Bacterial Intracellular Sodium Ion Measurement using CoroNa Green
使用CoroNa绿测定细菌细胞内钠离子
发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2092 浏览次数: 9744
评审: Arsalan DaudiKanika GeraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2016
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 (细菌)
Bacterial Flagellum (细菌鞭毛)
FlhA (FlhA)
Fluorescence microscopy (荧光显微镜检查)
Proton motive force (质子动力)
Sodium ion channel (钠离子通道)
PomAB complex (PomAB复合物)
Type III protein export (III型蛋白输出)
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
- 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 ProbesTM, 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 )
- 100x oil immersion objective lens (Olympus, model: UPLSAPO100XO , NA 1.4)
- sCMOS camera (Andor Technology, model: Zyla4.2 )
- Mercury light source system (Olympus, model: U-HGLGPS )
- 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
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- 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.
- 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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分类
微生物学 > 微生物细胞生物学 > 细胞活力
微生物学 > 微生物细胞生物学 > 基于细胞的分析方法
生物化学 > 其它化合物 > 离子
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