ArticlePloS one2013
Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 129 papers.
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129 citing papers in PubMed, 187 citations in OpenAlex.
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- The CspC:CspA heterodimer transduces germinant and co-germinant signals during Clostridioides difficile spore germination.PLoS biology · 2026Article
- Defining the order of assembly of thebioRxiv : the preprint server for biology · 2026Article
- In situ visualization of Clostridioides difficile phenotypic heterogeneity and single-cell morphology during gut infection.Nature communications · 2026Article
- Visualizing C. difficile During Murine Infection.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Xtr-mediated regulation of 4Fe-4S binding protein gene confers resistance to oxidative and antibiotic stress inFrontiers in microbiology · 2026Article
- Nanoluciferase-Based Reporters for Clostridioides difficile.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Generating Targeted Mutants Using a Riboswitch to Control Plasmid Behavior During Allelic Exchange Mutagenesis.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Penicillin-binding proteins exhibit functional redundancy during asymmetric cell division inJournal of bacteriology · 2025Article
- Engineered Clostridium butyricum: a promising application of synthetic biology in comprehensive health.Microbial cell factories · 2025Review
- Analysis of essential genes inJournal of bacteriology · 2025Article
- Molecular dissection of Class A PBP function uncovers novel features of the non-canonical Clostridioides difficile divisome complex.PLoS genetics · 2025Article
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- A targeted cell lysis mechanism facilitates toxin release in Clostridioides difficile.bioRxiv : the preprint server for biology · 2025Article
- Extracellular DNA filaments associated with surface polysaccharide II give Clostridioides difficile biofilm matrix a network-like structure.NPJ biofilms and microbiomes · 2025Article
- Analysis of Essential Genes inbioRxiv : the preprint server for biology · 2025Article
69 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 2 institutions in 2 countries.
Funding
Abstract
Sophisticated genetic tools to modify essential biological processes at the molecular level are pivotal in elucidating the molecular pathogenesis of Clostridium difficile, a major cause of healthcare associated disease. Here we have developed an efficient procedure for making precise alterations to the C. difficile genome by pyrE-based allelic exchange. The robustness and reliability of the method was demonstrated through the creation of in-frame deletions in three genes (spo0A, cwp84, and mtlD) in the non-epidemic strain 630Δerm and two genes (spo0A and cwp84) in the epidemic PCR Ribotype 027 strain, R20291. The system is reliant on the initial creation of a pyrE deletion mutant, using Allele Coupled Exchange (ACE), that is auxotrophic for uracil and resistant to fluoroorotic acid (FOA). This enables the subsequent modification of target genes by allelic exchange using a heterologous pyrE allele from Clostridium sporogenes as a counter-/negative-selection marker in the presence of FOA. Following modification of the target gene, the strain created is rapidly returned to uracil prototrophy using ACE, allowing mutant phenotypes to be characterised in a PyrE proficient background. Crucially, wild-type copies of the inactivated gene may be introduced into the genome using ACE concomitant with correction of the pyrE allele. This allows complementation studies to be undertaken at an appropriate gene dosage, as opposed to the use of multicopy autonomous plasmids. The rapidity of the 'correction' method (5-7 days) makes pyrE(-) strains attractive hosts for mutagenesis studies.
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