Removal of mobile genetic elements from the genome of Clostridioides difficile and the implications for the organism's biology
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 biology and contribution to...
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| Published in: | Frontiers in microbiology Vol. 15; p. 1416665 |
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| Main Authors: | , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Switzerland
Frontiers Media S.A
20-06-2024
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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
biology and contribution to the wider mobilome. Here, Tn
(21 kb) and ϕ027 (56 kb) were deleted from
630 and R20291, respectively, using allele replacement facilitated by CRISPR-Cas9. The 630 Tn
deletant transferred PaLoc at the same frequency (1 × 10
) as 630 harboring Tn
, indicating that Tn
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
. 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 Tn
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
630 and R20291. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by: Axel Cloeckaert, Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), France Reviewed by: Khald Blau, University of Applied Sciences Emden/Leer, Germany These authors share first authorship These authors have contributed equally to this work and share last authorship PRESENT ADDRESS: Amer Nubgan,Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq Nozomu Obana, University of Tsukuba, Japan |
| ISSN: | 1664-302X 1664-302X |
| DOI: | 10.3389/fmicb.2024.1416665 |