Table S1. Bacterial strains and plasmids used in the study Strain/plasmids Description Source/reference R20291 NAP1/027 ribotype C. difficile strain, isolated in 2006 following an outbreak in Stoke Mandeville Hospital, UK [27] CD646 078 ribotype C. difficile strain [28] R20291::tcdE mutant C. difficile R20291 with intron insertion within the tcdE gene This study CD646::tcdE mutant C. difficile CD646 with intron insertion within the tcdE gene This study DH5α E coli strain with endA1, recA1, deoR, and hsdR17 (r K - m K + ) New England Bio-Labs S17-1 E. coli strain that favors conjugation [30] RGL100 R20291 with pRGL209 This study RGL101 R20291::tcdE mutant with pRGL209 This study pMTL007C-E5 Clostron plasmid [31, 32] pMTL007E5:Cdi-tcdE-234a pMTL007C-E5 carrying the tcdE-specific intron This study pRPF185 Shuttle vector for C. difficile [39] pRGL60 pRPF185 carrying the tcdE ORF (TcdEWT) [15] pRGL126 pRPF185 derivative with tcdE166 carrying Met 25 and Met 27 mutated to Leu 25 and Leu 27 , respectively, to express TcdE 166 This study (Figure 2A) pRGL128 pRPF185 derivative with tcdE142 carrying Met 1 and Met 27 mutated to a stop codon and Leu 27 , respectively, to express TcdE 142 This study (Figure 2A) pRGL127 pRPF185 derivative with tcdE140 carrying Met 1 and Met 25 mutated to a stop codon and Leu 25 , respectively, to express TcdE 140 This study (Figure 2A) pRGL-TcdE 142N26A pRGL128 derivative with mutated tcdE142, in which Asn 26 was mutated to Ala 26 This study pRGL-TcdE 142N26Q pRGL128 derivative with mutated tcdE142, in which Asn 26 was mutated to Gln 26 This study pRGL-TcdE 142N26V pRGL128 derivative with mutated tcdE142, in which Asn 26 was mutated to Val 26 This study pRGL132 pRGL126 derivative with a deletion of the adenine at the -1 position from the Met 1 ATG start codon This study (Figure 3A) pRGL133 pRGL126 derivative with a deletion of the adenine at -1 and a mutation in the ATG at -3 from the Met 1 ATG start codon This study (Figure 3A) pRGL134 pRGL126 derivative with a deletion of the adenine at -1 and mutations in the ATGs at -3 and -6 from the Met 1 ATG start codon This study (Figure 3A) pRGL267 pRPF185 carrying tcdE166 with gus RBS This study pRGL209 pRPF185 carrying tcdR ORF under the tet-inducible promoter This study
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Table S1. Bacterial strains and plasmids used in the study
Strain/plasmids Description Source/reference
R20291 NAP1/027 ribotype C. difficile strain, isolated in 2006 following an outbreak in Stoke Mandeville Hospital, UK [27]
CD646 078 ribotype C. difficile strain [28]
R20291::tcdE mutant C. difficile R20291 with intron insertion within the tcdE gene This study
CD646::tcdE mutant C. difficile CD646 with intron insertion within the tcdE gene This study
DH5α E coli strain with endA1, recA1, deoR, and hsdR17 (rK- mK
+) New England Bio-Labs
S17-1 E. coli strain that favors conjugation [30]
RGL100 R20291 with pRGL209 This study
RGL101 R20291::tcdE mutant with pRGL209 This study
pMTL007C-E5 Clostron plasmid [31, 32]
pMTL007E5:Cdi-tcdE-234a pMTL007C-E5 carrying the tcdE-specific intron This study
pRPF185 Shuttle vector for C. difficile [39]
pRGL60 pRPF185 carrying the tcdE ORF (TcdEWT) [15]
pRGL126 pRPF185 derivative with tcdE166 carrying Met25 and Met27 mutated to Leu25 and Leu27, respectively, to express TcdE166 This study (Figure 2A)
pRGL128 pRPF185 derivative with tcdE142 carrying Met1 and Met27 mutated to a stop codon and Leu27, respectively, to express TcdE142 This study (Figure 2A)
pRGL127 pRPF185 derivative with tcdE140 carrying Met1 and Met25 mutated to a stop codon and Leu25, respectively, to express TcdE140 This study (Figure 2A)
pRGL-TcdE142N26A pRGL128 derivative with mutated tcdE142, in which Asn26 was mutated to Ala26 This study
pRGL-TcdE142N26Q pRGL128 derivative with mutated tcdE142, in which Asn26 was mutated to Gln26 This study
pRGL-TcdE142N26V pRGL128 derivative with mutated tcdE142, in which Asn26 was mutated to Val26 This study
pRGL132 pRGL126 derivative with a deletion of the adenine at the -1 position from the Met1 ATG start codon This study (Figure 3A)
pRGL133 pRGL126 derivative with a deletion of the adenine at -1 and a mutation in the ATG at -3 from the Met1 ATG start codon This study (Figure 3A)
pRGL134 pRGL126 derivative with a deletion of the adenine at -1 and mutations in the ATGs at -3 and -6 from the Met1 ATG start codon This study (Figure 3A)
pRGL267 pRPF185 carrying tcdE166 with gus RBS This study
pRGL209 pRPF185 carrying tcdR ORF under the tet-inducible promoter This study