Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis

ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, n...

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Autores principales:	Michael A. DeJesus, Elias R. Gerrick, Weizhen Xu, Sae Woong Park, Jarukit E. Long, Cara C. Boutte, Eric J. Rubin, Dirk Schnappinger, Sabine Ehrt, Sarah M. Fortune, Christopher M. Sassetti, Thomas R. Ioerger
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a77bd7f0cb4b428b9bb78ab3fcebcc71
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spelling	oai:doaj.org-article:a77bd7f0cb4b428b9bb78ab3fcebcc712021-11-15T15:51:07ZComprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis10.1128/mBio.02133-162150-7511https://doaj.org/article/a77bd7f0cb4b428b9bb78ab3fcebcc712017-03-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02133-16https://doaj.org/toc/2150-7511ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.Michael A. DeJesusElias R. GerrickWeizhen XuSae Woong ParkJarukit E. LongCara C. BoutteEric J. RubinDirk SchnappingerSabine EhrtSarah M. FortuneChristopher M. SassettiThomas R. IoergerAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 8, Iss 1 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
description	ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.
format	article
author	Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger
author_facet	Michael A. DeJesus Elias R. Gerrick Weizhen Xu Sae Woong Park Jarukit E. Long Cara C. Boutte Eric J. Rubin Dirk Schnappinger Sabine Ehrt Sarah M. Fortune Christopher M. Sassetti Thomas R. Ioerger
author_sort	Michael A. DeJesus
title	Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
title_short	Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
title_full	Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
title_fullStr	Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
title_full_unstemmed	Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis
title_sort	comprehensive essentiality analysis of the <italic toggle="yes">mycobacterium tuberculosis</italic> genome via saturating transposon mutagenesis
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/a77bd7f0cb4b428b9bb78ab3fcebcc71
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Comprehensive Essentiality Analysis of the <italic toggle="yes">Mycobacterium tuberculosis</italic> Genome via Saturating Transposon Mutagenesis

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