Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method
利用液相色谱-串联质谱法和萨尔科夫斯基氏法分析克雷伯菌中吲哚乙酸的产生   

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Plant and Soil
Mar 2018

 

Abstract

Many rhizobacteria isolated from plant rhizosphere produce various phytohormones in the form of secondary metabolites, the most common of which is Indole-3-acetic acid (IAA). Here, we detail analytical protocols of IAA detection and quantification, in vitro and in situ, as recently applied to Klebsiella SGM 81, a rhizobacterium isolated from the rhizosphere of Dianthus caryophyllus (a commercially important flower across the globe). Specifically, we describe a detailed protocol for a colorimetric assay using the Salkowski reagent method, which can be used to screen for the presence of Indole compounds. To further detect and quantify IAA, a highly accurate analytical approach of LC-MS/MS is used. To detect the presence of IAA around the root system of Dianthus caryophyllus, in situ staining of plant roots is done using Salkowski reagent.

Keywords: Indole-3-acetic acid (吲哚乙酸), Klebsiella (克雷伯菌), Salkowski reagent (萨尔科夫斯基试剂), LC-MS/MS (液相色谱-串联质谱法), Tryptophan (色氨酸), Spectrophotometer (分光光度计)

Background

The bacterial auxin in the form of Indole-3 Acetic Acid (IAA) is a product of L-tryptophan metabolized by bacteria (Lynch, 1985). The group of bacteria known as plant growth promoting rhizobacteria (PGPR) specifically residing in the vicinity of the roots depend on tryptophan being present in the root exudates of plants (Kravchenko et al., 2004; Kamilova et al., 2006). These PGPR use IAA as a signal to interact with plant roots and to colonize the plant parts. This signaling feature of IAA is thought to effect on the physiology of the bacteria (Spaepen et al., 2007).

Different methods are found in the literature to detect the biosynthesis of IAA. Gordon and Weber, (1951) were the first to provide a colorimetric assay using Salkowski reagent for the detection of IAA. This method has since been widely used for detecting IAA from microorganisms. Salkowski reagent is a mixture of 0.5 M ferric chloride (FeCl3 ) and 35% perchloric acid (HClO4) which upon reaction with IAA yields pink color, due to IAA complex formation with and reduction of Fe3+ (Kamnev et al., 2001). The color developed by positive reaction indicates the presence of various indole compounds as a product of tryptophan metabolism. Apart from the colorimetric assay, other methods for IAA estimation from bacteria and plant are High Performance Liquid Chromatography (HPLC) (Perrig et al., 2007), Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS/MS) (Chiwocha et al., 2003), and by High Performance Thin Layer Chromatography HPTLC (Goswami et al., 2015). Liquid Chromatography (LC) is the preferred approach to determine the concentration of IAA and to confirm its purity with high accuracy and standardization. LC coupled with various mass spectrometry detectors are powerful tools for IAA analysis. Because of the high sensitivity and selectivity, Mass Spectrometry detectors are most commonly coupled with LC. One of the important benefits of LC-MS is that analysis and separation of compounds can be achieved in a continuous manner eliminating the step of purification (Kallenbach et al., 2009). 

Materials and Reagents

  1. 100-1,000 μl pipette tips (Sigma-Aldrich)
  2. Test tube racks (Sigma-Aldrich, catalog number: Z334146)
  3. Screw-cap tubes and caps (Sigma-Aldrich, catalog number: AXYSCT10MLS)
  4. Pyrex test tube 20 ml (Sigma-Aldrich, catalog number: CLS980016)
  5. UV quartz cuvette semi-micro square, 1.4 ml, PTFE stopper (Sigma-Aldrich, catalog number: Z276707)
  6. Sterile syringe filters with a pore size of 0.2 μm (Fisher Scientific, catalog number: 720-1230)
  7. Centrifuge tube with screw cap, capacity 10 ml (Sigma-Aldrich, catalog number: SIAL301NN10R)
  8. Pyrex glass serological pipettes, capacity 5 ml (Sigma-Aldrich, catalog number: Z653829)
  9. Amber storage bottles (Sigma-Aldrich, Supelco, catalog number: 23230-U)
  10. Klebsiella SGM 81 strain (isolated from the rhizosphere of Dianthus caryophyllus)
  11. Distilled water
  12. Nutrient broth (Microbiology grade) (Sigma-Aldrich, catalog number: 70122-500G), its composition and final pH see below:


  13. FeCl3 reagent grade (Sigma-Aldrich, CAS: 7705-08-0)
  14. Perchloric acid 70% (Sigma-Aldrich, CAS: 7601-90-3)
  15. Methanol (laboratory grade) (Sigma-Aldrich, CAS: 67-56-1) 
  16. HCl (ACS grade) (Sigma-Aldrich, CAS: 7647-01-0)
  17. L-tryptophan (traceCERT grade) (Sigma-Aldrich, CAS: 73-22-3)
  18. Acetic acid glacial (Sigma-Aldrich, CAS: 64-19-7)
  19. Ethyl acetate (grade anhydrous) (Sigma-Aldrich, CAS: 141-78-6) 
  20. Indole-3-Acetic acid (IAA) (Sigma-Aldrich, CAS: 87-51-4)
  21. Murashige and Skoog medium (Sigma-Aldrich)
  22. Acetonitrile
  23. Ammonium formate
  24. CH3CN
  25. Formic acid
  26. Ethanol
  27. Agarose
  28. Culture media (see Recipes)
  29. Salkowski reagent (see Recipes)

Equipment

  1. Spatula (Thermo Fisher, catalog number: F36711-0012)
  2. Nichipet Eco pipette, 100-1,000 μl volume (Sigma-Aldrich, catalog number: Z710199)
  3. Measuring cylinder (Sigma-Aldrich, catalog number: Z324361-2EA)
  4. 250 ml conical flask (Sigma-Aldrich, catalog number: Z723088-1EA)
  5. Rotary Vacuum Flash Evaporator (Buchhi Type) (Jain Scientific Glass, 1188)
  6. Balance
  7. Agilent 1100 LC system and an ABSciex 6500 Qtrap MS [Chromatography was on a Phenomenex Luna C18 column (100 mm x 2 mm x 3 μm)]
  8. UV-spectrophotometer (Systronics, 166)
  9. Microcentrifuge (Thermo scientific, Pico 17)
  10. Magnetic stirrer (Remi, 1 MLH)
  11. Vortex cyclo mixer (Remi, CM 101)

Software

  1. Analyst 1.6.1 (AB Sciex)

Procedure

  1. IAA screening and quantification from Klebsiella SGM 81 using Salkowski reagent method
    1. Culture preparation
      1. Take preculture medium (without tryptophan) and inoculate it with a single colony of SGM 81 strain, or a loopful of glycerol stock of the strain.
      2. Incubate the inoculated pre-culture medium at 30 °C, 120 rpm overnight. This will result in the young culture of the Klebsiella SGM 81 strain which can be used for inoculating IAA production medium (test media). 
      3. Add 100 μl of the young culture in test medium and vortex it to get a uniform suspension.
      4. Incubate it under dark conditions (by wrapping the container with newspaper/aluminum foil) at 30 °C in shaking condition at 120 rpm. 
      5. Incubation lasts for 24-96 h in dark condition depending on IAA production ability of the strain. Klebsiella SGM 81 produces IAA till 72 h and subsequent decrease in the production in recorded after that.
    2. Indole quantification using Salkowaski reagent
      1. After 24 h, take out 1.5 ml of sample culture broth and transfer to an Eppendorf tube.
      2. Centrifuge at 16,278 x g for 5 min using a microcentrifuge.
      3. Carefully withdraw 1 ml supernatant and transfer to a new test tube.
      4. Mix equal volume (1 ml) of Salkowski reagent, vortex gently and incubate the reaction at 30 °C in a dark condition for 30 min. 
      5. In a new test tube replace the supernatant with 1 ml of control uninoculated medium and mix with 1 ml of Salkowski reagent. This serves as blank.
      6. The presence of IAA is detected by measuring pink color development after 30 min (some strains may develop red color as shown in Figure 1).


        Figure 1. Color development due to Indole-Salkowski reagent reaction. A. Klebsiella SGM 81. B. SGM09. C. Control uninoculated media.

      7. Use uninoculated medium to set the blank. Measure the color intensity spectrophotometrically at 536 nm using cuvette.
      8. Compare the optical density of the test sample with a standard IAA curve (10-100 μg·ml-1) to calculate the concentration. 
      9. Repeat the process at similar intervals until the optical density begins to decrease, indicating no further production of IAA.

  2. Analysis of IAA by liquid chromatography-mass spectrometry (LC-MS/MS)
    1. Sample preparation
      1. Prepare 100 ml nutrient broth medium supplemented with 0.5 g of L-tryptophan in a 250 ml Erlenmeyer flask.
      2. Add 100 μl of the young culture in test medium and vortex it to get a uniform suspension.
      3. Incubate it under dark conditions (by wrapping the container with newspaper/aluminum foil) at 30 °C in shaking condition at 120 rpm for 72 h. 
      4. To separate the cells, centrifuge at 4 °C for 20 min at 2,800 x g and collect 1,000 ml supernatant.
      5. Transfer the supernatant to a 250 ml screw-cap glass bottle. Acidify it by adding 2-3 drops of 1 N concentrated HCl to reach pH 2.5-3.
      6. To extract IAA, add double the volume of ethyl acetate to the acidified supernatant and shake vigorously for 5 min (alternatively, a separating funnel can be used). Let the mixture stand at room temperature for 10 min to get the top layer of ethyl acetate. Use this layer for further treatment. 
      7. In a rotary evaporator, set water bath temperature to boiling temperature of ethyl acetate i.e., 77.1 °C (alternatively, ethyl acetate phase can be vacuum dried in a rotational evaporator at 40 °C). The time for drying of the solvent system varies according to the sample volume. 
      8. Transfer the ethyl acetate layer to the round bottom flask and adjust its rotation to avoid any bumping of the liquid sample. “Bumping is the phenomenon in chemistry where homogenous liquids boiled in a test tube or other container will super heat and, upon nucleation rapid boiling will expel the liquid from the container (Wikipedia)”–This would leave the IAA escaping the flask into the condenser tube attached with the round bottom flask.
      9. Upon complete evaporation of the liquid ethyl acetate, pure IAA is left behind in the crystalline form attached to the bottom of the rotary flask. 
      10. Switch off all the units of the rotary evaporator and remove the round bottom flask. Re-dissolve the crystalline IAA in 5 ml of 20% methanol and store at -20 °C for future use.
    2. LC-MS/MS analysis
      1. Filter the stored methanol extract (100 µl) using sterile syringe filters of 0.2 µm so as to separate insoluble particles and larger compounds if any. This is the final analysis sample.
      2. Phenomenex Luna C18(2) column (100 mm x 2 mm x 3 µm) at temperature of 50 °C is used as chromatography system.
      3. A gradient solvent system of 10% solvent A (2% acetonitrile, 10 mM ammonium formate, pH 4.2) to 90% solvent B (94.9% CH3CN, 5% H2O, 0.1% formic acid) is used for the column over 10 min at a flow rate of 250 ml/min.
      4. Wash the column with 90% solvent B for 3 min and then re-equilibrate with 90% solvent A for 6 min. 
      5. Use 10 µl injections to load the sample for analysis.
      6. The MS is configured with a Turbo Spray Ion Drive source where the source temperature is set to 500 °C and the ion spray voltage to 5,500 V. 
      7. Analyze IAA by Multiple Reaction Monitoring (MRM) in positive mode using a transition of 176 > 130 with collision energy of 20 eV. Transition of 176 > 130 means the specific pairs of mass to charge (m/z) values associated to the precursor and fragment ions. In our case the Q1 is set to a value of 176 amu and the Q3 to 130 amu, the collision energy was 20 mV.
      8. Set the declustering potential, exit potential and collision cell exit potential at 30 V, 10 V and 10 V respectively.

  3. In Situ Salkowski staining
    1. Prepare Murashige and Skoog (MSM) basal salt medium with 0.8% agarose, devoid of plant hormone supplements. 
    2. Sterilize the D. caryophyllus seeds using three-step procedure: a 1 min wash in 70% ethanol, followed by a 4 min wash in 20% NaClO, and a final rinse in sterile distilled water 3 times.
    3. Allow the D. caryophyllus seeds to germinate on the MS media by incubating the plates at 25 °C in 14 h light and 10 h dark cycles in a plant culture room, until the root length reaches 2 cm (approximately six days). 
    4. Treat the germinated plant roots by immersing the root tips into 5 ml of the bacterial suspension with titre 105 CFU ml-1 in the universal tube. 
    5. Transfer the treated plants onto new MS agar medium devoid of IAA and sucrose in square Petri plates (120 mm x 120 mm x 15 mm).
    6. Again, incubate the plates at 25 °C in 14 h light and 10 h dark cycles in a plant culture room.
    7. After two weeks of infection, stain the treated roots with Salkowski reagent. 
    8. To stain the roots, add 400 μl of Salkowski reagent on each root and observe any visible color change. 
    9. The development of pink color as shown in the figure below indicates the presence of proximal IAA on and around the roots (Figure 2).


      Figure 2. In situ Salkowski staining on plant roots treated with Klebsiella SGM 81 of 105 CFU ml-1 titre

Data analysis

  1. Take the optical density of the samples at 536 nm and graphically calculate the concentration of IAA based on the standard curve. IAA estimation from Klebsiella SGM 81 strain, using Salkowski reagent shows a maximum yield to 215 μg·ml-1 detected after 72 h with 0.15% tryptophan (Figure 1a in Gang et al., 2018).
  2. For LC-MS/MS analysis, confirmation of the identity in the samples is done by Enhanced Product Ion scans. 
  3. Data acquisition and analysis is done with Analyst 1.6.1 (AB Sciex). 
  4. Quantification is carried out by comparing the test sample data with control standard IAA.
  5. Overlapping retention times during LC (5.90 min and 5.92 min) and identical m/z ratios (130.1) between a commercial IAA standard and sample supernatant showed Klebsiella SGM 81 to produce substantial amounts (960 μg·ml-1 after 72 h) homogeneous IAA (Gang et al., 2018).
  6. Development of pink color on the Salkowski treated roots indicates the presence of bacterial IAA proximal to the roots (Figure 3a in Gang et al., 2018).

Notes

  1. The Salkowski reagent method discussed in this paper is shown using nutrient broth liquid medium (supplemented with L-tryptophan) which is non-specific liquid medium for most gram-positive and gram-negative bacteria. However other media like Yeast extract mannitol (YEM), Pikovskaya’s broth (supplemented with L-tryptophan) can be used if IAA production and quantification is to be done from Rhizobium or phosphate solubilizing bacteria respectively. 
  2. The preculture preparation is important to normalize the initial cell count especially when a comparative study of IAA quantification is carried out for the optimization process.
  3. Salkowski reagent method quantifies the overall Indole present in the sample. To get the purity and conjugants of Indole-3-acetic acid (IAA), other analytical methods like HPLC, LC-MS/MS, HPTLC etc. can be used.

Recipes

  1. Culture media
    1. Making use of measuring cylinder, measure 100 ml distilled water and transfer it into a 250 ml Erlenmeyer flask or screw-capped glass bottle. Two other flasks of the same media, with and without tryptophan will be used as control and pre-culture media (used to activate the SGM 81 culture to yield young culture) respectively
    2. For simple liquid nutrient broth medium, weigh 1.3 g of Nutrient broth and dissolve it in 100 ml distilled water. Stir it properly so as to mix the powder in distilled water properly
    3. Weigh 0.15 g of L-tryptophan (0.15% w/v) and add to the prepared nutrient broth flask. This is liquid nutrient broth with tryptophan supplement
    4. Autoclave the prepared media at 121 °C for 15 min
    5. Allow the media to cool down and come to room temperature before inoculation so as to ensure the survival of inoculum
  2. Salkowski reagent
    1. 0.5 M of 100 ml ferric chloride (dissolve 8.125 g of FeCl3 in 100 ml of distilled water)
    2. To dilute readily available perchloric acid, measure 24.5 ml (v/v) of distilled water in a measuring cylinder and add 24.5 ml (v/v) of concentrated acid to it 
    3. Add 1 ml (v/v) of 0.5 M ferric chloride solution to 49 ml of 35% perchloric
    4. Mix well and store in a dark brown bottle at room temperature. It can be used for further experiments if stored under described conditions

Acknowledgments

We are thankful to the Department of Science and Technology, India for the financial assistance under INSPIRE Fellowship 2014-15 (IF 140042). This work was also supported by the BBSRC (BB/003608/1), UK. At Imperial College London, we thank Mark Bennett for helping with LC-MS/MS analysis. The protocol is adapted from Gang et al. (2018).

Competing interests

We declare no conflict of interest.

References

  1. Chiwocha, S. D., Abrams, S. R., Ambrose, S. J., Cutler, A. J., Loewen, M., Ross, A. R. and Kermode, A. R. (2003). A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J 35(3): 405-417.
  2. Gang, S., Saraf, M., Waite, C. J., Buck, M. and Schumacher, J. (2018). Mutualism between Klebsiella SGM 81 and Dianthus caryophyllus in modulating root plasticity and rhizospheric bacterial density. Plant Soil 424: 273-288. 
  3. Gordon, S. A. and Weber, R. P. (1951). Colorimetric estimation of indoleacetic acid. Plant Physiol 26(1): 192-195.
  4. Goswami, D., Thakker, J. N. and Dhandhukia, P. C. (2015). Simultaneous detection and quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) produced by rhizobacteria from l-tryptophan (Trp) using HPTLC. J Microbiol Methods 110: 7-14.
  5. Kallenbach, M., Baldwin, I. T. and Bonaventure, G. (2009). A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS. Plant Methods 5: 17.
  6. Kamilova, F., Kravchenko, L. V., Shaposhnikov, A. I., Azarova, T., Makarova, N. and Lugtenberg, B. (2006). Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria. Mol Plant Microbe Interact 19(3): 250-256.
  7. Kamnev, A., Shchelochkov, A., Perfiliev, Y. D. Tarantilis, P. A., and Polissiou, M. G. (2001). Spectroscopic investigation of indole-3-acetic acid interaction with iron (III). J Mol Struct 563:565-572.
  8. Kravchenko, L., Azarova, T., Makarova, N. and Tikhonovich, I. (2004). The effect of tryptophan present in plant root exudates on the phytostimulating activity of rhizobacteria. Microbiology 73:156-158.
  9. Lynch, J. (1985). Origin, nature and biological activity of aliphatic substances and growth hormones found in soil. In: Soil Organic Matter and Biological Activity. Springer 151-174.
  10. Perrig, D., Boiero, M. L., Masciarelli, O. A., Penna, C., Ruiz, O. A., Cassan, F. D. and Luna, M. V. (2007). Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculant formulation. Appl Microbiol Biotechnol 75(5): 1143-1150.
  11. Spaepen, S., Vanderleyden, J. and Remans, R. (2007). Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 31(4): 425-448.

简介

【摘要】从植物根际分离的许多根际细菌以次级代谢物的形式产生各种植物激素,其中最常见的是吲哚-3-乙酸(IAA)。 在这里,我们详细介绍了IAA检测和定量的分析方案,体外>和原位,>最近应用于 Klebsiella > SGM 81,一种分离的根际细菌 来自 Dianthus caryophyllus >(一种遍布全球的商业上重要的花)的根际。 具体而言,我们描述了使用Salkowski试剂方法进行比色测定的详细方案,该方法可用于筛选吲哚化合物的存在。 为了进一步检测和量化IAA,使用了一种高精度的LC-MS / MS分析方法。 为了检测 Dianthus caryophyllus >根系周围IAA的存在,使用Salkowski试剂进行植物根的原位>染色。

【背景】吲哚-3乙酸(IAA)形式的细菌生长素是由细菌代谢的L-色氨酸的产物(Lynch,1985)。被称为植物生长促进根际细菌(PGPR)的细菌群特别存在于根部附近,取决于色氨酸存在于植物的根系分泌物中(Kravchenko et al。>,2004; Kamilova 等人>,2006)。这些PGPR使用IAA作为信号与植物根部相互作用并定殖植物部分。 IAA的这种信号传导特征被认为影响细菌的生理学(Spaepen et al。>,2007)。

在文献中发现了不同的方法来检测IAA的生物合成。 Gordon和Weber,(1951)是第一个使用Salkowski试剂进行比色测定以检测IAA的人。此方法已被广泛用于从微生物中检测IAA。 Salkowski试剂是0.5 M氯化铁(FeCl 3 )和35%高氯酸(HClO 4 )的混合物,由于IAA复合物,与IAA反应产生粉红色形成和减少Fe 3 + (Kamnev et al。>,2001)。通过阳性反应产生的颜色表明存在各种吲哚化合物作为色氨酸代谢的产物。除了比色分析外,其他从细菌和植物中估算IAA的方法是高效液相色谱(HPLC)(Perrig et al。>,2007),液相色谱电喷雾电离串联质谱(LC-ESI) -MS / MS)(Chiwocha et al。>,2003),以及高效薄层色谱HPTLC(Goswami et al。>,2015)。液相色谱(LC)是确定IAA浓度并以高准确度和标准化确定其纯度的优选方法。 LC与各种质谱检测器配合使用是IAA分析的强大工具。由于高灵敏度和高选择性,质谱检测器最常与LC耦合。 LC-MS的一个重要优点是可以连续地分析和分离化合物,从而消除纯化步骤(Kallenbach et al。>,2009)。 

关键字:吲哚乙酸, 克雷伯菌, 萨尔科夫斯基试剂, 液相色谱-串联质谱法, 色氨酸, 分光光度计

材料和试剂

  1. 100-1,000μl移液器吸头(Sigma-Aldrich)
  2. 试管架(Sigma-Aldrich,目录号:Z334146)
  3. 螺帽管和盖帽(Sigma-Aldrich,目录号:AXYSCT10MLS)
  4. Pyrex试管20 ml(Sigma-Aldrich,目录号:CLS980016)
  5. 紫外石英比色杯,半微量方形,1.4 ml,PTFE塞子(Sigma-Aldrich,目录号:Z276707)
  6. 孔径为0.2μm的无菌注射器过滤器(Fisher Scientific,目录号:720-1230)
  7. 带螺旋盖的离心管,容量10 ml(Sigma-Aldrich,目录号:SIAL301NN10R)
  8. Pyrex玻璃血清移液器,容量5 ml(Sigma-Aldrich,目录号:Z653829)
  9. 琥珀色储存瓶(Sigma-Aldrich,Supelco,目录号:23230-U)
  10. Klebsiella > SGM 81菌株(从 Dianthus caryophyllus >的根际分离)
  11. 蒸馏水
  12. 营养肉汤(微生物级)(Sigma-Aldrich,目录号:70122-500G),其成分和最终pH值见下文:


  13. FeCl 3 试剂级(Sigma-Aldrich,CAS:7705-08-0)
  14. 高氯酸70%(Sigma-Aldrich,CAS:7601-90-3)
  15. 甲醇(实验室级)(Sigma-Aldrich,CAS:67-56-1) 
  16. HCl(ACS级)(Sigma-Aldrich,CAS:7647-01-0)
  17. L-色氨酸(traceCERT级)(Sigma-Aldrich,CAS:73-22-3)
  18. 醋酸冰醋酸(Sigma-Aldrich,CAS:64-19-7)
  19. 乙酸乙酯(无水级)(Sigma-Aldrich,CAS:141-78-6) 
  20. 吲哚-3-乙酸(IAA)(Sigma-Aldrich,CAS:87-51-4)
  21. Murashige和Skoog培养基(Sigma-Aldrich)
  22. 乙腈
  23. 甲酸铵
  24. CH <子> 3 CN
  25. 甲酸
  26. 乙醇
  27. 琼脂糖
  28. 文化媒体(见食谱)
  29. Salkowski试剂(见食谱)

设备

  1. 抹刀(Thermo Fisher,目录号:F36711-0012)
  2. Nichipet Eco移液器,体积100-1,000μl(Sigma-Aldrich,目录号:Z710199)
  3. 量筒(Sigma-Aldrich,目录号:Z324361-2EA)
  4. 250毫升锥形瓶(Sigma-Aldrich,目录号:Z723088-1EA)
  5. 旋转式真空闪蒸器(Buchhi型)(Jain Scientific Glass,1188)
  6. 平衡
  7. Agilent 1100液相色谱系统和ABSciex 6500 Qtrap MS [色谱法在Phenomenex Luna C18色谱柱上(100 mm x 2 mm x3μm)]
  8. 紫外分光光度计(Systronics,166)
  9. 微量离心机(Thermo scientific,Pico 17)
  10. 磁力搅拌器(Remi,1 MLH)
  11. Vortex环形混合器(Remi,CM 101)

软件

  1. 分析师1.6.1(AB Sciex)

程序

  1. 使用Salkowski试剂法从 Klebsiella > SGM 81进行IAA筛选和定量分析
    1. 文化准备
      1. 取预培养基(不含色氨酸),并用单菌落的SGM 81菌株或该菌株的一系列甘油原液接种。
      2. 将接种的预培养基在30℃,120rpm下孵育过夜。这将导致 Klebsiella > SGM 81菌株的幼龄培养物,其可用于接种IAA生产培养基(测试培养基)。&nbsp;
      3. 在测试培养基中加入100μl幼小培养物并涡旋以获得均匀的悬浮液。
      4. 在黑暗条件下(通过用报纸/铝箔包裹容器)在30°C,120rpm的摇动条件下孵育。&nbsp;
      5. 孵育在黑暗条件下持续24-96小时,这取决于菌株的IAA生产能力。 Klebsiella > SGM 81产生IAA直至72小时,之后记录的产量减少。
    2. 使用Salkowaski试剂进行吲哚定量分析
      1. 24小时后,取出1.5ml样品培养液并转移到Eppendorf管中。
      2. 使用微量离心机在16,278 离心5分钟离心5分钟。
      3. 小心取出1 ml上清液,转移到新的试管中。
      4. 混合等体积(1ml)的Salkowski试剂,轻轻涡旋并在30℃,黑暗条件下孵育反应30分钟。&nbsp;
      5. 在新的试管中,用1ml对照未接种的培养基替换上清液,并与1ml Salkowski试剂混合。这是空白。
      6. 通过在30分钟后测量粉红色显色来检测IAA的存在(一些菌株可能产生红色,如图1所示)。


        图1.由Indole-Salkowski试剂反应引起的显色。 A. Klebsiella SGM81。 B. SGM09。 C.控制未接种的培养基。

      7. 使用未接种的培养基设置空白。用比色皿在536nm处用分光光度法测量颜色强度。
      8. 将测试样品的光密度与标准IAA曲线(10-100μg·ml -1 )进行比较,以计算浓度。&nbsp;
      9. 以相似的间隔重复该过程,直到光密度开始降低,表明没有进一步产生IAA。

  2. 用液相色谱 - 质谱(LC-MS / MS)分析IAA
    1. 样品制备
      1. 在250ml Erlenmeyer烧瓶中制备100ml补充有0.5g L-色氨酸的营养肉汤培养基。
      2. 在测试培养基中加入100μl幼小培养物并涡旋以获得均匀的悬浮液。
      3. 将其在黑暗条件下(通过用报纸/铝箔包裹容器)在30℃下以120rpm的振荡条件孵育72小时。&nbsp;
      4. 为了分离细胞,在4℃下以2,800×g离心20分钟,并收集1,000ml上清液。
      5. 将上清液转移到250ml螺旋盖玻璃瓶中。加入2-3滴1N浓HCl使其酸化至pH2.5-3。
      6. 为了提取IAA,将双倍体积的乙酸乙酯加入到酸化的上清液中并剧烈摇动5分钟(或者,可以使用分液漏斗)。让混合物在室温下静置10分钟,得到顶层乙酸乙酯。使用此图层进行进一步处理。&nbsp;
      7. 在旋转蒸发器中,将水浴温度设定为乙酸乙酯的沸腾温度,即77.1℃(或者,乙酸乙酯相可在40℃的旋转蒸发器中真空干燥)。溶剂系统的干燥时间根据样品量而变化。&nbsp;
      8. 将乙酸乙酯层转移到圆底烧瓶中并调节其旋转以避免液体样品的任何撞击。 “碰撞是化学中的现象,其中在试管或其他容器中沸腾的均质液体将超热,并且在成核时快速沸腾将液体从容器中排出(维基百科)” - 这将使IAA从烧瓶中逸出进入冷凝器管附有圆底烧瓶。
      9. 在液体乙酸乙酯完全蒸发后,将纯IAA留在附着于旋转烧瓶底部的结晶形式中。&nbsp;
      10. 关闭旋转蒸发器的所有装置并取下圆底烧瓶。将结晶IAA重新溶解在5ml 20%甲醇中,并储存在-20℃以备将来使用。
    2. LC-MS / MS分析
      1. 使用0.2μm的无菌注射器过滤器过滤储存的甲醇提取物(100μl),以分离不溶性颗粒和较大的化合物(如果有的话)。这是最终的分析样本。
      2. 将Phenomenex Luna C18(2)柱(100mm×2mm×3μm)在50℃的温度下用作色谱系统。
      3. 10%溶剂A(2%乙腈,10mM甲酸铵,pH 4.2)至90%溶剂B的梯度溶剂系统(94.9%CH 3 CN,5%H 2 O,0.1%甲酸)用于该柱。
      4. 用90%溶剂B洗涤柱子3分钟,然后用90%溶剂A重新平衡6分钟。&nbsp;
      5. 使用10μl进样加载样品进行分析。
      6. MS配置了Turbo喷雾离子驱动源,其源温度设置为500°C,离子喷雾电压设置为5,500 V.&nbsp;
      7. 使用176> 1的转变以正模式通过多反应监测(MRM)分析IAA。 130的碰撞能量为20eV。 176的转变> 130表示与前体和碎片离子相关的特定质荷比(m / z)值对。在我们的例子中,Q1设置为176 amu,Q3设置为130 amu,碰撞能量为20 mV。
      8. 将分离电位,退出电位和碰撞池出口电位分别设置为30 V,10 V和10 V.

  3. 原位> Salkowski染色
    1. 准备Murashige和Skoog(MSM)基础盐培养基,含0.8%琼脂糖,不含植物激素补充剂。&nbsp;
    2. 对 D进行消毒。使用三步法制备caryophyllus种子:在70%乙醇中洗涤1分钟,然后在20%NaClO中洗涤4分钟,最后在无菌蒸馏水中漂洗3次。
    3. 允许 D。通过在25℃下在14小时光照和10小时黑暗循环中将平板在植物培养室中孵育直至根长度达到2cm(大约6天),使caryophyllus >种子在MS培养基上发芽。&nbsp;
    4. 通过将根尖浸入5ml细菌悬浮液中处理萌发的植物根,通孔管中滴定10 5 CFU ml -1 。&nbsp;
    5. 将处理过的植物转移到方形培养皿(120mm×120mm×15mm)中的缺乏IAA和蔗糖的新MS琼脂培养基上。
    6. 再次,在植物培养室中在25℃,14小时光照和10小时黑暗循环中孵育平板。
    7. 感染两周后,用Salkowski试剂对处理过的根进行染色。&nbsp;
    8. 为了染色根部,在每个根部添加400μlSalkowski试剂并观察任何可见的颜色变化。&nbsp;
    9. 粉红色的发展如下图所示,表明根部和周围存在近端IAA(图2)。


      图2.用克雷伯氏菌SGM 81处理的植物根部的原位Salkowski染色105 CFU ml-1滴度

数据分析

  1. 取样品的光密度为536nm,并根据标准曲线图形计算IAA的浓度。来自 Klebsiella > SGM 81菌株的IAA估计,使用Salkowski试剂显示在72小时后用0.15%色氨酸检测到最大产量至215μg·ml -1 (图1a中的Gang et al。>,2018)。
  2. 对于LC-MS / MS分析,通过增强产品离子扫描确认样品中的特性。&nbsp;
  3. 使用Analyst 1.6.1(AB Sciex)完成数据采集和分析。&nbsp;
  4. 通过将测试样品数据与对照标准IAA进行比较来进行定量。
  5. 在商业IAA标准品和样品上清液之间LC(5.90分钟和5.92分钟)和相同的m / z比(130.1)之间的重叠保留时间显示 Klebsiella > SGM 81产生大量(960μg·ml < 72小时后均相IAA(Gang et al。>,2018),sup> -1 。
  6. 在Salkowski处理的根上发展粉红色表明在根部附近存在细菌IAA(Gang 3a等图3a,2018)。

笔记

  1. 本文讨论的Salkowski试剂方法使用营养肉汤液体培养基(补充有L-色氨酸)显示,该培养基是大多数革兰氏阳性和革兰氏阴性细菌的非特异性液体培养基。然而,如果分别从根瘤菌或磷酸盐溶解细菌进行IAA生产和定量,则可以使用其他培养基如酵母提取物甘露醇(YEM),Pikovskaya肉汤(补充有L-色氨酸)。&nbsp;
  2. 预培养制剂对于初始细胞计数的标准化是重要的,尤其是当针对优化过程进行IAA定量的比较研究时。
  3. Salkowski试剂方法量化样品中存在的总吲哚。为了获得吲哚-3-乙酸(IAA)的纯度和结合物,可以使用其他分析方法,如HPLC,LC-MS / MS,HPTLC 等。>。

食谱

  1. 文化媒体
    1. 使用量筒,测量100毫升蒸馏水,并将其转移到250毫升锥形瓶或螺旋盖玻璃瓶中。使用和不使用色氨酸的相同培养基的另外两个烧瓶将分别用作对照和预培养基(用于激活SGM 81培养物以产生幼培养物)。
    2. 对于简单的液体营养肉汤培养基,称取1.3克营养肉汤,并将其溶于100毫升蒸馏水中。适当搅拌,使粉末在蒸馏水中适当混合
    3. 称取0.15g L-色氨酸(0.15%w / v)并加入到制备的营养肉汤烧瓶中。这是含有色氨酸补充剂的液体营养肉汤
    4. 将制备的培养基在121℃下高压灭菌15分钟
    5. 在接种前让培养基冷却并达到室温,以确保接种物的存活
  2. Salkowski试剂
    1. 0.5毫升100毫升氯化铁(溶解8.125克FeCl 3 在100毫升蒸馏水中)
    2. 为了稀释容易得到的高氯酸,在量筒中测量24.5ml(v / v)蒸馏水,并向其中加入24.5ml(v / v)浓酸。
    3. 将1ml(v / v)0.5M氯化铁溶液加入49ml 35%高氯酸中
    4. 在室温下充分混合并储存在深棕色瓶中。如果在所述条件下储存,它可用于进一步的实验

致谢

我们感谢印度科学和技术部在2014 - 15年INSPIRE奖学金(IF 140042)下提供的财政援助。这项工作也得到了英国BBSRC(BB / 003608/1)的支持。在伦敦帝国理工学院,我们感谢Mark Bennett帮助进行LC-MS / MS分析。该协议改编自Gang 等人>(2018)。

利益争夺

我们声明没有利益冲突。

参考

  1. Chiwocha,S.D.,Abrams,S.R.,Ambrose,S.J。,Cutler,A.J.,Loewen,M.,Ross,A.R。和Kermode,A.R。(2003)。 使用液相色谱 - 电喷雾电离串联质谱分析植物激素及其代谢物类别的方法:莴苣( Lactuca sativa > L.)种子的热调节激素调节分析。 植物> J 35(3):405-417。
  2. Gang,S.,Saraf,M.,Waite,C.J。,Buck,M。和Schumacher,J。(2018)。 Klebsiella > SGM 81与 Dianthus之间的相互关系caryophyllus >调节根系可塑性和根际细菌密度。 植物土壤> 424:273-288。&nbsp;
  3. Gordon,S。A.和Weber,R。P.(1951)。 吲哚乙酸的比色估算。 植物生理学> 26( 1):192-195。
  4. Goswami,D.,Thakker,J。N.和Dhandhukia,P。C.(2015)。 吲哚-3-乙酸(IAA)和吲哚-3-丁酸的同时检测和定量(IBA)使用HPTLC由来自1-色氨酸(Trp)的根际细菌产生。 J Microbiol Methods > 110:7-14。
  5. Kallenbach,M.,Baldwin,I。T.和Bonaventure,G。(2009)。 同时分析植物提取物中含有游离羧基的脂肪族和极性分子的快速灵敏方法通过LC-MS / MS。 植物方法> 5:17。
  6. Kamilova,F.,Kravchenko,L。V.,Shaposhnikov,A。I.,Azarova,T.,Makarova,N。和Lugtenberg,B。(2006)。 生长在石棉上的蔬菜分泌物中的有机酸,糖和L-色氨酸及其对活动的影响根际细菌。 Mol Plant Microbe Interact > 19(3):250-256。
  7. Kamnev,A.,Shchelochkov,A.,Perfiliev,Y。D. Tarantilis,P。A.和Polissiou,M。G.(2001)。 吲哚-3-乙酸与铁(III)相互作用的光谱研究。 J Mol Struct > 563:565-572。
  8. Kravchenko,L.,Azarova,T.,Makarova,N。和Tikhonovich,I。(2004)。 植物根系分泌物中存在的色氨酸对根际细菌的植物刺激活性的影响。< / a> 微生物学> 73:156-158。
  9. Lynch,J。(1985)。土壤中脂肪物质和生长激素的起源,性质和生物活性。在:土壤有机质和生物活性>。 Springer > 151-174。
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gang, S., Sharma, S., Saraf, M., Buck, M. and Schumacher, J. (2019). Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiella by LC-MS/MS and the Salkowski Method. Bio-protocol 9(9): e3230. DOI: 10.21769/BioProtoc.3230.
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