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An HPLC-based Method to Quantify Coronatine Production by Bacteria

采用基于HPLC的方法定量细菌的冠菌素生成

Shweta Panchal Shweta Panchal
ZB Zachary S. Breitbach
Maeli Melotto Maeli Melotto

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2147 浏览次数: 9421

评审: Jyotiska ChaudhuriHonghong WuDe Michele Roberto

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Abstract

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) search HPLC (HPLC) search Pseudomonas syringae (丁香假单胞菌) search Plant pathogen (植物病原体) search Virulence factor (毒力因子) search

Background

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)

Equipment

  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 )

Software

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

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

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分类

微生物学 > 微生物生物化学 > 其它化合物

植物科学 > 植物免疫 > 宿主-细菌相互作用

细胞生物学 > 细胞分离和培养 > 细胞分离

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