Fig 1. Exogenous calcium induces C. difficile germination in concert with taurocholate.

Cd630, VPI 10463, or R20291 spores were incubated with the indicated combinations of 0.2% Tc, 60 mM Ca-DPA, 50 mM glycine, 60 mM CaCl₂, or 60 mM DPA (A-G, I, J). Activation of SleC was assessed by western blot analyses. Cd630 spores were incubated for 15 minutes at 37°C with the indicated combinations of 1% Tc, 50 mM glycine, 60 mM CaCl₂, 60 mM DPA, or 60 mM Ca-DPA. Spores were subsequently lysed and assayed for levels of pro-SleC and SleC (K). Bacillus anthracis strain Sterne 34F₂ spores were incubated with the indicated combinations of either 60 mM CaCl₂, 60 mM DPA, or 60 mM CaDPA (H). Germination was measured either by tracking the loss of OD₆₀₀ over time (A-H), measuring loss of heat resistance at 37°C after 1 hour (I), or measuring release of DPA at 37°C after 1 hour (J). Germination assays were performed in triplicate. Germination assays and western blots are representative of three independent spore preps. Error bars are mean plus or minus SD. Statistical analysis was performed using one-way ANOVA. (****) p<0.0001.