USING FURA-2 TO MEASURE INTRACELLULAR FREE CALCIUM IN PROPIONIBACTERIUM-ACNES

The fluorescent probe fura-2 was used to measure the intracellular free calcium concentration [Ca2+](i) in the Gram-positive skin bacterium Propionibacterium acnes. Three methods for loading the probe into P. acnes were used. Two consisted of using the membrane permeable acetoxymethyl ester of fura-2, fura-2/AM, whereas in the third method, fura-2 was loaded directly into the bacteria by means of an acid shock procedure. One method using fura-2/AM was found to be satisfactory. In this case, a statistical experimental design (2(IV)(4-1)) was used to devise a suitable loading procedure. [Ca2+]i of P. acnes was dependent upon the external calcium concentration. At low external concentrations (0.2 < [Ca2+](o) < 5 mu M), [Ca2+](i) was 135 +/- 13 nM (n = 20). An increase in [Ca2+](o) to 1 mM resulted in an increase in [Ca2+](i) to approximately 280 +/- 40 nM whereas in a Ca-free solution [[Ca2+]](o) < 5 nM), [Ca2+](i) decreased to a lower resting value of 70 +/- 7 nM. The time constants for calcium regulation upon step changes in the external concentration were of the order of 10 min. Intracellular alkalinization induced by changes in the external pH (pH(o)) resulted in an increase in [Ca2+](i) when [Ca2+](o) was in the range 0.2-5 mu M. [Ca2+](i) was approximately constant in the range pH(o) 5.5-7.7 but increased when pH(o) > 8.0. This increase was partially reversed when pH(o) was again lowered to below 8.0. In a Ca-free solution, little or no increase in [Ca2+](i) was observed when pH(o) > 8.0.