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.

Figure 1. The developed single fluorescent protein (FP)-type ATP sensor (‘QUEEN') and its evaluation.

(a) Schematic illustration of the ATP sensing mechanism of QUEEN. (b) Schematic illustration of the genetic structure of QUEENs. (c, d) 400ex/494ex ratio of purified QUEEN versus ATP at 25°C (c) and 37°C (d). (e) Excitation spectra of QUEEN-2m at various ATP concentrations. (f, g) Response of QUEEN-2m to ATP concentration at different pH values. The buffer composition was the same as buffer C, except for different buffer reagents (MES for pH 6.2 and 7.0; HEPES for pH 7.0–7.8; Tricine for pH 8.2 and 8.6).

Figure 2. The comparison of single FP-type and FRET-type ATP indicators in vivo.

(a) Response of the FRET-type AT3.10 sensor to ATP at 25°C in vitro. Measurement was performed in buffer C. K_d = 1.8 × 10⁻³ mM. (b) Time courses of in vivo QUEEN-7µ and AT3.10 signals in cells grown in continuous culture (25°C). (c) Response of FRET-type AT1.03^YEMK sensor to ATP at 37°C in vitro. Measurement was performed in buffer B. K_d = 1.3 mM. (d) In vivo AT1.03^YEMK (37°C) and QUEEN-2m (25°C) cell suspension measurement results compared to in vitro measurements. Cell suspension was treated by KCN of various concentrations to change the intracellular ATP concentrations. Different markers indicate the different concentration of KCN used. The horizontal axis is the intracellular ATP value quantified by luciferase assay. The solid black lines indicate the expected values from the purified ATeam/QUEEN measurements. The solid red line indicates the response of QUEEN-2m in vivo after correction. Error bars indicate standard errors (see Supplementary Methods).

Figure 3. Evaluation of the factors that possibly affect the precision of the measurement by QUEEN.

All the cells in this figure are grown in a continuous culture condition. (a–d) The level of noise included in the experimental system. QUEEN variants with constant signal under physiological conditions were used. (a, b) Representative 405ex/480ex ratio images. Bars = 5 µm. (c, d) The distribution of the ratio in each cell population. The distribution was fitted to a normal distribution. The noise level was calculated as SD/mean × 100. (a, c) QUEEN-7µ cells. (b, d) QUEEN-NA cells. (e–f) The pH distribution in the cell population measured by ratiometric pHluorin. (e) The relationship between the ratio and pH value, measured by collapsing the ΔpH between inside and outside of the cell. Error bar = standard deviation (SD). 25–87 cells were examined for each pH value. The data points were fitted to the Hill equation. At pH 8.0, the distribution was fitted to N(1.86, 0.089). (f) The distribution of 405ex/480ex ratio in cells expressing ratiometric pHluorin. The main peak was fitted to a normal distribution N(1.90, 0.117). In the deconvolution of the noise, the SD of the experimental noise was assumed to be 0.089 (See Supplementary Methods).

Figure 4. ATP measurement in individual cells by using QUEEN-2m.

(a) A representative ratio image of cells expressing QUEEN-2m grown in continuous culture. Bar = 5 µm. (b) Distribution of 405ex/480ex ratio of QUEEN-2m cells in continuous culture in the steady state. (c) The raw (black) and corrected (red) response of 405ex/480ex ratio of purified QUEEN-2m to ATP concentration. Buffer C was used for the measurement. The corrected data show the response of QUEEN-2m in vivo. (d) Distribution of the ATP concentration. Error bars represent the standard deviation (SD) for each column calculated by bootstrapping. Note that 8 out of 216 cells in (b) showed smaller 405ex/480ex value than the ratio value at [ATP] = 0 mM in (c), and thereby assumed to contain 0 mM ATP in (d).