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Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa
从绿脓杆菌中提取并量化环鸟苷二磷酸   

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参见作者原研究论文

本实验方案简略版
Journal of Bacteriology
Jun 2012

Abstract

Cyclic di-GMP (c-di-GMP) has emerged as an important intracellular signaling molecule, controlling the transitions between planktonic (free-living) and sessile lifestyles, biofilm formation, and virulence in a wide variety of microorganisms. The following protocol describes the extraction and quantification of c-di-GMP from Pseudomonas aeruginosa samples. We have made every effort to keep the protocol as general as possible to enable the procedure to be applicable for the analysis of c-di-GMP levels in various bacterial species. However, some modifications may be required for the analysis of c-di-GMP levels in other bacterial species.

Keywords: HPLC (高效液相色谱法), Biofilm (生物膜), Standard curve (标准曲线), Calibration (校准)

Materials and Reagents

  1. Bacterial culture
  2. Ethanol (95-100%)
  3. Tris base
  4. EDTA
  5. NaCl
  6. KCl
  7. Na2HPO4.7H2O
  8. KH2PO4 (pH 7.2)
  9. 0.5 M Ethylenediaminetetraacetic acid (EDTA) disodium salt solution (pH 8.0)
  10. Bis-(3'-5')-cyclic diguanylic monophosphate (c-di-GMP) (Biolog)
  11. Ammonium acetate (MS grade)
  12. Methanol (HPLC grade)
  13. Nanopure water (18 Ohm)
  14. Protein determination reagents
  15. Phosphate-buffered saline (PBS) (see Recipes)
  16. HPLC Solvent A (see Recipes)
  17. HPLC Solvent B (see Recipes)
  18. TE buffer (see Recipes)

Equipment

  1. Syringes (1 ml)
  2. Syringe filters (2 μm) (Upchurch Scientific, catalog number: B-100 )
  3. Microfuge tubes (unpolished, to reduce static)
  4. Spectrophotometer
  5. Refrigerated microcentrifuge
  6. Vacuum concentrator/Centrifugal evaporator (e.g. SpeedVac)
  7. Reverse-phase C18 Targa column (2.1 x 40 mm, 5 μm) (The Nest Group, catalog number: TR-0421-C185 )
  8. Heat block or water bath
  9. High performance liquid chromatography (HPLC) system and software for HPLC peak analysis (e.g. Agilent 1100 HPLC)
  10. Sonicator
  11. Homogenizer

Software

  1. ChemStation for LC (Agilent Technologies)

Procedure

  1. Extraction of c-di-GMP
    1. Grow bacterial cells to desired growth stage under required experimental conditions. Proceed directly with the extraction, with no waiting periods or incubation of cells on ice, as this may drastically alter the c-di-GMP levels.
      Note: Extraction at mid-exponential phase is recommended, as using early exponential phase cells will yield c-di-GMP levels too low for accurate detection. To extract c-di-GMP from planktonic cells, allow P. aeruginosa to grow in either Lennox broth or Vogel-Bonner minimal medium (VBMM) for 6 h to mid-exponential phase in flasks at 37 °C and 220 rpm. Inoculate using a 5% inoculum size of an overnight culture. Biofilm c-di-GMP levels can be determined from biofilms grown for 3-5 days under flowing conditions; see References 2-4 for more detail.
    2. Determine the optical density of the bacterial culture at 600 nm (OD600).
      Note: If working with biofilm samples, include a step to homogenize (10 sec on high) the cultures to disrupt cell aggregates prior to OD600 determination.
    3. Obtain a bacterial culture volume equivalent to 1 ml of OD600 = 1.8 (e.g. If the OD600 = 0.9, spin down 2 ml of culture).
      Note: This biomass has been optimized for the analysis of c-di-GMP in P. aeruginosa strains PAO1 and PA14 planktonic and biofilm samples. Analysis of c-di-GMP levels in other strains or species may require the initial biomass harvested for extraction to be adjusted.
    4. Centrifuge (16,000 x g, 2 min, 4 °C) the respective culture volume. Discard the supernatant.
    5. Wash the cell pellet with 1 ml ice-cold PBS (16,000 x g, 2 min, 4 °C). Discard the supernatant.
    6. Repeat step A-5.
    7. Resuspend the cell pellet in 100 μl ice-cold PBS and incubate at 100 °C for 5 min.
    8. Add ice-cold ethanol (stored at -20 °C until use) to a final concentration of 65% (186 μl of 100% ethanol or 217 μl of 95% ethanol) and vortex for 15 sec.
    9. Centrifuge sample (16,000 x g, 2 min, 4 °C), and remove and retain the supernatant containing extracted c-di-GMP in a new microfuge tube. Store the supernatant on ice or at -80 °C until step A-10. Retain the cell pellet.
    10. Using the cell pellet, repeat twice the extraction procedure in steps A-7~A-9. Pool the supernatants obtained from the three extractions into one microfuge tube. Retain the cell pellet after the final extraction step. The cell pellet can be stored at -20 °C until step B-3-a.
    11. Dry the combined supernatants using a vacuum concentrator/centrifugal evaporator. Following evaporation, a white pellet should be visible. This sample, containing the extracted c-di-GMP, can be stored at -80 °C until step B-2-a.

  2. Quantification of c-di-GMP
    This procedure has been optimized for the detection of c-di-GMP using an Agilent 1100 HPLC equipped with an autosampler, degasser, pressure regulator, prefilter, and UV/Vis detector set to 253 nm. Separation was carried out using a reverse-phase C18 Targa column (2.1 x 40 mm; 5 μm) and a flow rate of 0.2 ml/min. Solvents containing methanol and ammonium acetate (see Recipes for solvents A and B) were used. The following gradient was used to elute c-di-GMP: 0 to 9 min, 1% B ( = 1% solvent B and 99% solvent A); 9 to 14 min, 15% B; 14 to 19 min, 25% B; 19 to 26 min, 90% B; 26 to 40 min, 1% B. This gradient resulted in the elution of c-di-GMP at approximately 14-15 min.
    Note: Analysis of c-di-GMP levels using a different reverse-phase column, flow rate, or HPLC system may require the optimization of HPLC separation gradients.
    1. Generation of a standard curve
      1. Using commercially available c-di-GMP, prepare the following standards in nanopure water: 1, 2, 5, 10 and 20 pmo/μl.
      2. To generate the standard curve, inject 20 μl per standard per HPLC run. Use 20 μl of nanopure water as a negative control (0 pmol μl-1 c-di-GMP). For an example of c-di-GMP detection (Figure 1).


        Figure 1. Example of c-di-GMP standard elution profile. 20 μl of a 10 pmo/μl c-di-GMP standard (200 pmol total) were separated using a reverse-phase C18 Targa column (2.1 x 40 mm; 5 μm) at a flow rate of 0.2 ml/min with the following gradient: 0 to 9 min, 1% B; 9 to 14 min, 15% B; 14 to 19 min, 25% B; 19 to 26 min, 90% B; 26 to 40 min, 1% B. Peak at 15 min corresponds to the elution of c-di-GMP. The c-di-GMP peak was found to have an area of 935.7.

      3. Prepare a standard curve by plotting the c-di-GMP amount in pmol (e.g. 200 pmol for the 20 μl of the 10 pmol/μl standard) vs the peak areas. An example of a standard curve is given in Figure 2. Peak areas can be determined using various programs, including ChemStation for LC, which was used here.


        Figure 2. Example of a c-di-GMP HPLC standard curve. The standard curve was generated by plotting the peak areas obtained following the separation of 20 μl aliquots of c-di-GMP standards (here: 0, 1, 2, 5, and 10 pmo/μl) versus the total c-di-GMP amounts in pmol. Peak areas were obtained using the ChemStation for LC software.

    2. Analysis of samples
      1. Resuspend the dried extracts from step A-11 in 200 μl nanopure water and vortex for 1 min.
      2. Centrifuge the solution at max speed (≥ 16,000 x g) to remove insoluble material.
      3. Using a 2 μm HPLC syringe filter attached to a 1 ml syringe, filter the sample supernatants into a new microfuge tube.
        Note: Small sample volume loss may occur, but will not interfere with downstream application, as only a limited sample volume (20 μl) is subjected to HPLC analysis.
      4. Analyze 20 μl per sample using the HPLC program used for the standards above. Repeat using biological replicates.
        Note: This procedure has been optimized for P. aeruginosa PAO1 and PA14. Analysis of c-di-GMP levels in other strains or species may require the adjustment of sample volumes.
      5. Determine the peak area for each sample and determine the c-di-GMP amounts using the standard curve established with the commercially available c-di-GMP in step B-1-c.
    3. Calculations
      To normalize the c-di-GMP levels, total cellular protein levels from the extraction procedure must be determined.
      1. Resuspend the cell pellet from step A-10 in 500 μl TE buffer.
      2. Sonicate the suspension for a total of 1 min using 10 sec bursts at 5 W followed by 15 sec off. Sonication should be carried out on ice.
      3. Determine the protein concentrations using a protein determination assay.
      4. Normalize the c-di-GMP levels to total cellular protein levels (i.e. pmol/mg) using the following calculations:
        1. Total c-di-GMP in pmol
          = (pmol c-di-GMP) x 10
          A factor of 10 is used here, as only 1/10th of the c-di-GMP extract (20 μl out of 200 μl) was used for HPLC analysis
        2. Total protein in mg
          = (mg/ml protein) * 0.5 ml
          →Normalized c-di-GMP (pmol/mg)
          = Total c-di-GMP/Total protein

Recipes

  1. PBS
    137 mM NaCl
    2.7 mM KCl
    4.3 mM Na2HPO4·7H2O
    1.4 mM KH2PO4 (pH 7.2)
  2. HPLC Solvent A
    10 mM ammonium acetate in water
    Do not adjust pH
  3. HPLC Solvent B
    10 mM ammonium acetate in methanol
    Dissolve ammonium acetate salt in methanol
    Do not adjust pH
  4. TE buffer
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA

Acknowledgments

The c-di-GMP extraction procedure using heat and ethanol is based on previously published protocols (Amikam et al., 1995; Simm et al., 2004). The HPLC-based method for the detection and quantitation of c-di-GMP is a modification of protocols published by Thormann and Spormann (Thormann et al., 2006) and Ueda and Wood (2009). This work was supported by a grant from NIH (1RO1 A107525701A2).

References

  1. Amikam, D., Steinberger, O., Shkolnik, T. and Ben-Ishai, Z. (1995). The novel cyclic dinucleotide 3'-5' cyclic diguanylic acid binds to p21ras and enhances DNA synthesis but not cell replication in the Molt 4 cell line. Biochem J 311 ( Pt 3): 921-927
  2. Morgan, R., Kohn, S., Hwang, S. H., Hassett, D. J. and Sauer, K. (2006). BdlA, a chemotaxis regulator essential for biofilm dispersion in Pseudomonas aeruginosa. J Bacteriol 188(21): 7335-7343
  3. Petrova, O. E., Schurr, J. R., Schurr, M. J. and Sauer, K. (2012). Microcolony formation by the opportunistic pathogen Pseudomonas aeruginosa requires pyruvate and pyruvate fermentation. Mol Microbiol 86(4): 819-835.
  4. Simm, R., Morr, M., Kader, A., Nimtz, M. and Romling, U. (2004). GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53(4): 1123-1134.
  5. Thormann, K. M., Duttler, S., Saville, R. M., Hyodo, M., Shukla, S., Hayakawa, Y. and Spormann, A. M. (2006). Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP. J Bacteriol 188(7): 2681-2691.
  6. Ueda, A. and Wood, T. K. (2009). Connecting quorum sensing, c-di-GMP, pel polysaccharide, and biofilm formation in Pseudomonas aeruginosa through tyrosine phosphatase TpbA (PA3885). PLoS Pathog 5(6): e1000483.

简介

环状di-GMP(c-di-GMP)已经作为重要的细胞内信号传导分子出现,控制在各种微生物中浮游生物(自由生命)和固着的生活方式,生物膜形成和毒力之间的转变。 以下方案描述了从绿脓假单胞菌样品中提取和定量c-di-GMP。 我们已尽一切努力使方案尽可能一般,以使该程序适用于分析各种细菌物种中的c-di-GMP水平。 然而,对于其他细菌物种中c-di-GMP水平的分析可能需要一些修饰。

关键字:高效液相色谱法, 生物膜, 标准曲线, 校准

材料和试剂

  1. 细菌培养
  2. 乙醇(95-100%)
  3. Tris碱
  4. EDTA
  5. NaCl
  6. KCl
  7. Na2HPO4 7H2O
  8. KH2PO4(pH7.2)
  9. 0.5M乙二胺四乙酸(EDTA)二钠盐溶液(pH8.0)
  10. 双 - (3'-5') - 环二磷酸单磷酸酯(c-di-GMP)(Biolog)
  11. 乙酸铵(MS级)
  12. 甲醇(HPLC级)
  13. 纳米纯水(18欧姆)
  14. 蛋白质测定试剂
  15. 磷酸盐缓冲盐水(PBS)(见配方)
  16. HPLC溶剂A(参见配方)
  17. HPLC溶剂B(参见配方)
  18. TE缓冲区(参见配方)

设备

  1. 注射器(1 ml)
  2. 注射器过滤器(2μm)(Upchurch Scientific,目录号:B-100)
  3. Microfuge管(未抛光,以减少静电)
  4. 分光光度计
  5. 冷藏微量离心机
  6. 真空浓缩器/离心蒸发器(如 SpeedVac)
  7. 反相C18Targa柱(2.1×40mm,5μm)(Nest Group,目录号:TR-0421-C185)
  8. 热块或水浴
  9. 高效液相色谱(HPLC)系统和用于HPLC峰分析的软件(例如Agilent 1100 HPLC)
  10. 超声波仪
  11. 均质器

软件

  1. LC化学工作站(安捷伦科技公司)

程序


I.   c-di-GMP的提取

  1. 在所需的实验条件下将细菌细胞生长至所需的生长阶段。直接进行提取,没有等待期或细胞在冰上孵育,因为这可能会大大改变c-di-GMP水平。
    注意:推荐在中指数期提取,因为使用早期指数期细胞将产生c-di-GMP水平太低而无法进行精确检测。为了从浮游细胞提取c-di-GMP,允许铜绿假单胞菌在Lennox肉汤或Vogel-Bonner基本培养基(VBMM)中在37℃和220rpm下在烧瓶中生长6小时至中指数期。使用5%接种物大小的过夜培养物接种。生物膜c-di-GMP水平可以从在流动条件下生长3-5天的生物膜确定;有关更多详细信息,请参阅参考文献2-4。
  2. 确定细菌培养物在600nm的光密度(OD600)。
    注意:如果使用生物膜样品,包括在OD600测定之前将培养物匀浆(高10秒钟)以破坏细胞聚集体的步骤。
  3. 获得相当于1ml OD 600 = 1.8的细菌培养物体积(例如,如果OD 600 = 0.9,旋转2ml培养物)。
    注意:该生物质已经被优化用于分析铜绿假单胞菌菌株PAO1和PA14浮游和生物膜样品中的c-di-GMP。在其他菌株或物种中的c-di-GMP水平的分析可能需要调整用于提取的初始生物量。
  4. 离心(16,000×g,2分钟,4℃)相应的培养物体积。弃去上清液。
  5. 用1ml冰冷的PBS(16,000×g,2分钟,4℃)洗涤细胞沉淀。 弃去上清液。
  6. 重复步骤A-5。
  7. 重悬细胞沉淀在100μl冰冷的PBS中,并在100℃孵育5分钟
  8. 加入冰冷的乙醇(储存于-20℃直至使用)至最终浓度为65%(186μl的100%乙醇或217μl的95%乙醇)并涡旋15秒。
  9. 离心样品(16,000×g,2分钟,4℃),并且在新的微量离心管中除去并保留含有提取的c-di-GMP的上清液。 将上清液保存在冰上或-80℃下,直到步骤A-10。 保持细胞沉淀。
  10. 使用细胞沉淀,重复步骤A-7〜A-9中的提取程序两次。 将从三次提取获得的上清液混合到一个微量离心管中。 在最后的提取步骤后保留细胞沉淀。 细胞沉淀可以储存在-20℃直到步骤B-3-a。
  11. 使用真空浓缩器/离心蒸发器干燥合并的上清液。 蒸发后,白色沉淀应可见。 包含提取的c-di-GMP的该样品可以储存在-80℃直到步骤B-2-a。


II。  c-di-GMP的定量

这个程序已经优化用于使用a的c-di-GMP的检测   Agilent 1100 HPLC,配备自动进样器,脱气器,压力 调节器,预滤器和设置为253nm的UV/Vis检测器。 分离 使用反相C 18 Targa柱(2.1×40mm;5μm)进行, 和流速为0.2ml/min。 含甲醇和铵的溶剂 (参见溶剂A和B的配方)。 下列 梯度用于洗脱c-di-GMP:0至9分钟,1%B(= 1%溶剂B) 和99%溶剂A); 9至14分钟,15%B; 14至19分钟,25%B; 19〜26 min,90%B; 26至40分钟,1%B。该梯度导致洗脱   c-di-GMP在约14-15分钟。

注意:使用不同的反相柱,流速或HPLC系统分析c-di-GMP水平可能需要优化HPLC分离梯度。

  1. 生成标准曲线
    1. 使用市售的c-di-GMP,在纳米纯水中制备以下标准:1,2,5,10和20 pmo /μl。
    2. 为了产生标准曲线,每个HPLC运行注射20μl每标准。 使用20微升纳米纯水作为阴性对照(0 pmolμl -1 c-di-GMP)。 对于c-di-GMP检测的实例(图1)。


      图1.c-di-GMP标准洗脱图谱的实施例使用反相C 分离20μl的10 pmo /μlc-di-GMP标准品(总共200pmol) min,流速为0.2ml/min,梯度为0至9分钟,1%B;流速为0.2ml/min。 9至14分钟,15%B; 14至19min,25%B; 19至26分钟,90%B; 26至40分钟,1%B。15分钟的峰对应于c-di-GMP的洗脱。发现c-di-GMP峰的面积为935.7
    3. 通过绘制c-di-GMP量(以pmol计)(例如 200pmol对于20pm 10pmol /μl标准物)峰面积来制备标准曲线。在图2中给出了标准曲线的实例。峰面积可以使用各种程序确定,包括用于LC的化学工作站,其在此使用。


      图2.c-di-GMP HPLC标准曲线的实施例通过绘制在c-di-GMP标准品的20μl等分试样分离后获得的峰面积(这里: 0,1,2,5和10 pmo /μl)相对于以pmol计的总c-di-GMP量。使用ChemStation for LC软件获得峰面积。

  2. 样品分析
    1. 将来自步骤A-11的干燥的提取物重悬在200μl纳米纯水中并涡旋1分钟
    2. 以最大速度(≥16,000×g)离心溶液以除去不溶物质。
    3. 使用连接到1ml注射器的2μmHPLC注射器过滤器,将样品上清液过滤到新的微量离心管中。
      注意:样品体积可能会减少,但不会影响下游应用,因为只有有限的样品体积(20μl)可用于HPLC分析。
    4. 使用用于上述标准的HPLC程序分析每个样品20μl。使用生物复制重复。
      注意:这个程序已经针对铜绿假单胞菌PAO1和PA14进行了优化。对其他菌株或物种中c-di-GMP水平的分析可能需要调整样品体积。
    5. 确定每个样品的峰面积,并使用在步骤B-1-c中使用市售c-di-GMP建立的标准曲线确定c-di-GMP量。
  3. 计算
    为了使c-di-GMP水平正常化,必须测定来自提取程序的总细胞蛋白水平
    1. 将步骤A-10的细胞沉淀重悬在500μlTE缓冲液中。
    2. 超声处理悬浮液共1分钟,使用10秒爆发在5W,然后15秒关闭。 超声应该在冰上进行。
    3. 使用蛋白质测定测定法确定蛋白质浓度。
    4. 使用以下计算将c-di-GMP水平标准化为总细胞蛋白水平(i pmol/mg):
      1. 总c-di-GMP以pmol计
        =(pmol c -di-GMP)×10
        在此使用因子10,因为只有1/10的c-di-GMP提取物(200μl中的20μl)用于HPLC分析。
      2. 总蛋白(mg)
        =(mg/ml蛋白质)* 0.5ml
        →归一化c-di-GMP(pmol/mg)
        =总c-di-GMP /总蛋白质

食谱

  1. PBS
    137 mM NaCl 2.7 mM KCl
    4.3mM Na 2 HPO 4 7H·7H 2 O 1.4mM KH 2 PO 4(pH 7.2)
  2. HPLC溶剂A
    10mM乙酸铵水溶液
    不要调整pH值
  3. HPLC溶剂B
    10mM乙酸铵的甲醇溶液
    将乙酸铵盐溶于甲醇中 不要调整pH值
  4. TE缓冲区
    10mM Tris-HCl(pH8.0) 1mM EDTA

致谢

使用热和乙醇的c-di-GMP提取程序基于先前公开的方案(Amikam等人,1995; Simm等人,2004)。 用于检测和定量c-di-GMP的基于HPLC的方法是Thormann和Spormann(Thormann等人,2006)和Ueda和Wood(2009)公布的方案的修改。 这项工作得到了NIH(1RO1 A107525701A2)的资助。

参考文献

  1. Amikam,D.,Steinberger,O.,Shkolnik,T。和Ben-Ishai,Z。(1995)。 新的环状二核苷酸3'-5'环状二乙酸与p21ras结合,增强DNA合成,但不能 在Molt 4细胞系中的细胞复制。 Biochem J 311(Pt 3):921-927
  2. Morgan,R.,Kohn,S.,Hwang,S.H.,Hassett,D.J。和Sauer,K。(2006)。 BdlA,一种对绿脓假单胞菌中生物膜分散必不可少的趋化性调节剂。 J Bacteriol 188(21):7335-7343
  3. Petrova,O.E.,Schurr,J.R.,Schurr,M.J.and Sauer,K。(2012)。 机会性病原体铜绿假单胞菌形成小菌落需要丙酮酸和丙酮酸的发酵。 Mol Microbiol 86(4):819-835。
  4. Simm,R.,Morr,M.,Kader,A.,Nimtz,M.and Romling,U.(2004)。 GGDEF和EAL结构域逆向调节循环di-GMP水平,并从无生命转变为运动。 Mol Microbiol 53(4):1123-1134。
  5. Thormann,K.M.,Duttler,S.,Saville,R.M.,Hyodo,M.,Shukla,S.,Hayakawa,Y.and Spormann,A.M。 控制希瓦氏菌病的形成和细胞分离 nsis MR-1 biofilms by cyclic di-GMP。 J Bacteriol 188(7):2681-2691。
  6. Ueda,A。和Wood,T.K。(2009)。 在假单胞菌中连接群体感应,c-di-GMP,荚膜多糖和生物膜形成 通过酪氨酸磷酸酶TpbA(PA3885) uginos (6):e1000483。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Roy, A. B., Petrova, O. E. and Sauer, K. (2013). Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa. Bio-protocol 3(14): e828. DOI: 10.21769/BioProtoc.828.
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jayalekshmi haripriyan
Amrita School of Biotechnology
In this protocols what is the volume of bacterial growth media used for the cultivation of P.aeruginosa
8/13/2014 9:31:10 PM Reply