Maintenance of intracellular calcium in Escherichia coli.

Abstract

Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells.Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli.Cells of E. coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid.The concentration of free [Ca2+Ii in unstarved cells was maintained at 90 f 10 nM, irrespective of the Ca2+ concentration in the extracellular medium.Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium.In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration.In starved cells [Ca2+]i varied from 0.2 to 0.7 PM when the loading Ca2+ concentration varied from 10 PM to 10 mM.Addition of glucose lowered the Ca2+ levels to 90 nM.Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux.Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool.These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free [Ca2+Ii is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.Calcium ion plays a pivotal role in the regulation of various cellular processes in eukaryotic cells.Although little is known about the physiological role of the ion in prokaryotes, calcium transport systems have been found in all bacterial species investigated so far (1,2).The transport of calcium in bacterial cells was first demonstrated in Escherichia coli, where it was demonstrated that Ca2+ is extruded from the cells in a temperature-dependent fashion (3).The existence of a metabolically linked efflux system was suggested.Tsujibo and Rosen (4) showed that calcium extrusion is driven by a proton motive force.Detailed studies with everted membrane vesicles of E. coli (5-7) demonstrated that the calcium extrusion occurs via a secondary Ca2+/H+ exchange catalyzed by a calcium/proton antiporter.A second system for calcium extrusion, a calcium phosphate symporter/proton antiporter, has also been iden-