title: 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.
authors.affiliation: 1] Graduate School of Engineering, the University of Tokyo, Japan [2] Graduate School of Frontier Biosciences, Osaka University, Japan [3] CREST, JST, Japan [4] QBiC, RIKEN, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yaginuma
authors.affiliation: 1] Research Institute for Electronic Science, Hokkaido University, Japan [2] Faculty of Science, Shizuoka University.
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Kawai
authors.affiliation: 1] Graduate School of Engineering, the University of Tokyo, Japan [2] PRESTO, JST, Japan.
authors.forename: Kazuhito V
authors.initials: KV
authors.lastname: Tabata
authors.affiliation: Graduate School of Frontier Biosciences, Osaka University, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Tomiyama
authors.affiliation: Graduate School of Biostudies, Kyoto University, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kakizuka
authors.affiliation: Research Institute for Electronic Science, Hokkaido University, Japan.
authors.forename: Tamiki
authors.initials: T
authors.lastname: Komatsuzaki
authors.affiliation: 1] Graduate School of Engineering, the University of Tokyo, Japan [2] Graduate School of Frontier Biosciences, Osaka University, Japan [3] CREST, JST, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.affiliation: 1] Graduate School of Biostudies, Kyoto University, Japan [2] The Hakubi Center for Advanced Research, Kyoto University, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Imamura
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D003952:Diagnostic Imaging; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate