PCR screening of double crossover candidate clones for complementation of the <i>cwp84</i> gene in <i>C. difficile</i> 630 Δ<i>erm</i> Δ<i>cwp84</i>.

<p>(A) Schematic diagram of the complementation of <i>cwp84</i>, with a single nucleotide change to base 2280 of <i>cwp84</i> from a T to an A, without changing the corresponding valine amino acid residue and at the same time creating a <i>Sca</i>I site. The purpose of this single nucleotide change was to prove the occurrence of the complementation event. (B) PCR screening of candidate clones of the complemented <i>cwp84</i> gene. Primers cwp84-F4 and cwp84-R4 anneal to the internal sequence of the knockout cassette and the downstream sequence of <i>cwp84</i>, respectively, resulting in a 1, 026 bp PCR product from double-crossover complemented clones and wild-type, while no PCR product is expected from Δ<i>cwp84</i> mutants. MW is a 2-Log DNA Ladder (NEB) molecular weight marker, WT is a wild-type <i>C. difficile</i> DNA control, and 1–3 are the candidate clones. All candidates 1 to 3 show the expected complemented 1, 026 bp band, thereby confirmed as <i>cwp84</i> complemented clones, as seen in the wildtype control. (C) PCR products amplified using primers cwp84-F4 and cwp84-R4 from candidates clones and wildtype were analysed by RE digestion with <i>Sca</i>I. PCR products amplified from cwp84 complemented clones were cut into two fragments (786 and 240 bp), whereas PCR products amplified from the wildtype control did not.</p

Allelic Exchange vectors for manipulation of <i>C. difficile</i> 630Δ<i>erm</i> (pMTL-YN3) and R20291 (pMTL-YN4).

<p>Common plasmid components are: CatP, the <i>catP</i> gene of <i>Clostridium perfringens</i> conferring thiamphenicol resistance; PyrE, the <i>pyrE</i> gene of <i>Clostridium sporogenes</i>; ColE1, the replication region of the <i>E.coli</i> plasmid ColE1, and; TraJ, transfer function of the RP4 <i>oriT</i> region; Z, the <i>lacZ'</i> gene encoding the alpha fragment of the <i>E.coli</i> β-galactosidase (and containing a multiple cloning site, MCS, region derived from plasmid pMTL20); T1, a transcriptional terminator isolated from downstream of the <i>Clostridium difficile</i> strain 630 CD0164 gene, and; T2, a transcriptional terminator of the ferredoxin gene of <i>Clostridium pasteurianum</i>. The position of the frame-shift generated at the NsiI site is indicated by an asterick. Plasmid pMTL-ME2 is identical to plasmid pMTL-YN3, except it carries an NsiI site at the 3′-end of RepH at the position marked by an asterick.</p

Segregational stability of pMTL83*251 or pMTL83251 in <i>C. difficile</i> strain 630Δ<i>erm</i>.

<p>The two plasmids differ only in that pMTL83*251 has a frame shift in the pCB102 RepH gene, introduced by blunt-end ligation following cleavage with NsiI. Cells carrying the two plasmids were grown in BHIS media in the absence of antibiotic and then CFUs estimated on agar media supplemented with thiamphenicol after 6, 12 and 24 h of growth. The illustrated data was derived from three independent experiments.</p

Glycin extracts analysis of 630Δerm and R20291 WT, mutant and complemented strains.

<p>(A) Immunoblot analysis with anti-Cwp84 antibodies of glycin extracts, showing complete absence of Cwp84 in the mutants compared to WT and complemented strains. (B) SDS-PAGE of glycin extracts of 630Δerm and R20291 WT, mutant and complemented strains, showing no processing of SlpA precursor in the 630Δ<i>cwp84</i> mutant, and in contrast, an incomplete processing of SlpA in the R20291Δ<i>cwp84</i> mutant. (C) Identification of the HMW-SLP in the glycin extract of the R20291Δ<i>cwp84</i>, showing that a partial processing of SlpA takes place in this mutant even in absence of the Cwp84 protease. Lanes 1, 630Δ<i>erm</i>; lanes 2, 630Δ<i>erm</i>Δ<i>cwp84</i>; lanes 3, 630Δ<i>erm</i>Δ<i>cwp84</i> complemented; lanes 4, R20291; lanes 5, R20291Δ<i>erm</i>Δ<i>cwp84</i>; lanes 6, R20291Δ<i>erm</i>Δ<i>cwp84</i> complemented; MW, molecular weight standard.</p

List of strains and plasmids used in this study.

<p>List of strains and plasmids used in this study.</p

PyrE ACE correction vectors for <i>C. difficile</i> 630Δ<i>erm</i> (pMTL-YN1) and R20291 (pMTL-YN2).

<p>Both vectors carry identical components between their FseI and SbfI restriction sites. These are: CatP, the <i>catP</i> gene of <i>Clostridium perfringens</i> conferring thiamphenicol resistance; ColE1, the replication region of the <i>E.coli</i> plasmid ColE1, and; TraJ, transfer function of the RP4 <i>oriT</i> region. Plasmids pMTL-YN1C and pMTL-YN2C have an additional segment of DNA inserted between the left-hand homology arm (LHA) and the right-hand homology arm (RHA) which carries: a transcriptional terminator (T1) of the ferredoxin gene of <i>Clostridium pasteurianum</i>; a copy of the <i>lacZ'</i> gene encoding the alpha fragment of the <i>E.coli</i> β-galactosidase, and; a multiple cloning site (MCS) region derived from plasmid pMTL20 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056051#pone.0056051-Chambers1" target="_blank">[19]</a>. Plasmids pMTL-YN1X and pMTL-YN2X differ from pMTL-YN1C and pMTL-YN2C, respectively, in that they carrying the promoter region (P<i>_fdx</i>) of the <i>Clostridium sporogenes</i> ferredoxin gene.</p

Growth of <i>C. difficile</i> 630Δerm strains with mannitol as the sole carbon source.

<p>(A) Clock-wise from top-left, <i>C. difficile</i> 630Δ<i>erm</i> (1) 630Δ<i>erm</i>Δ<i>mtlD</i> mutant (2), and 630Δ<i>erm</i>Δ<i>mtlD</i>-complemented (3) and 630Δ<i>erm</i>Δ<i>mtlD</i>-overexpressed (4) were streaked onto minimal media agar with mannitol as the sole carbon source and incubated for 48 h to observe growth. In contrast to the wild type, complemented and overexpressed strains, no growth was evident for the 630ΔermΔ <i>mtlD</i> mutant. (B) The growth of Δ<i>mtlD</i> was limited in mannitol broth, while growth of the Δ<i>mtlD</i> complemented and <i>mtlD</i> overexpressed strains were restored to wildtype levels. (C) The pH of the media broth showed a dip in pH caused by the fermentation of mannitol for the wildtype, Δ<i>mtlD</i> complemented and Δ<i>mtlD</i> overexpressed strains, which correlate to their growth. The 630 Δ<i>erm</i> Δ<i>mtlD</i> mutant strain grew very weakly in mannitol broth, which was reflected in the sustained pH levels of the media. All experiments were undertaken in triplicate. The data, complete with error bars is provided in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056051#pone.0056051.s001" target="_blank">Supporting Information File S1</a>.</p

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p

Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles

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