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Removal of mobile genetic elements from the genome of Clostridioides difficile and the implications for the organism's biology.
Hussain, Haitham; Nubgan, Amer; Rodríguez, César; Imwattana, Korakrit; Knight, Daniel R; Parthala, Valerija; Mullany, Peter; Goh, Shan.
Afiliación
  • Hussain H; Department of Microbial Diseases, Eastman Dental Institute, University College London, London, United Kingdom.
  • Nubgan A; Department of Clinical, Pharmaceutical and Biological Sciences, University of Hertfordshire, Hatfield, United Kingdom.
  • Rodríguez C; Facultad de Microbiología and Centro de Investigación en Enfermedades Tropicales (CIET), Universidad de Costa Rica, San José, Costa Rica.
  • Imwattana K; School of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia.
  • Knight DR; Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Salaya, Thailand.
  • Parthala V; School of Biomedical Sciences, The University of Western Australia, Perth, WA, Australia.
  • Mullany P; Department of Microbiology, PathWest Laboratory Medicine WA, Queen Elizabeth II Medical Centre, Nedlands, WA, Australia.
  • Goh S; Department of Clinical, Pharmaceutical and Biological Sciences, University of Hertfordshire, Hatfield, United Kingdom.
Front Microbiol ; 15: 1416665, 2024.
Article en En | MEDLINE | ID: mdl-38966395
ABSTRACT
Clostridioides difficile is an emerging pathogen of One Health significance. Its highly variable genome contains mobile genetic elements (MGEs) such as transposons and prophages that influence its biology. Systematic deletion of each genetic element is required to determine their precise role in C. difficile biology and contribution to the wider mobilome. Here, Tn5397 (21 kb) and ϕ027 (56 kb) were deleted from C. difficile 630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn5397 deletant transferred PaLoc at the same frequency (1 × 10-7) as 630 harboring Tn5397, indicating that Tn5397 alone did not mediate conjugative transfer of PaLoc. The R20291 ϕ027 deletant was sensitive to ϕ027 infection, and contained two unexpected features, a 2.7 kb remnant of the mutagenesis plasmid, and a putative catalase gene adjacent to the deleted prophage was also deleted. Growth kinetics of R20291 ϕ027 deletant was similar to wild type (WT) in rich medium but marginally reduced compared with WT in minimal medium. This work indicates the commonly used pMTL8000 plasmid series works well for CRISPR-Cas9-mediated gene deletion, resulting in the largest deleted locus (56.8 kb) described in C. difficile. Removal of MGEs was achieved by targeting conjugative/integrative regions to promote excision and permanent loss. The deletants created will be useful strains for investigating Tn5397 or ϕ027 prophage contribution to host virulence, fitness, and physiology, and a platform for other mutagenesis studies aimed at functional gene analysis without native transposon or phage interference in C. difficile 630 and R20291.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Microbiol Año: 2024 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Suiza
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Front Microbiol Año: 2024 Tipo del documento: Article País de afiliación: Reino Unido Pais de publicación: Suiza