Application of Bioluminescent Methods to Study the Effect of the Membrane-active Antibiotic Colistin on Bacterial Cells.

Abstract

For the first time, recombinant Escherichia coli cells expressing thermostable Luciola mingrelica firefly luciferase were used to study the effect of the membrane-active antibiotic colistin on live cells. Simple, fast, and highly sensitive bioluminescent methods were developed for measurement of luciferase activity and ATP concentration inside and outside E.coli cells incubated in a nutrient medium, or in saline. Luciferase proved to be an informative protein marker for detecting the irreversible changes in cell membrane permeability. The study of kinetics of intra- and extracellular ATP concentration at different concentrations of colistin showed that the rate of decrease in intracellular ATP concentration significantly exceeded the rate of accumulation of extracellular ATP concentration. This fact could not be explained only by the release of ATP from the cell with an increase in the permeability of the outer cell membrane under the action of colistin. The loss of a significant part of intracellular ATP in presence of the colistin is probably due to a decrease in the activity of the respiratory chain enzymes and ATP synthase which operate in the cytoplasmic cell membrane, which leads to a decrease in the rate of ATP synthesis or even to its halt.