Changes in the ratio of adenyl nucleotides in Escherichia coli cells as an integral indicator of stress

The paper describes changes in the concentration of ATP and ADP in Escherichia coli cells subjected to sublethal stress. Sublethal stress did not lead to a decrease in the number of colony-forming units relative to the moment of the onset of stress exposure, but inhibited growth. Bacteria were cultured in minimal M9 medium supplemented with 0.4% glucose at 37˚C and stirring at 120 rpm. Stressors (50 g/L sodium chloride, 1 g/L acetic acid, 7% ethanol) were added at the exponential growth phase (OD600=0.3). Nucleotides were extracted with 0.4 N HClO4 on ice bath from bacterial cells previously washed from the medium, followed by neutralization with 2 M K2CO3. Quantitative analysis of nucleotides was performed by reversed-phase high-performance liquid chromatography with tetrabutylammonium hydrogen sulfate as an ion-pair reagent. Sodium chloride caused an increase in [ATP] and [ADP], ethanol caused an increase in [ATP] and an increase followed by a decrease in [ADP], and acetic acid caused a decrease followed by an increase in [ATP] and a decrease in [ADP] relative to the unstressed culture. Despite the dissimilar changes in the concentration of ATP and ADP in cells subjected to different types of stress, all of them were characterized by an increased level of the [ATP]/[ADP] compared to the control culture.