大肠杆菌中胆汁盐/H+反向协同运输蛋白的电性活力测定

Scarlett R. Holdsworth; Christopher J. Law

doi:10.21769/BioProtoc.1279

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Peer-reviewed

Measurement of the Electrogenicity of Bile Salt/H⁺ Antiport in Escherichia coli

大肠杆菌中胆汁盐/H+反向协同运输蛋白的电性活力测定

SH Scarlett R. Holdsworth

Christopher J. Law email

发布: 2014年11月05日第4卷第21期 DOI: 10.21769/BioProtoc.1279 浏览次数: 7493

评审: Aksiniya AsenovaKanika GeraAnonymous reviewer(s)

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Abstract

The transmembrane proton gradient (ΔpH) is the primary source of energy exploited by secondary active substrate/H⁺ antiporters to drive the electroneutral transport of substrates across the Escherichia coli (E. coli) inner membrane. Such electroneutral transport results in no net movement of charges across the membrane. The charge on the transported substrate and the stoichiometry of the exchange reaction, however, can result in an electrogenic reaction which is driven by both the ΔpH and the electrical (∆Ψ) components of the proton electrochemical gradient, resulting in a net movement of electrical charges across the membrane. We have shown that the major facilitator superfamily transporter MdtM - a multidrug efflux protein from E. coli that functions physiologically in protection of bacterial cells against bile salts - 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 electrogenic antiport reaction (Paul et al., 2014). This protocol describes, using fluorometry, how to detect electrogenic antiport activity of MdtM in inverted membrane vesicles of an antiporter-deficient strain of E. coli TO114 cells by measuring transmembrane ∆Ψ. The method exploits changes that occur in the intensity of the fluorescence signal (quenching and dequenching) of the probe Oxonol V in response to changes in membrane potential due to the MdtM-catalysed sodium cholate/H⁺ exchange reaction. The protocol can be adapted to detect activity of any secondary active antiporter that couples the electrogenic translocation of H⁺ across a biological membrane to that of its counter-substrate, and may be used to unmask otherwise camouflaged transport activities and physiological roles.

Keywords: Membrane transport (膜转运)

Exchange (交换)

Acridine orange (吖啶橙)

Fluorescence quenching (荧光猝灭)

Antiporter (逆向转运蛋白)

Materials and Reagents

E. coli TO114 (gift of Prof. Hiroshi Kobayashi, Chiba University, Japan)
E. coli BW25113 (National BioResource Project, Japan)
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 )
Bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol (Oxonol V) (Life Technologies, Molecular Probes^®, catalog number: O-266 )
Note: In our original work we used Oxonol V supplied by Cambridge Bioscience Ltd. However, this product no longer appears in their catalogue.
Nigericin (Sigma-Aldrich, catalog number: N7143 )
Valinomycin (Sigma-Aldrich, catalog number: 94675 )
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, catalogue number: 27927.298 )
Magnesium sulphate heptahydrate (MgSO₄^.7H₂O) (Thermo Fisher Scientific, catalogue number: M/1000/60 )
Sodium cholate hydrate (Sigma-Aldrich, catalog number: C1254 )
Sodium D-gluconate (Sigma-Aldrich, catalog number: S2054 )
Potassium D-gluconate (Sigma-Aldrich, catalog number: G4500 )
Absolute ethanol (Thermo Fisher Scientific, catalogue number: E/0650DF/17 )
High purity (18 MΩ) Millipore or AnalR water
D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
Sucrose (Sigma-Aldrich, catalog number: 84097 )
DL-dithiothreitol(Sigma-Aldrich, catalog number: 43815 )
TRIZMA base (Sigma-Aldrich, catalog number: T1503 )
Sulphuric acid (Sigma-Aldrich, catalog number: 320501 )
LBK agar (see Recipes)
LBK liquid medium (see Recipes)
Tris/sorbitol/dithiothreitol/sucrose (TSDS) buffer (see Recipes)
Transport assay buffer (see Recipes)
2 M sodium D-gluconate stock solution (see Recipes)
2 M potassium D-gluconate stock solution (see Recipes)

Equipment

Temperature-controlled shaking incubator for bacterial growth
Large ice bucket
Petri dishes for bacterial colony growth
100 ml conical flasks
250 ml conical flasks (x2)
5,000 ml conical flask
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 Electron Corp, catalogue number: FA-078 )
Standard pressure cell (40 kpsi; 35 ml capacity) (Thermo Electron Corp, catalogue 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

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Holdsworth, S. R. and Law, C. J. (2014). Measurement of the Electrogenicity of Bile Salt/H⁺ Antiport in Escherichia coli. Bio-protocol 4(21): e1279. DOI: 10.21769/BioProtoc.1279.