Probing the transmembrane potential of bacterial cells by voltage-sensitive dyes

Fluorescent dyes have been widely employed as optical indicators of the membrane potential difference in cells, isolated organelles and lipid vesicles that are too small to make microelectrode measurements feasible. We describe here the application of a carbocyanine dye, 3,3'-dipropylthiodicarbocyanine iodide [DiS-C-3-(5)], to monitor the transmembrane potential changes induced by a variation of the K, concentration for the cells of Escherichia (E.) coli and photosynthetic bacterium Rhodospirillum (R.) rubrum. The cells were first incubated in buffers containing DiS-C-3-(5) and K+ ions of various concentrations until the fluorescence intensity reached a constant value. Valinomycin was then added to the solution, which caused changes in the fluorescence intensity, depending on the K+ concentrations. The membrane potential is shown to have a linear relationship with the fluorescence intensity of DiS-C3-(5). The results demonstrate that the K+ concentrations inside intact cells are 4.6 mM and 5.3 mM for E. coli and R. rubrum, respectively. The diffusion potentials of K+ ions were determined using the Nernst equation over the range of -1.3 mV to 44 mV, corresponding to K+ concentrations of 5 mM -25 mM outside of the cells.