ATP-DEPENDENT CALCIUM RELEASE FROM BINDING SITE IN STREPTOCOCCUS BOVIS: BOUND VERSUS FREE POOLS

The introduction of the acetoxymethyl ester of fura-2 (fura-2/AM) has made possible the measurement of the concentration of intracellular free calcium (free [Ca 2+ ] i ) in bacteria. We report here a study of intracellular calcium levels and exclusion mechanisms in Streptococcus bovis using fura-2/AM and 45 CaCl 2 . The total [Ca 2+ ] i in cells not energized with glucose increased in proportion to external Ca 2+ concentration over the range 0.1-0.5 mM, the intra- and extracellular levels being approximately equal. In contrast, the free [Ca 2+ ] i remained at 71.1±1.9 nM in the face of extracellular concentrations from 0.1 to 5 mM. Energizing by the addition of glucose produced a dramatic increase in the intracellular concentration of ATP and a marked decrease in total [Ca 2+ ] i , but had no effect on the free [Ca 2+ ] i regardless of whether extracellular calcium was high or low. Total [Ca 2+ ] i was affected little, if at all, by the metabolic inhibitors FCCP and DCCD, implying that neither proton motive force nor F 0 F 1 H + -ATPase is necessary for calcium exclusion. In contrast, total [Ca 2+ ] i and ATP were both greatly reduced by iodoacetate. These results suggest that 1) S. bovis has a remarkable calcium buffering capacity, and 2) most of its intracellular calcium exists in a bound form which can be released by energizing via a mechanism coupled to ATP hydrolysis.