Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

ABSTRACT Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, method...

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Autores principales:	JoAnn M. Tufariello, Adel A. Malek, Catherine Vilchèze, Laura E. Cole, Hannah K. Ratner, Pablo A. González, Paras Jain, Graham F. Hatfull, 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/1268cf3ded4a4c76a1dc124fd7328d8a
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spelling	oai:doaj.org-article:1268cf3ded4a4c76a1dc124fd7328d8a2021-11-15T15:47:39ZEnhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>10.1128/mBio.01179-142150-7511https://doaj.org/article/1268cf3ded4a4c76a1dc124fd7328d8a2014-07-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01179-14https://doaj.org/toc/2150-7511ABSTRACT Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. IMPORTANCE Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.JoAnn M. TufarielloAdel A. MalekCatherine VilchèzeLaura E. ColeHannah K. RatnerPablo A. GonzálezParas JainGraham F. HatfullMichelle 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 JoAnn M. Tufariello Adel A. Malek Catherine Vilchèze Laura E. Cole Hannah K. Ratner Pablo A. González Paras Jain Graham F. Hatfull Michelle H. Larsen William R. Jacobs Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
description	ABSTRACT Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria. IMPORTANCE Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.
format	article
author	JoAnn M. Tufariello Adel A. Malek Catherine Vilchèze Laura E. Cole Hannah K. Ratner Pablo A. González Paras Jain Graham F. Hatfull Michelle H. Larsen William R. Jacobs
author_facet	JoAnn M. Tufariello Adel A. Malek Catherine Vilchèze Laura E. Cole Hannah K. Ratner Pablo A. González Paras Jain Graham F. Hatfull Michelle H. Larsen William R. Jacobs
author_sort	JoAnn M. Tufariello
title	Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_short	Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full	Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_fullStr	Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_full_unstemmed	Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>
title_sort	enhanced specialized transduction using recombineering in <named-content content-type="genus-species">mycobacterium tuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2014
url	https://doaj.org/article/1268cf3ded4a4c76a1dc124fd7328d8a
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Enhanced Specialized Transduction Using Recombineering in <named-content content-type="genus-species">Mycobacterium tuberculosis</named-content>

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