Fig. 9. Dynamic response of the cytosolic pH to an increase in acid loading in wild-type cells and VatMpr cells. Cells expressing ratiometric pHluorin were washed and placed in a fluorimeter cuvette, and the fluorescence of the `acid' pHluorin peak (_ex=475 nm, _em=510 nm) was monitored continuously while acetate was added to the medium without a change in extracellular pH. An increase in fluorescence indicates a decrease in cytosolic pH. (A) Bacterially grown VatMpr and AX2 cells at pH 6. Acetate was added at T₀ to a final concentration of 15 mM. The pH decreased over at least 200 seconds in AX2, but reached an abrupt plateau at 30-45 seconds in VatMpr. (B) AX2 cells grown axenically at pH 7. Half of the cells were shifted to pH 5 at T minus 4 hours; acetate was added at T₀ (2 mM final concentration). The pH decreased for both cultures, but in cells that had been pre-incubated at pH 5, the curve reached an abrupt plateau, similar to that observed for bacterially grown VatMpr. The behavior of axenically grown VatMpr is identical to that of AX2 (data not shown).