FIG. 4.

(A) Protease activity of V. anguillarum strains (M93Sm [•], M99 [empA mutant] [▴], M101 [epp mutant] [▾], M99 plus M101 after first mixing [▪], and M99 plus M101 after second mixing [○]). Cultures (10 ml) were grown with shaking for 16 h in LB20 at 27°C. Cells were harvested by centrifugation, washed two times with NSS, and then resuspended in NSSM (∼2 × 10⁹ CFU/ml) at 27°C. Cells from all cultures were harvested at 180 min and the supernatants were sterile filtered through a 0.22-μm filter. Aliquots (5 ml) of M99 and M101 culture supernatant were mixed 1:1 (mix 1) and incubated at 27°C with shaking. Remaining M99 and M101 culture supernatants (4 ml) were incubated separately at 27°C with shaking. Samples (750 μl) of M99/M101 mix 1 supernatant were taken at 0, 1, 2, 3, and 4 h. Samples (750 μl) of individual M99 and M101 supernatants were taken at 0, 1, and 2 h. At 2 h, M99 and M101 supernatants were mixed 1:1 (mix 2) and incubated at 27°C. Samples (600 μl) of mix 2 were taken at 0, 1, and 2 h after mixing (2, 3, and 4 h after the start of the experiment). M93Sm was used as a control. Protease activity was determined by OD readings at 442 nm (OD₄₄₂). The data presented are from a representative experiment. Experiments were performed in triplicate and repeated at least three times. Error bars indicate 1 standard deviation. (B) Western blot of individual supernatants. TCA-precipitated proteins of cell-free supernatants from NSSM-grown cultures of M93Sm, M99 (empA mutant), and M101 (epp mutant) were separated by SDS-PAGE, transferred to nitrocellulose, probed with rabbit anti-LasB antibody followed by immunoglobulin G-labeled goat anti-rabbit antibody, and visualized using TMB liquid substrate as described in Materials and Methods. Lanes: 1, molecular size protein ladder; 2, M93Sm; 3, M99 (empA mutant); 4, M101 (epp mutant). (C) Western blot of M99/M101 mix 1 and M99/M101 mix 2. Lanes: 1, molecular size protein ladder; 2, M99/M101 mix 1 supernatant; 3, M99/M101 mix 2 supernatant. Positions of the secreted pro-EmpA (∼48 kDa) and mature EmpA enzyme (∼36 kDa) are indicated with arrows on the right. The data presented are from a representative experiment. Experiments were repeated at least three times.