Figure 1.

Ca²⁺-dependent growth restriction and YopE/GFP levels in the YPT/yopE::gfp strain. (A) The YPT/yopE::gfp strain was grown to saturation at 26 °C in a defined media supplemented with 2.5 mM CaCl₂, diluted 50-fold in the same media, and then propagated for 2 hrs at 26 °C followed by 1 hr at 37 °C by which time the titer was approximately 1 × 10⁸ cfu/ml. The culture was then divided into fourths (time 0), with each part receiving EGTA at a final concentration of either 0, 1.25, 2.5, or 5 mM (resulting in molar ratios of EGTA/Ca²⁺ of 0, 0.5, 1, and 2, respectively). At the indicated time points the turbidity of each culture was determined by measuring the OD₆₀₀. (B) In the same experiment shown in (A) and at the same time points, samples of each culture were removed and YopE protein levels from combined whole cell and supernatant fractions derived from an equivalent number of bacterial cells (determined by viable plating) were assessed by immunoblotting. Dilutions of the 5 hr sample from the [EGTA/Ca²⁺] = 2 culture are shown on the lowest blot. (C) The blots shown in (B) were stripped and re-probed with a GFP-specific antiserum.