FIG. 2.
The LcrV dependence of YopE synthesis and localization is relaxed under Dam-overproducing conditions in Y. pseudotuberculosis. Whole-cell (WC), membrane (Memb), and supernatant (Sup) fractions (12) were prepared from dam wild-type (WT) and Dam-overproducing (OP) Y. pseudotuberculosis containing (A) or lacking (B) the lcrV gene. For each growth condition (12, 44, 45), total protein extracts corresponding to 2.0 × 106 cells (∼20 μg of protein/well) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene difluoride membrane (Pierce), and probed with rabbit anti-YopE polyclonal antibodies (1:50,000 dilution). Peroxidase-conjugated donkey anti-rabbit immunoglobulin G was used as the secondary antibody (1:20,000 dilution; Amersham Biosciences), and hybridization was detected by chemiluminescence using Supersignal West Femto Maximum Sensitivity Substrate (Pierce) followed by a 30-s (A) or 2-min (B) exposure to film. Inspection of corresponding Coomassie-stained gels showed similar band intensities of nonregulated proteins under all conditions tested (data not shown). Western analysis of yopE+ and yopE− strains confirmed that the 23-kDa protein was YopE (23).