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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   

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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

  1. E. coli TO114 (gift of Prof. Hiroshi Kobayashi, Chiba University, Japan)
  2. E. coli BW25113 (National BioResource Project, Japan)
  3. pBAD/Myc-His A expression vector (Life Technologies, catalog number: V440-01 )
  4. L-(+)-arabinose (Sigma-Aldrich, catalog number: A3256 )
  5. Carbenicillin (Carbenicillin Direct)
  6. Agar (Sigma-Aldrich, catalog number: A1296 )
  7. Tryptone (Fluka, catalog number: T7293 )
  8. Yeast extract (Fluka, catalog number: 92114)
  9. Potassium chloride (Thermo Fisher Scientific, catalog number: BP366 )
  10. 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.
  11. Nigericin (Sigma-Aldrich, catalog number: N7143 )
  12. Valinomycin (Sigma-Aldrich, catalog number: 94675 )
  13. BisTris propane (BTP) (Sigma-Aldrich, catalog number: B6755 )
  14. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  15. Deoxyribonuclease I (DNase) from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  16. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich, catalog number: C2759 )
  17. Sodium DL-lactate solution 50% aqueous (VWR International, catalogue number: 27927.298 )
  18. Magnesium sulphate heptahydrate (MgSO4.7H2O) (Thermo Fisher Scientific, catalogue number: M/1000/60 )
  19. Sodium cholate hydrate (Sigma-Aldrich, catalog number: C1254 )
  20. Sodium D-gluconate (Sigma-Aldrich, catalog number: S2054 )
  21. Potassium D-gluconate (Sigma-Aldrich, catalog number: G4500 )
  22. Absolute ethanol (Thermo Fisher Scientific, catalogue number: E/0650DF/17 )
  23. High purity (18 MΩ) Millipore or AnalR water
  24. D-sorbitol (Sigma-Aldrich, catalog number: S1876 )
  25. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  26. DL-dithiothreitol (Sigma-Aldrich, catalog number: 43815 )
  27. TRIZMA base (Sigma-Aldrich, catalog number: T1503 )
  28. Sulphuric acid (Sigma-Aldrich, catalog number: 320501 )
  29. LBK agar (see Recipes)
  30. LBK liquid medium (see Recipes)
  31. Tris/sorbitol/dithiothreitol/sucrose (TSDS) buffer (see Recipes)
  32. Transport assay buffer (see Recipes)
  33. 2 M sodium D-gluconate stock solution (see Recipes)
  34. 2 M potassium D-gluconate stock solution (see Recipes)

Equipment

  1. Temperature-controlled shaking incubator for bacterial growth
  2. Large ice bucket
  3. Petri dishes for bacterial colony growth
  4. 100 ml conical flasks
  5. 250 ml conical flasks (x2)
  6. 5,000 ml conical flask
  7. Refrigerated, large capacity centrifuge and rotor for harvesting bacterial cells
  8. 1,000 ml or 500 ml centrifuge pots and lids
  9. Benchtop vortexer
  10. 100 ml beaker and stir bar to fit
  11. Magnetic stirrer
  12. 25 ml disposable plastic pipettes
  13. Selection of single channel pipettes (1,000 µl, 100 µl, 20 µl, 10 µl)
  14. Pipette tips for above
  15. Refrigerated centrifuge and rotor capable of spinning ~50 ml tubes at 18k x g.
  16. Refrigerated ultracentrifuge, rotor and polycarbonate ultracentrifuge tubes capable of handling ~30 - 50 ml volumes
  17. French Press (Thermo Electron Corp, catalogue number: FA-078 )
  18. Standard pressure cell (40 kpsi; 35 ml capacity) (Thermo Electron Corp, catalogue number: FA-031 )
  19. 50 ml syringes (for filtering solutions)
  20. 0.22 µm sterile filters to fit 50 ml syringe
  21. 1 L and 500 ml Duran bottles with lids
  22. 1.5 ml Eppendorf tubes
  23. Medical wipes (Kimwipes®)
  24. Parafilm
  25. UV/vis spectrophotometer and 10 mm pathlength quartz cuvette
  26. 10 x 4 mm, 1,400 µl volume quartz cuvette for fluorescence spectroscopy (Hellma, catalog number: 104F-QG )
  27. Small magnetic stir bar to fit inside quartz cuvette
  28. Fluorometer e.g. Fluoromax-4 (Horiba) capable of performing time-based acquisition measurements and fitted with a temperature controlled cuvette holder and stirrer
  29. Source of compressed air (either aerosol can or fixed supply) for drying cuvette

Procedure

  1. Growth and harvesting of bacterial cultures and preparation of inverted membrane vesicles
    1. Due to typically low levels of endogenous transporter expression, this method necessitates that the transporter of interest be overexpressed from a multicopy plasmid. We therefore begin the protocol with a description of the conditions used specifically for overexpression of MdtM from the pBAD/Myc-His A expression vector; these conditions (for example, the vector type, inducer concentration, and growth temperature) will vary depending upon the transporter that requires to be overexpressed. Furthermore, although the protocol described here pertains to measurement of electrogenic bile salt (sodium cholate) transport by MdtM specifically, it can be readily adapted for measurement of the electrogenicity of transport by other secondary active antiporters. Typically, a 1 L culture of bacterial cells will provide sufficient material for these experiments.
    2. Perform a fresh transformation with the pBAD/Myc-His A plasmid DNA encoding wild type or mutant transporter (or your vector of choice encoding your transporter of interest) into chemically competent E. coli TO114 cells. Plate the cells onto Luria Bertani Potassium (LBK) agar containing 100 μg/ml carbenicillin for selection. LBK agar must be used instead of regular Luria Bertani (LB) agar because the latter contains NaCl and the TO114 strain is sensitive to this salt due to deletion of the chromosomally encoded Na+/H+ antiporter NhaA. Incubate the plate overnight (~15 to ~18 h) at 37 °C.
    3. Pick a few single colonies from the agar plate and use to inoculate 2 x 250 ml conical flasks, each containing 100 ml of LBK liquid medium supplemented with 100 μg/ml carbenicillin. Grow the cultures overnight (~15 h) at 30 °C with 250 rpm shaking in a temperature-controlled shaking incubator. Measure the optical density at 600 nm (OD600) of the culture using a spectrophotometer; it should be between 3.0-3.3. If the OD600 is less than 3.0, then incubate for a further 0.5-1.0 h and re-measure. If the OD600 is greater than 3.3, the cultures have overgrown and fresh cultures will need to be prepared.
    4. Inoculate a 5 L conical flask containing 1,000 ml of LBK liquid medium supplemented with 100 μg/ml carbenicillin with 15 ml of overnight culture. Incubate at 32 °C with 270 rpm shaking for ~2.5 hrs in a temperature-controlled shaking incubator. The OD600 should be ~0.6. Drop the temperature to 25 °C and grow with 270 rpm shaking until the OD600 is 1.0. This usually takes between 0.5 to 1.0 h.
    5. Induce overexpression of MdtM by addition of 0.1% w/v L-(+)-arabinose [5 ml of 20% w/v L-(+)-arabinose] to the culture. After addition of arabinose, grow the cells for a further 1.5 h at 25 °C with 270 rpm shaking prior to harvesting.
    6. Transfer the E. coli TO114 cells that contain overexpressed transporter into 500 ml or 1,000 ml capacity centrifuge pots that have been pre-chilled on ice for at least 15 min. Pre-cool the centrifuge to 4 °C and harvest cells by centrifugation at 5,000 x g for 20 min.
    7. Decant the supernatant and wash the pelleted cells by resuspending (either by gentle vortexing using a benchtop vortexer or by gentle aspiration using a 25 ml sterile plastic pipette) in chilled (4 °C) TSDS buffer. Use 30 ml of TSDS buffer for each one litre of cell culture that was pelleted. Maintain the cells on ice during this procedure. Harvest the washed cells by centrifugation as described in step A1 (above) and repeat the washing procedure. Resuspend the resultant cell pellet in 30 ml of chilled TSDS buffer containing 2 mM PMSF (Which should be made up as a 100 mM stock in ethanol and stored at -20 °C; ensure the solution is thawed thoroughly and vortexed vigorously before use.) and 5 µM DNase and maintain the mixture on ice.
    8. Decant the resuspended cells into a 100 ml beaker containing a stir bar, place on a magnetic stirrer and stir in a cold room at 4 °C for 20 min. Alternatively, place the beaker on ice in an ice bucket, place on the magnetic stirrer and stir for 20 min.
    9. The method for production of inverted vesicles relies on a combination of the fluid shear forces and decompression created as the cell mixture passes through the needle valve of a French pressure cell. Generate inverted membrane vesicles by a single passage of the resuspended cell mixture through a French pressure cell at a minimum of 4,000 psi. If the pressure is too low, inverted vesicles will not be formed. The pressure cell should be chilled on ice for ~30 min prior to use. The resulting inverted vesicle mixture should be collected in a 100 ml conical flask kept on ice.
    10. Decant the mixture into a pre-chilled ~50 ml centrifuge tube and remove any unbroken cells and cell debris by centrifugation at 18,000 x g for 10 min at 4 °C. Carefully decant the supernatant containing the cell membrane vesicles (do not disturb the pellet that contains unbroken cells and cell debris) into a pre-chilled 30-50 ml volume polycarbonate ultracentrifuge tube on ice.
    11. Harvest the inverted vesicles by ultracentrifugation at 100,000 x g for 1 h at 4 °C. Carefully decant the supernatant and retain the pellet. Place the ultracentrifuge tube containing the pelleted vesicles on ice.
    12. Thoroughly resuspend the inverted vesicle pellet in 1 ml of ice-cold TSDS buffer by gentle aspiration using a 1,000 µl pipette. Transfer the resuspended vesicles to a pre-chilled 1.5 ml Eppendorf tube on ice for use in the transport assay. In our experience, vesicles stored on ice are stable for several hours.
    13. Quantify the total membrane protein content of the inverted vesicles by UV absorbance spectroscopy at 280 nm. Blank the spectrophotometer using a 10 mm pathlength quartz cuvette containing 1,000 µl of TSDS buffer. The buffer should be at room temperature to prevent frosting of the cuvette faces. Clean the faces of the cuvette using a fresh paper wipe prior to measurement. Once the spectrophotometer is blanked, remove 5 µl of buffer from the cuvette using a 10 µl pipette and replace with 5 µl of vesicles. Cover the opening of the cuvette with a square of Parafilm and invert the cuvette a few times to ensure a homogeneous distribution of vesicles. Record the absorbance of the vesicle mixture at 280 nm and calculate the total membrane protein concentration assuming that an A280 of 1.0 is equivalent to a protein concentration of 1.0 mg/ml. Remember to multiply the 280 nm absorbance value you obtain by a factor of 200 to calculate the concentration of the undiluted vesicle mixture in mg/ml.

  2. Measurement of electrogenic transport
    1. In this section we describe the set-up parameters for a Fluoromax-4 fluorometer. These parameters, however, can form the basis for the set-up of other fluorometers. Once the instrument is switched on and the software booted up, set the temperature of the cuvette holder to 25 °C. Open the instrument software and select for time-based data acquisition with excitation and emission wavelengths of 599 nm and 634 nm, respectively. Set the excitation and emission slit widths to 10 nm and 20 nm, respectively.
    2. Add an aliquot of inverted vesicles (which should be maintained on ice in TSDS buffer) to room temperature transport assay buffer containing the Oxonol V probe in a 10 mm x 4 mm quartz cuvette to a final concentration of 0.5 mg/ml membrane protein in a total volume of 1,500 µl. The longest pathlength of the cuvette should face the excitation light source. Place a small magnetic flea into the cuvette and stir the contents gently. Allow the vesicles and assay buffer to equilibrate for ~200 sec.
    3. Start recording the fluorescence emission.
    4. After approximately 50 sec, add 15 µl of 200 mM stock solution of sodium DL-lactate to the cuvette contents to give a final sodium DL-lactate concentration of ~2.0 mM. Addition of lactate initiates respiration-dependent generation of ∆Ψ that causes a dequench of the Oxonol V fluorescence signal (see Figure 1).
    5. If the fluorescence signal does not quench, or enhances, the vesicles have either not maintained integrity or are not inverted and their preparation needs to be repeated.
    6. Following the establishment of ΔΨ, monitor the Oxonol V fluorescence dequench for a further ~150 sec until it stabilises. Initiate MdtM-mediated antiport by adding substrate (in this case the bile salt sodium cholate) to the inverted vesicle mixture. We added 12.5 µl of 250 mM stock solution made up in high purity water to give a final concentration of sodium cholate in the cuvette of ~2.0 mM. For other substrates we suggest testing a range varying from 1 mM to 100 mM to establish the concentration that gives the best dequench signal. If the transport reaction is electrogenic there should be an immediate dequench (enhancement) of the Oxonol V fluorescence emission signal as the established membrane potential, ΔΨ, is consumed by the transport reaction (see Figure 1a).
    7. Record the fluorescence dequench signal for ~60 s to allow the antiport reaction to achieve a steady state (as observed by a plateauing of the fluorescence signal). Addition of the protonophore CCCP to a final concentration of ~100 µM (1.6 µl of a 100 mM stock made up in ethanol) in the assay mixture should be performed to abolish transport by collapsing both the membrane potential and ΔpH. Addition of CCCP should result in a further dequench of the fluorescence signal (see Figure 1a). Record the fluorescence signal for a further ~40 sec then terminate the acquisition and save the electronic data.
    8. Decant the cuvette contents into a suitable waste container and wash the cuvette thoroughly with ethanol then high-purity water. Dry the cuvette carefully with compressed air.
    9. As a further control, and to provide evidence that the inverted vesicles retain integrity and are therefore able to maintain an electrochemical potential across the membrane during the assay lifetime, the fluorescence response of Oxonol V upon addition of the ionophore nigericin (which at low concentrations selectively consumes ΔpH in the presence of potassium ions via electroneutral K+/H+ exchange) in place of substrate should be measured. For this experiment, inverted vesicles of TO114 cells containing the transporter of interest are incubated in assay buffer that contains 50 mM potassium D-gluconate. After addition of sodium DL-lactate to initiate respiration as described in step B3 above, 1.5 µl of 1 mM nigericin stock solution made up in ethanol (to give a final concentration of ~1 µM) is added to the cuvette. Concentrations of nigericin > 1 µM must be avoided to prevent electrogenic exchange that can occur under conditions of high nigericin concentrations and abolish both components of the electrochemical gradient. Record the fluorescence dequench signal for a further ~60 s then add the ionophore valinomycin (to selectively abolish ΔΨ) to a final concentration of ~5 μM by adding ~1.6 μl of 5 mM stock made up in ethanol (see Figure 1d). Record data for a further 40 sec prior to termination of the acquisition.

Representative data


Figure 1. Representative data illustrating typical changes in the fluorescence signal of the ΔΨ-sensitive fluorophore Oxonol V in response to MdtM-catalysed electrogenic transport in inverted membrane vesicles. Addition of lactate to energise vesicles results in generation of a respiratory ΔΨ, as evidenced by a rapid quench of the Oxonol V fluorescence signal. (a) Addition of 2.5 mM cholate to inverted vesicles generated from TO114 cells enriched with wild-type MdtM results in a partial depolarization of ΔΨ, represented as a dequenching of the Oxonol V fluorescence, as the ΔΨ was consumed by the MdtM-mediated bile salt/H+ transport reaction. (b) Addition of cholate to negative control vesicles generated from TO114 cells enriched with dysfunctional MdtM D22A results in a small but perceptible dequench arising from residual electrogenic Na+/H+ antiport activity of the mutant. (c) Positive control assay in which Na+ ions are added to inverted vesicles that contain a full complement of electrogenic antiporters in order to measure electrogenic Na+/H+ activity of NhaA transporter. (d) Response of Oxonol V fluorescence to addition of the nigericin. In the presence of K+ ions, this ionophore selectively dissipates ΔpH and converts it into ΔΨ, resulting in a further quench of the fluorescence signal. Valinomycin collapses the ΔΨ. In assays (a, b and c), addition of the protonophore CCCP at the time indicated resulted in almost complete dissipation of ΔΨ. The fluorescence intensity is measured in counts per second (cps). The fluorescence intensity you measure may differ depending upon how your instrument is set up.

Notes

  1. Resuspended inverted vesicles from step A8 can be transferred to tubes in aliquots of 25-100 µl, snap-frozen in liquid nitrogen and stored at -80 °C for subsequent use. Vesicles frozen in this way will retain their integrity for several months. However, if frozen vesicle stocks are used for the subsequent transport measurements, the vesicles must be thawed very slowly on ice prior to use to prevent their fracture.
  2. To ensure the suitability of the experimental conditions for detection of electrogenic antiport, a positive control should first be performed using inverted vesicles produced from E. coli cells e.g. strain BW25113 that contain a full complement of electrogenic antiporters. This control experiment should be performed by following the protocol below except that the transport assay buffer pH should be adjusted to pH 8.5 with H2SO4, and sodium gluconate (to a final concentration in the cuvette of ~100 mM by adding 80 µl of a 2 M stock of sodium gluconate) should be added in place of sodium cholate to specifically enable detection of electrogenic Na+/H+ exchange catalysed by the NhaA transporter (Figure 1c).
  3. To ensure reproducibility, the assays should be performed in triplicate on at least two separate preparations of inverted vesicles.
  4. As with all assays that rely on detection of fluorescence, robust controls must be in place to ensure that any detected transport activity can be attributed unambiguously to the protein of interest. In our experiments, we used inverted vesicles that overexpressed MdtM D22A, a dysfunctional point mutant of MdtM, as a negative control (see Figure 1b).
  5. If the method is to be used for detection of metal ion/H+ antiport activity, the use of inverted vesicles generated from the antiporter-deficient TO114 strain of E. coli is important because at least four other transporters (NhaA, NhaB, ChaA and MdfA) present in the bacterium can catalyse a monovalent metal cation/H+ exchange.
  6. Because chloride ions can depolarise the membrane potential, all buffer systems must be maintained as chloride-free. Therefore, pH of buffers should be adjusted using H2SO4 instead of HCl.
  7. Finally, if this protocol is used for comparison of antiport activities of wild type and mutant transporters, the amount of target protein present in the inverted vesicle membranes must be quantified (usually by immunodetection methods) to ensure that any measured differences in H+ uptake are due solely to differences in the activity of the transporters and not to differences in expression levels.

Recipes

  1. LBK agar (100 ml)
    1.0 g tryptone
    0.5 g yeast extract
    0.745 g KCl
    1.5 g agar
    Make up to 100 ml with high purity water then autoclave
    Add 100 μg/ml carbenicillin for selection when the solution is still liquid and warm to the touch. Add to Petri dishes under sterile conditions
  2. LBK liquid medium (1 L)
    10 g tryptone
    5 g yeast extract
    7.45 g KCl
    Make up to 1,000 ml with high purity water then autoclave
  3. Tris/sorbitol/dithiothreitol/sucrose (TSDS) buffer (1 L)
    Consisting of 10 mM Tris (pH 7.5), 280 mM sorbitol, 0.5 mM dithiothreitol and 250 mM sucrose
    1 M Tris (pH 7.5) 10 ml
    1 M sorbitol 280 ml
    1 M sucrose 250 ml
    Make up to 999.5 ml with high purity water then check pH and adjust if necessary with H2SO4 Sterile filter and store at 4 °C
    Add 0.5 ml dithiothreitol from frozen 1 M stocks immediately before use
  4. Transport assay buffer (100 ml)
    Consisting of 10 mM BisTris propane (pH 7.2) (or pH 8.5 for the initial control experiment), 5 mM MgSO4, 5 μM Oxonol V
    100 mM BisTris propane (pH 7.2 or pH 8.5) 10 ml
    2 M MgSO4 0.25 ml
    1 mM Oxonol V (made up in ethanol) 0.5 ml
    Make up to 100 ml with high purity water then sterile filter.
    For control experiments that use nigericin, potassium D-gluconate (2.5 ml of 2 M stock) to a final concentration of 50 mM should be added to the assay buffer
    Oxonol V is light sensitive so both the transport assay buffer and Oxonol V stocks should be stored either in amber bottles or in a container protected from light
  5. 2 M sodium D-gluconate stock solution (10 ml)
    4.36 g made up to 10 ml with high purity water
    Sterile filter and store at 4 °C
  6. 2 M potassium D-gluconate stock solution (10 ml)
    6.68 g made up to 10 ml with high purity water
    Sterile filter and store at 4 °C

Acknowledgements

This work was supported by BBSRC grant BB/K014226/1 (to CJL). The protocol described above is adapted from one reported previously (Resch et al., 2010).

References

  1. Paul, S., Alegre, K. O., Holdsworth, S. R., Rice, M., Brown, J. A., McVeigh, P., Kelly, S. M. and Law, C. J. (2014). A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress. Mol Microbiol 92(4): 872-884.
  2. Resch, C. T., Winogrodzki, J. L., Patterson, C. T., Lind, E. J., Quinn, M. J., Dibrov, P. and Hase, C. C. (2010). The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo. Biochemistry 49(11): 2520-2528.

简介

跨膜质子梯度(ΔpH)是由次级活性底物/H sup +反转录子开发的能量的主要来源,以驱动底物穿过大肠杆菌的电中性转运( >大肠杆菌)内膜。这种电中性转运导致电荷没有跨膜的净移动。然而,运输的底物上的电荷和交换反应的化学计量可以导致由ΔpH和质子电化学梯度的电(ΔΨ)分量驱动的电致反应,导致电移动的净移动电荷穿过膜。我们已经显示主要促进子超家族转运蛋白MdtM-来自E的多药物外排蛋白。在保护细菌细胞对抗胆汁盐中起到生理作用的大肠杆菌通过偶联外部质子(H + +)与胆汁盐的外流的交换而赋予细菌细胞对胆汁盐的抗性通过电致反应反应的细胞内部(Paul et al。,2014)。该协议使用荧光测定法描述了如何检测Ed的逆转子缺陷菌株的倒置膜囊泡中MdtM的电致逆转运蛋白活性。通过测量跨膜ΔΨ测定大肠杆菌 TO114细胞。该方法利用响应于由于MdtM催化的胆酸钠/H +交换反应引起的膜电位变化而发生在探针Oxonol V的荧光信号强度(淬灭和去淬灭)中发生的变化。该方案可以适于检测任何辅助活性反转运蛋白的活性,其将H sup +跨越生物膜的电致易位与其对应底物的电转运偶联,并且可以用于解偶联否则伪装的转运活性和生理作用。

关键字:膜转运, 交换, 吖啶橙, 荧光猝灭, 逆向转运蛋白

材料和试剂

  1. E。 大肠杆菌 TO114(日本千叶大学Kobayashi教授的礼物)
  2. E。 大肠杆菌BW25113(日本国家生物资源计划)
  3. pBAD/myc -His表达载体(Life Technologies,目录号:V440-01)
  4. L - (+) - 阿拉伯糖(Sigma-Aldrich,目录号:A3256)
  5. 羧苄青霉素(Carbenicillin Direct)
  6. 琼脂(Sigma-Aldrich,目录号:A1296)
  7. 胰蛋白胨(Fluka,目录号:T7293)
  8. 酵母提取物(Fluka,目录号:92114)
  9. 氯化钾(Thermo Fisher Scientific,目录号:BP366)
  10. 双 - (3-苯基-5-氧代异恶唑-4-基)五次甲基氧杂醇(Oxonol V)(Life Technologies,Molecular Probes ,目录号:O-266) 在我们原来的工作中,我们使用了由Cambridge Bioscience Ltd.提供的Oxonol V。
  11. 尼日利亚霉素(Sigma-Aldrich,目录号:N7143)
  12. 灭菌霉素(Sigma-Aldrich,目录号:94675)
  13. BisTris丙烷(BTP)(Sigma-Aldrich,目录号:B6755)
  14. 苯基甲磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  15. 来自牛胰腺的脱氧核糖核酸酶I(DNase)(Sigma-Aldrich,目录号:DN25)
  16. 羰基氰化物3-氯苯基腙(CCCP)(Sigma-Aldrich,目录号:C2759)
  17. DL-乳酸钠溶液50%水溶液(VWR International,目录号:27927.298)
  18. 硫酸镁七水合物(MgSO 4·7H 2 O 7H 2 O)(Thermo Fisher Scientific,目录号:M/1000/60)
  19. 胆酸钠水合物(Sigma-Aldrich,目录号:C1254)
  20. D-葡萄糖酸钠(Sigma-Aldrich,目录号:S2054)
  21. D-葡萄糖酸钾(Sigma-Aldrich,目录号:G4500)
  22. 无水乙醇(Thermo Fisher Scientific,目录号:E/0650DF/17)
  23. 高纯度(18MΩ)Millipore或AnalR水
  24. D-山梨醇(Sigma-Aldrich,目录号:S1876)
  25. 蔗糖(Sigma-Aldrich,目录号:84097)
  26. DL二硫苏糖醇(Sigma-Aldrich,目录号:43815)
  27. TRIZMA碱(Sigma-Aldrich,目录号:T1503)
  28. 硫酸(Sigma-Aldrich,目录号:320501)
  29. LBK琼脂(见配方)
  30. LBK液体介质(见配方)
  31. Tris /山梨醇/二硫苏糖醇/蔗糖(TSDS)缓冲液(参见配方)
  32. 运输测定缓冲液(参见配方)
  33. 2 M D-葡萄糖酸钠储备溶液(见配方)
  34. 2 M D-葡萄糖酸钾储备溶液(见配方)

设备

  1. 温控的细菌生长摇动培养箱
  2. 大冰桶
  3. 用于细菌菌落生长的培养皿
  4. 100ml锥形瓶
  5. 250ml锥形瓶(×2)
  6. 5,000 ml锥形瓶
  7. 冷冻,大容量离心机和转子收获细菌细胞
  8. 1,000 ml或500 ml离心机罐和盖
  9. 台式涡流器
  10. 100毫升烧杯和搅拌棒以适应
  11. 磁力搅拌器
  12. 25 ml一次性塑料移液器
  13. 选择单通道移液器(1,000μl,100μl,20μl,10μl)
  14. 以上的移液器提示
  15. 冷冻离心机和转子,能够以18k x g旋转〜50 ml管。
  16. 冷冻超速离心机,转子和聚碳酸酯超速离心管,能够处理〜30 - 50 ml的体积
  17. French Press(Thermo Electron Corp,目录号:FA-078)
  18. 标准压力室(40kpsi; 35ml容量)(Thermo Electron Corp,目录号:FA-031)
  19. 50 ml注射器(用于过滤溶液)
  20. 0.22μm无菌过滤器,适合50 ml注射器
  21. 1升和500毫升杜兰瓶盖子
  22. 1.5 ml Eppendorf管
  23. 医用湿巾(Kimwipes ®
  24. parafilm
  25. UV/vis分光光度计和10mm长度石英比色皿
  26. 10×4mm,用于荧光光谱的1400μl体积石英比色杯(Hellma,目录号:104F-QG)
  27. 小型磁力搅拌棒,以适应石英比色杯
  28. 例如能够进行基于时间的采集测量并装配有温度受控的试管架和搅拌器的Fluoromax-4(Horiba)。
  29. 用于干燥比色皿的压缩空气源(气溶胶罐或固定供应源)

程序

  1. 细菌培养物的生长和收获和倒置膜囊泡的制备
    1. 由于通常低水平的内源性转运蛋白表达,这 方法需要过量表达感兴趣的转运蛋白 从多拷贝质粒。因此,我们开始协议与a 描述专门用于过表达的条件 来自pBAD/emc -His A表达载体的MdtM;这些条件(for 例如,载体类型,诱导剂浓度和生长温度)  将根据需要的运输者而变化 过表达。此外,虽然这里描述的协议 属于电致胆汁盐(胆酸钠) 特别是MdtM的运输,它可以很容易地适应 测量其他次级运输的电活性 活性抗寄生虫。通常,1L细菌细胞培养物 为这些实验提供足够的材料
    2. 执行新鲜  用pBAD/Myc转化 - 编码野生型的质粒DNA 或突变运输者(或你的选择编码你的矢量 感兴趣的转运蛋白)转化为化学感受态E。大肠杆菌 TO114细胞。 将细胞铺板到含有100的Luria Bertani钾(LBK)琼脂上 μg/ml羧苄青霉素。必须使用LBK琼脂代替 常规的Luria Bertani(LB)琼脂,因为后者含有NaCl和  TO114菌株对该盐敏感,由于缺失 染色体编码的Na +/+/H + +反向转运体NhaA。孵育平板 在37℃过夜(〜15至约18小时)
    3. 从中选择几个单一的殖民地  琼脂板上并用于接种2×250ml锥形瓶 含有100ml补充有100μg/ml的LBK液体培养基 羧苄青霉素。在30℃下以250rpm生长培养物过夜(〜15h)  在温度控制的摇动培养箱中振荡。测量 使用a。在600nm处的光密度(OD <600) 分光光度计;它应该在3.0-3.3之间。如果OD <600> 较小 比3.0,然后再孵育0.5-1.0小时并重新测量。如果 OD 600大于3.3,培养物长满和新鲜 文化将需要准备
    4. 接种5升锥形瓶 其含有补充有100μg/ml的1,000ml LBK液体培养基 羧苄青霉素与15ml过夜培养物。在32°C孵育 在温度控制的振荡中摇动约2.5小时 孵化器。 OD <600> 应为〜0.6。将温度降至25°C和 以270rpm摇动生长直至OD 600为1.0。这通常需要 在0.5至1.0小时之间
    5. 通过加入诱导MdtM的过表达  0.1%w/v L - (+) - 阿拉伯糖[5ml 20%w/v L - (+) - 阿拉伯糖] 文化。加入阿拉伯糖后,将细胞再培养1.5小时  在25℃,270rpm摇动,然后收获
    6. 传输 E。大肠杆菌 TO114细胞,其含有过表达的转运蛋白至500ml 或已经在冰上预冷却的1,000ml容量的离心机罐 至少15分钟。将离心机预冷至4°C并收获细胞 通过在5,000xg离心20分钟。
    7. 滗析上清液   并通过重悬(通过温和涡旋)洗涤沉淀的细胞   使用台式涡旋器或通过使用25ml无菌的温和抽吸   塑料移液管)在冷冻(4℃)TSDS缓冲液中。 使用30毫升TSDS 缓冲液用于每一升沉淀的细胞培养物。 保持 在此过程中冰上的细胞。 收获洗过的细胞 离心,如步骤A1(上述)中所述,并重复洗涤 程序。 将所得细胞沉淀重悬在30ml冷冻的TSDS中 含有2mM PMSF的缓冲液(其应当制成100mM储备液 在乙醇中并储存于-20℃; 确保溶液解冻 彻底并在使用前剧烈涡旋)和5μMDNA酶和 将混合物保持在冰上
    8. 将重悬的细胞倒入a 100毫升含有搅拌棒的烧杯中,置于磁力搅拌器上 在冷室中在4℃下搅拌20分钟。 或者,放置烧杯 在冰桶中放置在磁力搅拌器上并搅拌20分钟 min。
    9. 用于生产倒囊泡的方法依赖于a 组合的流体剪切力和减压作用 细胞混合物通过弗氏压力室的针阀。 通过单次通过产生反向膜囊泡 重悬细胞混合物通过法式压力池至少 4,000psi。 如果压力太低,倒置的囊泡将不会 形成。 压力室应在冰上冷却〜30分钟 使用。 得到的倒囊泡混合物应收集在100℃   ml锥形瓶置于冰上
    10. 将混合物倒入a 预冷〜50 ml离心管,并取出所有未破碎的细胞 细胞碎片通过在4℃以18,000×g离心10分钟。 小心倾析含有细胞膜囊泡的上清液 (不要打扰含有未破坏的细胞和细胞的沉淀 碎片)倒入预冷的30-50ml体积的聚碳酸酯中 超速离心管在冰上。
    11. 收获倒置的囊泡 在100,000xg超速离心在4℃下1小时。 小心倾倒   上清液并保留沉淀。 放置超速离心管 包含在冰上的颗粒囊泡
    12. 彻底重悬   倒置的囊泡沉淀在1ml冰冷的TSDS缓冲液中 吸取使用1000微升移液器。 转移重悬的囊泡 到在冰上预冷的1.5ml Eppendorf管中用于运输 测定。 根据我们的经验,存储在冰上的囊泡几个是稳定的 小时。
    13. 量化总膜蛋白含量 通过在280nm的UV吸收光谱测定反相囊泡。空白 分光光度计使用10毫米长径石英比色杯 1000μlTSDS缓冲液。缓冲液应在室温至室温 防止反应杯面的结霜。清洁试管的表面 在测量前使用新鲜的纸巾。一旦 分光光度计为空白,从比色杯中取出5μl缓冲液 使用10μl移液管并用5μl的囊泡替换。封面 打开具有Parafilm正方形的比色皿并倒置比色皿a  几次以确保囊泡的均匀分布。记录 在280nm处的囊泡混合物的吸光度并计算总量 膜蛋白浓度,假设A 280为1.0 相当于蛋白质浓度为1.0mg/ml。记住乘法  你得到的280 nm吸光度值乘以因子200来计算 未稀释的囊泡混合物的浓度(mg/ml)。

  2. 电致运输的测量
    1. 在本节中,我们描述了Fluoromax-4的设置参数 荧光计。 然而,这些参数可以形成基础 设置其他荧光计。 一旦仪器开启,   软件启动,将试管架的温度设置为25°C。   打开仪器软件并选择基于时间的数据采集   激发和发射波长为599nm和634nm, 分别。 设置激发和发射缝隙宽度为10 nm和 20nm。
    2. 添加一等分的倒囊泡( 应在冰上保持在TSDS缓冲液中)至室温 运输测定缓冲液中含有Oxonol V探针,在10mm×4mm 石英比色皿至终浓度为0.5 mg/ml膜蛋白   总体积为1500μl。 比色皿的最长路径长度 应面向激发光源。 放置一个小磁蚤 进入比色杯并轻轻搅拌内容物。 允许囊泡和 测定缓冲液平衡〜200秒
    3. 开始记录荧光发射。
    4. 大约50秒后,加入15μl的200 mM储备液 DL-乳酸钠到比色皿内容物中,得到最终的钠 DL-乳酸盐浓度为〜2.0mM。 加入乳酸盐引发 呼吸依赖性的ΔΨ的产生导致的dequench Oxonol V荧光信号(见图1)
    5. 如果荧光 信号不淬灭或增强,囊泡没有 保持完整性或不被反转和他们的准备需要 重复。
    6. 在建立ΔΨ后,监测Oxonol  V荧光淬灭进一步〜150秒直到它稳定。 通过添加底物(在这种情况下,启动MdtM介导反运输 胆汁盐胆酸钠)至倒置的囊泡混合物。我们添加了12.5  μl的250mM储备溶液在高纯度水中制成 在比色皿中的胆酸钠的最终浓度为〜2.0mM。对于 其他底物我们建议测试范围从1 mM到100 mM不等 以建立给出最佳脱猝信号的浓度。如果 运输反应是电生的,应该有即时的 脱氧(增强)Oxonol V荧光发射信号 建立的膜电位,ΔΨ,被运输所消耗 反应(见图1a)。
    7. 记录荧光淬灭信号 约60秒,以允许反向反应达到稳定状态(如 通过荧光信号的平台观察)。加入 质子载体CCCP至〜100μM的最终浓度(1.6μl的100μM) mM储备在乙醇中制备)在测定混合物中进行  通过破坏膜电位和ΔpH来消除运输。 添加CCCP应导致荧光的进一步脱色  信号(参见图1a)。记录荧光信号进一步 〜40秒,然后终止采集并保存电子数据
    8. 将比色杯内容物倒入合适的废物容器中并洗涤 比色杯用乙醇,然后用高纯水彻底。干燥 用压缩空气小心地清洗试管
    9. 作为进一步的控制,和 以提供反转囊泡保持完整性的证据 因此能够维持跨越的电化学电势 膜在测定寿命期间,Oxonol V的荧光响应  加入离子载体尼日利亚菌素(其浓度低) 在存在钾离子通道时选择性地消耗ΔpH 应当测量代替衬底的电中性(阳极/阴极/阳极/阴极交换)。  对于这个实验,倒置的囊泡的TO114细胞含有 感兴趣的转运蛋白在含有50μL的测定缓冲液中孵育 mM D-葡萄糖酸钾。加入DL-乳酸钠后 如上述步骤B3中所述启动呼吸,1.5μl的1mM 尼古丁素储备溶液在乙醇中(得到最终的 浓度〜1μM)加入比色皿中。浓度 尼日利亚菌素必须避免1μM以防止电子交换 其可以在高尼日利亚浓度的条件下发生 废除电化学梯度的两个组分。记录 荧光淬灭信号进一步〜60秒,然后添加离子载体 缬氨霉素(以选择性地消除ΔΨ)至终浓度〜5 μM,通过加入〜1.6μl在乙醇中制备的5mM储备液(参见图1d)。 在终止之前再记录数据40秒 收购。

代表数据


图1.代表性数据,说明 Ψ 敏感荧光团Oxonol V的荧光信号对MdtM-催化电转运膜反转膜囊泡。加入乳酸以激活囊泡导致产生呼吸性 Ψ ,如通过Oxonol V荧光信号的快速淬灭所证明的。/a>(a)向从富含野生型MdtM的TO114细胞产生的倒置泡囊中加入2.5mM胆酸盐导致ΔΨ的部分去极化,表示为Oxonol V荧光的去淬灭,因为ΔΨ被MdtM消耗 - 介导的胆汁盐/H 运输反应。 (b)将胆酸盐添加到从富含功能障碍的MdtM D22A的TO114细胞产生的阴性对照囊泡中,导致由残留的生电Na + +/+/+对照物引起的小的但是可感知的脱活性的突变体。 (c)阳性对照测定,其中将Na +离子加入到含有完全补体的电致反义植物的倒置囊泡中,以便测量产电Na + +/+ 活性的NhaA转运蛋白。 (d)Oxonol V荧光对添加尼日利亚菌素的反应。在K on +离子的存在下,该离子载体选择性地耗散ΔpH并将其转化为ΔΨ,导致荧光信号的进一步淬灭。丝氨霉素破坏ΔΨ。在测定(a,b和c)中,在所示的时间加入质子载体CCCP导致几乎完全耗散ΔΨ。荧光强度以每秒计数(cps)测量。您测量的荧光强度可能因仪器设置的不同而有所不同。

笔记

  1. 来自步骤A8的重悬的倒置囊泡可以以25-100μl的等分试样转移到试管中,在液氮中快速冷冻并储存在-80℃下供随后使用。以这种方式冷冻的囊泡将保持其完整性几个月。然而,如果冷冻的囊泡原料用于随后的运输测量,则在使用之前必须在冰上非常缓慢地解冻囊泡以防止其破裂。
  2. 为了确保用于检测电致逆转运体的实验条件的适合性,应首先使用从大肠杆菌细胞例如BW25113产生的倒置泡囊进行阳性对照,所述菌株BW25113含有全部的电致逆转录剂。该对照实验应当通过遵循以下方案进行,除了转运测定缓冲液pH应当用H 2 SO 4 4调节至pH 8.5,并且葡萄糖酸钠(至通过加入80μl的2M葡萄糖酸钠储备液,在比色皿中的最终浓度〜100mM)以代替胆酸钠,以特异性地检测产生电的Na + + 由NhaA转运体催化的交换(图1c)
  3. 为了确保重现性,测定应至少进行两次单独的倒置泡囊制备,一式三份。
  4. 与依赖于荧光检测的所有测定一样,必须进行强有力的对照以确保任何检测到的转运活性可以明确地归因于感兴趣的蛋白质。在我们的实验中,我们使用过表达MdtM D22A(MdtM的功能失调点突变体)的反转囊泡作为阴性对照(见图1b)。
  5. 如果该方法用于检测金属离子/H sup +反向运动活性,则使用从大肠杆菌的逆转子缺陷型TO114菌株产生的反向囊泡是重要的,因为至少另外四种 细菌中存在的转运蛋白(NhaA,NhaB,ChaA和MdfA)可以催化单价金属阳离子/H + 交换。
  6. 因为氯离子可以使膜电位去极化,所有缓冲系统必须保持无氯化物。 因此,缓冲液的pH应使用H 2 SO 4来代替HCl来调节。
  7. 最后,如果该方案用于比较野生型和突变转运蛋白的反向运动活性,则必须定量(通常通过免疫检测方法)反向囊泡膜中存在的靶蛋白的量,以确保H + 吸收仅由于转运蛋白活性的差异,而不是表达水平的差异。

食谱

  1. LBK琼脂(100ml) 1.0克胰蛋白酶
    0.5g酵母提取物
    0.745克KCl
    1.5克琼脂 用高纯水补充至100ml,然后高压灭菌
    加入100μg/ml羧苄青霉素进行选择,当溶液仍然是液体和温暖的触摸。 加入到无菌条件下的培养皿中
  2. LBK液体培养基(1 L) 10g胰蛋白胨
    5g酵母提取物
    7.45克KCl
    用高纯水补充至1000ml,然后高压灭菌
  3. Tris /山梨醇/二硫苏糖醇/蔗糖(TSDS)缓冲液(1L) 由10mM Tris(pH7.5),280mM山梨醇,0.5mM二硫苏糖醇和250mM蔗糖组成
    1M Tris(pH7.5)10ml 1μM山梨醇280ml
    1M蔗糖250ml
    用高纯度水补充至999.5ml,然后检查pH,如果需要,用H 2 SO 4 4无菌过滤器调节,并在4℃下贮存。
    在使用前立即从冷冻的1M储备液中加入0.5ml二硫苏糖醇
  4. 运输测定缓冲液(100 ml)
    由10mM BisTris丙烷(pH 7.2)(或初始对照实验的pH 8.5),5mM MgSO 4,5μMOxonol V / 100mM BisTris丙烷(pH7.2或pH8.5)10ml
    2M MgSO 4 0.25ml
    1mM Oxonol V(在乙醇中制备)0.5ml
    用高纯水补充至100ml,然后无菌过滤。
    对于使用尼日利亚菌素的对照实验,应当将将终浓度为50mM的D-葡萄糖酸钾(2.5ml的2M储备液)加入到测定缓冲液中
    Oxonol V是光敏感的,所以运输测定缓冲液和Oxonol V储液应储存在琥珀色瓶子或保存在避光的容器中
  5. 2 M D-葡萄糖酸钠储备溶液(10ml) 4.36g用高纯水
    补充至10ml 无菌过滤并在4°C下保存
  6. 2 M D-葡萄糖酸钾储备溶液(10ml) 6.68g用高纯水加至10ml 无菌过滤并在4°C下保存

致谢

这项工作由BBSRC拨款BB/K014226/1(向CJL)支持。 以上描述的方案改变自之前报道的方案(Resch等人,2010)。

参考文献

  1. Paul,S.,Alegre,K.O.,Holdsworth,S.R.,Rice,M.,Brown,J.A.,McVeigh,P.,Kelly,S.M.and Law,C.J。(2014)。 主要促进者超家族的单组分多药转运蛋白是网络的一部分,可保护 Escherichia coli from bile salt stress。 Mol Microbiol 92(4):872-884。
  2. Resch,C.T.,Winogrodzki,J.L.,Patterson,C.T.,Lind,E.J.,Quinn,M.J.,Dibrov,P.and Hase,C.C。(2010)。 推定的Na + /H + 霍乱弧菌的转运体,Vc-NhaP2在体内介导特异性K sup/+/sup/H交换/em>。 49(11):2520-2528。
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引用: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.
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