An HPLC-based Method to Quantify Coronatine Production by Bacteria

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Frontiers in Plant Science
Jun 2016


Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Keywords: Coronatine (冠菌素), HPLC (HPLC), Pseudomonas syringae (丁香假单胞菌), Plant pathogen (植物病原体), Virulence factor (毒力因子)


Coronatine (COR), a potent bacterial phytotoxin, is a molecular mimic of the plant hormone jasmonoyl-L isoleucine (JA-Ile). As such, COR activates jasmonic acid (JA) signaling, induces JA-responsive genes, and antagonizes the action of the immune signal salicylic acid. COR consists of two components, coronafacic acid (CFA) and coronamic acid (CMA). The genes that encode for CMA and CFA biosynthesis are not constitutively expressed in the bacterium. Instead, these genes are induced on the plant leaf surface, in planta or in vitro when the bacterium is grown in inducing medium (Palmer and Bender, 1993; Panchal et al. 2016). This article describes a method adapted from Panchal et al. (2016) to determine the ability of bacteria to produce coronatine, which can be used as an indication of virulence.

Materials and Reagents

  1. 1.5 ml microfuge tubes (VWR, catalog number: 20170-650 )
  2. Centrifuge tubes, polypropylene, sterile, 50 ml (Corning, Falcon®, catalog number: 352070 )
  3. Micropipettes (Mettler-Toledo, Rainin, catalog number: KitPR-START )
  4. Petri dishes, 100 x 15 mm (VWR, catalog number: 25384-088 )
  5. Plastic bags
  6. Square cuvette, 10 mm path length (Thermo Fisher Scientific, catalog number: 331709 )
  7. Pasteur pipette (VWR, catalog number: 14673-010 )
  8. Pseudomonas syringae bacterial culture (can be maintained in 25% glycerol at -80 °C)
  9. Coronatine (Sigma-Aldrich, catalog number: C8115 )
  10. Glycerol (VWR, BDH®, catalog number: BDH1172-1LP )
  11. Sterile distilled ultrapure water
  12. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 )
  13. Acetonitrile (Sigma-Aldrich, catalog number: 34998 )
  14. Rifampicin
  15. 3 N HCl (VWR, BDH®, catalog number: BDH7375-1 )
  16. Ethyl acetate (VWR, BDH®, catalog number: BDH1123-4LG )
  17. Sodium hydroxide (NaOH) (Fisher Scientific, catalog number: S318-1 )
  18. BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
  19. Tryptone (IBI Scientific, catalog number: 41116105 )
  20. Yeast extract (U.S. Biotech Sources, BTS, catalog number: Y01PD-500 )
  21. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
  22. Bacteriological agar (IBI Scientific, catalog number: IB49171 )
  23. Ammonium chloride (NH4Cl) (VWR, catalog number: 470300-196 )
  24. Magnesium sulfate heptahydrate (MgSO4·7H2O) (VWR, catalog number: 97062-134 )
  25. Potassium phosphate (KH2PO4) (VWR, catalog number: 97062-350 )
  26. Potassium hydrogen phosphate trihydrate (K2HPO4·3H2O) (VWR, catalog number: ALFA33365.A1 )
  27. Potassium nitrate (KNO3) (VWR, catalog number: 97065-038 )
  28. Iron(III) chloride (FeCl3) (VWR, catalog number: 470301-584 )
  29. Glucose (VWR, catalog number: 101094-092 )
  30. Low-sodium Luria Bertani medium (see Recipes)
  31. Liquid HSC medium (see Recipes)


  1. HPLC system: Agilent 1200 HPLC (Agilent Technologies, model: Agilent 1200 HPLC) equipped with quaternary pump, autosampler, thermostated column compartment, and a diode array
  2. C8 column, 4.6 x 250 mm, 5 µm (ASTEC, Whippany, NJ, USA or equivalent)
  3. Shaker incubator (Eppendorf, model: New BrunswickTM Innova® 42R )
  4. Spectrophotometer (Thermo Fisher Scientific, Thermo Scientific, model: Spectronic 20D+ )
  5. Microcentrifuge (Eppendorf, model: 5418 )
  6. Water bath (Thermo Fisher Scientific, Thermo ScientificTM, model: Precision GP 2S )
  7. Vortex (VWR, catalog number: 945300 )
  8. Autoclave (VWR, catalog number: 97002-402 )
  9. Laminar flow hood (ESCO, model: AC1-4E8 )
  10. Centrifuge (Eppendorf, model: 5810 )
  11. Synergy water purification system (EMD Millipore, catalog number: SYNS0HFWW )


  1. Chromatography data system software (Agilent Technologies, Palo Alto, CA)
  2. Microsoft Excel


  1. Calibration curve
    1. Calibration curves must be created using purified COR to determine the exact concentration of COR in biological samples. Prepare five COR solutions with different concentrations (5-50 µg/ml) in 1.5 ml microfuge tubes; so that 5 µl of the solutions represents a range of COR amounts between 25-250 ng. COR is detected by HPLC at 208 nm using a C8 column (4.6 x 250 mm, 5 µm). Isocratic separations are performed using a 0.05% TFA:acetonitrile (60:40 v/v) mobile phase with a flow rate of 1 ml/min. The column temperature is set at 25 °C. Inject 5 µl aliquots of purified COR into the HPLC C8 column. Pure coronatine must produce a peak at 9.4 min.
    2. Using the chromatography data system software, integrate the area under the peak for every concentration of COR. Plot a graph of peak area versus ng of coronatine injected as shown in Figure 1. Calculate the linear equation of the trend line obtained using linear regression analysis.

      Figure 1. Standard curve for 5-50 μg/ml of coronatine (COR). Purified COR was injected in different concentrations and the peak area (in arbitrary units) for each injection was quantified with the chromatography data system software. Note that the red error bars for each data point are very small and do not clearly appear in the graph.

  2. Determining bacterial production of COR
    1. From the glycerol stock, streak the bacterial culture on low-salt Luria Bertani (LS-LB) medium supplemented with appropriate antibiotic and incubate the medium plate at 28 °C for approximately 30 h. For example, 100 µg/ml rifampicin is used to culture COR-producing Pseudomonas syringae pv. tomato DC3000.
    2. Inoculate a small part of a single colony (just touch with tip) from this fresh plate into 10 ml of LS-LB medium with appropriate antibiotic in a 50 ml tube and incubate at 28 °C and 200 rpm. Grow until mid to late log phase is reached (OD600 of 0.8). It will take approximately 10-12 h. Check optical density (OD) at 600 nm using a spectrophotometer.
    3. Centrifuge the bacterial suspension at 2,600 x g for 20 min in 50 ml tube. Remove the supernatant and re-suspend the pellets in 10 ml of sterile water. Vortex thoroughly to wash the cells. Vortex continuously at full speed until the cells are completely re-suspended and there is no visible cell clumps. Repeat this step twice. Finally, re-suspend the total bacterial cells in 40 ml sterile water to obtain a 0.2 OD600 inoculum that corresponds to 1 x 108 cells for Pseudomonas syringae pv. tomato. The calculated approximation of the OD600 is sufficient.
    4. Transfer 1 ml from this culture to 10 ml of HSC medium in a 50 ml tube and incubate for 24 h at 18 °C and 200 rpm. More volume of culture can be used if greater concentrations of COR are required. However, 1 ml is adequate for COR detection. From 10 ml of bacterial culture, one can obtain a concentration of COR in the range of 0.5-2 µg/mg of total protein.
    5. After 24 h, transfer 0.6 ml of culture to microfuge tubes. Spin at a maximum speed (or 16,000 x g) at room temperature in a microcentrifuge for 3 min to pellet cells.
    6. Transfer 0.5 ml of supernatant to fresh microfuge tubes and store pelleted cells at -80 °C. These cells are used to estimate total protein content as described in step B14 below.
    7. To the supernatant, add 25 µl of 3 N HCl and 0.5 ml ethyl acetate to each tube. Vortex the tubes briefly for 30 sec at maximum speed. Allow phases to separate. The phases should separate out within two minutes.
    8. Transfer 0.4 ml of the ethyl acetate phase (upper layer) to a fresh microfuge tube.
    9. Repeat steps B7-B8 twice (Figure 2).

      Figure 2. Flowchart of the COR extraction. Steps for extracting COR from bacterial culture supernatant using ethyl acetate. The three ethyl acetate phases are combined in a single tube and let dry. Dried COR is dissolved in TFA:acetonitrile and this solution is now ready for injection into HPLC.

    10. Dry contents of tubes under a stream of compressed air. One method is to float tubes in a 55 °C water bath, using a Pasteur pipette to gently blow air into each tube (see Figure 3 for a representative setup). Alternatively, tubes may be left open on the bench at room temperature overnight.

      Figure 3. Set up for drying the contents of microfuge tubes in a water bath. After COR is extracted from bacterial culture with ethyl acetate, the ethyl acetate is evaporated by floating the tube on water bath set at 55 °C and blowing air using Pasteur pipette. Alternatively, the tubes can be left open to dry overnight.

    11. For HPLC analysis, add 250 µl of 0.05% TFA:acetonitrile (90:10 v/v) to each dry tube. Vortex each tube for 15 sec. Samples are now ready for direct injection into the HPLC system.
    12. Determine the presence of COR by HPLC using a C8 column (4.6 x 250 mm, 5 µm) with detection at 208 nm. Isocratic separations are performed using a 0.05% TFA:acetonitrile (60:40 v/v) mobile phase with a flow rate of 1 ml/min. The injection volume is 5 µl and the column temperature is set at 25 °C.
    13. Inject the unknown sample into the HPLC system. If a peak is observed at 9.4 min, determine the area under the peak. Using the peak area, apply the standard curve and linear equation to determine the ng of coronatine injected in 5 µl. Then, calculate µg/ml coronatine for the sample.
    14. The amount of COR produced can be expressed as a function of protein concentration. The cells obtained in step B6 are lysed by suspending the pellet in 1 ml of 1 N NaOH followed by boiling and freezing three times. The total protein content in bacterial cell lysates is determined with the Pierce BCA Protein Assay Kit according to manufacturer’s instructions. Express COR concentration as µg/mg protein as shown by Panchal et al., (2016).

Data analysis

  1. The COR-producing bacterium P. syringae pv. tomato strain DC3000 and its derivative COR-defective mutant strains DC3118 or DB29 must be used as positive and negative controls for this assay, respectively.
  2. At least three technical replicates and three biological replicates should be used for quantification of COR. Statistical analysis should be done by calculating the average and standard error of COR production across replicates. Significance of the difference between two samples can be obtained by performing a Student’s t-test. These analyses and linear regression can be done with Microsoft Excel or other comparable statistical analysis software.


  1. Always use bacterial pathogen isolates that have been freshly streaked from glycerol stock. Multiple sub-culturing of isolates may lead to loss of virulence and COR production. Ideally, the bacterium pathogen is isolated from the plant host every two years.
  2. At least 24 h of incubation in HSC medium is required for consistent production of COR.


  1. Low-sodium Luria Bertani medium
    10 g/L tryptone
    5 g/L yeast extract
    5 g/L NaCl
    2.5% agar
    Adjust pH to 7.0 with NaOH
    Autoclave medium at 15 psi, 120 °C for 15 min. Allow the medium to cool down to about 55 °C and add appropriate antibiotic as needed. Pour medium into plates in a laminar flow hood. Store plates in plastic bags at 4 °C to avoid medium dehydration
  2. Liquid HSC medium
    Part A:
    1.0 g NH4Cl
    0.2 g MgSO4·7H2O
    4.1 g KH2PO4
    3.6 g K2HPO4·3H2O
    0.3 g KNO3
    10 ml 2 mM FeCl3
    890 ml ddH2O
    Adjust pH to 6.8
    Autoclave at 10 psi, 120 °C for 20 min
    Part B:
    20 g glucose
    100 ml ddH2O
    Filter-sterilize the solution
    Store parts A and B separately at 4 °C. Mix part A and part B (9:1 ratio) right before use


We thank Dr. Carol Bender for useful advice on the coronatine extraction method. This study was supported by a grant from the US National Institute of Allergy and Infectious Disease (5R01AI068718) to Dr. Maeli Melotto.


  1. Palmer, D. A. and Bender, C. L. (1993). Effects of environmental and nutritional factors on production of the polyketide phytotoxin coronatine by Pseudomonas syringae pv. glycinea. Appl Environ Microbiol 59(5): 1619-1626.
  2. Panchal, S., Roy, D., Chitrakar, R., Price, L., Breitbach, Z. S., Armstrong, D. W. and Melotto, M. (2016). Coronatine facilitates Pseudomonas syringae infection of Arabidopsis leaves at night. Front Plant Sci 7: 880.



背景 Coronatine(COR)是有效的细菌植物毒素,是植物激素茉莉酮酸-L异亮氨酸(JA-Ile)的分子模拟物。因此,COR激活茉莉酸(JA)信号传导,诱导JA响应基因,并拮抗免疫信号水杨酸的作用。 COR由两种成分组成:冠心病(CFA)和冠状氨酸(CMA)。编码CMA和CFA生物合成的基因在细菌中不是组成型表达的。相反,当细菌在诱导培养基中生长时,这些基因在植物叶片表面或植物体内或/或体外诱导(Palmer和Bender,1993; Panchal等等,2016)。本文介绍了从Panchal等人改编的方法。 (2016),以确定细菌产生冠心病的能力,可用作毒性指征。

关键字:冠菌素, HPLC, 丁香假单胞菌, 植物病原体, 毒力因子


  1. 1.5ml微量离心管(VWR,目录号:20170-650)
  2. 离心管,聚丙烯,无菌,50ml(Corning,Falcon ®,目录号:352070)
  3. 微量移液器(Mettler-Toledo,Rainin,目录号:KitPR-START)
  4. 培养皿,100 x 15毫米(VWR,目录号:25384-088)
  5. 塑料袋
  6. 方形比色皿,10 mm路径长度(Thermo Fisher Scientific,目录号:331709)
  7. 巴斯德移液器(VWR,目录号:14673-010)
  8. 细菌培养物(可在-80℃下保持在25%甘油中)
  9. 冠心病(Sigma-Aldrich,目录号:C8115)
  10. 甘油(VWR,BDH ,目录号:BDH1172-1LP)
  11. 无菌蒸馏超纯水
  12. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:302031)
  13. 乙腈(Sigma-Aldrich,目录号:34998)
  14. 利福平
  15. 3 N HCl(VWR,BDH ®,目录号:BDH7375-1)
  16. 乙酸乙酯(VWR,BDH ,目录号:BDH1123-4LG)
  17. 氢氧化钠(NaOH)(Fisher Scientific,目录号:S318-1)
  18. BCA蛋白测定试剂盒(Thermo Fisher Scientific,Thermo Scientific TM,目录号:23225)
  19. Tryptone(IBI Scientific,目录号:41116105)
  20. 酵母提取物(U.S.Biotech Sources,BTS,目录号:Y01PD-500)
  21. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-500)
  22. 细菌琼脂(IBI Scientific,目录号:IB49171)
  23. 氯化铵(NH 4 Cl)(VWR,目录号:470300-196)
  24. 七水硫酸镁(MgSO 4·7H 2 O)(VWR,目录号:97062-134)
  25. 磷酸钾(KH 2 PO 4)(VWR,目录号:97062-350)
  26. 磷酸氢二钾三水合物(K 2 HPO 4·3H 2 O)(VWR,目录号:ALFA33365.A1)
  27. 硝酸钾(KNO 3)(VWR,目录号:97065-038)
  28. 氯化铁(III)(FeCl 3)(VWR,目录号:470301-584)
  29. 葡萄糖(VWR,目录号:101094-092)
  30. 低钠Luria Bertani培养基(见食谱)
  31. 液体HSC介质(见配方)


  1. HPLC系统:配有四元泵,自动进样器,恒温柱室和二极管阵列的Agilent 1200 HPLC(Agilent Technologies,型号:Agilent 1200 HPLC)
  2. C8柱,4.6×250mm,5μm(ASTEC,Whippany,NJ,USA或等同物)
  3. Shaker孵化器(Eppendorf,型号:New Brunswick TM Innova ® 42R)
  4. 分光光度计(Thermo Fisher Scientific,Thermo Scientific,型号:Spectronic 20D +)
  5. 微量离心机(Eppendorf,型号:5418)
  6. 水浴(Thermo Fisher Scientific,Thermo Scientific TM,型号:Precision GP 2S)
  7. 涡旋(VWR,目录号:945300)
  8. 高压釜(VWR,目录号:97002-402)
  9. 层流罩(ESCO,型号:AC1-4E8)
  10. 离心机(Eppendorf,型号:5810)
  11. 协同净水系统(EMD Millipore,目录号:SYNS0HFWW)


  1. 色谱数据系统软件(Agilent Technologies,Palo Alto,CA)
  2. Microsoft Excel


  1. 校准曲线
    1. 必须使用纯化的COR产生校准曲线,以确定生物样品中COR的精确浓度。在1.5ml微量离心管中制备不同浓度(5-50μg/ml)的五种COR溶液;使得5μl的溶液代表在25-250ng之间的COR量的范围。使用C8柱(4.6×250mm,5μm)通过HPLC在208nm检测到COR。使用流速为1ml/min的0.05%TFA:乙腈(60:40v/v)流动相进行等度分离。柱温度设定在25℃。将5μl等分的纯化的COR注入HPLC C8柱。纯冠心病必须在9.4分钟内产生峰值
    2. 使用色谱数据系统软件,将每个浓度的COR峰值下的面积进行整合。绘制如图1所示的冠状面积相对于冠状动脉注射的ng图。计算使用线性回归分析获得的趋势线的线性方程。


  2. 确定COR的细菌生产
    1. 从甘油储备液中,在补充有适当抗生素的低盐Luria Bertani(LS-LB)培养基上条件培养细菌,并将培养基板在28℃下孵育约30小时。例如,使用100μg/ml的利福平来培养产生COR的Pseudomonas syringae pv。西蒙 DC3000。
    2. 将一小部分单一菌落(只是与尖端接触)从这个新鲜的板中接种到10ml具有合适的抗生素的10ml LS-LB培养基中,并在28℃和200rpm下孵育。生长直至达到中期至晚期对数期(OD 600)为0.8)。大约需要10-12小时。使用分光光度计检查600 nm处的光密度(OD)。
    3. 将细菌悬液以2,600×g离心在50ml管中20分钟。取出上清液,并将颗粒重新悬浮在10ml无菌水中。彻底冲洗漩涡细胞。以全速连续旋转,直到细胞完全重新悬浮,没有可见的细胞团。重复此步骤两次。最后,将总细菌细胞重新悬浮在40ml无菌水中,得到对应于1×10 8个细胞的0.2 OD 600接种物,用于 pv。 番茄。 OD 600的计算的近似是足够的。
    4. 从50ml培养瓶中将1ml转移到10ml的HSC培养基中,并在18℃和200rpm下孵育24小时。如果需要更大浓度的COR,可以使用更多的培养体积。然而,1毫升足以进行COR检测。从10ml细菌培养物中,可以得到0.5-2μg/mg总蛋白质的COR浓度。
    5. 24小时后,将0.6ml培养物转移到微量离心管中。在室温下以微量离心机旋转最大速度(或16,000×g g)3分钟以沉淀细胞。
    6. 将0.5ml上清液转移到新鲜的离心管中,并将沉淀的细胞储存在-80℃。这些细胞用于估计总蛋白质含量,如下文步骤B14所述。
    7. 向上清液中加入25μl3N HCl和0.5ml乙酸乙酯。以最大速度短暂旋转管30秒。允许阶段分离。这两个阶段应在两分钟内分开。
    8. 将0.4ml乙酸乙酯相(上层)转移到新鲜的离心管中。
    9. 重复步骤B7-B8两次(图2)。

      图2. COR提取流程图使用乙酸乙酯从细菌培养上清液中提取COR的步骤。将三个乙酸乙酯相在单个管中混合并干燥。将干燥的COR溶解在TFA:乙腈中,此溶液现在可以注入HPLC。

    10. 在压缩空气流下干管内容物。一种方法是使用巴斯德吸管轻轻地将空气吹入每个管中(参见图3中的代表性设置)在55°C水浴中漂浮管。或者,管可以在室温下在室温下放置过夜。


    11. 对于HPLC分析,向每个干燥管中加入250μl的0.05%TFA:乙腈(90:10v/v)。涡旋每管15秒。样品现在可以直接注入HPLC系统。
    12. 通过HPLC测定COR的存在,使用C8色谱柱(4.6×250mm,5μm),在208nm检测。使用流速为1ml/min的0.05%TFA:乙腈(60:40v/v)流动相进行等度分离。注射体积为5μl,柱温设定在25℃。
    13. 将未知样品注入HPLC系统。如果在9.4分钟观测到峰值,则确定峰值下的面积。使用峰面积,应用标准曲线和线性方程来确定5μl注射的冠状动脉的摄取。然后,计算样品的μg/ml coronatine
    14. 产生的COR的量可以表示为蛋白质浓度的函数。将步骤B6中获得的细胞通过将沉淀悬浮在1ml 1N NaOH中,然后沸腾和冷冻三次来裂解。根据制造商的说明书,用Pierce BCA蛋白测定试剂盒测定细菌细胞裂解物中的总蛋白质含量。表达如Panchal等人(2016)所示的COR浓度为μg/mg蛋白质。


  1. 产生COR的细菌丁香草 pv。必须使用番茄株DC3000及其衍生的COR缺陷型突变菌株DC3118或DB29作为本试验的阳性和阴性对照。
  2. 应使用至少三项技术重复和三项生物重复进行定量的COR。统计分析应通过计算复制品中COR生产的平均值和标准误差来进行。通过执行学生的测试可以获得两个样本之间的差异的显着性。这些分析和线性回归可以用Microsoft Excel或其他可比较的统计分析软件完成。


  1. 始终使用从甘油原液新鲜划分的细菌病原体分离物。分离物的多次培养可能导致毒力和COR生产的丧失。理想情况下,每两年从植物宿主中分离细菌病原体。
  2. 需要在HSC培养基中培养至少24小时才能连续生产COR。


  1. 低钠Luria Bertani培养基
    用NaOH调节pH至7.0 高压灭菌介质在15psi,120℃下15分钟。让培养基冷却至约55℃,并根据需要加入适当的抗生素。将介质在层流罩中倒入板中。在4°C的塑料袋中放置板,以避免中度脱水
  2. 液体HSC培养基
    1.0g NH 4 Cl
    0.2g MgSO 4·7H 2 O→// 4.1g KH 2 PO 4
    3.6g K 2 HPO 4 <3h 2=""> O
    0.3g KNO 3
    10ml 2mM FeCl 3
    890毫升ddH 2 O -/- 将pH调节至6.8
    高压灭菌10 psi,120°C 20分钟
    20 g葡萄糖 100ml ddH 2 O 过滤灭菌溶液


我们感谢Carol Bender博士关于冠状动脉提取方法的有用建议。这项研究得到了美国国家过敏和传染病研究所(5R01AI068718)给Maeli Melotto医生的资助。


  1. Palmer,DA和Bender,CL(1993)。  效果的环保和营养因素通过丁香假单胞菌生产聚酮化合物植物毒素冠状病毒。 Appl Environ Microbiol 59(5):1619-1626。
  2. Panchal,S.,Roy,D.,Chitrakar,R.,Price,L.,Breitbach,ZS,Armstrong,DW和Melotto,M。(2016)。  Coronatine促进了晚期的拟南芥叶片的感染丁香假单胞菌。/a> 前植物科学 7:880.
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引用:Panchal, S., Breitbach, Z. S. and Melotto, M. (2017). An HPLC-based Method to Quantify Coronatine Production by Bacteria. Bio-protocol 7(5): e2147. DOI: 10.21769/BioProtoc.2147.