FIG 3.

LytM contributes to release of protein A by LAC*. (A and B) Culture supernatants from LAC* sbi and LAC* sbi lytM grown to the OD₆₀₀ indicated were probed with HRP-labeled rabbit IgG in Western immunoblotting. Supernatants harvested at an OD₆₀₀ of 0.3 were concentrated 8-fold before being loaded on a gel, and supernatants harvested at an OD₆₀₀ of 1.2 were concentrated 2-fold. Size markers are indicated (kDa). (C) Protein A on the surface of LAC* sbi and LAC* sbi lytM was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured by flow cytometry. Values are plotted as a percentage of the mean fluorescence intensity measured for LAC* sbi grown to an OD₆₀₀ of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. (D) Protein was captured from culture supernatants using chicken anti-SpA polyclonal IgY and detected using biotin-conjugated mouse monoclonal anti-SpA IgG followed by streptavidin-HRP in an ELISA. Values are expressed as a percentage of total released SpA measured for LAC* sbi grown to an OD₆₀₀ of 0.3. Bars represent the mean percent release from four independent experiments. Error bars represent the standard errors of the means. **, P = 0.006; ***, P = 0.0003; ns, not significant (P > 0.05).