Measurement of Proton-driven Antiport in Escherichia coli
大肠杆菌质子逆向运输蛋白活性的测定
发布: 2014年11月05日第4卷第21期 DOI: 10.21769/BioProtoc.1278 浏览次数: 9126
评审: Aksiniya AsenovaKanika GeraAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2014
Abstract
Secondary active transport of substrates across the inner membrane is vital to the bacterial cell. Of the secondary active transporter families, the ubiquitous major facilitator superfamily (MFS) is the largest and most functionally diverse (Reddy et al., 2012). Recently, it was reported that the MFS multidrug efflux protein MdtM from Escherichia coli (E. coli) functions physiologically in protection of bacterial cells against bile salts (Paul et al., 2014). The MdtM transporter imparts bile salt resistance to the bacterial cell by coupling the exchange of external protons (H+) to the efflux of bile salts from the cell interior via an antiport reaction. This protocol describes, using fluorometry, how to detect the bile salt/H+ antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆pH. This method exploits the changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the pH-sensitive dye acridine orange in response to changes in [H+] in the vesicular lumen. Due to low levels of endogenous transporter expression that would normally make the contribution of individual transporters such as MdtM to proton-driven antiport difficult to detect, the method typically necessitates that the transporter of interest be overexpressed from a multicopy plasmid. Although the first section of the protocol described here is very specific to the overexpression of MdtM from the pBAD/Myc-His A expression vector, the protocol describing the subsequent measurement of bile salt efflux by MdtM can be readily adapted for measurement of antiport of other substrates by any other antiporter that exchanges protons for countersubstrate.
Materials and Reagents
- pBAD/Myc-His A expression vector (Life Technologies, catalog number: V440-01 )
- L-(+)-arabinose (Sigma-Aldrich, catalog number: A3256 )
- Carbenicillin (Carbenicillin Direct)
- Agar (Sigma-Aldrich, catalog number: A1296 )
- Tryptone (Fluka, catalog number: T7293 )
- Yeast extract (Fluka, catalog number: 92114 )
- Potassium chloride (Thermo Fisher Scientific, catalog number: BP366 )
- Escherichia coli (E. coli) TO114 (gift of Prof. Hiroshi Kobayashi, Chiba University, Japan)
- Acridine orange hemi (zinc chloride) salt (Sigma-Aldrich, catalog number: A6014 )
- BisTris propane (BTP) (Sigma-Aldrich, catalog number: B6755 )
- Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
- Deoxyribonuclease I (DNase) from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
- Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
- Sodium DL-lactate solution 50% aqueous (VWR International, catalog number: 27927.298 )
- Magnesium sulphate heptahydrate (MgSO4.7H2O) (Thermo Fisher Scientific, catalog number: M/1000/60 )
- Sodium cholate hydrate (Sigma-Aldrich, catalog number: C1254 )
- Absolute ethanol (Thermo Fisher Scientific, catalog number: E/0650DF/17 )
- High purity (18 MΩ) Millipore or AnalR water
- Choline chloride (Sigma-Aldrich, catalog number: C1879 )
- Sucrose (Sigma-Aldrich, catalog number: 84097 )
- DL-dithiothreitol(Sigma-Aldrich, catalog number: 43815 )
- TRIZMA base (Sigma-Aldrich, catalog number: T1503 )
- 32% hydrochloric acid (VWR International, catalog number: 20254.321 )
- LBK agar (see Recipes)
- LBK liquid medium (see Recipes)
- Tris/choline/dithiothreitol/sucrose (TCDS) buffer (see Recipes)
- Transport assay buffer (see Recipes)
- 200 mM sodium DL-lactate solution (see Recipes)
Equipment
- Temperature-controlled shaking incubator for bacterial growth
- Petri dishes for bacterial colony growth
- 100 ml conical flasks
- 250 ml conical flasks (x2)
- 5,000 ml conical flasks (x2)
- Large ice bucket
- Refrigerated, large capacity centrifuge and rotor for harvesting bacterial cells
- 1,000 ml or 500 ml centrifuge pots and lids
- Benchtop vortexer
- 100 ml beaker and stir bar to fit
- Magnetic stirrer
- 25 ml disposable plastic pipettes
- Selection of single channel pipettes (1,000 µl, 100 µl, 20 µl, 10 µl)
- Pipette tips for above
- Refrigerated centrifuge and rotor capable of spinning ~50 ml tubes at 18k x g
- Refrigerated ultracentrifuge, rotor and polycarbonate ultracentrifuge tubes capable of handling ~30 - 50 ml volumes
- French Press (Thermo Fisher Scientific, catalogue number: FA-078 )
- Standard pressure cell (40 kpsi; 35 ml capacity) (Thermo Fisher Scientific, catalog number: FA-031 )
- 50 ml syringes (for filtering solutions)
- 0.22 µm sterile filters to fit 50 ml syringe
- 1 L and 500 ml Duran bottles with lids
- 1.5 ml Eppendorf tubes
- Medical wipes (Kimwipes®)
- Parafilm
- UV/vis spectrophotometer and 10 mm pathlength quartz cuvette
- 10 x 4 mm, 1,400 µl volume quartz cuvette for fluorescence spectroscopy (Hellma, catalog number: 104F-QG )
- Small magnetic stir bar to fit inside quartz cuvette
- Fluorometer e.g. Fluoromax-4 (Horiba) capable of performing time-based acquisition measurements and fitted with a temperature controlled cuvette holder and stirrer
- Source of compressed air (either aerosol can or fixed supply) for drying cuvette
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Holdsworth, S. R. and Law, C. J. (2014). Measurement of Proton-driven Antiport in Escherichia coli. Bio-protocol 4(21): e1278. DOI: 10.21769/BioProtoc.1278.
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分类
微生物学 > 微生物生物化学 > 其它化合物
微生物学 > 微生物新陈代谢 > 营养运输
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