Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis
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 sub...
| Main Authors: | , , , , , , |
|---|---|
| Format: | info:ar-repo/semantics/artículo |
| Language: | Inglés |
| Published: |
Elsevier
2020
|
| Subjects: | |
| Online Access: | 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 |
| _version_ | 1855035914801119232 |
|---|---|
| author | Alonso, Maria Natalia Malaga, Wladimir Mc Neil, Michael Jackson, Mary Romano, Maria Isabel Guilhot, Christophe Santangelo, María De La Paz |
| author_browse | Alonso, Maria Natalia Guilhot, Christophe Jackson, Mary Malaga, Wladimir Mc Neil, Michael Romano, Maria Isabel Santangelo, María De La Paz |
| author_facet | Alonso, Maria Natalia Malaga, Wladimir Mc Neil, Michael Jackson, Mary Romano, Maria Isabel Guilhot, Christophe Santangelo, María De La Paz |
| author_sort | Alonso, Maria Natalia |
| collection | INTA Digital |
| 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. |
| format | info:ar-repo/semantics/artículo |
| id | INTA7753 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | INTA77532020-08-21T12:49:57Z Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis Alonso, Maria Natalia Malaga, Wladimir Mc Neil, Michael Jackson, Mary Romano, Maria Isabel Guilhot, Christophe Santangelo, María De La Paz Mycobacterium Mycobacterium avium subsp. paratuberculosis Triglicéridos Mutación Genética Triglycerides Mutation Genetics 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 2020-08-21T12:45:54Z 2020-08-21T12:45:54Z 2020-09 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion 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 eng info:eu-repo/semantics/restrictedAccess application/pdf Elsevier Research in Microbiology 171 (5–6) : 203-210 (July–September 2020) |
| spellingShingle | Mycobacterium Mycobacterium avium subsp. paratuberculosis Triglicéridos Mutación Genética Triglycerides Mutation Genetics Alonso, Maria Natalia Malaga, Wladimir Mc Neil, Michael Jackson, Mary Romano, Maria Isabel Guilhot, Christophe Santangelo, María De La Paz 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 |
| 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_short | 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 |
| topic | Mycobacterium Mycobacterium avium subsp. paratuberculosis Triglicéridos Mutación Genética Triglycerides Mutation Genetics |
| 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 |
| work_keys_str_mv | AT alonsomarianatalia efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT malagawladimir efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT mcneilmichael efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT jacksonmary efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT romanomariaisabel efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT guilhotchristophe efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis AT santangelomariadelapaz efficientmethodfortargetedgenedisruptionbyhomologousrecombinationinmycobacteriumaviumsubspecieparatuberculosis |