Fagan, R orcid.org/0000-0002-8704-4828 and Kirk, J orcid.org/0000-0002-8117-8120 (2016) Heat shock increases conjugation efficiency in Clostridium difficile. Anaerobe, 42. pp. 1-5. ISSN 1075-9964
Abstract
Clostridium difficile infection has increased in incidence and severity over the past decade, and poses a unique threat to human health. However, genetic manipulation of C. difficile remains in its infancy and the bacterium remains relatively poorly characterised. Low-efficiency conjugation is currently the only available method for transfer of plasmid DNA into C. difficile. This is practically limiting and has slowed progress in understanding this important pathogen. Conjugation efficiency varies widely between strains, with important clinically relevant strains such as R20291 being particularly refractory to plasmid transfer. Here we present an optimised conjugation method in which the recipient C. difficile is heat treated prior to conjugation. This significantly improves conjugation efficiency in all C. difficile strains tested including R20291. Conjugation efficiency was also affected by the choice of media on which conjugations were performed, with standard BHI media giving most transconjugant recovery. Using our optimised method greatly increased the ease with which the chromosome of R20291 could be precisely manipulated by homologous recombination. Our method improves on current conjugation protocols and will help speed genetic manipulation of strains otherwise difficult to work with.
Metadata
Item Type: | Article |
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Authors/Creators: | |
Copyright, Publisher and Additional Information: | © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
Keywords: | Clostridium difficile; PCR ribotype 027; Conjugation |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Funding Information: | Funder Grant number MEDICAL RESEARCH COUNCIL MR/N000900/1 |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 11 Jul 2016 08:05 |
Last Modified: | 23 Jun 2023 22:09 |
Published Version: | http://dx.doi.org/10.1016/j.anaerobe.2016.06.009 |
Status: | Published |
Publisher: | Elsevier Masson |
Refereed: | Yes |
Identification Number: | 10.1016/j.anaerobe.2016.06.009 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:101978 |