Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

ABSTRACT Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a tempe...

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Autores principales:	Paras Jain, Tsungda Hsu, Masayoshi Arai, Karolin Biermann, David S. Thaler, Andrew Nguyen, Pablo A. González, Joann M. Tufariello, Jordan Kriakov, Bing Chen, Michelle H. Larsen, William R. Jacobs
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2014
Materias:	Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/c26cd4668a5142cca824b53be638ee1f
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spelling	oai:doaj.org-article:c26cd4668a5142cca824b53be638ee1f2021-11-15T15:47:38ZSpecialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>10.1128/mBio.01245-142150-7511https://doaj.org/article/c26cd4668a5142cca824b53be638ee1f2014-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01245-14https://doaj.org/toc/2150-7511ABSTRACT Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. IMPORTANCE This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.Paras JainTsungda HsuMasayoshi AraiKarolin BiermannDavid S. ThalerAndrew NguyenPablo A. GonzálezJoann M. TufarielloJordan KriakovBing ChenMichelle H. LarsenWilliam R. JacobsAmerican Society for MicrobiologyarticleMicrobiologyQR1-502ENmBio, Vol 5, Iss 3 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Microbiology QR1-502
spellingShingle	Microbiology QR1-502 Paras Jain Tsungda Hsu Masayoshi Arai Karolin Biermann David S. Thaler Andrew Nguyen Pablo A. González Joann M. Tufariello Jordan Kriakov Bing Chen Michelle H. Larsen William R. Jacobs Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
description	ABSTRACT Specialized transduction has proven to be useful for generating deletion mutants in most mycobacteria, including virulent Mycobacterium tuberculosis. We have improved this system by developing (i) a single-step strategy for the construction of allelic exchange substrates (AES), (ii) a temperature-sensitive shuttle phasmid with a greater cloning capacity than phAE87, and (iii) bacteriophage-mediated transient expression of site-specific recombinase to precisely excise antibiotic markers. The methods ameliorate rate-limiting steps in strain construction in these difficult-to-manipulate bacteria. The new methods for strain construction were demonstrated to generalize to all classes of genes and chromosomal loci by generating more than 100 targeted single- or multiple-deletion substitutions. These improved methods pave the way for the generation of a complete ordered library of M. tuberculosis null strains, where each strain is deleted for a single defined open reading frame in M. tuberculosis. IMPORTANCE This work reports major advances in the methods of genetics applicable to all mycobacteria, including but not limited to virulent M. tuberculosis, which would facilitate comparative genomics to identify drug targets, genetic validation of proposed pathways, and development of an effective vaccine. This study presents all the new methods developed and the improvements to existing methods in an integrated way. The work presented in this study could increase the pace of mycobacterial genetics significantly and will immediately be of wide use. These new methods are transformative and allow for the undertaking of construction of what has been one of the most fruitful resources in model systems: a comprehensive, ordered library set of the strains, each of which is deleted for a single defined open reading frame.
format	article
author	Paras Jain Tsungda Hsu Masayoshi Arai Karolin Biermann David S. Thaler Andrew Nguyen Pablo A. González Joann M. Tufariello Jordan Kriakov Bing Chen Michelle H. Larsen William R. Jacobs
author_facet	Paras Jain Tsungda Hsu Masayoshi Arai Karolin Biermann David S. Thaler Andrew Nguyen Pablo A. González Joann M. Tufariello Jordan Kriakov Bing Chen Michelle H. Larsen William R. Jacobs
author_sort	Paras Jain
title	Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_short	Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full	Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_fullStr	Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full_unstemmed	Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_sort	specialized transduction designed for precise high-throughput unmarked deletions in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/c26cd4668a5142cca824b53be638ee1f
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Specialized Transduction Designed for Precise High-Throughput Unmarked Deletions in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

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