Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology

ABSTRACT During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by t...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Yu Tanouchi, Markus W. Covert
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR/Cas 9 DNA sequencing phage lambda Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/522da07a5e1d4183898daef6f10ab1f7
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:522da07a5e1d4183898daef6f10ab1f7
record_format	dspace
spelling	oai:doaj.org-article:522da07a5e1d4183898daef6f10ab1f72021-11-15T15:51:51ZCombining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology10.1128/mBio.01038-172150-7511https://doaj.org/article/522da07a5e1d4183898daef6f10ab1f72017-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01038-17https://doaj.org/toc/2150-7511ABSTRACT During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study—sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection—are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems.Yu TanouchiMarkus W. CovertAmerican Society for MicrobiologyarticleCRISPR/Cas 9DNA sequencingphage lambdaMicrobiologyQR1-502ENmBio, Vol 8, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas 9 DNA sequencing phage lambda Microbiology QR1-502
spellingShingle	CRISPR/Cas 9 DNA sequencing phage lambda Microbiology QR1-502 Yu Tanouchi Markus W. Covert Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
description	ABSTRACT During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study—sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection—are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems.
format	article
author	Yu Tanouchi Markus W. Covert
author_facet	Yu Tanouchi Markus W. Covert
author_sort	Yu Tanouchi
title	Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
title_short	Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
title_full	Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
title_fullStr	Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
title_full_unstemmed	Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology
title_sort	combining comprehensive analysis of off-site lambda phage integration with a crispr-based means of characterizing downstream physiology
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/522da07a5e1d4183898daef6f10ab1f7
work_keys_str_mv	AT yutanouchi combiningcomprehensiveanalysisofoffsitelambdaphageintegrationwithacrisprbasedmeansofcharacterizingdownstreamphysiology AT markuswcovert combiningcomprehensiveanalysisofoffsitelambdaphageintegrationwithacrisprbasedmeansofcharacterizingdownstreamphysiology
_version_	1718427334519816192

Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology

Ejemplares similares