FIG. 5.

Expression of the YopN-TyeA hybrid protein is not required for the calcium-dependent regulation of Yop secretion in Y. pestis. Y. pestis KIM8-3002 (parent) and KIM8-3002.P55 (ΔyopN ΔtyeA) carrying plasmids pYopN2, pTyeA1, pYopN2, and pTyeA1 or carrying pYopN-TyeA were grown in TMH medium with (+) or without (−) 2.5 mM CaCl₂ for 5 h at 37°C. Individual bacterial cultures were harvested by centrifugation and dissolved in SDS-PAGE sample buffer volumes corresponding to equal numbers of bacteria. Respective culture supernatants were precipitated with 10% trichloroacetic acid and resuspended in an equivalent volume of sample buffer. Samples were separated by SDS-PAGE, transferred to Immobilon-P membranes, and probed with antiserum specific for YopN (α-YopN) or YopE (α-YopE).