Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile  by induction of DNA Double-strand Breaks

Elena-Stella Theophilou, Prerna Vohra, Maurice P Gallagher, Ian R Poxton, Garry W Blakely

Research output: Contribution to journalArticlepeer-review


Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterisation of such clinically important bacteria is often hampered by availability of suitable tools. Here we describe the use of I-SceI to induce DNA double-strand breaks which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile. The usefulness of the system is illustrated by deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultra violet light and the antibiotic metronidazole, indicating a role in homologous recombination and repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyl transferase.

IMPORTANCE 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 likelyhood of polar effects, although does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homology 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 role of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

Original languageEnglish
Number of pages12
JournalApplied and Environmental Microbiology
Issue number3
Early online date26 Nov 2018
Publication statusPublished - 23 Jan 2019


  • AddAB
  • Clostridium difficile
  • SOS system
  • allelic exchange
  • deletions
  • double-strand-break repair
  • fliC
  • mutagenesis
  • nosocomial


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