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.
Materials and Reagents
Equipment
Software
Procedure
Note: Carry out procedures at ca. 23 °C unless otherwise specified.
Data analysis
Analyze fluorescent images by an image analysis software ImageJ (National Institutes of Health).
Representative data
Figure 2. Measurement of intracellular Na+ concentrations using CoroNa Green. Effect of overexpression of FlhA or PomAB∆plug on intracellular Na+ concentrations in E. coli cells. Intracellular Na+ concentrations were measured with CoroNa Green in the presence and absence of 100 mM NaCl at an external pH of 7.0. The E. coli BL21(DE3) strain was transformed with pBAD24 (Vector, V), pNH319 (FlhA) or pBAD-Pom∆plug (PomAB∆plug). For each transformant, 200 cells were measured. Vertical bars indicate standard errors. (Modified from Minamino et al., 2016)
Notes
Recipes
Acknowledgments
This protocol was modified from a previous work (Lo et al., 2006). This research has been supported in part by JSPS KAKENHI Grant Numbers JP15K14498 and JP15H05593 to YVM, JP21227006 and JP25000013 to KN and JP26293097 to TM and MEXT KAKENHI Grant Numbers JP26115720 and JP15H01335 to YVM and JP23115008, JP24117004, JP25121718 and JP15H01640 to TM.
References
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