Figure 2.

Mutations in flg RIR affect C. difficile motility and toxin production. (a) Representative image of swimming motility in soft agar medium of C. difficile R20291 (WT), flg-3sub ON and OFF, flg-ΔRIR ON and OFF, and flg-Δ3 ON and OFF, and sigD-null non-motile control, incubated for 48 hours. (b) Quantification of swimming motility after 48 h of strains in (a). (c) Immunoblot detection of TcdA and toxin titers after growth in TY broth. For immunoblot, a representative image of three independent experiments is shown. Green bands indicate TcdA detection; the red band is the 250 kDa protein in the molecular weight marker. Toxin titers of supernatants from overnight bacterial cultures were calculated as the reciprocal of the highest dilution that causes ≥80% rounding of Vero cells, expressed after log-transformation and normalization to OD₆₀₀ of the cultures. (b, c) Each symbol represents one biological replicate, and dotted line represents the limit of detection. *p < 0.05 by one-way ANOVA with Dunnett’s posttest comparing values to flg-Δ3 ON. (d) Quantitative orientation-specific PCR of the flagellar switch in WT, flg-3sub ON, flg-ΔRIR ON, and flg-Δ3 ON mutants expressing recV (pRecV) or bearing vector. Means and standard deviations are shown. ****p < 0.0001, ***p < 0.001, **p < 0.01 by one-way ANOVA and Dunnett’s posttest comparing values to WT pRecV. p value for comparison of WT pRecV and flg-3sub ON pRecV was determined by unpaired two-tailed t-test. (b–d) Means and standard deviations are shown.