FIG. 2.
Ethanol does not affect the stability of Spo0A-Sad67. Western blot experiment showing the in vivo stability of the Spo0A∼P equivalent form Spo0A-Sad67 in ethanol-treated and untreated cultures. A 100-ml culture of B. subtilis strain Sik31 (Δspo0A::Ermr Pspac-sad67) was grown in SSM at 37°C until mid-exponential phase. At this point IPTG (1 mM) was added, and growth was continued for another 2 h. After this induction period (production of Spo0A-Sad67), the 100-ml culture was washed three times and growth was resumed in fresh prewarmed SSM without IPTG. Five minutes later, ethanol (0.7 M) was added to one-half of the washed culture, and both halves (with and without ethanol) were further incubated at 37°C. At the indicated times, samples were removed to prepare the cell extracts. Lanes 1 and 2, levels of Spo0A-Sad67 after the 2-h incubation period with IPTG before and immediately after its removal (IPTG washing), respectively. Lanes 3 to 10, levels of Spo0A-Sad67 at the indicated times after resumption of growth in fresh IPTG-free SSM with or without ethanol supplementation. Protein extracts, electrophoresis conditions, and reaction with anti-Spo0A antibodies were as indicated in Materials and Methods.