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Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria
采用荧光探针标记细胞研究乳酸菌中的胞内酸稳态   

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Abstract

Here we describe a protocol which we have used to study the homeostasis intracellular in vivo in lactic acid bacteria (LAB) using a fluorescent probe. This type of probes can be used for determining changes in the pH of cytoplasm with high sensitivity, temporal resolution and technical simplicity as well as accessing the rate of change of intracellular pH in response to a stimulus from kinetic measurements on short time scales (Breeuwer et al., 1996; Molenaar et al., 1991). This protocol has been designed to measure the intracellular pH using the pH-sensitive fluorescent probe 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) in LAB, Enterococcus faecalis (E. faecalis), Lactococcus lactis (L. lactis) and Lactobacillus casei (L. casei).

Keywords: Fluorescent probe(荧光探针), Loading cell(负载细胞), Ph measurement(pH值测定), Lactic acid bacteria(乳酸菌), Intracellular acid-base homeostasis(细胞内酸碱平衡)

Materials and Reagents

  1. Lactococcus lactis IL1403
  2. Enterococcus faecalis JH2-2
  3. Lactobacillus casei (ATCC, catalog number: 334 )
  4. 2´,7´-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF) (acid form) (Life Technologies, Molecular Probes®)
  5. Luria-Bertani broth (Sigma-Aldrich, catalog number: L3147 )
  6. MRS broth (Sigma-Aldrich, catalog number: 69966 )
  7. Citrate (Sigma-Aldrich, catalog number: C7254 )
  8. Malic acid (Supelco, catalog number: 46940U )
  9. Pyruvate (Sigma-Aldrich, catalog number: P2256 )
  10. D-(+)-glucose (Sigma-Aldrich, catalog number: G8270 )
  11. D-(+)-galactose (Sigma-Aldrich, catalog number: G0750 )
  12. Electrode filling solution (Orion, catalog number: 900011 )
  13. Na2HPO4 (Merck KGaA, catalog number: 1006559 )
  14. NaH2PO4 (Merck KGaA, catalog number: 106349 )
  15. HCl (Merck KGaA, catalog number: 100317 )
  16. NaOH (Merck KGaA, catalog number: 106462 )
  17. Triton X100 detergent (Merck KGaA, catalog number: 648466 )
  18. Valinomycin (Sigma-Aldrich, catalog number: V0627 )
  19. Nigericin (Sigma-Aldrich, catalog number: N7143 )
  20. BCECF stock solution (see Recipes)
  21. 50 mM potassium phosphate (KPi) buffer (see Recipes)

Equipment

  1. Stove Lab Tech (LIB-080M)
  2. Centrifuge for Eppendorf tubes (Eppendorf, model: 5418 )
  3. Centrifuge Sorvall ST 16R for centrifugation of Falcon tube under refrigerated conditions (Thermo Fisher Scientific)
  4. Fluorescence spectrophotometer (PerkinElmer, model: LS55 )
  5. Thermostatic bath (Lauda Alpha, model: RA8 )
  6. pH/MV meters (Orion, model: 420A )
  7. 4 M KCl saturated with AgCl for combination Ag/AgCl pH electrode (Orion, catalog number: 900011)
  8. 5 ml Quartz cuvette (optical path 1 cm)
  9. pH microelectrode (Horiba, model: 9669-10D )

Procedure

  1. Growth conditions
    1. Enterococcus faecalis cultures were grown at 37 °C without agitation in 100 ml stoppered bottles that contained 20 to 50 ml of LB medium (LB), initial pH 7.0 (pH adjusted by adding 5 M HCl or 5 M NaOH as correspond employing a pH meter) supplemented with organic acid (citrate, malate or pyruvate) and/or different carbon source.
    2. Lactococcus lactis cultures were routinely grown at 30 °C without shaking in 100 ml sealed bottles containing 20 ml of lactose free M17 medium, supplemented with 25 mM glucose (M17G). Overnight cultures prepared in this way were used to inoculate fresh M17 adjusted to different initial pH values as indicated. The medium was supplemented with organic acid and/or different carbon source.
    3. Lactobacillus casei was grown overnight at 37 °C in modified MRS broth adjusted to pH 6.0. Cells were grown in screw-capped tubes of 50 ml without shaking at 37 °C for about 15 hours inoculated with 15 μl of a preculture in stationary phase mMRS supplemented with 25 mM glucose (mMRSG) stored at -80 °C.
    4. Growth of the microbial cultures was followed by measurement of the optical density at a wavelength of 660 nm (OD660).
    5. Cells were harvested in mid-exponential growth phase when the optical density was 0.6-0.8, centrifugate for 10 min at 1,600 x g at 4 °C.
    6. The cells were washed twice with 50 mM potassium phosphate (KPi) (pH 5.5) at 4 °C, and finally resuspended in 1 ml the same buffer. For each wash, we used the same volume of solution that the initial culture volume.
      The same KPi buffer condition (50 mM KPi, pH 5.5, kept at 4 °C) is used in the rest of steps in loading of cells with the BCECF probe.

  2. Loading of cells with the BCECF probe
    1. The different OD-cultures were matched in such way to start with the loading-probe protocol with the same amount of cells.
    2. Thus, the cells were centrifugated at maximum speed 24,900 x g for 1 min at room temperature.
    3. Finally the cells were resuspended in 20 µl of KPi solution. To be loaded with BCECF, 1 μl of 10 mM BCECF was added to said resuspension cells. Then 2.5 µl of 0.5 M HCl was added and incubated for 10 min in the dark at room temperature.
    4. It was then neutralized with the addition of 1 ml of KPi ice solutions and washed twice with 1 ml of KPi. The centrifugation between wash steps was at maximum speed 24,900 x g for 1 min at room temperature.
    5. Centrifugated between each wash step at 24,900 x g for 1 min. Finally the cells were resuspended in 100-150 μl of KPi solutions and stored on ice until use.

  3. Fluorescent determination
    1. For each experiment, 10 µl of charged BCECF cells were resuspended in 3 ml of 50 mM KPi solution at indicated pH in a 5 ml quartz cuvette (optical path 1 cm) and equilibrated at 37 °C for 2 min.
    2. The sample was stirred with a suitable magnetic strip and the fluorescence signal was monitored every 1 sec in a fluorometer.
    3. Fluorescence emissions were recorded at 525 nm with excitation wavelength at 503 nm (slit widths were 16 and 4 nm, respectively). The opening of the sample chamber causes data loss during the first 6-7-8 sec after addition of each substrate to be tested that was made in the cuvette.

  4. Conversion of the fluorescence signal in pH
    1. The conversion of the fluorescence signal to cytoplasm pH was performed by titration curves as shown in the Note 1. This procedure was performed for each batch of cells loaded. The loaded cells were resuspended in buffer 50 mM KPi at pH 3.0.
    2. In the same way, the determinations were performed with addition of 2% (v/v) Triton X-100, 75 μM valinomycin and nigericin for permeabilizing the membranes to reach the ionic balance between the intracellular and the external solution. This cell mixture was incubated for 2 min, at 37 °C (work temperature) with agitation until the fluorescent signal was stabilized. Thus, the measurement of fluorescence and pH (with a pH microelectrode) is performed per each addition of 5 of 0.1 N NaOH solutions. To each pH value we get a fluorescent signal value, measured in separate steps. Each determination is a point of the titration curves and covers the pH range from 3.0 to 11.0.
      Figure 1 shows titration curves for fluorometric probe BCECF in L. lactis and E. faecalis. The cells loaded were titrated prior permeabilization of the cells with 2% (v/v) Triton X-100, 75 mM valinomycin and 75 mM nigericin. The pKa, Ifmín and Ifmax parameters were determined from nonlinear fit of experimental data.


      Figure 1. Fluorometric titration curves of the BCECF probe, fluorescence intensity as a function of external pH of 50 mM KPi buffer where the cells were resuspended. Wavelength excitation: 503 nm, wavelength emission: 525 nm. The pKa of BCECF was determined in cells of wild type L. lactis IL1403 (- ▲ -), L. lactis ILGR1 (IL1403 isogenic derivative als -α acetolactate sinthase- mutant) (- ● -) and E. faecalis JH2-2 (- ■ -), properly loaded with BCECF.

    3. This permeabilization allowed the electrochemical decoupling of the cell so that the pHint and pHext are equilibrated. Table 1 shows the four parameters of the non-linear fit curve (If vs pH) obtained for BCECF in each microorganism.
    4. Parameters a, d (Ifmin and Ifmax) and c (pKa) were then used in the transformation of the of fluorescence intensity to pH values.

      Table 1. Parameter values of nonlinear adjustment from titration curves of BCECF probe

      L. lactis
      IL1403
      L. lactis
      ILGR1
      E. faecalis
      JH2-2
      a (Ifmax) (AUifa)
      277 ± 4
      181 ± 4
      213 ± 10
      b (if.pH-1)
      0.46 ± 0.02
      0.48 ± 0.03
      0.51 ± 0.08
      c (pKa)
      7.20 ± 0.02
      7.24 ± 0.04
      7.00 ± 0.08
      d (Ifmin) (AUifa)
      48 ± 2
      43 ± 2
      17 ± 7
      aAUif: Arbitrary units of fluorescence intensity

  5. Intracellular pH measurement
    The following are examples of using BCECF for determination of intracellular pH in LAB as result the metabolism of organic acids and/or sugars. Figure 2 shows the intracellular pH variation during the metabolism of different carbon sources in E. faecalis JH2-2 (Espariz et al., 2011; Repizo et al., 2013).


    Figure 2. Effect of citrate and malate metabolism on the intracellular pH of E. faecalis JH2-2. A and B, cells were grown in LB supplemented with: 28 mM malate (- ♦ -); 28 mM malate and 10 mM arginine (- ● -); 28 mM glucose (- ▼ -); and 28 mM glucose and malate (- ■ -). C, cells were grown in LB: basal (- ■ -); and supplemented with 30 mM citrate (- ● -). Arrows indicated the substrates additions: 10 mM malate (Mal); 10 mM glucose (Glc) or 10 mM citrate (Cit) in loaded cell with BCECF and resuspended in 50 mM KPi solution (pH 6.5).

    Figure 3 shows the intracellular pH variation during the metabolism of different carbon sources in L. casei ATCC 334 (Mortera et al., 2013).


    Figure 3. Energetics of carbohydrate and Ca2+-citrate metabolism in Lb. casei. Lb. casei ATCC 334 was grown in mMRSGlc A, mMRSGal B and mMRSCitCa C. The cells were harvested at an OD660 of 0.6 and loaded with BCECF for intracellular pH (pHin) measurements (A, B, and C). For intracellular pH measurements, 5 mM glucose (solid line), 5 mM galactose (dashed line), or 2 mM citrate and then 2 mM Ca2+ (dotted line) was added at the time point indicated by the arrow.

    Figure 4 shows the intracellular pH variation in L. lactis IL1403 and the mutant ILGR1 product the metabolism of different carbon source (Zuljan et al., 2014).


    Figure 4. L. lactis intracellular pH variations in response to pyruvate. L. lactis IL1403 (dotted line, wt) and L. lactis ILGR1 (continuous line) strains were grown in M17G up to exponential phase. Then, cells were loaded with the BCECF fluorescent probe and resuspended in phosphate buffer at 4.5. Pulses of 50 mM pyruvate (Pyr), 3 mM glucose (Glu) or 0.1 N NaOH (*) were added at the times indicated by the arrows (gray line).

Notes

  1. We can represent the fluorescent activity of the probe from its acid-base equilibrium; Where SF is the fluorescent specie, which is the one that allows us follow the pH changes in the environment:
    HSF ↔ SF- + H+ Ka = [SF-]x [H+]x/[HSF]x
    Figure 5A shows a titration curve of a generic probe where fluorescence intensity versus pH is plotted. For a given value Ifx fluorescence intensity corresponds to a pHx value within the range in the titration curve (pHx; Ifx). In this case the concentrations of both species will be given by:
    [SF-]x = Ifx – Ifmin
    [HSF]x = Ifmax – Ifx
    Replacing in the expression of the Ka, have:
    pHx = pKa + log {(Ifx – Ifmin)/(Ifmax – Ifx)}
    This is an expression which allows us to transform each Ifx at pHint scale at a certain time x (tx; Ifx) in a kinetic experiment, as shown in Figure 5B.


    Figure 5. If conversion to pH scale. Schematic representation of the correlation between of a titration curve A and fluorescence emission kinetics, for a generic fluorescent probe in BAL strain. A point (pHx; Ifx) indicated in A that is linked to the point (tx; Ifx) to B as explained in the text.

    The pKa, Ifmax and Ifmín parameters of (1) associated with the titration curve are obtained from nonlinear four parameter fit using the following expression:
    f (x) = {(ad) / [1 + (x/c)b]} + d
    where a and d are the asymptotic maximum and minimum values corresponding to Ifmax and Ifmin for the probe, c the inflection point corresponding to the pKa of the probe and b the slope that gives us an idea the sensitivity of the system. The pH range for the use of the probe will be set to the value of pKa and system sensitivity.
  2. Measurement of the pH intracellular in E. faecalis loaded with BCECF when the extracellular pH is below to 5.5. As shown in Figure 6, the response of JH2-2/BCECF cells was to be maximal at pHext 7.5 while is to virtually unable to be detected at pHext under 6.0. Thus, it is impossible to study organic acid metabolism in E. faecalis with BCECF probe at pHext less than 6.0 (data not shown).


    Figure 6. Fluorescence intensity of E. faecalis cells loaded with BCECF at different pHext. pHext: 6.0 (- Δ -), 6.5 (- ▼ -), 7.0 (- ○ -) and 7.5 (- ● -). The arrow indicates a pulse of glucose (Glc) 5 mM.

    E. faecalis, unlike other species of BAL, are unable to maintain a ΔpH with the more acidic external media when the cells are not energized. These results demonstrate that for E. faecalis in very acidic conditions, when pH below 5.5, it is necessary to use different probes. In those conditions we used the CDCFD fluorescent probe in our experiment (Espariz et al., 2011).

Recipes

  1. BCECF stock solution
    10 mM BCECF in dimethyl sulfoxide (DMSO) anhydrous
    Stored at -20 °C
  2. 50 mM potassium phosphate (KPi) buffer
    Mix 1 M K2HPO4 and 1 M KH2PO4 to a desired final pH (4.5, 5.8, 6.0, 6.5, 7.0 or 7.5) and dilute 20x with distilled H2O
    mMRS: Original composition without glucose, ammonium citrate, and Tween 80
    mMRSCitCa: mMRS supplemented with 30mM sodium citrate and 10mM chloride calcium
    mMRSGlc: mMRS supplemented 0.5% (wt/vol) glucose
    mMRSGal: mMRS supplemented 0.5% (wt/vol) galactose
    The medium was adjusted to pH 6.0

Acknowledgments

This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and CONICET (PICT2010-1828 and PIP/2012). F.S. is a fellow of ANPCyT, P. M. is a fellow of CONICET, S. H. A. and C. M. are Career Investigators of the same institution.

References

  1. Breeuwer, P., Drocourt, J., Rombouts, F. M. and Abee, T. (1996). A novel method for continuous determination of the intracellular pH in bacteria with the internally conjugated fluorescent probe 5 (and 6-)-carboxyfluorescein succinimidyl ester. Appl Environ Microbiol 62(1): 178-183.
  2. Espariz, M., Repizo, G., Blancato, V., Mortera, P., Alarcon, S. and Magni, C. (2011). Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis. FEBS J 278(12): 2140-2151.
  3. Molenaar, D., Abee, T. and Konings, W. N. (1991). Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator. Biochim Biophys Acta 1115(1): 75-83.
  4. Mortera, P., Pudlik, A., Magni, C., Alarcon, S. and Lolkema, J. S. (2013). Ca2+-citrate uptake and metabolism in Lactobacillus casei ATCC 334. Appl Environ Microbiol 79(15): 4603-4612.
  5. Repizo, G. D., Blancato, V. S., Mortera, P., Lolkema, J. S. and Magni, C. (2013). Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex. Appl Environ Microbiol 79(9): 2882-2890.
  6. Zuljan, F. A., Repizo, G. D., Alarcon, S. H. and Magni, C. (2014). alpha-Acetolactate synthase of Lactococcus lactis contributes to pH homeostasis in acid stress conditions. Int J Food Microbiol 188: 99-107.

简介

在这里我们描述了一个协议,我们已经用来研究乳酸细菌(LAB)使用荧光探针在体内细胞内体内平衡。 这种类型的探针可用于以高灵敏度,时间分辨率和技术简单性确定细胞质pH的变化,以及响应于来自短时间尺度上的动力学测量的刺激,获得细胞内pH的变化速率(Breeuwer& et al。,1996; Molenaar et al。,1991)。 该方案已经设计为使用pH敏感性荧光探针在LAB中的2'-,7'-双 - (2-羧乙基)-5(和-6) - 羧基荧光素(BCECF),粪肠球菌 ( E.cfaecalis ),乳酸乳球菌(<乳酸乳球菌)和干酪乳杆菌 em> L。casei )。

关键字:荧光探针, 负载细胞, pH值测定, 乳酸菌, 细胞内酸碱平衡

材料和试剂

  1. 乳酸乳杆菌 IL1403
  2. 粪肠球菌 JH2-2
  3. <干酪乳杆菌(ATCC,目录号:334)
  4. 2'-7'-双 - (2-羧乙基)-5(和-6) - 羧基荧光素(BCECF)(酸形式)(Life Technologies,Molecular Probes )
  5. Luria-Bertani肉汤(Sigma-Aldrich,目录号:L3147)
  6. MRS肉汤(Sigma-Aldrich,目录号:69966)
  7. 柠檬酸盐(Sigma-Aldrich,目录号:C7254)
  8. 苹果酸(Supelco,目录号:46940U)
  9. 丙酮酸(Sigma-Aldrich,目录号:P2256)
  10. D - (+) - 葡萄糖(Sigma-Aldrich,目录号:G8270)
  11. D - (+) - 半乳糖(Sigma-Aldrich,目录号:G0750)
  12. 电极填充液(Orion,目录号:900011)
  13. Na 2 HPO 4(Merck KGaA,目录号:1006559)
  14. NaH 2 PO 4(Merck KGaA,目录号:106349)
  15. HCl(Merck KGaA,目录号:100317)
  16. NaOH(Merck KGaA,目录号:106462)
  17. Triton X100洗涤剂(Merck KGaA,目录号:648466)
  18. 伏立诺霉素(Sigma-Aldrich,目录号:V0627)
  19. 尼日利亚霉素(Sigma-Aldrich,目录号:N7143)
  20. BCECF储备溶液(见配方)
  21. 50 mM磷酸钾(KPi)缓冲液(参见配方)

设备

  1. Stove Lab Tech(LIB-080M)
  2. 离心机用于Eppendorf管(Eppendorf,型号:5418)
  3. 离心Sorvall ST 16R,用于在冷藏条件下(Thermo Fisher Scientific)离心Falcon管
  4. 荧光分光光度计(PerkinElmer,型号:LS55)
  5. 恒温浴(Lauda Alpha,型号:RA8)
  6. pH/MV计(Orion,型号:420A)
  7. 用AgCl饱和的4M KCl用于组合Ag/AgCl pH电极(Orion,目录号:900011)
  8. 5毫升石英比色杯(光程1厘米)
  9. pH微电极(Horiba,型号:9669-10D)

程序

  1. 生长条件
    1. 粪肠球菌培养物在37℃下不搅拌生长 将100ml含有20至50ml LB培养基(LB)的塞紧瓶子, 初始pH 7.0(通过加入5M HCl或5M NaOH调节pH) 使用pH计),补充有机酸(柠檬酸盐,苹果酸盐或   丙酮酸盐)和/或不同的碳源。
    2. 乳酸乳球菌培养物在100ml密封的容器中不摇动地在30℃下常规生长 瓶中装有20ml无乳糖的M17培养基,补充 25mM葡萄糖(M17G)。 使用以这种方式制备的过夜培养物 以接种调节至不同初始pH值的新鲜M17 表示。 培养基补充有机酸和/或 不同的碳源
    3. 干酪乳杆菌(Lactobacillus casei)生长过夜 在37℃下在改良的MRS肉汤中调节至pH 6.0。 细胞生长 螺旋盖的管,在37℃下振摇约15小时 在固定相mMRS中接种15μl的预培养物 补充有储存在-80℃的25mM葡萄糖(mMRSG)。
    4. 微生物培养物的生长之后是在660nm的波长(OD 660)测量光密度。
    5. 在中期指数生长期收获细胞 光密度为0.6-0.8,在4℃以1,600×g离心10分钟 C。
    6. 用50mM磷酸钾洗涤细胞两次 (KPi)(pH 5.5)在4℃,最后重悬于1ml相同的缓冲液中。   对于每次洗涤,我们使用与初始溶液相同体积的溶液 文化量 相同的KPi缓冲液条件(50mM KPi, 保持在4℃)用于其余的用细胞装载的步骤 BCECF探针

  2. 用BCECF探针装载细胞
    1. 以这种方式使不同的OD-培养物与装载 - 探针方案以相同量的细胞开始匹配。
    2. 因此,将细胞在室温下以最大速度24,900×g离心1分钟。
    3. 最后,将细胞重悬于20μlKPi溶液中。 成为 加载BCECF,向所述重悬浮液中加入1μl10mM BCECF 细胞。 然后加入2.5μl的0.5M HCl并孵育10分钟 在室温下黑暗。
    4. 然后用中和 加入1ml KPi冰溶液,并用1ml KPi洗涤两次。   洗涤步骤之间的离心在室温下以最大速度24,900×g下离心1分钟。
    5. 在每次洗涤之间离心 步骤为24,900 x g ,持续1分钟。 最后,将细胞重悬浮 100-150微升KPi溶液,并储存在冰上直至使用

  3. 荧光测定
    1. 对于每个实验,将10μl带电的BCECF细胞重悬于3中 ml的指定pH的50mM KPi溶液在5ml石英比色皿中 (光程1cm),在37℃下平衡2分钟。
    2. 的 样品用合适的磁条和荧光进行搅拌 在荧光计中每1秒监测信号。
    3. 荧光 在525nm处用在503nm的激发波长记录发射 (狭缝宽度分别为16和4nm)。 样品的打开 室在添加后的前6-7-8秒内导致数据丢失 在试管中制备的每个待测试的底物。

  4. 在pH值为
    的荧光信号转换
    1. 进行荧光信号向细胞质pH的转化 通过如注释1中所示的滴定曲线进行   为每批加载的单元格。 将装载的细胞重悬浮 缓冲液50mM KPi,pH3.0。
    2. 以相同的方式,确定 加入2%(v/v)Triton X-100,75μM缬氨霉素  和尼日利亚菌素,用于使膜透化以达到离子 平衡细胞内和外部溶液。这个单元格 混合物在37℃(工作温度)下温育2分钟 搅拌直至荧光信号稳定。因此, 荧光和pH(具有pH微电极)的测量 每次添加5μL0.1N NaOH溶液。至每个pH 值,我们得到荧光信号值,在单独的步骤中测量。 每次测定是滴定曲线的一点,并覆盖pH 范围为3.0至11.0。
      图1显示了滴定曲线 荧光探针BCECF。 lactis 和 E。粪便。单元格已加载 预先用2%(v/v)Triton透化细胞 X-100,75mM缬氨霉素和75mM尼日利亚菌素。通过实验数据的非线性拟合确定pKa,If mín和If max 参数。

      图1. BCECF探针的荧光滴定曲线,荧光 强度作为50mM KPi缓冲液的外部pH的函数, 细胞重悬。波长激发:503nm,波长 发射:525nm。 在野生型L的细胞中测定BCECF的pKa。 乳酸IL1403( - ▲ - ),L。 乳酸乳杆菌ILGR1(IL1403同基因衍生物α-α-乙酰乳酸乙酯合酶突变体)( - ● - )和E。 粪肠 JH2-2( - ■   - ),正确装入BCECF
    3. 这种透化 允许细胞的电化学解耦,使得pH <   pH平衡。 表1显示了四个参数 非线性拟合曲线(If对pH) 微生物
    4. 参数a,d(如果 min 和If max )和c(pK a )   然后用于将荧光强度转化为pH 值。

      表1. BCECF探针
      滴定曲线的非线性调整的参数值

      L。 乳酸
      IL1403
      L。 乳酸
      ILGR1
      E。 粪便
      JH2-2
      a(If max )(AU if a
      277±4
      181±4
      213±10
      b(if.pH -1
      0.46±0.02
      0.48±0.03
      0.51±0.08
      c(pK a
      7.20±0.02
      7.24±0.04
      7.00±0.08
      d(If min )(AU if a
      48±2
      43±2
      17±7
      a AU if :荧光强度的任意单位

  5. 细胞内pH测量
    以下是使用BCECF测定LAB中的细胞内pH,结果是有机酸和/或糖的代谢的实例。图2显示了E中不同碳源代谢期间的细胞内pH变化。粪便 JH2-2(Espariz等人,2011; Repizo等人,2013)。


    图2.柠檬酸盐和苹果酸代谢对细胞内pH的影响。粪便 JH2-2。 A和B,细胞在补充有28mM苹果酸( - ◆ - )的LB中生长。 28mM苹果酸和10mM精氨酸( - ● - ); 28 mM葡萄糖( - ▼ - );和28mM葡萄糖和苹果酸( - ■ - )。 C,细胞在LB:基础( - ■ - )中生长;并补充有30mM柠檬酸盐( - ● - )。箭头表示底物添加:10mM苹果酸(Mal); 10mM葡萄糖(Glc)或10mM柠檬酸盐(Cit),并重悬于50mM KPi溶液(pH 6.5)中。

    图3显示在不同碳源的代谢期间细胞内pH变化。 casei ATCC 334(Mortera等人,2013)。


    图3.碳水化合物和Ca 2 + - 柠檬酸代谢的能量学 干酪。 Lb。在mMRSGlc A,mMRSGal B和mMRSCitCa C中生长。在0.6的OD 660下收获细胞,并用BCECF装载用于细胞内pH(pHin)测量(A, B和C)。对于细胞内pH测量,在指示的时间点加入5mM葡萄糖(实线),5mM半乳糖(虚线)或2mM柠檬酸盐,然后加入2mM Ca 2+(虚线)由箭头。

    图4显示了细胞内pH变化。乳酸IL1403和突变体ILGR1产物的不同碳源的代谢(Zuljan等人,2014)。


    图4. L。乳酸 响应丙酮酸的细胞内pH变化。 乳酸IL1403(虚线,wt)和L。乳酸 ILGR1(连续株)菌株在M17G中生长直至指数期。然后,将细胞用BCECF荧光探针装载并在4.5重悬于磷酸盐缓冲液中。在由箭头(灰色线)指示的时间加入50mM丙酮酸(Pyr),3mM葡萄糖(Glu)或0.1N NaOH(*)的脉冲。

笔记

  1. 我们可以从其酸碱平衡表示探针的荧光活性;其中SF是荧光物质,其是允许我们遵循环境中的pH变化的物质:
    HSF↔SF - + H + K a = [SF - ] [H + ] x /[HSF] x
    图5A显示通用探针的滴定曲线,其中绘制荧光强度对pH。对于给定值如果荧光强度对应于在滴定曲线的范围内的pH值(pH值),如果 x )。在这种情况下,两种物质的浓度将由下式给出:
    [SF - ] x =如果 x - 如果 min
    [HSF] x =如果 max - 如果 x
    在Ka的表达式中替换,具有:
    (如果 min )/(If max

    - 如果 x )}
    这是一种表达式,其允许我们在某个时间x(t x
    ),如图5B所示

    图5.如果转化为pH标度。对于BAL菌株中的通用荧光探针,滴定曲线A和荧光发射动力学之间的相关性的示意图。在A中指示的与点(t sub)相关的点(pH ; x sub>)到B,如文中所述
    与滴定曲线相关联的(1)的pK sub和a max和sub参数通过使用以下的非线性四参数拟合获得表达式:
    em>/ c ) b 其中 和 d 是对应于探测的If max 和If min 的渐近最大值和最小值, c 对应于探针的pKa的拐点和 b 斜率,其给出了系统灵敏度的想法。使用探头的pH范围将设置为pKa和系统灵敏度的值。
  2. 在E中测量细胞内的pH。当细胞外pH低于5.5时,加载有BCECF的粪便。如图6所示,JH2-2/BCECF细胞的响应在pH sub 7.5下最大,而在pH小于6.0时几乎不能检测到。因此,不可能研究E中的有机酸代谢。粪便与pHEC sub 小于6.0的BCECF探针接触(数据未显示)。


    图6. E的荧光强度。粪便 在不同pH ext 下装载BCECF的细胞。 pHext:6.0( - Δ - ),6.5( - ▼ - ),7.0( - ○ - )和7.5( - ● - )。箭头表示葡萄糖(Glc)5mM的脉冲
    E。粪便不同于其他种类的BAL,当细胞不通电时,不能用更酸性的外部培养基维持ΔpH。这些结果表明对于E。粪便在非常酸性的条件下,当pH低于5.5时,必须使用不同的探针。在这些条件下,我们在我们的实验中使用CDCFD荧光探针(Espariz et al。,2011)。

食谱

  1. BCECF储液
    10mM BCECF的二甲基亚砜(DMSO)无水
    储存于-20°C
  2. 50mM磷酸钾(KPi)缓冲液
    将1×MK 2 HPO 4和1M KH 2 PO 4混合至所需的最终pH(4.5,5.8) ,6.0,6.5,7.0或7.5)并用蒸馏的H 2 O稀释20倍 mMRS:不含葡萄糖,柠檬酸铵和吐温80的原始组合物
    mMRSCitCa:补充有30mM柠檬酸钠和10mM氯化钙的mMRS mMRSGlc:补充0.5%(wt/vol)葡萄糖的mMRS mMRSGal:补充有0.5%(wt/vol)半乳糖的mMRS 将介质调节至pH6.0v/v

致谢

这项工作得到了Agencia Nacional dePromociónCientíficayTecnológica(ANPCyT)和CONICET(PICT2010-1828和PIP/2012)的资助。 F.S. 是ANPCyT的研究员,P. M.是CONICET的研究员,S. H. A.和C. M.是同一机构的职业调查员。

参考文献

  1. Breeuwer,P.,Drocourt,J.,Rombouts,F.M.and Abee,T。(1996)。 使用内部偶联的荧光探针5连续测定细菌中细胞内pH的新方法(和6 - ) - 羧基荧光素琥珀酰亚胺酯。 Appl Environ Microbiol 62(1):178-183。
  2. Espariz,M.,Repizo,G.,Blancato,V.,Mortera,P.,Alarcon,S.and Magni,C.(2011)。 在粪肠球菌中鉴定苹果酸和可溶性草酰乙酸脱羧酶。 a> FEBS J 278(12):2140-2151。
  3. Molenaar,D.,Abee,T.and Konings,W.N。(1991)。 用荧光pH指示剂连续测量乳酸乳球菌中的细胞质pH值 。 Biochim Biophys Acta 1115(1):75-83。
  4. Mortera,P.,Pudlik,A.,Magni,C.,Alarcon,S.and Lolkema,J.S。(2013)。在干酪乳杆菌中的 Ca 2+ /柠檬酸摄取和代谢 ATCC 334. Appl Environ Microbiol 79(15):4603-4612。
  5. Repizo,G.D.,Blancato,V. S.,Mortera,P.,Lolkema,J.S.and Magni,C。(2013)。 粪肠球菌草酰乙酸脱羧酶复合物的生物化学和遗传表征。 Appl> Environ Microbiol 79(9):2882-2890。
  6. Zuljan,F.A.,Repizo,G.D.,Alarcon,S.H.and Magni,C.(2014)。 乳酸乳球菌的α-乙酰乳酸合酶有助于酸胁迫中的pH稳态 。 Int J Food Microbiol 188:99-107。
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引用:Mortera, P., Zuljan, F., Magni, C. and Alarcón, S. H. (2015). Loading of Cells with Fluorescent Probe to Study Intracellular Acid-base Homeostasis in Lactic Acid Bacteria. Bio-protocol 5(2): e1380. DOI: 10.21769/BioProtoc.1380.
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