title: Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production.
abstract: Clostridium difficile causes a potentially fatal diarrheal disease through the production of its principal virulence factors, toxin A and toxin B. The tcdC gene is thought to encode a negative regulator of toxin production. Therefore, increased toxin production, and hence increased virulence, is often inferred in strains with an aberrant tcdC genotype. This report describes the first allele exchange system for precise genetic manipulation of C. difficile, using the codA gene of Escherichia coli as a heterologous counterselection marker. It was used to systematically restore the Δ117 frameshift mutation and the 18-nucleotide deletion that occur naturally in the tcdC gene of C. difficile R20291 (PCR ribotype 027). In addition, the naturally intact tcdC gene of C. difficile 630 (PCR ribotype 012) was deleted and then subsequently restored with a silent nucleotide substitution, or "watermark," so the resulting strain was distinguishable from the wild type. Intriguingly, there was no association between the tcdC genotype and toxin production in either C. difficile R20291 or C. difficile 630. Therefore, an aberrant tcdC genotype does not provide a broadly applicable rationale for the perceived notion that PCR ribotype 027 strains are "high-level" toxin producers. This may well explain why several studies have reported that an aberrant tcdC gene does not predict increased toxin production or, indeed, increased virulence.
authors.affiliation: Clostridia Research Group, Centre for Biomolecular Sciences, School of Molecular Medical Sciences, Nottingham Digestive Diseases Centre, NIHR Biomedical Research Unit, The University of Nottingham, University Park, United Kingdom. stephen.cartman@nottingham.ac.uk
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Cartman
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Kelly
authors.forename: Daniela
authors.initials: D
authors.lastname: Heeg
authors.forename: John T
authors.initials: JT
authors.lastname: Heap
authors.forename: Nigel P
authors.initials: NP
authors.lastname: Minton
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001427:Bacterial Toxins / Q000096:biosynthesis*; D016360:Clostridioides difficile / Q000235:genetics* / Q000472:pathogenicity*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016368:Frameshift Mutation; D017353:Gene Deletion; D005827:Genetics, Microbial / Q000379:methods; D005838:Genotype; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C105615:TcdC protein, Clostridium difficile