Table 1.
Strains, plasmids and oligonucleotides used in this study.
| Strain | Characteristics | Source |
|---|---|---|
| R20291 | C. difficile ribotype 027 strain isolated during an outbreak at Stoke Mandeville hospital, UK in 2006. | [34] |
| R20291ΔpyrE | An R20291 pyrE deletion mutant. | [26] |
| R20291ΔspoVD | R20291 with the entire spoVD gene, except the first and last three codons, deleted. | This study |
| R20291ΔspoVD pyrE::spoVD | R20291ΔspoVD complemented by simultaneous restoration of the wild type pyrE gene and insertion of spoVD under the native promoter between pyrE and the downstream R20291_0189. | This study |
| R20291 SNAP-spoVD | R20291 with the native spoVD locus modified by homologous recombination to add the coding sequence of SNAP to the 5′ end of spoVD. | This study |
| CA434 | E. coli conjugative donor. HB101 carrying R702. | [35] |
| NEB5α |
fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17. |
New England Biolabs |
|
Plasmid |
Characteristics |
Source |
| pMTL960 | E. coli-C. difficile shuttle vector. | Nigel Minton |
| pRPF150 | Pcwp2-Strep-tag II-secA2 cassette cloned between KpnI and BamHI sites in pMTL960. | [36] |
| pJAK012 | pRPF150 modified to introduce an XhoI site between Strep-tag II encoding sequence and the secA2 gene. | This study |
| pJAK032 | Strep Tag II coding sequence in pJAK012 replaced with a codon-optimized CLIP gene. | This study |
| pFT46 | Plasmid containing a C. difficile codon-optimized copy of the SNAP gene under the control of a tetracycline inducible promoter. | [37] |
| pMTL-YN4 | Allele exchange vector for pyrE-based selection. | [26] |
| pMTL-YN2C | pyrE restoration vector allowing simultaneous insertion of cargo DNA between pyrE and R20291_0189. | [26] |
| pMTL-SC7215 | Allele exchange vector for codA-based selection. | [25] |
| pYAA024 | spoVD deletion: 1200 bp homology arms representing the sequence upstream and downstream of R20291_2544 (spoVD) cloned into pMTL-YN4. Designed to delete all but the first and last 9 bp of spoVD. | This study |
| pYAA027 | SpoVD complementation: spoVD and its native promoter cloned into pMTLYN2C. | This study |
| pYAA031 | Constitutive CLIP-SpoVD: spoVD cloned between XhoI and BamHI sites in pJAK032. | This study |
| pYAA047 | SNAP-SpoVD: 1200 bp upstream of spoVD was fused to the coding sequence of SNAP and the first 1200 bp of spoVD and the subsequent recombination cassette cloned into pMTL-SC7215. | This study |
| pYAA048 | SpoVD(ΔDimerization): pYAA031 modified by deletion of the sequence encoding the SpoVD PBP dimerization domain. | This study |
| pYAA049 | SpoVD(ΔPASTA): pYAA031 modified by deletion of the sequence encoding the SpoVD PASTA domain. | This study |
| pYAA050 | SpoVD(ΔTranspeptidase): pYAA031 modified by deletion of the sequence encoding the SpoVD transpeptidase domain. | This study |
| pYAA051 | SpoVD(ΔDimer & PASTA): pYAA031 modified by deletion of the sequence encoding the SpoVD PBP dimerization and PASTA domains. | This study |
| pYAA052 |
His-SpoVD: spoVD cloned into pET-28a between NcoI and XhoI sites. |
This study |
|
Oligonucleotide |
Sequence |
Use |
| NF1957 | GAGTCAGTTATAGATTCGATACTTGAC | To introduce an XhoI site into pRPF150 by inverse PCR with NF1958 |
| NF1958 | GAGTTTTTCAAATTGTGGATGACTCCAC | To introduce an XhoI site into pRPF150 by inverse PCR with NF1957 |
| RF20 | AAACTCCTTTTTGATAATCTCATGACC | To linearize pMTL-SC7215 with RF311 |
| RF139a | GTCAGAGCTCGTTCTTTATTTAGATTAAATAAAGTCAATG | To clone spoVD into pMTL-YN4 with RF187 |
| RF187a | GTCAGGATCCCTTAGGAATCAGAGAGTAGATAG | To clone spoVD into pMTL-YN4 with RF139 |
| RF226 | GATCGAGCTCGGAGGAACTACTATGGATAAAGATTGTGAAATGAAAAG | To add a 5′ SacI site onto a codon optimized clip gene fragment |
| RF227 | GATCCTCGAGAGCAGCTGCTCCTAATCCTGGTTTTCCTAATC | To add 3xAla codons and a 3′ XhoI site onto a codon optimized clip gene fragment |
| RF311 | TAGGGTAACAAAAAACACCG | To linearize pMTL-SC7215 with RF20 |
| RF323a | GTCAGGATCCGTTTATGGGTATATGTTAATTATCTGTTAC | To clone R20291_2545 and spoVD into pMTL-YN2C with RF324 |
| RF324a | GTCAGAGCTCCTTAGGAATCAGAGAGTAGATAG | To clone R20291_2545 and spoVD into pMTL-YN2C with RF323 |
| RF374a | GATCCTCGAGAGAAAAGTAAAGAGGATAAGTAAGAAAAGG | To clone spoVD into pJAK032 with RF375 |
| RF375a | GTCAGGATCCTTAGTTTTCAAAATATAGGGTTATACTTGAG | To clone spoVD into pJAK032 with RF374 |
| RF461 | CTCAAATCTATTCCCCCTAGTTATCC | To amplify spoVD promoter probe with RF462 for Southern blotting |
| RF462 | GAATCTATGTGGTTATTCAAAAATCTCG | To amplify spoVD promoter probe with RF462 for Southern blotting |
| RF528 | aaatacggtgttttttgttaccctaagtttAAGCTAGAATAGATGGACC | To amplify 1200 bp homology arm upstream of spoVD |
| RF529 | acaatctttatccatATCTATTCCCCCTAGTTATCC | To amplify 1200 bp homology arm upstream of spoVD |
| RF530 | ctagggggaatagatATGGATAAAGATTGTGAAATGAAGAGAACCAC | To amplify SNAP |
| RF531 | cctctttacttttctAGCAGCTGCCCCAAGTCC | To amplify SNAP |
| RF532 | cttggggcagctgctAGAAAAGTAAAGAGGATAAGTAAGAAAAG | To amplify first 1200 bp of spoVD |
| RF533 | tttggtcatgagattatcaaaaaggagtttTAAATCTATACCTGTCTTATCCATAAG | To amplify first 1200 bp of spoVD |
| RF582 | TATATCTCTTGTTTGTTGTTCTAGTGCTTTTG | To delete the coding sequence of the SpoVD PBP Dimerization domain with RF583 |
| RF583 | GCAAAAAAGGTTACTGCAATAGCTATG | To delete the coding sequence of the SpoVD PBP Dimerization domain with RF582 |
| RF584 | GGTTTAACTCCCAAATATTTTAAAGAGTCATTC | To delete the coding sequence of the SpoVD PASTA domain with RF585 |
| RF585 | TAAGGATCCACTAGTAACGGCC | To delete the coding sequence of the SpoVD PASTA domain with RF584 |
| RF586 | AGTATATAAAGAAGAAGAAAAAGCTGAGTATG | To delete the coding sequence of the SpoVD Transpeptidase domain with RF587 |
| RF587 | ATTATTTAACTCATAAGCTTTCTGTACTGC | To delete the coding sequence of the SpoVD Transpeptidase domain with RF586 |
a
Restriction endonuclease sites are underlined.