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Bacterial Intracellular Sodium Ion Measurement using CoroNa Green   

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Abstract

The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H+ and Na+ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na+ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na+ concentration quantitatively.

Keywords: Bacteria, Bacterial Flagellum, FlhA, Fluorescence microscopy, Proton motive force, Sodium ion channel, PomAB complex, Type III protein export

Background

Measurements of intracellular Na+ concentrations by fluorescence imaging techniques are able to be more accurately and quantitatively performed at single cell levels, because background noise of each cell can be removed by image analysis procedures. Lo et al. have established a protocol for measurement of the cytoplasmic Na+ concentrations in living E. coli cells using a sodium-sensitive fluorescent dye, Sodium Green and have shown that the cytoplasmic Na+ concentration maintains around 10 mM in E. coli over a wide range of 0 to 100 mM of the external Na+ concentrations (Lo et al., 2006). Because CoroNa Green, which is a sodium-sensitive fluorescent dye too, shows much higher cell permeability than Sodium Green, we have developed a CoroNa Green-based protocol to measure the intracellular Na+ concentrations in E. coli. (Minamino et al., 2016). This protocol allows us to quite easily and reproducibly measure the intracellular Na+ concentration of E. coli cells overexpressing FlhA or PomAB complex, both of which have the Na+ channel activity.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Morimoto, Y. V., Namba, K. and Minamino, T. (2017). Bacterial Intracellular Sodium Ion Measurement using CoroNa Green. Bio-protocol 7(1): e2092. DOI: 10.21769/BioProtoc.2092.
  2. Minamino, T., Morimoto, Y. V., Hara, N., Aldridge, P. D. and Namba, K. (2016). The bacterial flagellar type III export gate complex is a dual fuel engine that can use both H+ and Na+ for flagellar protein export. PLoS Pathog 12(3): e1005495
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