Figure 5.

 Accumulation of biotinylated pre-AmyQ–PSBT in the absence of functional SipS and SipT. An overnight culture of B. subtilis ΔSTxS–D146A (pKTH10-BT) was diluted 100-fold in fresh TY medium without xylose and incubated at 37°C. When the culture reached an OD₆₀₀ of ∼0.6, a sample (t = −2) was taken. Subsequently, the culture was divided in two equal moieties A and B. Culture A was kept for 2 hr at 37°C, and samples were taken at t = −1 and t = 0. Culture B was incubated for 1 hr at 42°C and, after a sample (t = −1) had been taken, incubation was continued for 1 hr at 48°C before a second sample (t = 0) was taken. Cells were collected by centrifugation and analyzed by SDS-PAGE and Western blotting. The presence of AmyQ–PSBT was visualized with specific antibodies. Biotinylated pre-AmyQ–PSBT was visualized with streptavidin–HRP. (p) Precursor; (m) mature.