Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging

Abstract

Recent advances in quantitative single-cell analysis revealed large diversity in gene expression levels between individual cells, which could affect the physiology and/or fate of each cell. In contrast, for most metabolites, the concentrations were only measureable as ensemble averages of many cells. In living cells, adenosine triphosphate (ATP) is a critically important metabolite that powers many intracellular reactions. Quantitative measurement of the absolute ATP concentration in individual cells has not been achieved because of the lack of reliable methods. In this study, we developed a new genetically-encoded ratiometric fluorescent ATP indicator “QUEEN”, which is composed of a single circularly-permuted fluorescent protein and a bacterial ATP binding protein. Unlike previous FRET-based indicators, QUEEN was apparently insensitive to bacteria growth rate changes. Importantly, intracellular ATP concentrations of numbers of bacterial cells calculated from QUEEN fluorescence were almost equal to those from firefly luciferase assay. Thus, QUEEN is suitable for quantifying the absolute ATP concentration inside bacteria cells. Finally, we found that, even for a genetically-identical Escherichia coli cell population, absolute concentrations of intracellular ATP were significantly diverse between individual cells from the same culture, by imaging QUEEN signals from single cells.

Information Theoretical Study of Cross-Talk Mediated Signal Transduction in MAPK Pathways

“…In future, we plan to address the influence and physiological relevance of cross-talk in other network motifs. Our theoretical observations in the present work could be verified upon the quantification of phosphorylated kinase protein in a single cell using flow cytometry and time lapse microscopy [72][73][74]. These experimental approaches can be implemented to measure the amount of intra-cellular phosphorylated kinases by treating cells with external stimuli, fixing and permeabilizing cells with appropriate chemicals, and then staining with phospho-specific antibodies for different kinases.…”

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

“…However, whereas ADP changes only moderately, ATP decreases significantly in stationary phase, leading to a variation in the ATP/ ADP ratio from 10 to 3 depending on the growth conditions (28). Single-cell measurements in continuously growing bacterial cultures showed a distribution of ATP concentrations with a mean value of 1.5 mM (29). The concentration of inorganic phosphate in the bacterial cytosol lies at about 10 mM (30).…”

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

“…When performing the ATPase studies, we observed unexpected substrate inhibition of the ATPase activity. Interestingly, inhibition starts at about 2.5 mM ATP, which is in the range of its intracellular concentration (about 1 to 10 mM) (36). To confirm that the inhibition was not caused by the reagents used in the colorimetric ATPase assay, we incubated 10 M E. faecalis GdpP with 2 and 6 mM ATP at 28°C, respectively, and analyzed the products directly by reverse-phase HPLC (see Fig.…”