FIG. 4.

Analysis of the presence of the 47-kDa enzyme in cell-free supernatants of representative Azo⁺ (Y. ruckeri 150) and Azo⁻ (Y. ruckeri 146) strains. Culture supernatants (20 ml) from cells grown on NB for 24 h were dialyzed, freeze-dried, and resuspended in 0.5 ml of Tris buffer (protein concentration, 50 μg/ml). Aliquots (10 to 40 μl) were used to analyze the presence of the 47-kDa protein by SDS–12% PAGE, (A) caseinolytic activity by zymograms by using 1% sodium caseinate (B) and the immunoblot probed with antibodies (1:500) raised against the 47-kDa protein (C). The details are described in the Materials and Methods section. Molecular mass markers (expressed in kilodaltons) are indicated on the left. Lanes 1, Azo⁺ strain (Y. ruckeri 150); lanes 2: Azo⁻ strain (Y. ruckeri 146).