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. 2019 Mar 12;10(2):e01991-18. doi: 10.1128/mBio.01991-18

FIG 6.

FIG 6

A hybrid rstA construct containing the C. perfringens DNA-binding domain with the C. difficile Spo0F-like and quorum-sensing-like domains complements C. difficile rstA toxin production and sporulation. (A) Western blot analysis of TcdA in 630Δerm pMC211 (MC282; vector control), rstA::erm pMC211 (MC505; vector control), rstA::erm pPcprA-rstA3XFLAG (MC1004), rstA::erm pPcprA-Cp-rstA3XFLAG (MC1324), and rstA::erm pPcprA-CpHTHCdCterminal3XFLAG (MC1257) grown in TY medium, pH 7.4, supplemented with 2 µg/ml thiamphenicol and 1 µg/ml nisin, at H24. The corresponding image showing total protein is shown in Fig. S8B. (B) qRT-PCR analysis of tcdR, tcdA, and tcdB transcript levels in 630Δerm pMC211 (MC282; vector control), rstA::erm pMC211 (MC505; vector control), rstA::erm pPcprA-rstA3XFLAG (MC1004), rstA::erm pPcprA-Cp-rstA3XFLAG (MC1324), and rstA::erm pPcprA-CpHTHCdCterminal3XFLAG (MC1257) grown in TY medium, pH 7.4, supplemented with 2 µg/ml thiamphenicol and 1 µg/ml nisin, at T3 (three hours after the entry into stationary phase). (C) Ethanol-resistant spore formation of 630Δerm pMC211 (MC282; vector control), rstA::erm pMC211 (MC505; vector control), rstA::erm pPcprA-rstA3XFLAG (MC1004), rstA::erm pPcprA-Cp-rstA3XFLAG (MC1324), and rstA::erm pPcprA-CpHTHCdCterminal3XFLAG (MC1257) grown on 70:30 sporulation agar supplemented with 2 µg/ml thiamphenicol and 1 µg/ml nisin. Sporulation frequency is calculated as the number of ethanol-resistant spores divided by the total number of cells enumerated at H24 as detailed in Materials and Methods. The means and standard errors of the means for at least three independent biological replicates are shown; asterisks represent P ≤ 0.05 by one-way ANOVA, followed by Dunnett’s multiple-comparison test compared to rstA pMC211 (MC505).