Overproduction of SycO represses secretion and expression of Yops. Y. enterocolitica parental strain WA-314 and the isogenic deletion mutants ΔyopO and ΔyopP were transformed with plasmids pWS-sycO and pWS, respectively. After culturing at 37°C in the presence of 0.2 mM CaCl2 for 2 h, Yop secretion was induced with EGTA. Simultaneously, the cultures were supplemented with 1 mM IPTG and grown for another 2 h. (A) TCA-precipitated culture supernatants were analyzed by Coomassie-staining of an SDS gel (upper panel), and by immunoblotting of YopO and YopP (lower panels). Two independent cultures of each strain are represented. Calibration of gel loads was obtained by measurement of the optical density of the cultures. (B) Concentration-dependency of the SycO overproduction phenotype. Yop secretion was induced in Y. enterocolitica WA-314 harboring plasmids pWS-sycO and pWS, respectively, and simultaneously SycO overproduction was induced by addition of IPTG in increasing amounts (indicated by the filled triangles). The IPTG concentrations were 0, 0.001, 0.01, 0.1, and 1 mM, respectively. The upper panel shows a Coomassie-stained SDS gel after separation of TCA-precipitated supernatants. The lower panel shows an immunoblot of bacterial pellets to detect the expression of SycO. (C) Cell lysates corresponding to the secretion analysis presented in (A) were analyzed by immunoblotting of SycO, YopO, YopH, and YopE.