FIG 6.

Toxin production in the ΔCD1492 mutant. (A) Transcriptional analysis of the primary toxins, tcdA and tcdB in the ΔCD1492 mutant (MC674) relative to the parent strain, 630Δerm. Cultures were grown on 70:30 agar medium for 12 h, RNA was harvested, cDNA was prepared, and qRT-PCR was performed with gene-specific primers as outlined in Materials and Methods. WT, wild type. (B) A representative Western blot analysis of TcdA in 630Δerm, ΔCD1492 (MC674), 630Δerm pPcpr (MC282, vector control), ΔCD1492 pPcpr (MC729, vector control), ΔCD1492 pPcprA::CD1492 (MC730), and ΔCD1492 pPcprA::CD1492-H668A (MC771) grown in TY medium for 24 h. The mean values and the standard errors of the means of three independent experiments are shown at the bottom; bold values are statistically significantly different from those of the parent strain by a two-tailed Student t test or by a one-way ANOVA, followed by Dunnett's multiple-comparison test, as described in Materials and Methods. (C) qRT-PCR analysis of tcdA and tcdB transcript levels in cecal contents of hamsters infected with 630Δerm (n = 5) or MC674 (ΔCD1492; n = 5). The mean values and the standard errors of the means are shown (*, P ≤ 0.05 by two-tailed Student t test).