Figure 1.
The ACE vector pMTL-YN18 is designed to create a deletion mutant specifically in the C. difficile strain R20191. Plasmid components are: CatP, the catP gene of Clostridium perfringens conferring thiamphenicol resistance; ColE1, the replication region of the E.coli plasmid ColE1; TraJ, transfer function of the RP4 oriT region; RepA and Orf2, the replication region of the Clostridium botulinum plasmid pBP1; LHA, left-hand homology arm encompassing a 300 bp internal fragment of the R20291 pyrE gene lacking 50 nucleotides from the 5′-end, and 235 bp from the 3′-end; RHA, right-hand homology arm comprising encompassing the 1200 bp region of DNA immediately downstream of pyrE, and; lacZ', gene encoding the alpha fragment of the E.coli β-galactosidase (and containing a multiple cloning site region derived from plasmid pMTL20 [19]).
Figure 2.
PyrE ACE correction vectors for C. difficile 630Δerm (pMTL-YN1) and R20291 (pMTL-YN2).
Both vectors carry identical components between their FseI and SbfI restriction sites. These are: CatP, the catP gene of Clostridium perfringens conferring thiamphenicol resistance; ColE1, the replication region of the E.coli plasmid ColE1, and; TraJ, transfer function of the RP4 oriT 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 Clostridium pasteurianum; a copy of the lacZ' gene encoding the alpha fragment of the E.coli β-galactosidase, and; a multiple cloning site (MCS) region derived from plasmid pMTL20 [19]. Plasmids pMTL-YN1X and pMTL-YN2X differ from pMTL-YN1C and pMTL-YN2C, respectively, in that they carrying the promoter region (Pfdx) of the Clostridium sporogenes ferredoxin gene.
Figure 3.
Segregational stability of pMTL83*251 or pMTL83251 in C. difficile strain 630Δerm.
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.
Figure 4.
Allelic Exchange vectors for manipulation of C. difficile 630Δerm (pMTL-YN3) and R20291 (pMTL-YN4).
Common plasmid components are: CatP, the catP gene of Clostridium perfringens conferring thiamphenicol resistance; PyrE, the pyrE gene of Clostridium sporogenes; ColE1, the replication region of the E.coli plasmid ColE1, and; TraJ, transfer function of the RP4 oriT region; Z, the lacZ' gene encoding the alpha fragment of the E.coli β-galactosidase (and containing a multiple cloning site, MCS, region derived from plasmid pMTL20); T1, a transcriptional terminator isolated from downstream of the Clostridium difficile strain 630 CD0164 gene, and; T2, a transcriptional terminator of the ferredoxin gene of Clostridium pasteurianum. 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.
Figure 5.
PCR screening of double crossover candidate clones for complementation of the cwp84 gene in C. difficile 630 Δerm Δcwp84.
(A) Schematic diagram of the complementation of cwp84, with a single nucleotide change to base 2280 of cwp84 from a T to an A, without changing the corresponding valine amino acid residue and at the same time creating a ScaI 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 cwp84 gene. Primers cwp84-F4 and cwp84-R4 anneal to the internal sequence of the knockout cassette and the downstream sequence of cwp84, respectively, resulting in a 1, 026 bp PCR product from double-crossover complemented clones and wild-type, while no PCR product is expected from Δcwp84 mutants. MW is a 2-Log DNA Ladder (NEB) molecular weight marker, WT is a wild-type C. difficile 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 cwp84 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 ScaI. 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.
Figure 6.
Glycin extracts analysis of 630Δerm and R20291 WT, mutant and complemented strains.
(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 630Δcwp84 mutant, and in contrast, an incomplete processing of SlpA in the R20291Δcwp84 mutant. (C) Identification of the HMW-SLP in the glycin extract of the R20291Δcwp84, showing that a partial processing of SlpA takes place in this mutant even in absence of the Cwp84 protease. Lanes 1, 630Δerm; lanes 2, 630ΔermΔcwp84; lanes 3, 630ΔermΔcwp84 complemented; lanes 4, R20291; lanes 5, R20291ΔermΔcwp84; lanes 6, R20291ΔermΔcwp84 complemented; MW, molecular weight standard.
Figure 7.
Growth of C. difficile 630Δerm strains with mannitol as the sole carbon source.
(A) Clock-wise from top-left, C. difficile 630Δerm (1) 630ΔermΔmtlD mutant (2), and 630ΔermΔmtlD-complemented (3) and 630ΔermΔmtlD-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 mtlD mutant. (B) The growth of ΔmtlD was limited in mannitol broth, while growth of the ΔmtlD complemented and mtlD 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, ΔmtlD complemented and ΔmtlD overexpressed strains, which correlate to their growth. The 630 Δerm ΔmtlD 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 Supporting Information File S1.
Table 1.
List of oligonucleotides used in this study.
Table 2.
List of strains and plasmids used in this study.