FIG 9.

Secretion of extracellular degradative enzymes by tSEC6 strain. (A) Cells from overnight cultures were transferred to BSA medium and incubated for 24 h to induce secretion of aspartyl proteases. The cells were then washed and concentrated into fresh BSA medium with or without DOX and incubated for 24 h with shaking. Supernatants collected from these cultures were analyzed by SDS-PAGE and subsequently stained with Coomassie blue to visualize the extent of BSA degradation that occurred as a result of proteolytic cleavage by Saps released into the medium. Liquid BSA medium alone was used as a control. The triple deletion mutant sapΔ(1-3) was also included as a negative control. Bands of intact BSA are indicative of reduced secretion of Saps. BSA degradation was markedly reduced in the tSEC6 strain grown with DOX compared to wild-type controls. (B) Secretion of degradative lipases was also assessed. Cells were grown overnight in liquid YPD and transferred to Tween 80 medium for 24 h to induce production and secretion of lipases. The cells were then washed and concentrated in fresh Tween 80 medium with or without DOX and incubated for 24 h at 37°C. The supernatant was collected and used in a turbidimetric kinetic assay that measured increases in optical density (500 nm) as a result of the precipitation of calcium salts formed when degradative products are combined with CaCl₂. The rate of increase in the OD₅₀₀ is proportional to the concentration of lipase present in the supernatant. Three biological replicates are represented and show a significant difference between the tSEC6 strain grown with DOX and the controls (P < 0.05). Error bars indicate standard deviations.