This study reports on the construction, calibration and use of recombinant cells of Rhodobacter capsulatus expressing the
luciferase gene of the North American firefly Photinus pyralis to detect, by bioluminescence, variations of endogenous
ATP levels under various physiological conditions. We show that the
antibiotic polymyxin B allows
luciferin to rapidly move into cell cytosol, but does not make external
ATP freely accessible to intracellular
luciferase. Notably, in
toluene:
ethanol-permeabilized cells, the apparent K(mATP) for
luciferase (50 microM) is similar to that measured in soluble cell fractions. This finding limits the applicability of the
firefly luciferase for monitoring intracellular maximal
ATP concentration because dark/aerobic-grown recombinant cells of Rba. capsulatus contain approximately 1.3-2.6+/-0.5 mM
ATP. Therefore, the effects of chemical and physical factors such as
oxygen, light,
carbonyl cyanide m-chlorophenyl hydrazone and
antimycin A on
ATP synthesis were examined in cells subjected to different
starvation periods to reduce the endogenous
ATP pool below the
luciferase ATP saturation level (< or =0.2 mM). We conclude that the amount of endogenous
ATP generated by light is maximal in the presence of
oxygen, which is required to optimize the membrane redox poise.