Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource

ABSTRACT Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in ord...

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Autores principales:	Katlyn Borgers, Kristof Vandewalle, Annelies Van Hecke, Gitte Michielsen, Evelyn Plets, Loes van Schie, Sandrine Vanmarcke, Laurent Schindfessel, Nele Festjens, Nico Callewaert
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Mycobacterium tuberculosis complex characterized transposon library optimized Himar1 transposon transposon mutagenesis unmarked mutants Microbiology QR1-502
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spelling	oai:doaj.org-article:a1527f59eb684ae5b37df81ba59b4dbf2021-12-02T19:47:35ZDevelopment of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource10.1128/mSystems.00180-202379-5077https://doaj.org/article/a1527f59eb684ae5b37df81ba59b4dbf2020-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00180-20https://doaj.org/toc/2379-5077ABSTRACT Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated. IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.Katlyn BorgersKristof VandewalleAnnelies Van HeckeGitte MichielsenEvelyn PletsLoes van SchieSandrine VanmarckeLaurent SchindfesselNele FestjensNico CallewaertAmerican Society for MicrobiologyarticleMycobacterium tuberculosis complexcharacterized transposon libraryoptimized Himar1 transposontransposon mutagenesisunmarked mutantsMicrobiologyQR1-502ENmSystems, Vol 5, Iss 4 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Mycobacterium tuberculosis complex characterized transposon library optimized Himar1 transposon transposon mutagenesis unmarked mutants Microbiology QR1-502
spellingShingle	Mycobacterium tuberculosis complex characterized transposon library optimized Himar1 transposon transposon mutagenesis unmarked mutants Microbiology QR1-502 Katlyn Borgers Kristof Vandewalle Annelies Van Hecke Gitte Michielsen Evelyn Plets Loes van Schie Sandrine Vanmarcke Laurent Schindfessel Nele Festjens Nico Callewaert Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
description	ABSTRACT Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated. IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.
format	article
author	Katlyn Borgers Kristof Vandewalle Annelies Van Hecke Gitte Michielsen Evelyn Plets Loes van Schie Sandrine Vanmarcke Laurent Schindfessel Nele Festjens Nico Callewaert
author_facet	Katlyn Borgers Kristof Vandewalle Annelies Van Hecke Gitte Michielsen Evelyn Plets Loes van Schie Sandrine Vanmarcke Laurent Schindfessel Nele Festjens Nico Callewaert
author_sort	Katlyn Borgers
title	Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
title_short	Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
title_full	Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
title_fullStr	Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
title_full_unstemmed	Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource
title_sort	development of a counterselectable transposon to create markerless knockouts from an 18,432-clone ordered <named-content content-type="genus-species">mycobacterium bovis</named-content> bacillus calmette-guérin mutant resource
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a1527f59eb684ae5b37df81ba59b4dbf
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Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered <named-content content-type="genus-species">Mycobacterium bovis</named-content> Bacillus Calmette-Guérin Mutant Resource

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