参见作者原研究论文

本实验方案简略版
Nov 2017

本文章节


 

Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria
从革兰氏阴性菌中提取和测定多聚磷酸盐   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Polyphosphate (polyP), a universally conserved biomolecule, is composed of up to 1,000 phosphate monomers linked via phosphoanhydride bonds. Reaching levels in bacteria that are in the high nmoles per mg protein range, polyP plays important roles in biofilm formation and colonization, general stress protection and virulence. Various protocols for the detection of polyP in bacteria have been reported. These methods primarily differ in the ways that polyP is extracted and/or detected. Here, we report an improved method, in which we combine polyP extraction via binding to glassmilk with a very sensitive PolyP kinase/luciferase-based detection system. By using this procedure, we significantly enhanced the sensitivity of polyP detection, making it potentially applicable for mammalian tissues.

Keywords: Polyphosphate (多聚磷酸盐), PolyP extraction method (多聚磷酸盐提取方法 ), PolyP quantification (多聚磷酸盐测定), Luciferase-based ATP quantification (基于荧光素酶ATP定量), Bacterial virulence (细菌毒力), Bacterial stress response (细菌应激反应)

Background

Polyphosphate (polyP), a biopolymer composed of linear chains of up to 1,000 inorganic phosphate monomers, is found in cells of all three domains of life. Yet, bacteria are the only organisms for which the enzymes of polyP metabolism have been well studied. Bacterial polyP kinase (PPK), which converts ATP into polyP, catalyzes both forward and reverse reactions. While synthesis of polyP is clearly the favored reaction in the cell, by providing sufficient amounts of ADP in vitro, the enzyme can be used to generate ATP from polyP, making a luciferase-based ATP detection possible (Ault-Riché et al., 1998). Bacteria lacking PPK are defective in biofilm formation, motility, persistence, and various stress responses, and show significantly increased sensitivity towards hypohalous acids (i.e., bleach) stress or phosphate starvation (Figure 1) (Rao et al., 2009; Gray et al., 2014; Maisonneuve and Gerdes, 2014; Gray and Jakob, 2015; Groitl et al., 2017).


Figure 1. Synthesis of polyP and its role in Gram-negative Bacteria. The bacteria-specific polyphosphate kinase (PPK) reversibly catalyzes the conversion from ATP into polyP and ADP. Various functions for polyP have been described in Gram-negative bacteria, including its involvement in biofilm formation, colonization, motility, and formation of antibiotic-resistant persister cells. PolyP also contributes to the resistance of bacteria towards various stresses, including oxidative stress and starvation, and serves as metal chelator and Pi reservoir.

Given the many roles that polyP plays in Gram-negative bacteria, PPK became attractive as drug target to interfere with biofilm formation, make bacteria less persistent, and sensitize them towards physiological oxidants such as bleach (Dahl et al., 2017). Therefore, reliable and sensitive methods to determine the polyP levels in vivo are necessary. Several methods for the extraction and detection of polyP have been reported in Bio-protocol, including extraction of polyP with (i) perchloric acid, (ii) sodium hypochlorite, and (iii) phenol/chloroform and detection of polyP via visualization with urea-PAGE or colorimetric assays using malachite green or molybdenum blue (Gomez Garcia, 2014; Canadell et al., 2016; Ota and Kawano, 2017). In this protocol, we combined extraction of polyP via binding to glassmilk (Ault-Riché et al., 1998) with a very sensitive two-step enzyme-based detection system. First, the extracted polyP is converted into ATP by E. coli PPK in the presence of ultra-pure ADP. The ATP levels are then quantified using a luciferase-based detection system and corrected for cellular ATP. In comparison to the urea-PAGE or the colorimetric methods, the luciferase-based detection allows the quantification of much lower levels of polyP. This protocol has been successfully applied to quantify polyP levels from Pseudomonas aeruginosa.

Materials and Reagents

  1. Microcentrifuge tubes, 1.5 ml, clear (BioExpress, catalog number: C-3260-1 )
  2. Silica membrane spin columns (Epoch Life Science, EconoSpinTM, catalog number: 1920-250 )
  3. TempPlate non-skirted 96-well PCR plate, low profile, natural (USA Scientific, catalog number: 1402-9500 )
  4. 96-well plate solid white (Corning, catalog number: 3912 )
  5. 96-well plate clear flat bottom (Corning, catalog number: 3596 )
  6. Bradford reagent (Bio-Rad Laboratories, catalog number: 5000006 )
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A3059-100G )
  8. Tris (Fisher Scientific, catalog number: BP152-5 )
  9. Ultra-pure ADP (Cell Technology, catalog number: ADP100-2 )
  10. E. coli polyphosphate kinase (PPK) (for expression and purification of E. coli PPK, see Gray et al., 2014)
  11. Dithiothreitol (DTT), > 99% pure, protease-free (Gold Biotechnology, catalog number: DTT25 )
  12. Sodium-dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771-1KG )
  13. 95% v/v Ethanol
  14. QuantiLum® Recombinant Luciferase (Promega, catalog number: E1701 ; exact concentration of aliquots may vary)
    Notes:
    1. Prepare aliquots of the enzyme and store in small glass vials at -80 °C.
    2. Thaw and use the enzyme for each experiment.
    3. Discard all unused product.
    4. Do not subject to freeze-thaw cycles.
  15. Guanidine thiocyanate (Sigma-Aldrich, catalog number: G6639-250G )
  16. Silicon dioxide (Sigma-Aldrich, catalog number: S5631 )
  17. NaCl (Fisher Scientific, catalog number: S271-10 )
  18. EDTA (Fisher Scientific, catalog number: BP120-500 )
  19. HEPES (pH 7.5) (Fisher Scientific, catalog number: BP310-100 )
  20. Ammonium sulfate (Fisher Scientific, catalog number: A702-500 )
  21. Tricine buffer (pH 7.8) (Sigma-Aldrich, catalog number: T0377 )
  22. MgSO4 (Thermo Fisher Scientific, catalog number: M63500 )
  23. Sodium azide (MP Biomedicals, catalog number: 210289125 )
  24. Luciferin ( Biotium, catalog number: 10101-1 )
  25. Glycylglycine (pH 7.8) (MP Biomedicals, catalog number: 210185610 )
  26. ddH2O
  27. Hydrochloric acid (Fisher Scientific, catalog number: A144212 )
  28. GITC lysis buffer (see Recipes)
  29. Glassmilk (see Recipes)
  30. New Wash (NW-) Buffer (see Recipes)
  31. Elution Buffer (see Recipes)
  32. PPK buffer (see Recipes)
  33. Luciferase reaction buffer (see Recipes)
  34. Luciferin working solution (see Recipes)

Equipment

  1. Eppendorf Pipettes: 100-1,000 μl, 20-200 μl, 2-20 μl, 1-10 μl
  2. Thermomixer (Eppendorf, model: 5350 )
  3. Microcentrifuge (Eppendorf, model: 5415D )
  4. Incubator 37 °C (VWR, model: 1555 )
  5. -80 °C Freezer (Eppendorf, New BrunswickTM, model: Innova® U725 , catalog number: U9440-0002)
  6. Fluostar Omega Plate Reader with luminescence reading function and injector module (BMG Labtech, catalog number: 0415-102 )
  7. pH meter
  8. Vortexer
  9. Refrigerator (4 °C)

Software

  1. Fluostar Omega Control and Evaluation Software (BMG Labtech, catalog number: 1300-501)

Procedure

The procedure is ideally suited to extract and quantify polyP from gram-negative PPK-containing bacteria, including P. aeruginosa, which accumulate polyP under stress, e.g., sublethal concentration of hypohalous acids or nutrient starvation (for details regarding growth and stress treatment, see Ault-Riché et al., 1998; Groitl et al., 2017).


  1. Prepare samples for the extraction of polyP
    Note: As GITC is considered as being moderately toxic, keep separate GITC waste container for tips and flow-through.
    1. Collect bacterial cells sufficient to yield 100-300 μg of total cellular protein (approximately 1 ml of P. aeruginosa culture at OD600 = 1), and spin down cells at 13,000 x g, 4 °C.
    2. Discard supernatant.
    3. Resuspend cell pellets in 0.25 ml GITC lysis buffer.
    4. Incubate the samples in a Thermomixer for 10 min at 95 °C without mixing.
    5. Take 20 μl samples for determining the protein concentration (see Step B2).
    6. Store samples at -80 °C after cooling down to RT until further procedure. Degradation is no concern as GITC denatures proteins including proteases.
      Note: We always processed the samples within 7 days.

  2. Determine the protein concentration of the samples
    1. Prepare a standard curve by dissolving BSA in GITC lysis buffer to a final concentration of 0, 0.25, 0.5, 1, 1.5 or 2 μg/μl.
    2. Mix 195 μl Bradford Reagent with 5 μl of the BSA standard or 5 μl of the lysed samples, and incubate for 5-10 min at room temperature (RT) in microcentrifuge tubes. Use triplicates for each sample.
    3. Transfer samples into 96-well clear plates with flat bottom and measure absorbance at 595 nm in the Fluostar Omega Plate Reader.
    4. Calculate the protein concentration as described in the Data analysis section. Samples will be normalized at the end.

  3. Extract polyP
    1. Thaw samples from Step A6 and vortex to mix. 
    2. Add subsequently to the samples: 15 μl 10% w/v SDS, 0.5 ml 95% v/v ethanol, and 5 μl glassmilk (see Recipes) and vortex in between.
    3. Transfer samples to silica membrane spin columns and spin down in microcentrifuge for 3 min at 3,000 x g, RT. Note that we observed a pellet formation in the flow-through at velocities higher than 3,000 x g.
    4. Discard flow-through.
    5. Add 0.75 ml NW-Buffer (see Recipes).
    6. Spin down for 3 min at 3,000 x g at RT and discard flow-through.
    7. Repeat Steps C5 and C6.
    8. Dry samples by centrifugation at 11,000 x g for 3 min at RT.
    9. Transfer column to a clean 1.5 ml microcentrifuge tube.
    10. Add 50 μl Elution buffer to the column, incubate for 15 min at RT and elute polyP by centrifugation at 11,000 x g for 3 min at RT.
    11. Samples can be stored at -80 °C.
      Note: Processing should be done within 1-3 days.

  4. Convert polyP into ATP
    1. Thaw samples from Step C11 and vortex to mix.
    2. Prepare two PPK Assay Master Mixes according to the number of samples. Master mix I (+PPK) contains 50 nM of E. coil PPK, whereas master mix II (-PPK) does not. The reactions without PPK are used to correct for cellular ATP that was extracted during the process. The following volumes refer to the amount required for one sample.
      PPK Assay Master Mix I (+ PPK) (total 20 μl per sample):
      7.5 μl
      4-fold PPK Buffer
      0.9 μl
      ultra-pure ADP [stock concentration of 8.4 mM (may vary among batches); final concentration of 250 μM]
      0.7 μl
      E. coli PPK [stock concentration of 2.4 μM (may vary among batches); final concentration of 50 nM]
      10.9 μl
      H2O

      PPK Assay Master Mix II (-PPK) (total 20 μl per sample):
      7.5 μl
      4-fold PPK Buffer
      0.9 μl
      ultra-pure ADP ultra-pure ADP [stock concentration of 8.4 mM (may vary among batches); final concentration of 250 μM ]
      11.6 μl
      H2O

    3. Combine 20 μl PPK Assay Master Mix I or II with 10 μl of the sample by pipetting up and down twice.
    4. Incubate the reaction for 1 h at 37 °C in the incubator to allow PPK to fully convert polyP to ATP.
    5. Inactivate PPK by incubating samples at 95 °C for 2 min in the thermomixer.
    6. Cool samples on ice or at 4 °C, then spin 1 min at 11,000 x g and continue with the supernatant.

  5. Detect ATP
    Note: Much of this is specific to the Omega plate reader. Modify as necessary for the instrument you have available.
    1. Take 10 μl aliquots of the PPK-digested samples (incubated in PPK Assay Master Mix I) and undigested samples (incubated in PPK Assay Master Mix II), and transfer them into wells of white flat bottom 96-well plate.
    2. Prepare Luciferase Reaction Master Mix as follows and keep it at RT.
      Luciferase Reaction Master Mix (total 10 ml):
      0.5 ml
      20-fold Luciferase Reaction Buffer
      10 μl
      1 M DTT (final concentration of 1 mM) (Thaw on ice)
      1 ml
      Luciferin working solution (final concentration of 100 μM)
      1 μl
      luciferase [stock concentration of 244 μM (may vary among batches), final concentration of 25 nM]
      8.5 ml
      H2O
      Note: The reaction is very temperature sensitive, therefore do not store the reaction master mix on ice. Because luciferase loses its activity quickly, use the reaction master mix within 15-20 min of preparation.
    3. Prime injector of Omega plate reader with 2 ml Luciferase Reaction Master Mix.
    4. Set the temperature of the Omega plate reader to 25 °C and the gain to 3,000 (This is specific to the OMEGA plate reader and may vary in other plate readers).
    5. Inject 90 μl of Luciferase Reaction Master Mix to each sample, mix for 10 sec fast mixing and measure luminescence over 2-3 min. The length of each cycle depends on the amount of samples.
    6. Measure the PPK-digested (incubated with PPK-Assay Master Mix I) samples in the same order as the undigested samples (incubated with PPK-Assay Master Mix II). Measure luminescence over 2-3 min.
    7. When finished, wash injector by priming with 4 ml dH2O.

Data analysis

  1. Calculate protein amount in each sample:
    1. Standard Curve: plot the measured absorbances against the respective BSA concentrations of the standard curve.
    2. Determine the trend line and calculate a linear equation.
    3. Calculate the protein amount for each sample using the equation of the trend line.
      Note: Protein content of the samples should be in the range of 100-300 μg. Dilute if necessary.

  2. Calculate fold-change of polyP in treated relative to untreated cells
    1. Subtract the relative luminescence units (RLU) values of samples incubated in the absence of PPK (incubated with PPK-Assay Master Mix II) from the values of the same samples incubated in the presence of PPK (incubated with PPK-Assay Master Mix I), usually of cycle 2.
      Note: The RLU values of the undigested samples represent the baseline.
    2. Normalize the resulting RLU for each sample to the respective protein amount.
    3. Calculate the fold-change in polyP accumulation in each sample by normalizing the values of samples after treatment (i.e., treatment with HOCl, HOBr and HOSCN, respectively) to those without treatment.


      Figure 2. Extraction and quantification of polyP from P. aeruginosa PA14 wild-type and ∆ppk cells. PA14 wild-type (WT) and ∆ppk cells were grown to mid-log phase and incubated with sublethal concentrations of HOCl (0.5 mM), HOBr (0.15 mM) or HOSCN (0.25 mM) for 2.5 h. Then, polyP was extracted by binding to glassmilk and quantified via the luciferase-based detection system after conversion of the extracted polyP to ATP. The resulting amount was normalized to the respective protein amount in each sample. Fold-change in polyP levels was calculated relative to the untreated cells. Experiments were performed independently for at least three times. Data represent means ± S.D. [Copyright© 2017, Wiley Online Library (Groitl et al., 2017)].

Notes

  1. Short chains of polyP (< 60 Pi units) are considered to bind less efficiently to glassmilk than longer chains and most likely are lost during this extraction method. While bacteria produce polyP of up to 1,000 Pi molecules per chain, the average chain length of polyP in eukaryotes is considerably shorter. For commercially available polyP45, which is a mix of different polyP with enrichment in polyP chains with 45 phosphate residues, we determined a recovery rate of polyP between 24% and 38% using polyP concentrations in a range between 500 nM and 10 mM. In comparison to previously described methods, the previously described methods allow a similar recovery, however, the PPK-based detection system appears to be more sensitive (Canadell et al., 2016; Ota and Kawano, 2017).
  2. The present protocol was designed for the extraction and quantification of polyP from bacteria after treatment with sublethal concentrations of hypohalous acids or after nutrient starvation (shift from rich media into minimal media with low phosphate and lack of amino acids). These conditions are known to result in the accumulation of high amounts of long-chain polyP (Ault-Riché et al., 1998; Gray et al., 2014). For samples that contain lower polyP levels, two additional steps can be incorporated after extraction of polyP by glassmilk to reduce the risk of interference during PPK reaction and/or ATP detection: (i) Treatment with apyrase to decrease the background ATP level, and (ii) Benzonase treatment to remove DNA/RNA (Ault-Riché et al., 1998).

Recipes

  1. GITC lysis buffer (500 ml)
    4 M guanidine thiocyanate
    50 mM Tris-HCl (pH 7.0)
    Store at RT for up to several months 
  2. Glassmilk (25 ml)
    Note: Binding capacity ≥ 100 nmol polyphosphates (in Pi equivalents) per μl. Glassmilk will also bind DNA and RNA.
    1. Suspend 5 g of silicon dioxide in 40 ml of 0.1 M HCl and mix thoroughly
    2. Pellet the glassmilk by centrifugation at 2,000 x g for 5 min at RT
    3. Remove supernatant and repeat Steps a and b
    4. Wash the pellet with 10 ml ddH2O
    5. Repeat Step d at least once and check pH (pH has to be neutral)
    6. Store at room temperature for up to several months
  3. New Wash (NW) Buffer (500 ml)
    5 mM Tris-HCl (pH 7.5)
    50 mM NaCl
    5 mM EDTA
    50% v/v ethanol
    Store at RT for up to several months
  4. Elution Buffer (25 ml)
    50 mM Tris-HCl (pH 8)
    Store at RT for up to several months
  5. PPK Buffer (4-fold) (25 ml)
    200 mM HEPES (pH 7.5)
    200 mM ammonium sulfate
    20 mM MgCl2
    Store at RT for up to several months
  6. Luciferase Reaction Buffer (20-fold) (25 ml, storage time several months at RT)
    500 mM Tricine buffer (pH 7.8)
    100 mM MgSO4
    2 mM EDTA
    2 mM Sodium azide
    2 mg/ml BSA
    Store at RT for up to several months
  7. Luciferin working solution (50x 1 ml)
    1 mM luciferin dissolved in 25 mM glycylglycine (pH 7.8)
    Aliquot in 1 ml aliquots and store at -80 °C for up to several months

Acknowledgments

This work was funded by the National Institute of Health grants GM065318 and GM116582 to U.J. J.-U. D. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (DA 1697/1-1). We thank Dr. Michael J. Gray (University of Alabama) for helping to establish the assay and fruitful discussions.

Competing interests

The authors declare no conflict of interest.

References

  1. Ault-Riché, D., Fraley, C. D., Tzeng, C. M. and Kornberg, A. (1998). Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli. J Bacteriol 180(7): 1841-1847.
  2. Canadell, D., Bru, S., Clotet, J. and Ariño, J. N. (2016). Extraction and quantification of polyphosphate in the budding yeast Saccharomyces cerevisiae. Bio-protocol 6(14): e1874.
  3. Dahl, J. U., Gray, M. J., Bazopoulou, D., Beaufay, F., Lempart, J., Koenigsknecht, M. J., Wang, Y., Baker, J. R., Hasler, W. L., Young, V. B., Sun, D. and Jakob, U. (2017). The anti-inflammatory drug mesalamine targets bacterial polyphosphate accumulation. Nat Microbiol 2: 16267.
  4. Gomez Garcia, M. R. (2014). Extraction and quantification of poly P, poly P analysis by Urea-PAGE. Bio-protocol 4(9): e1113.
  5. Gray, M. J. and Jakob, U. (2015). Oxidative stress protection by polyphosphate--new roles for an old player. Curr Opin Microbiol 24: 1-6.
  6. Gray, M. J., Wholey, W. Y., Wagner, N. O., Cremers, C. M., Mueller-Schickert, A., Hock, N. T., Krieger, A. G., Smith, E. M., Bender, R. A., Bardwell, J. C. and Jakob, U. (2014). Polyphosphate is a primordial chaperone. Mol Cell 53(5): 689-699.
  7. Groitl, B., Dahl, J. U., Schroeder, J. W. and Jakob, U. (2017). Pseudomonas aeruginosa defense systems against microbicidal oxidants. Mol Microbiol 106(3): 335-350. 
  8. Maisonneuve, E. and Gerdes, K. (2014). Molecular mechanisms underlying bacterial persisters. Cell 157(3): 539-548.
  9. Ota, S. and Kawano, S. (2017). Extraction and molybdenum blue-based quantification of total phosphate and polyphosphate in Parachlorella. Bio-protocol 7(17): e2539.
  10. Rao, N. N., Gomez-Garcia, M. R. and Kornberg, A. (2009). Inorganic polyphosphate: essential for growth and survival. Annu Rev Biochem 78: 605-647.

简介

多磷酸盐(polyP)是一种普遍保守的生物分子,由多达1,000个通过磷酸酐键连接的磷酸盐单体组成。 达到每毫克蛋白质高纳摩尔细菌的水平,polyP在生物膜形成和定植,一般应力保护和毒力中起重要作用。 已经报道了用于检测细菌中polyP的各种方案。 这些方法主要在于提取和/或检测polyP的方式不同。 在这里,我们报告了一种改进的方法,其中我们结合polyP提取通过结合到玻璃奶与非常敏感的PolyP激酶/荧光素酶检测系统。 通过使用该程序,我们显着增强了polyP检测的灵敏度,使其可能适用于哺乳动物组织。

【背景】聚磷酸盐(polyP)是一种由多达1,000种无机磷酸盐单体的直链组成的生物聚合物,存在于生命的所有三个领域的细胞中。然而,细菌是唯一已经充分研究了polyP代谢酶的生物。将ATP转化为polyP的细菌polyP激酶(PPK)催化正向和反向反应。虽然polyP的合成显然是细胞中有利的反应,但通过在体外提供足够量的ADP ,该酶可用于从polyP产生ATP,使得基于荧光素酶的ATP检测成为可能(Ault -Riché et al。,1998)。缺乏PPK的细菌在生物膜形成,运动性,持久性和各种应激反应方面存在缺陷,并显示出对次卤酸(即,漂白)应激或磷酸盐饥饿的显着增加的敏感性(图1)(Rao et al。,2009; Gray et al。,2014; Maisonneuve and Gerdes,2014; Gray和Jakob,2015; Groitl et al。,2017 )。


图1. polyP的合成及其在革兰氏阴性细菌中的作用细菌特异性多磷酸激酶(PPK)可逆地催化从ATP转化为polyP和ADP。已经在革兰氏阴性细菌中描述了polyP的各种功能,包括其参与生物膜形成,定植,运动和抗生素抗性持留细胞的形成。 PolyP还有助于细菌抵抗各种压力,包括氧化应激和饥饿,并用作金属螯合剂和Pi储库。

鉴于polyP在革兰氏阴性细菌中发挥的许多作用,PPK作为药物靶标变得具有吸引力,干扰生物膜形成,使细菌不那么持久,并使它们对生理氧化剂如漂白剂敏感(Dahl 等。 >,2017)。因此,确定体内polyP水平的可靠且灵敏的方法是必要的。在Bio-protocol中已经报道了几种提取和检测polyP的方法,包括用(i)高氯酸,(ii)次氯酸钠和(iii)苯酚/氯仿提取polyP,并通过尿素可视化检测polyP-使用孔雀石绿或钼蓝的PAGE或比色测定(Gomez Garcia,2014; Canadell 等人,2016; Ota和Kawano,2017)。在该方案中,我们通过结合玻璃乳(Ault-Riché et al。,1998)将polyP的提取与非常灵敏的两步酶基检测系统相结合。首先,通过 E将提取的polyP转化为ATP。在超纯ADP存在下的大肠杆菌 PPK。然后使用基于荧光素酶的检测系统对ATP水平进行定量,并校正细胞ATP。与尿素-PAGE或比色法相比,基于荧光素酶的检测允许定量更低水平的polyP。该方案已成功应用于量化来自铜绿假单胞菌的polyP水平。

关键字:多聚磷酸盐, 多聚磷酸盐提取方法 , 多聚磷酸盐测定, 基于荧光素酶ATP定量, 细菌毒力, 细菌应激反应

材料和试剂

  1. 微量离心管,1.5 ml,透明(BioExpress,目录号:C-3260-1)
  2. 二氧化硅膜旋转柱(Epoch Life Science,EconoSpin TM ,目录号:1920-250)
  3. TempPlate非裙边96孔PCR板,薄型,自然(USA Scientific,目录号:1402-9500)
  4. 96孔板实心白(康宁,目录号:3912)
  5. 96孔板透明平底(康宁,目录号:3596)
  6. Bradford试剂(Bio-Rad Laboratories,目录号:5000006)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A3059-100G)
  8. Tris(Fisher Scientific,目录号:BP152-5)
  9. 超纯ADP(Cell Technology,目录号:ADP100-2)
  10. 电子。大肠杆菌多磷酸激酶(PPK)(用于表达和纯化大肠杆菌 PPK,参见Gray et al。,2014)
  11. 二硫苏糖醇(DTT),&gt; 99%纯度,无蛋白酶(Gold Biotechnology,目录号:DTT25)
  12. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771-1KG)
  13. 95%v / v乙醇
  14. QuantiLum ®重组荧光素酶(Promega,目录号:E1701;等分试样的精确浓度可能不同)
    N otes:
    1. 准备酶的等分试样并储存在-80°C的小玻璃瓶中。
    2. 解冻并在每次实验中使用酶。
    3. 丢弃所有未使用的产品。
    4. 不要进行冻融循环。
  15. 硫氰酸胍(Sigma-Aldrich,目录号:G6639-250G)
  16. 二氧化硅(Sigma-Aldrich,目录号:S5631)
  17. NaCl(Fisher Scientific,目录号:S271-10)
  18. EDTA(Fisher Scientific,目录号:BP120-500)
  19. HEPES(pH 7.5)(Fisher Scientific,目录号:BP310-100)
  20. 硫酸铵(Fisher Scientific,目录号:A702-500)
  21. Tricine缓冲液(pH 7.8)(Sigma-Aldrich,目录号:T0377)
  22. MgSO 4 (赛默飞世尔科技,目录号:M63500)
  23. 叠氮化钠(MP Biomedicals,目录号:210289125)
  24. Luciferin(Biotium,目录号:10101-1)
  25. 甘氨酰甘氨酸(pH 7.8)(MP Biomedicals,目录号:210185610)
  26. 双蒸<子> 2 0
  27. 盐酸(Fisher Scientific,目录号:A144212)
  28. GITC裂解缓冲液(见食谱)
  29. Glassmilk(见食谱)
  30. 新洗(NW-)缓冲液(见食谱)
  31. 洗脱缓冲液(见食谱)
  32. PPK缓冲液(见食谱)
  33. 荧光素酶反应缓冲液(见食谱)
  34. Luciferin工作解决方案(见食谱)

设备

  1. Eppendorf移液器:100-1,000μl,20-200μl,2-20μl,1-10μl
  2. Thermomixer(Eppendorf,型号:5350)
  3. 微量离心机(Eppendorf,型号:5415D)
  4. 孵化器37°C(VWR,型号:1555)
  5. -80°C冰箱(Eppendorf,New Brunswick TM ,型号:Innova ® U725,目录号:U9440-0002)
  6. 具有发光读数功能和进样器模块的Fluostar Omega板读数器(BMG Labtech,目录号:0415-102)
  7. pH计
  8. 涡流混合器
  9. 冰箱(4°C)

软件

  1. Fluostar Omega控制和评估软件(BMG Labtech,目录号:1300-501)

程序

该方法非常适用于从革兰氏阴性PPK细菌中提取和定量polyP,包括 P.铜绿假单胞菌,在压力下积累polyP,例如,亚致死浓度的次卤酸或营养饥饿(有关生长和压力处理的详细信息,请参阅Ault-Riché等。,1998; Groitl et al。,2017)。


  1. 准备用于提取polyP的样品
    注意:由于GITC被认为具有中度毒性,因此请保留单独的GITC废液容器以便提示和流通。
    1. 收集足以产生100-300μg总细胞蛋白的细菌细胞(OD 600 = 1时约1 ml的铜绿假单胞菌培养物),并将细胞分解为13,000 xg ,4°C。
    2. 丢弃上清液。
    3. 将细胞沉淀重悬于0.25ml GITC裂解缓冲液中。
    4. 将样品在Thermomixer中在95°C孵育10分钟而不混合。
    5. 取20μl样品测定蛋白质浓度(参见步骤B2)。
    6. 冷却至室温后,将样品保存在-80℃,直至进一步操作。由于GITC使包括蛋白酶在内的蛋白质变性,因此降解无关紧要。
      注意:我们总是在7天内处理样品。

  2. 确定样品的蛋白质浓度
    1. 通过将BSA溶解在GITC裂解缓冲液中至终浓度0,0.25,0.5,1,1.5或2μg/μl来制备标准曲线。
    2. 将195μlBradford试剂与5μlBSA标准品或5μl裂解的样品混合,并在室温(RT)下在微量离心管中孵育5-10分钟。每个样品使用一式三份。
    3. 将样品转移到具有平底的96孔透明板中,并在Fluostar Omega板读数器中测量595nm处的吸光度。
    4. 如数据分析部分所述计算蛋白质浓度。样品将在最后标准化。

  3. 提取polyP
    1. 从步骤A6解冻样品并涡旋。&nbsp;
    2. 随后添加到样品中:15μl10%w / v SDS,0.5ml 95%v / v乙醇和5μl玻璃乳(参见配方)并在其间涡旋。
    3. 将样品转移至二氧化硅膜旋转柱并在微量离心机中以3,000 x g ,RT进行旋转3分钟。请注意,我们观察到流速中的颗粒形成速度高于3,000 x g 。
    4. 丢弃流通。
    5. 加入0.75毫升NW-Buffer(见食谱)。
    6. 在室温下以3,000 x g 旋转3分钟并丢弃流过物。
    7. 重复步骤C5和C6。
    8. 通过在室温下以11,000 x g 离心3分钟来干燥样品。
    9. 将柱子转移到干净的1.5ml微量离心管中。
    10. 向柱中加入50μl洗脱缓冲液,在室温下孵育15分钟,并在室温下以11,000 x g 离心3分钟洗脱polyP。
    11. 样品可以在-80°C下储存。
      注意:处理应在1-3天内完成。

  4. 将polyP转换为ATP
    1. 从步骤C11解冻样品并涡旋混合。
    2. 根据样品数量准备两个PPK Assay Master Mixes。 Master mix I( + PPK)包含50 nM的 E.线圈 PPK,而主混合物II( -PPK)则没有。没有PPK的反应用于校正在该过程中提取的细胞ATP。以下卷是指一个样本所需的数量。
      PPK Assay Master Mix I(+ PPK)(每个样品总共20μl):
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:1100px;” border =“0”cellspacing =“0”cellpadding =“2”>7.5μl
      4倍PPK缓冲液
      0.9μl
      超纯ADP [原料浓度8.4 mM(批次间可能不同);最终浓度为250μM]
      0.7μl E.大肠杆菌 PPK [原液浓度2.4μM(批次间可能不同);最终浓度为50 nM]
      10.9μl H 2 0 < br />
      PPK Assay Master Mix II(-PPK)(每个样品总共20μl):
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:1100px;” border =“0”cellspacing =“0”cellpadding =“2”>7.5μl
      4倍PPK缓冲液
      0.9μl
      超纯ADP超纯ADP [原液浓度8.4 mM(批次间可能不同);最终浓度为250μM]
      11.6μl
      H 2 0 < br />
    3. 将20μlPPKAssay Master Mix I或II与10μl样品混合,上下移液两次。
    4. 将反应在37℃下在培养箱中孵育1小时,以使PPK将polyP完全转化为ATP。
    5. 通过在热式混合器中将样品在95℃下孵育2分钟来灭活PPK。
    6. 在冰上或4℃下冷却样品,然后在11,000 x g 旋转1分钟并继续上清液。

  5. 检测ATP
    注意:其中大部分是Omega读板器特有的。根据您可用的仪器进行必要的修改。
    1. 取10μl等份的PPK消化样品(在 PPK Assay Master Mix I中孵育)和未消化的样品(在 PPK Assay Master Mix II中孵育),并将它们转移到白色平底96孔板的孔。
    2. 如下准备荧光素酶反应主混合物并保持在室温下。
      荧光素酶反应混合物(总共10毫升):
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:1100px;” border =“0”cellspacing =“0”cellpadding =“2”>0.5毫升
      20倍荧光素酶反应缓冲液
      10μl
      1 M DTT(终浓度为1 mM)(在冰上解冻)
      1毫升
      荧光素工作溶液(终浓度为100μM)
      1μl
      荧光素酶[原液浓度244μM(批次间可能不同),最终浓度为25 nM]
      8.5毫升
      H 2 0 注意:反应对温度非常敏感,因此不要将反应主混合物储存在冰上。由于荧光素酶很快失去其活性,因此在制备后15-20分钟内使用反应主混合物。
    3. Omega酶标仪的主要注射器,含2 ml Luciferase Reaction Master Mix。
    4. 将Omega读板器的温度设置为25°C,增益设置为3,000(这是OMEGA读板器特有的,可能在其他读板器中有所不同)。
    5. 向每个样品注射90μl荧光素酶反应主混合物,混合10秒快速混合并在2-3分钟内测量发光。每个周期的长度取决于样品的数量。
    6. 以与未消化的样品相同的顺序测量PPK消化的(用 PPK-Assay Master Mix I孵育)样品(用 PPK-Assay Master Mix II孵育)。在2-3分钟内测量发光。
    7. 完成后,用4 ml dH 2 O灌注清洗注射器。

数据分析

  1. 计算每个样品中的蛋白质含量:
    1. 标准曲线:将测量的吸光度与标准曲线的相应BSA浓度作图。
    2. 确定趋势线并计算线性方程。
    3. 使用趋势线的等式计算每个样品的蛋白质量。
      注意:样品的蛋白质含量应在100-300μg范围内。必要时稀释。
  2. 计算相对于未处理细胞处理的polyP的倍数变化
    1. 减去在不存在PPK的情况下孵育的样品的相对发光单位(RLU)值(用 PPK-Assay Master Mix II孵育)从孵育的相同样品的值中减去PPK的存在(与 PPK-Assay Master Mix I一起孵育),通常是第2周期。
      注意:未消化样本的RLU值代表基线。
    2. 将每个样品的所得RLU标准化为相应的蛋白质量。
    3. 通过将处理后样品的值(即,分别用HOCl,HOBr和HOSCN处理)标准化为未处理的样品,计算每个样品中polyP积累的倍数变化。


      图2. P中polyP的提取和定量。铜绿假单胞菌 PA14野生型和Δ ppk 细胞。 PA14野生型(WT)和Δ ppk 细胞生长至对数中期,并与亚致死浓度的HOCl(0.5mM),HOBr(0.15mM)或HOSCN(0.25mM)一起温育2.5小时。然后,通过与玻璃乳结合提取polyP,并在将提取的polyP转化为ATP后通过基于荧光素酶的检测系统进行定量。将得到的量标准化为各样品中的各蛋白质量。相对于未处理的细胞计算polyP水平的倍数变化。实验独立进行至少三次。数据代表平均值±S.D。 [版权所有© 2017,Wiley Online Library(Groitl et al。,2017)]。

笔记

  1. 认为polyP短链(<60Pi单位)与较长链的结合效率低于玻璃乳,并且在该提取方法期间很可能丢失。虽然细菌每链产生多达1,000个Pi分子的polyP,但真核生物中polyP的平均链长相当短。对于市售的polyP 45 ,它是不同polyP的混合物,富含polyP链,含有45个磷酸酯残基,我们使用polyP浓度测定polyP的回收率在24%和38%之间。 500 nM和10 mM。与先前描述的方法相比,先前描述的方法允许类似的恢复,然而,基于PPK的检测系统似乎更敏感(Canadell 等人,2016; Ota和Kawano,2017) 。
  2. 本方案设计用于在用亚致死浓度的次卤酸处理后或在营养物饥饿后从富含培养基中提取和定量polyP(从富含培养基转变为具有低磷酸盐和缺乏氨基酸的基本培养基)。已知这些条件导致大量长链polyP的积累(Ault-Riché et al。,1998; Gray et al。,2014)。对于含有较低polyP水平的样品,可在玻璃奶提取polyP后加入两个额外步骤,以降低PPK反应和/或ATP检测过程中干扰的风险:(i)用腺苷三磷酸双磷酸酶处理降低背景ATP水平,和( ii)Benzonase处理以去除DNA / RNA(Ault-Riché et al 。,1998)。

食谱

  1. GITC裂解缓冲液(500毫升)
    4 M硫氰酸胍
    50mM Tris-HCl(pH 7.0)
    在室温下存放长达数月的时间&nbsp;
  2. Glassmilk(25毫升)
    注意:结合容量≥100nmol多磷酸盐(以Pi当量计)/μl。 Glassmilk也会结合DNA和RNA。
    1. 将5克二氧化硅悬浮在40毫升0.1M HCl中并充分混合
    2. 通过在室温下以2,000 x g 离心5分钟来沉淀玻璃奶
    3. 去除上清液并重复步骤a和b
    4. 用10ml ddH 2 O洗涤沉淀
    5. 重复步骤d至少一次并检查pH值(pH值必须为中性)
    6. 在室温下储存长达数月
  3. 新洗(NW)缓冲液(500毫升)
    5 mM Tris-HCl(pH 7.5)
    50 mM NaCl
    5 mM EDTA
    50%v / v乙醇
    在室温下储存长达数月
  4. 洗脱缓冲液(25毫升)
    50mM Tris-HCl(pH 8)
    在室温下储存长达数月
  5. PPK缓冲液(4倍)(25毫升)
    200 mM HEPES(pH 7.5)
    200 mM硫酸铵
    20mM MgCl 2
    在室温下储存长达数月
  6. 荧光素酶反应缓冲液(20倍)(25毫升,在室温下储存几个月)
    500 mM Tricine缓冲液(pH 7.8)
    100mM MgSO 4
    2 mM EDTA
    2 mM叠氮化钠
    2 mg / ml BSA
    在室温下储存长达数月
  7. Luciferin工作溶液(50x 1 ml)
    1mM荧光素溶于25mM甘氨酰甘氨酸(pH 7.8)中 分装成1 ml等分试样并在-80°C下储存长达数月。

致谢

这项工作由美国国立卫生研究院拨款GM065318和GM116582资助到联合国。 J.-U. D.得到了Deutsche Forschungsgemeinschaft(DA 1697 / 1-1)的博士后奖学金。我们感谢Michael J. Gray博士(阿拉巴马大学)帮助建立分析和富有成效的讨论。

利益争夺

作者宣称没有利益冲突。

参考

  1. Ault-Riché,D.,Fraley,C。D.,Tzeng,C。M. and Kornberg,A。(1998)。 新型检测揭示了在大肠杆菌中调节应激诱导的无机多磷酸盐积累的多种途径。 J Bacteriol 180(7):1841-1847。
  2. Canadell,D.,Bru,S.,Clotet,J。和Ariño,J。N.(2016)。 芽殖酵母中多磷酸盐的提取和定量 Saccharomyces cerevisiae 。 Bio -protocol 6(14):e1874。\
  3. Dahl,JU,Gray,MJ,Bazopoulou,D.,Beaufay,F.,Lempart,J.,Koenigsknecht,MJ,Wang,Y.,Baker,JR,Hasler,WL,Young,VB,Sun,D。和Jakob ,U。(2017)。 抗炎药物美沙拉嗪靶向细菌多磷酸盐积累。 Nat Microbiol 2:16267。
  4. Gomez Garcia,M。R.(2014)。 通过尿素-PAGE提取和定量poly P,poly P分析。 Bio-protocol 4(9):e1113。
  5. Gray,M。J.和Jakob,U。(2015)。 多磷酸盐的氧化应激保护 - 老玩家的新角色。 Curr Opin Microbiol 24:1-6。
  6. Grey,MJ,Wholey,WY,Wagner,NO,Cremers,CM,Mueller-Schickert,A.,Hock,NT,Krieger,AG,Smith,EM,Bender,RA,Bardwell,JC和Jakob,U。(2014) 。 多磷酸盐是一种原始的伴侣。 Mol Cell 53( 5):689-699。
  7. Groitl,B.,Dahl,J.U.,Schroeder,J。W.和Jakob,U。(2017)。 铜绿假单胞菌抗微生物氧化剂的防御系统。 Mol Microbiol 106(3):335-350。&nbsp;
  8. Maisonneuve,E。和Gerdes,K。(2014年)。 细菌持久性的分子机制。 细胞 157(3 ):539-548。
  9. Ota,S。和Kawano,S。(2017年)。 提取和钼蓝量化 Parachlorella 中磷酸盐和多磷酸盐的总量。 生物协议 7(17):e2539。
  10. Rao,N.N.,Gomez-Garcia,M.R。和Kornberg,A。(2009)。 无机多磷酸盐:对生长和生存至关重要。 Annu Rev Biochem 78:605-647。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Dahl, J. U., Xie, L. and Jakob, U. (2018). Extraction and Quantification of Polyphosphate (polyP) from Gram-negative Bacteria. Bio-protocol 8(18): e3011. DOI: 10.21769/BioProtoc.3011.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。