Materials and Reagents

1.5 ml Eppendorf tubes
Aluminum foil
Slide
24 x 32 mm coverslip (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C024321 )
18 x 18 mm coverslip (thickness: 0.12-0.17 mm) (Matsunami Glass, catalog number: C018181 )
Double-sided tape (NICHIBAN, catalog number: NW-5 )
Pipette tips
Filter paper
E. coli BL21(DE3) cell (Novagen)
pBAD24 expression vector (Guzman et al., 1995)
pNH319 (pBAD24/ N-His-FLAG-FlhA) (Minamino et al., 2016)
pBAD-PomΔplug (pBAD24/ PomA + PomB[∆41-120]) (Minamino et al., 2016)
Ampicillin sodium (Wako Pure Chemical Industries, catalog number: 014-23302 )
L-arabinose (Wako Pure Chemical Industries, catalog number: 010-04582 )
CoroNa Green-AM (Thermo Fisher Scientific, Molecular Probes^TM, catalog number: C36676 )
Ethylenediamine-N, N, N', N'-tetraacetic acid, dipotassium salt, dihydrate (EDTA·2K) (Dojindo, catalog number: 340-01511 )
Sodium chloride (Wako Pure Chemical Industries, catalog number: 192-13925 )
Gramicidin (Thermo Fisher Scientific, catalog number: G6888 )
Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
Bacto tryptone (BD, catalog number: 211705 )
Potassium dihydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-22635 )
Dipotassium hydrogenphosphate (Wako Pure Chemical Industries, catalog number: 164-04295 )
T-broth (see Recipes)

Equipment

haking incubator (30 °C, at 200 rpm) (TAITEC, model: BR-40LF )
Centrifuge (able to hold 1.5 ml tube, spin at 6,000 x g) (TOMY SEIKO, model: MX-305 )
Tube rotator (able to hold 1.5 ml tubes, rotate at 5 rpm) (WAKENBTECH, model: WKN-2210 )
Single channel pipettes (1,000 µl, 100 µl) (Gilson, model: P-1000 , P-100 )
Spectrophotometer (able to measure OD₆₀₀) (Shimadzu, model: UV-1800 )
Inverted fluorescence microscope (Olympus, model: IX-73 )
1. 100x oil immersion objective lens (Olympus, model: UPLSAPO100XO , NA 1.4)
2. sCMOS camera (Andor Technology, model: Zyla4.2 )
3. Mercury light source system (Olympus, model: U-HGLGPS )
4. Fluorescence mirror unit (Olympus, model: U-FGFP [Excitation BP 460-480; Emission BP 495-540])

Software

Image J (National Institutes of Health, https://imagej.nih.gov/ij/)
KaleidaGraph (Synergy Software)
Microsoft Excel (Microsoft)

Procedure

Note: Carry out procedures at ca. 23 °C unless otherwise specified.

Transform competent cells of the E. coli BL21(DE3) strain, which are treated with calcium chloride method, with a pBAD24-based plasmid encoding FlhA or the PomAB∆plug, which is a Na⁺ channel complex of the marine Vibrio flagellar motor (Morimoto and Minamino, 2014).
Grow the resulting transformants overnight in duplicate in 5 ml T-broth containing 100 µg/ml ampicillin at 30 °C.
Inoculate 50 µl of overnight culture of the E. coli BL21 (DE3) cells carrying the pBAD24-based plasmid into 5 ml of fresh T-broth containing 100 µg/ml ampicillin and incubate at 30 °C for 4 h with shaking at 150 rpm. (The cell density reaches an OD₆₀₀ of ca. 1.0.)
Add 0.2% arabinose (final concentration, w/v) to induce over-expression of FlhA or PomAB∆plug derived by a pBAD promotor on the pBAD24 vector and keep shaking for another 1 h.
After 1 h: Transfer 200 µl of each culture to a 1.5 ml Eppendorf tube and then collect the cells by centrifugation (6,000 x g, 2 min).
Suspend the cell pellet gently in 1.0 ml of fresh TB using a pipette (P-1000).
Centrifuge at 6,000 x g for 2 min.
Discard supernatant.
Repeat steps 6-8 (2 x).
Resuspend the cells in 100 µl of T-broth containing 40 µM CoroNa Green (Invitrogen) and 10 mM EDTA.
Cover the tubes with aluminum foil to keep them in the dark.
Rotate the tubes at 5 rpm for 60 min using a tube rotator.
Centrifuge at 6,000 x g for 2 min.
Discard supernatant.
Suspend the cell pellet in 1.0 ml of fresh T-broth.
Centrifuge at 6,000 x g for 2 min.
Discard supernatant.
Repeat steps 14-16 (3 x).
Resuspend the cells in 500 µl of fresh T-broth.
Make a tunnel slide by sandwiching double-sided tape between 24 x 32 mm coverslip (bottom side) and 18 x 18 mm coverslip (top side) (see Video 1).

Video 1. Preparation for a tunnel slide
Add the cell suspension to the tunnel slide and leave for 5-10 min to attach the cells onto the coverslip surface.
Wash out unbound cells by supplying 100 µl of fresh T-broth using a pipette (P-100). Absorb the excess amount of the medium with a piece of filter paper.
Put the sample on an inverted fluorescence microscope.
Select a 100x objective.
Capture fluorescence images of CoroNa Green using a mirror unit for GFP (U-FGFP, Ex: 460–480, Em: 495–540, Olympus) by an sCMOS camera every 100 msec.
Measure the fluorescence intensity of the cells stained with CoroNa Green in T-broth with 0 mM, 5 mM, 10 mM, 20 mM, 50 mM or 100 mM NaCl in the presence of 20 µM gramicidin and 5 µM CCCP.

Data analysis

Analyze fluorescent images by an image analysis software ImageJ (National Institutes of Health).

Apply a rectangular mask for the fluorescent image of the bacterial cell body of 8 x 8 pixels to the ROI (region of interest).
Subtract the total background intensity from each pixel value. The instrumental background intensity is defined as the mean pixel intensity within the ROI of a nearby cell-less region.
Measure the intensity of CoroNa Green from more than 100 cells at each condition.
Make the calibration curve from the data acquired at various external Na⁺ concentrations in the presence of 20 µM gramicidin and 5 µM CCCP, both of which equilibrate Na⁺ concentrations inside and outside E. coli cells (Figure 1).

Figure 1. Calibration curve for intracellular Na⁺ concentrations using CoroNa Green. The fluorescence intensity of the cells stained with CoroNa Green were measured in T-broth containing 20 µM gramicidin and 5 µM CCCP for 30 min at various Na⁺ concentrations (5 mM, 10 mM, 20 mM, 50 mM and 100 mM NaCl). The fluorescent intensities of intracellular CoroNa Green were plotted against external Na⁺ concentrations and then were fitted by the Hill equation (black line). Vertical bars indicate standard deviations. Because the staining efficiency with CoroNa Green is quite variable among E. coli cells, the standard deviations of fluorescence intensity become large at high Na⁺ concentrations.
Fit the calibration curve to the Hill equation by KaleidaGraph (Synergy Software).
Estimate the intracellular Na⁺ concentration through the fluorescent intensity of CoroNa Green of each cell using the calibration curve.
Calculate the average of the intracellular Na⁺ concentration and standard error of the mean from the data of more than 100 cells using Microsoft Excel (Microsoft).

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

To investigate the effect of extracellular sodium concentration, whole experiments are done using T-broth with or without 100 mM NaCl.
10 mM EDTA is required to allow CoroNa Green to be incorporated into the E. coli cells.
To avoid contamination of sodium, EDTA·2K must be used (Do not use EDTA·2Na).

Recipes

T-broth
1% Bacto tryptone, 10 mM potassium phosphate, pH 7.0 with or without 100 mM NaCl

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

Guzman, L. M., Belin, D., Carson, M. J., Beckwith. J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177(14): 4121-4130.
Lo, C. J., Leake, M. C., Berry, R. M. (2006). Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells. Biophys J 90(1): 357-365.
Minamino, T. (2014). Protein export through the bacterial flagellar type III export pathway. Biochim Biophys Acta 1843(8): 1642-1648.
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.
Morimoto, Y. V. and Minamino, T. (2014). Structure and function of the bi-directional bacterial flagellar motor. Biomolecules 4(1): 217-234.