FIG. 1.

Dam^OP affects T3S in Y. enterocolitica. While Dam^OP affects the Ca²⁺ regulation of Yop secretion, it does not influence the temperature regulation of Yop expression or the polarity of Yop translocation into the host cell cytoplasm, which is in contrast to a Ca²⁺-blind ΔlcrG strain. To analyze T3S, supernatants of strains grown under different secretion-inducing and noninducing conditions were precipitated by TCA, separated by SDS-PAGE, and stained with Coomassie brilliant blue. Yop expression was analyzed by Western blotting. Translocated YopE protein was collected from the cytoplasm (CP) of infected CHO cells by immunoprecipitation. Apolarly secreted YopE was TCA precipitated from the culture supernatant. Proteins were subjected to SDS-PAGE and transferred to nitrocellulose. YopE was detected using anti-YopE polyclonal antiserum. Lanes are labeled as follows: OP, GHY150 (Dam^OP); WT, GHY147 (wild-type control); ΔG, GHY347 (Y. enterocolitica ΔlcrG); CP, immunoprecipitated YopE from the cytoplasm of Y. enterocolitica-infected CHO cells; SN, TCA-precipitated supernatant of Y. enterocolitica-infected CHO cells, corresponding to 3 ml of supernatant (only 250 μl in the case of the ΔlcrG strain).