Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile by Induction of DNA Double-Strand Breaks

• Elena-Stella Theophilou ^[2] ; Prerna Vohra ^[1] ; Maurice P. Gallagher ^[1] ; Ian R. Poxton ^[1] ; Garry W. Blakely ^[1]
  1. [1] University of Edinburgh

     University of Edinburgh

     Reino Unido

     
  2. [2] a Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, United Kingdom; b Microbial Pathogenicity Research Laboratory Medical Microbiology, University of Edinburgh, Edinburgh, United Kingdom
• Localización: Applied and Environmental Microbiology, ISSN 0099-2240, Vol. 85, Nº 3, 2019
• Idioma: inglés
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• Resumen

  • Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.