai:doaj.org-article:944e443574194337893472bae4c81c022021-11-15T15:45:10ZGenome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development10.1128/mBio.00926-132150-7511https://doaj.org/article/944e443574194337893472bae4c81c022014-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00926-13https://doaj.org/toc/2150-7511ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.Madeleine G. MouleClaudia M. HemsleyQihui SeetJosé Afonso Guerra-AssunçãoJiali LimMitali Sarkar-TysonTaane G. ClarkPatrick B. O. TanRichard W. TitballJon CuccuiBrendan W. WrenAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 1 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Madeleine G. Moule Claudia M. Hemsley Qihui Seet José Afonso Guerra-Assunção Jiali Lim Mitali Sarkar-Tyson Taane G. Clark Patrick B. O. Tan Richard W. Titball Jon Cuccui Brendan W. Wren Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
description	ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infectious disease for which there is no vaccine. B. pseudomallei is listed as a tier 1 select agent, and as current therapeutic options are limited due to its natural resistance to most antibiotics, the development of new antimicrobial therapies is imperative. To identify drug targets and better understand the complex B. pseudomallei genome, we sought a genome-wide approach to identify lethal gene targets. As B. pseudomallei has an unusually large genome spread over two chromosomes, an extensive screen was required to achieve a comprehensive analysis. Here we describe transposon-directed insertion site sequencing (TraDIS) of a library of over 106 transposon insertion mutants, which provides the level of genome saturation required to identify essential genes. Using this technique, we have identified a set of 505 genes that are predicted to be essential in B. pseudomallei K96243. To validate our screen, three genes predicted to be essential, pyrH, accA, and sodB, and a gene predicted to be nonessential, bpss0370, were independently investigated through the generation of conditional mutants. The conditional mutants confirmed the TraDIS predictions, showing that we have generated a list of genes predicted to be essential and demonstrating that this technique can be used to analyze complex genomes and thus be more widely applied. IMPORTANCE Burkholderia pseudomallei is a lethal human pathogen that is considered a potential bioterrorism threat and has limited treatment options due to an unusually high natural resistance to most antibiotics. We have identified a set of genes that are required for bacterial growth and thus are excellent candidates against which to develop potential novel antibiotics. To validate our approach, we constructed four mutants in which gene expression can be turned on and off conditionally to confirm that these genes are required for the bacteria to survive.
format	article
author	Madeleine G. Moule Claudia M. Hemsley Qihui Seet José Afonso Guerra-Assunção Jiali Lim Mitali Sarkar-Tyson Taane G. Clark Patrick B. O. Tan Richard W. Titball Jon Cuccui Brendan W. Wren
author_facet	Madeleine G. Moule Claudia M. Hemsley Qihui Seet José Afonso Guerra-Assunção Jiali Lim Mitali Sarkar-Tyson Taane G. Clark Patrick B. O. Tan Richard W. Titball Jon Cuccui Brendan W. Wren
author_sort	Madeleine G. Moule
title	Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
title_short	Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
title_full	Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
title_fullStr	Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
title_full_unstemmed	Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development
title_sort	genome-wide saturation mutagenesis of <named-content content-type="genus-species">burkholderia pseudomallei</named-content> k96243 predicts essential genes and novel targets for antimicrobial development
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/944e443574194337893472bae4c81c02
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Genome-Wide Saturation Mutagenesis of <named-content content-type="genus-species">Burkholderia pseudomallei</named-content> K96243 Predicts Essential Genes and Novel Targets for Antimicrobial Development

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