Effect of pH up-shift on AmyK38 production. (A) B. subtilis strains 168 and MGB874 harboring pHYK38 were grown in 2xL-Mal medium at 30°C. At the transition phase (12 h), a sodium carbonate solution (Na2CO3) was added to the growth medium at the indicated final concentrations (w/v). Culture OD600 (open circles) and external pH (open triangles) were measured after 24 h of cultivation, and α-amylase activities (black bars) were measured after 72 h cultivation. All results presented are the averages of three individual experiments. Error bars represent standard deviations (n = 3). (B) Secretion and cellular amounts of AmyK38 in strains 168 and MGB874 harboring pHYK38. Cells were grown in 2xL-Mal medium at 30°C until the transition phase (12 h), at which point Na2CO3 was added to the growth medium at a final concentration of 0.9% (w/v). The amounts of AmyK38 in supernatants and cellular fractions after 48 h of cultivation were analyzed by Western blotting. Supernatants were diluted 240-fold and 3 μl sample volumes were used for analysis. Cells were re-suspended to an OD600 of 3.0, corresponding to an approximately 10-fold dilution, and 12 μl sample volumes (cell fraction) were analyzed. The positions of the protein standards are indicated in kDa to the left of the gels. Lanes: 1, purified AmyK38 (10 ng); 2, strain 168 harboring pHYK38 without Na2CO3 addition; 3, strain 168 harboring pHYK38 with Na2CO3 addition; 4, strain MGB874 harboring pHYK38 without Na2CO3 addition; and 5, strain MGB874 harboring pHYK38 with Na2CO3 addition.