FIG. 3.

Complementation of YopD secretion. Y. enterocolitica sycD expressing SycD-FT, Scc1-FT, Scc2-FT, and Scc3-FT or Y. enterocolitica WT and sycD (ΔsycD) were cultivated in the presence (+) or absence (−) of 2.5 mM Ca²⁺. Cultures were incubated at 26°C for 2 h and then shifted to 37°C. IPTG was added to 0.1 mM at the time of temperature shift to achieve induction of trans genes. After 4 h of growth at 37°C, 1.0-ml volumes were harvested and directly precipitated with TCA for TC samples or fractionated into samples representing CS prior to concentration. Material corresponding to 0.05 OD₆₂₀/ml of original cultures for CS and 0.02 OD₆₂₀/ml for TC was resolved in 12% (wt/vol) polyacrylamide gels. Flag-tagged SycD, Scc1, Scc2, and Scc3 were detected by immunoblotting with α-Flag M2, and YopD and YopE were detected with α-YopD and α-YopE, respectively. Immunoblots were probed with β-lactamase-specific antibodies as a control for cell lysis. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and development with NBT-BCIP.