*These authors contributed equally to this work.Clostridium difficile infection is the leading cause of healthcare associated diarrhoea in Europe and North America 1, 2 . During infection, C. difficile produces two key virulence determinants, toxin A and toxin B. Experiments with purified toxins have suggested that toxin A alone is able to evoke the symptoms of C. difficile infection, but toxin B is unable to do so unless it is mixed with toxin A, or there is prior damage to the gut mucosa 3 . However, a recent study suggested that toxin B is essential for C. difficile virulence and that a strain producing toxin A alone was avirulent 4 . This creates a paradox over the individual importance of toxin A and toxin B. Here we show that isogenic mutants of C. difficile producing either toxin A or toxin B alone can cause fulminant disease in the hamster model of infection. By using a gene knock-out system 5, 6 to permanently inactivate the toxin genes, we found that C. difficile producing either one or both toxins displayed cytotoxic activity in vitro, which translated directly into virulence in vivo.Furthermore, by constructing the first ever double mutant strain of C. difficile, in which both toxin genes were inactivated, we were able to completely attenuate virulence. Our findings re-establish the importance of both toxin A and toxin B and highlight the need to continue considering both toxins in the development of diagnostic tests and effective counter-measures against C. difficile.2 Toxin A and toxin B both catalyse the glucosylation, and hence inactivation, of Rho-GTPases; small regulatory proteins of the eukaryotic actin cell cytoskeleton. This leads to disorganisation of the cell cytoskeleton and cell death 7 . The toxin genes, tcdA and tcdB, are situated on the C. difficile chromosome in a 19.6 kilobase pathogenicity locus (PaLoc), along with the three accessory genes, tcdC, tcdR and tcdE (Fig. 1a). To address the individual importance of toxin A and toxin B, we used the ClosTron gene knock-out system 6 to inactivate the toxin genes of C. difficile. This system inactivates genes by inserting an intron into the protein-encoding DNA sequence of a gene, thus resulting in a truncated and non-functional protein. The intron sequence itself encompasses an erythromycin resistance determinant which permits selective isolation of mutants. Furthermore, it has been shown experimentally that the insertions are completely stable, meaning that inactivation of a gene is permanent 5 .Using the ClosTron system, we targeted insertions to tcdA and tcdB at nucleotide positions 1584 and 1511, respectively (Fig. 1a). In both cases, this placed the intron within DNA sequence encoding the toxin catalytic domain. Three separate isogenic The genotype of each toxin mutant was characterised by PCR and DNA sequence analysis to confirm the exact location of each intron insertion made (data not shown).Southern blot analysis of EcoRV-digested genomic DNA samples, using an intron-3 specific probe, confirmed that the A -B + and A + B -mutants ...

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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.

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 ⌬117 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.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms.

Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal pseudomembranous colitis. Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and colonisation are essential for disease onset. Previously it has been suggested that flagella might be implicated in colonisation. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonisation in vivo using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis.