FIG 4.

Extracellular proteases are not required for the release of protein A. (A) Protein A on the surface of LAC* and LAC* PD was detected using FITC-labeled rabbit IgG, and the fluorescence intensity was measured by flow cytometry. Values are expressed as a percentage of the mean fluorescence intensity measured for LAC* harvested at an OD₆₀₀ of 0.3. Bars represent the mean values, and error bars indicate the standard errors of the means of three independent experiments. ns, not significant (P > 0.05). (B and C) Cell wall extracts (CW) and culture supernatants (SN) from LAC* and LAC* PD grown to the OD₆₀₀ indicated were probed with HRP-labeled rabbit IgG in a Western immunoblot. Supernatants harvested at an OD₆₀₀ of 0.3 were concentrated 8-fold, those at an OD₆₀₀ of 0.6 were concentrated 4-fold, and those at an OD₆₀₀ of 1.2 were concentrated 2-fold before being loaded on a gel. (D) LAC* PD was grown to an OD₆₀₀ of 0.8 in broth alone or in broth supplemented with V8 (1 U/ml) and DCI (200 μM), and cell wall extracts were probed with biotin-labeled fibronectin in a ligand affinity blot. Bound fibronectin was detected using streptavidin-HRP. (E) The same cell wall extracts and supernatants from the same cultures were probed with HRP-conjugated rabbit IgG to detect protein A. Size markers are indicated (kDa).