The orientation of the flagellar switch impacts toxin production.

<p>(A) qRT-PCR was used to determine the abundance of the indicated transcripts in <i>flg</i> ON and OFF isolates of <i>C</i>. <i>difficile</i> R20291. Four independent isolates were tested, and C<i>t</i> values for each gene were normalized to those of the housekeeping gene <i>rpoC</i>; the <i>flg</i> ON samples were arbitrarily chosen as the reference condition. Shown are means and standard deviations. * <i>p</i> < 0.05 by t-tests comparing mean transcript abundances between <i>flg</i> ON and OFF samples, n = 4. (B) TcdA protein levels in cell lysates of <i>flg</i> ON and OFF isolates were evaluated by western blot. Shown is a representative image for three independent experiments each with at least three replicates of each <i>flg</i> phase. (C) The <i>flg</i> ON and OFF isolates, as well as the <i>sigD</i> mutant control, were grown to stationary phase in TY medium. The supernatants were serially diluted and applied to Vero cells for 24 hours. Cell viability was assessed using the CellTiter Glo assay. Data are combined from two independent experiments each with four replicates of <i>sigD</i> mutant and <i>flg</i> phase variants, and means and standard deviations are shown. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by one-way ANOVA comparing the means for each dilution.