Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis

Autores
Alonso, Maria Natalia; Malaga, Wladimir; Mc Neil, Michael; Jackson, Mary; Romano, Maria Isabel; Guilhot, Christophe; Santangelo, María De La Paz
Año de publicación
2020
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.
Instituto de Biotecnología
Fil: Alonso, Maria Natalia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Malaga, Wladimir. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; Francia
Fil: Mc Neil, Michael. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados Unidos
Fil: Jackson, Mary. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados Unidos
Fil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Guilhot, Christophe. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; Francia
Fil: Santangelo, María De La Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fuente
Research in Microbiology 171 (5–6) : 203-210 (July–September 2020)
Materia
Mycobacterium
Mycobacterium avium subsp. paratuberculosis
Triglicéridos
Mutación
Genética
Triglycerides
Mutation
Genetics
Nivel de accesibilidad
acceso restringido
Condiciones de uso
Repositorio
INTA Digital (INTA)
Institución
Instituto Nacional de Tecnología Agropecuaria
OAI Identificador
oai:localhost:20.500.12123/7753
Acceder
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oai_identifier_str oai:localhost:20.500.12123/7753
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repository_id_str l
network_name_str INTA Digital (INTA)
spelling Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosisAlonso, Maria NataliaMalaga, WladimirMc Neil, MichaelJackson, MaryRomano, Maria IsabelGuilhot, ChristopheSantangelo, María De La PazMycobacteriumMycobacterium avium subsp. paratuberculosisTriglicéridosMutaciónGenéticaTriglyceridesMutationGeneticsTargeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.Instituto de BiotecnologíaFil: Alonso, Maria Natalia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malaga, Wladimir. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Mc Neil, Michael. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados UnidosFil: Jackson, Mary. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados UnidosFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Guilhot, Christophe. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; FranciaFil: Santangelo, María De La Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaElsevier2020-08-21T12:45:54Z2020-08-21T12:45:54Z2020-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfhttp://hdl.handle.net/20.500.12123/7753https://www.sciencedirect.com/science/article/pii/S09232508203003830923-2508https://doi.org/10.1016/j.resmic.2020.04.001Research in Microbiology 171 (5–6) : 203-210 (July–September 2020)reponame:INTA Digital (INTA)instname:Instituto Nacional de Tecnología Agropecuariaenginfo:eu-repo/semantics/restrictedAccess2025-09-04T09:48:36Zoai:localhost:20.500.12123/7753instacron:INTAInstitucionalhttp://repositorio.inta.gob.ar/Organismo científico-tecnológicoNo correspondehttp://repositorio.inta.gob.ar/oai/requesttripaldi.nicolas@inta.gob.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:l2025-09-04 09:48:36.994INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuariafalse
dc.title.none.fl_str_mv Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
title Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
spellingShingle Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
Alonso, Maria Natalia
Mycobacterium
Mycobacterium avium subsp. paratuberculosis
Triglicéridos
Mutación
Genética
Triglycerides
Mutation
Genetics
title_short Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
title_full Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
title_fullStr Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
title_full_unstemmed Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
title_sort Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
dc.creator.none.fl_str_mv Alonso, Maria Natalia
Malaga, Wladimir
Mc Neil, Michael
Jackson, Mary
Romano, Maria Isabel
Guilhot, Christophe
Santangelo, María De La Paz
author Alonso, Maria Natalia
author_facet Alonso, Maria Natalia
Malaga, Wladimir
Mc Neil, Michael
Jackson, Mary
Romano, Maria Isabel
Guilhot, Christophe
Santangelo, María De La Paz
author_role author
author2 Malaga, Wladimir
Mc Neil, Michael
Jackson, Mary
Romano, Maria Isabel
Guilhot, Christophe
Santangelo, María De La Paz
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv Mycobacterium
Mycobacterium avium subsp. paratuberculosis
Triglicéridos
Mutación
Genética
Triglycerides
Mutation
Genetics
topic Mycobacterium
Mycobacterium avium subsp. paratuberculosis
Triglicéridos
Mutación
Genética
Triglycerides
Mutation
Genetics
dc.description.none.fl_txt_mv Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.
Instituto de Biotecnología
Fil: Alonso, Maria Natalia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Malaga, Wladimir. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; Francia
Fil: Mc Neil, Michael. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados Unidos
Fil: Jackson, Mary. Colorado State University. Department of Microbiology, Immunology and Pathology. Mycobacteria Research Laboratories; Estados Unidos
Fil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Guilhot, Christophe. University of Toulouse. Institut de Pharmacologie et de Biologie Structurale; Francia
Fil: Santangelo, María De La Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
description Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.
publishDate 2020
dc.date.none.fl_str_mv 2020-08-21T12:45:54Z
2020-08-21T12:45:54Z
2020-09
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12123/7753
https://www.sciencedirect.com/science/article/pii/S0923250820300383
0923-2508
https://doi.org/10.1016/j.resmic.2020.04.001
url http://hdl.handle.net/20.500.12123/7753
https://www.sciencedirect.com/science/article/pii/S0923250820300383
https://doi.org/10.1016/j.resmic.2020.04.001
identifier_str_mv 0923-2508
dc.language.none.fl_str_mv eng
language eng
dc.rights.none.fl_str_mv info:eu-repo/semantics/restrictedAccess
eu_rights_str_mv restrictedAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv Research in Microbiology 171 (5–6) : 203-210 (July–September 2020)
reponame:INTA Digital (INTA)
instname:Instituto Nacional de Tecnología Agropecuaria
reponame_str INTA Digital (INTA)
collection INTA Digital (INTA)
instname_str Instituto Nacional de Tecnología Agropecuaria
repository.name.fl_str_mv INTA Digital (INTA) - Instituto Nacional de Tecnología Agropecuaria
repository.mail.fl_str_mv tripaldi.nicolas@inta.gob.ar
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score 12.623145

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