HlyA activity of <i>E. coli</i> SE5000 expressing wild-type or mutant HlyD.

<p>A. Hemolytic activity of <i>E. coli</i> cells expressing wild-type or mutant HlyD. The absorbance at 450 and 543 nm was detected using a spectrophotometer, and hemolytic activities were calculated according to the following formula: Percent Hemolysis = (1-ODs/ODt)×100, where ODs and ODt are the differences in optical density at 543 nm between the samples and 100% hemolyzed SRBC solution, and differences in optical density at 450 nm between nonhemolyzed (control) and 100% hemolyzed sheep red blood cell solution, respectively. Filled symbols and lines indicate hemolytic activity detected in culture supernatants (OD₅₄₃/OD₄₅₀). B. Effects of expression of HlyD mutants (HlyD-R131A, HlyD-L186A, HlyD-T190A, HlyD-T197Y, HlyD-null) on HlyA secretion. <i>E. coli</i> SE5000 coexpressing HlyA, HlyB, HlyC, and wild-type or HlyD mutants (HlyD-R186A, HlyD-L190A, HlyD-T197A, HlyD-T197Y, or HlyD-Stop) were grown exponentially in LB with 10 mM CaCl₂ at 37°C, and proteins in the culture supernatant (equivalent to 3.5 OD₆₀₀ of cells) were TCA precipitated. The precipitated protein samples were separated by SDS-PAGE and subjected to immunoblotting with HlyA antibody (upper panel). Total cell lysate was used for the western blot analysis of HlyA to detect its expression levels in the cell (lower panel).