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Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum
植物病原体茄青枯病菌胞外多糖的定量分析   

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Abstract

Soluble exopolysaccharide is a major virulence factor produced by the plant pathogen Ralstonia solanacearum. Its massive production during plant infection is associated with the arrest of water flow in xylem vessels leading eventually to plant death. The composition of this heavy macromolecule includes mainly N-acetylgalactosamine. Here we describe a colorimetric method for quantitative determination of the soluble exopolysaccharide present in culture supernatant of R. solanacearum.

Keywords: Ralstonia solanacearum(青枯雷尔氏菌), Exopolysaccharide(胞外多糖), Virulence factor(毒力因子), Plant pathogen(植物病原体), Hexoseamine(氨基己糖)

Background

The plant pathogen Ralstonia solanacearum produces exopolysaccharide under the control of quorum sensing system, i.e., at high cell density, above 5 x 107 cell ml-1 (Flavier et al., 1997). The sugar content of the exopolysaccharide includes galactosamine, glucose, and rhamnose in the ratio of 10:2.5:1 (Drigues et al., 1985). A protocol for a reliable extraction and quantification of the exopolysaccharide from culture supernatant was initially developed by Brumbley and Denny (1990) and was updated recently by Peyraud et al. (2016). The quantification is based on the determination of hexoseamine content of the macromolecule using an adapted Elson and Morgan assay (Elson and Morgan, 1933; Gatt and Berman, 1966). Exopolysaccharides containing N-acetyl-D-galactosamine are produced by diverse Gram-negative or Gram-positive bacteria (Vaningelgem et al., 2004; Balzaretti et al., 2017) and also some fungi (Lee et al., 2015), so this protocol may also be applicable to such organisms.

Materials and Reagents

  1. Millex®-GP 33 mm syringe filter with a polyethersulfone membrane and 0.22 µm pore size (EMD Millipore, catalog number: SLGP033RB )
  2. Polypropylene microcentrifuge tubes of 2.0 ml Eppendorf® Safe-Lock (Eppendorf, catalog number: 0030120094 )
  3. Paper towel
  4. Aluminum foil
  5. Pipette tips
  6. 15-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 188271 )
  7. 50-ml conical polypropylene Greiner centrifuge tubes (Greiner Bio One International, catalog number: 227261 )
  8. Bridges for microcentrifuge tube (Milian, catalog number: 045427 )
  9. Gloves
  10. R. solanacearum strain GMI1000 (Salanoubat et al., 2002). This strain can be retrieved from the CIRM Biological Resource Center (http://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria) in the CIRM-BP collection (code CFBP6924)
  11. Acetone > 99.8% AnalaR NORMAPUR® (VWR, catalog number: 20066.296 )
  12. Milli-Q water obtained at a resistivity of 18.2 MOhm cm at 25 °C
  13. Acetyl acetone > 99% AnalaR NORMAPUR® (VWR, catalog number: 20092.23 0)
  14. Ethanol, 99.8% (Sigma-Aldrich, catalog number: 02851 )
  15. 4-(dimethylamino)benzaldehyde, 98% (Erlich’s reagent) (Sigma-Aldrich, catalog number: 109762 )
  16. Sodium L-glutamic acid monohydrate (Sigma-Aldrich, catalog number: 49621 )
  17. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: F8263 )
  18. Ammonium sulfate, (NH4)2SO4 (Sigma-Aldrich, catalog number: A4418 )
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: M5921 )
  20. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  21. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60377 )
  22. Sodium chloride (NaCl) > 99.5% AnalaR NORMAPUR® (VWR, catalog number: 27810.295 )
  23. N-acetylgalactosamine, 98% (Sigma-Aldrich, catalog number: A2795 )
  24. Hydrochloric acid (HCl), 37%, AnalaR NORMAPUR® (VWR, catalog number: 20252.29 0)
  25. Sodium carbonate (Na2CO3) > 99.9 AnalaR NOR MAPUR® (VWR, catalog number: 27771.29 0)
  26. Minimal medium (see Recipes)
  27. 5 M NaCl solution (see Recipes)
  28. Standard samples of N-acetyl galactosamine (see Recipes)
  29. N-acetylgalactosamine standard stock solution (see Recipes)
  30. 2% acetyl acetone, 1.5 M Na2CO3 solution (see Recipes)
  31. Erlich’s reagent (see Recipes)

Equipment

  1. Dry bath heater with dual block Corning® (Corning, model: Corning® LSETM Digital Dry Bath Heater, catalog number: 6786-DB )
  2. Fume hood
  3. Eppendorf® microcentrifuge 5415 R (Eppendorf, model: 5415 R )
  4. Mini microcentrifuges Corning® (Corning, model: Corning® LSETM Mini Microcentrifuge, catalog number: 6766 )
  5. Vortex for microcentrifuge tubes
  6. UltrospecTM 2100 pro UV/Visible spectrophotometer (GE Healthcare, model: UltrospecTM 2100 pro UV/Visible Spectrophotometer , catalog number: 80211221)
  7. 10 ml volumetric flask

Software

  1. Appropriate software (SciDavis, Excel, etc.)

Procedure

  1. Culture and sample preparation
    1. Run a liquid R. solanacearum culture. Culture is performed in an Erlenmeyer containing 100 ml liquid minimal medium inoculated at 0.01 optical density at 600 nm, agitated at 180 rpm at a temperature of 28 °C.
    2. Collect 1 ml culture broth in the exponential or stationary phase. The cell culture must be at least above an optical density of 0.1 (ideally around 0.6) at 600 nm or 5 x 108 cell ml-1 to ensure passing the quorum sensing threshold and thus production of exopolysaccharide.
    3. Filter the cell broth with a 0.22 µm filter and collect the supernatant in a 2.0 ml microcentrifuge tube.
    4. Store the sample at -20 °C or run the extraction right away.

  2. Exopolysaccharides extraction
    1. To precipitate exopolysaccharide, mix 0.2 ml supernatant + 0.004 ml 5 M NaCl solution + 0.8 ml acetone in a 2.0 ml microcentrifuge tube. Mix well with a vortex for 10 sec and place the sample at 4 °C overnight (around 12 h).
    2. Centrifuge the sample for 10 min at 16,000 x g (13,000 rpm) and at 4 °C and then discard the supernatant. Invert the tube on a paper towel to drain the pellet well during 15 min. Then, add 0.2 ml Milli-Q water to dissolve the pellet.
    3. The sample can be stored at 4 °C for few days (optional) until quantification.
    4. On the same day that the assay is to be performed, heat samples at 65 °C on dry bath heater for 10 min. Vortex well and then spin for 5 min at 16,000 x g (13,000 rpm), 4 °C. Collect the supernatant and place it in a new 2.0 ml microcentrifuge tube. The preparation is now referred to as the EPS (exopolysaccharide) sample.

  3. Exopolysaccharide quantification
    1. Turn on the dry bath heater positioned in a fume hood. Temperature needs to be at 115 °C before adding the samples.
    2. Use the 200 μl of the EPS sample or you can gather EPS samples up to 450 ml. The chosen volume of the sample i is referred to be Vi in the data treatment step, see below.
    3. Add 0.15 ml concentrated HCl (37%) to i) the EPS sample or gathered EPS sample up to 0.45 ml, ii) the standard samples, see Table 1. Add enough Milli-Q water to make 0.6 ml total. Mix with a vortex and close the tubes tightly by adding bridges.

      Table 1. Preparation of N-acetylgalactosamine standard samples. We recommend performing a standard curve at each run of the experiment, or at least running the 10 µg standard sample and the blank, see Table 1. The optical density at 530 nm in the 10 µg standard sample should be around 0.2.


    4. Transfer the tubes to the dry bath heater. Maintain temperatures in the dry bath heater at 115 °C for 30 min. Cover the tubes and the dry bath heater with an aluminum foil if necessary.
    5. Transfer the tubes to a rack and cool to room temperature. Then centrifuge for 5 sec the tubes at 2,000 x g (6,000 rpm) using a mini microcentrifuge so the liquid on the top falls back.
    6. SLOWLY add, drop by drop using a pipette, 0.4 ml of 2 M Na2CO3 solution to each tube. Addition of this solution to the sample tubes will cause a very bubbly reaction. Mix gently but completely.
    7. Add 0.5 ml of a freshly made solution containing 2% acetyl acetone in 1.5 M Na2CO3. Vortex each tube, then open them to remove last CO2 released before heat in order to avoid opening of the tubes. Well close the tubes by adding bridges. Heat at 100 °C in a dry bath heater for 20 min.
    8. Transfer the tubes to a rack and cool to room temperature. Then centrifuge the tubes quickly so the liquid on the top falls back.
    9. Transfer liquid into a 15 ml tube and then add 1.0 ml 99.8% ethanol. Mix well.
    10. SLOWLY add 0.5 ml Erlich’s reagent solution. Addition of this solution to the sample tubes will cause a very bubbly reaction, like champagne. Once bubbles slow down, first mix gently and then vortex well.
    11. Let the tubes stand for about 30 min in the dark or with aluminum fold around. Then, read optical density of the solution at 530 nm. Use the water sample of the standard samples as blank, see Table 1.

Data analysis

  1. Plot the measured OD at 530 nm (OD530) by N-acetylgalactosamine mass (mNgal in µg) in the standard samples using appropriate software (SciDavis, Excel, etc.)
  2. Determine the regression equation with the following equation and the value of the correlation factor, α:



  3. Determine the concentration of [Ngal] in mM in the sample i using the following equation:



    where,
    ω is the mass weight of N-acetylgalactosamine, 221.2090 g mol-1,
    Vi is the volume of the sample supernatant i, usually from 0.2 ml to 0.450 ml depending on the step C2.

Notes

  1. We recommend extreme carefulness during this essay since i) the protocol requires usage of concentrated HCl, ii) the acidic solutions are heated over 100 °C and the Eppendorf tube may open, iii) mixing the Na2CO3 and acidic solutions leads to a strong bubbling, and thus liquid may overflow out of the tubes. Hence, working under a fume hood and using gloves is required. Hence, we recommend usage of microcentrifuge tube bridges to avoid opening of the tubes during heat bath.
  2. Using two dry bath heaters with 2 heat blocks each is convenient to avoid temperature adjustment between the different protocol steps and also for running the standard samples plus the replicates.

Recipes

  1. Minimal medium
    20 mM sodium L-glutamic acid monohydrate (carbon and energy source)
    Salt composition:
    1.25 x 10-4 g/L FeSO4·7H2O,
    0.5 g/L (NH4)2SO4
    0.05 g/L MgSO4·7H2O
    3.4 g/L KH2PO4
    The pH is adjusted to 7 with 10 N KOH
    Note: Sodium L-glutamic acid monohydrate is the sole source of carbon and energy in Minimal medium (Plener et al., 2010).
  2. 5 M NaCl solution (10 ml)
    2.92 g NaCl
    Milli-Q water
    Dissolve 2.92 g NaCl in Milli-Q water using a 10 ml volumetric flask
  3. N-acetylgalactosamine standard stock solution at 500 µg ml-1 concentration (100 ml)
    50 mg N-acetylgalactosamine
    100 ml Milli-Q water
    Dissolve 50 mg N-acetylgalactosamine in Milli-Q water using a 100 ml volumetric flask. Prepare 1 ml aliquots in 2.0 ml microcentrifuge tubes and store at -20 °C until use
  4. Standard samples of N-acetyl galactosamine (10 samples with different concentration)
    N-acetyl galactosamine standard stock solution at 500 µg ml-1 concentration
    Milli-Q water
    Prepare the standards samples by diluting various quantity of the N-acetylgalactosamine standard stock solution at 500 µg ml-1 concentration with Milli-Q water to reach 0.450 ml final volume, see Table 1
  5. 2% acetyl acetone, 1.5 M Na2CO3 solution (20 ml)
    3.18 g of Na2CO3
    19.6 ml Milli-Q water
    0.4 ml acetyl acetone
    Dissolve 3.18 g of Na2CO3 in 19.6 ml Milli-Q water in a 50 ml Falcon tube. Then, add 0.4 ml acetyl acetone
  6. Erlich’s reagent (12 ml, for 24 samples)
    0.4 g 4-(dimethylamino)benzaldehyde (Erlich’s reagent)
    6.0 ml 99.8% ethanol
    6.0 ml HCl (37%)
    Under a fume hood, dissolve the 4-(dimethylamino)benzaldehyde into 6 ml ethanol 99.8%. Then, carefully, add 6.0 ml of 37% HCl. Make fresh, just before use and protect from the light

Acknowledgments

This work was supported by EMBO (Long-Term Fellowship ALTF 1627-2011), Marie Curie Actions (EMBOCOFUND2010, GA-2010-267146), the Institut National de la Recherche Agronomique (Plant Health Division grant AAP SPE 2012) and the French Laboratory of Excellence project TULIP (ANR-10-LABX-41; ANR-11-IDEX-0002-02).

References

  1. Balzaretti, S., Taverniti, V., Guglielmetti, S., Fiore, W., Minuzzo, M., Ngo, H. N., Ngere, J. B., Sadiq, S., Humphreys, P. N. and Laws, A. P. (2017). A novel rhamnose-rich hetero-exopolysaccharide isolated from Lactobacillus paracasei DG activates THP-1 human monocytic cells. Appl Environ Microbiol 83(3) pii: e02702-16.
  2. Brumbley, S. M. and Denny, T. P. (1990). Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence. J Bacteriol 172(10): 5677-5685.
  3. Drigues, P., Demery-Lafforgue, D., Trigalet, A., Dupin, P., Samain, D. and Asselineau, J. (1985). Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants. J Bacteriol 162(2): 504-509.
  4. Elson, L. A. and Morgan, W. T. (1933). A colorimetric method for the determination of glucosamine and chondrosamine. Biochem J 27(6): 1824-1828.
  5. Flavier, A. B., Clough, S. J., Schell, M. A. and Denny, T. P. (1997). Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum. Mol Microbiol 26(2): 251-259.
  6. Gatt, R. and Berman, E. R. (1966). A rapid procedure for the estimation of amino sugars on a micro scale. Anal Biochem 15(1): 167-171.
  7. Lee, M. J., Liu, H., Barker, B. M., Snarr, B. D., Gravelat, F. N., Al Abdallah, Q., Gavino, C., Baistrocchi, S. R., Ostapska, H., Xiao, T., Ralph, B., Solis, N. V., Lehoux, M., Baptista, S. D., Thammahong, A., Cerone, R. P., Kaminskyj, S. G., Guiot, M. C., Latge, J. P., Fontaine, T., Vinh, D. C., Filler, S. G. and Sheppard, D. C. (2015). The fungal exopolysaccharide galactosaminogalactan mediates virulence by enhancing resistance to neutrophil extracellular Traps. PLoS Pathog 11(10): e1005187.
  8. Peyraud, R., Cottret, L., Marmiesse, L., Gouzy, J. and Genin, S. (2016). A resource allocation trade-off between virulence and proliferation drives metabolic versatility in the plant pathogen Ralstonia solanacearum. PLoS Pathog 12(10): e1005939.
  9. Plener, L., Manfredi, P., Valls, M. and Genin, S. (2010). PrhG, a transcriptional regulator responding to growth conditions, is involved in the control of the Type III secretion system regulon in Ralstonia solanacearum. J Bacteriol 192(4): 1011-1019.
  10. Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Arlat, M., Billault, A., Brottier, P., Camus, J. C., Cattolico, L., Chandler, M., Choisne, N., Claudel-Renard, C., Cunnac, S., Demange, N., Gaspin, C., Lavie, M., Moisan, A., Robert, C., Saurin, W., Schiex, T., Siguier, P., Thebault, P., Whalen, M., Wincker, P., Levy, M., Weissenbach, J. and Boucher, C. A. (2002). Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415(6871): 497-502.
  11. Vaningelgem, F., Zamfir, M., Mozzi, F., Adriany, T., Vancanneyt, M., Swings, J. and De Vuyst, L. (2004). Biodiversity of exopolysaccharides produced by Streptococcus thermophilus strains is reflected in their production and their molecular and functional characteristics. Appl Environ Microbiol 70(2): 900-912.

简介

可溶性外多糖是由植物病原体Ralstonia solanacearum产生的主要毒力因子。植物感染期间的大量生产与木质部血管中的水流停滞有关,最终导致植物死亡。该重大分子的组成主要包括N-乙酰半乳糖胺。这里我们描述了用于定量测定R中培养上清液中可溶性外多糖的比色法。雷尔氏菌

背景 植物病原体罗勒氏菌(Ralstonia solanacearum)在群体感应系统的控制下产生胞外多糖,即高细胞密度,高于5×10 7细胞ml -1 (Flavier等人,1997)。外多糖的糖含量包括比例为10:2.5:1的半乳糖胺,葡萄糖和鼠李糖(Drigues等人,1985)。 Brumbley和Denny(1990)最初开发了用于从培养上清液中可靠地提取和定量外多糖的方案,并且最近由Peyraud等人(2016)更新。定量基于使用适应的Elson和Morgan测定法测定大分子的己糖胺含量(Elson和Morgan,1933; Gatt和Berman,1966)。含有N-乙酰基-D-半乳糖胺的外多糖由不同的革兰氏阴性或革兰氏阳性细菌(Vaningelgem等人,2004; Balzaretti等人,2017)产生,以及一些真菌(Lee等人,2015),因此该方案也可适用于这些生物。

关键字:青枯雷尔氏菌, 胞外多糖, 毒力因子, 植物病原体, 氨基己糖

材料和试剂

  1. Millex ® -GP 33mm聚醚砜膜和0.22μm孔径的注射器过滤器(EMD Millipore,目录号:SLGP033RB)
  2. 2.0ml Eppendorf Safe-Lock(Eppendorf,目录号:0030120094)的聚丙烯微量离心管
  3. 纸巾
  4. 铝箔
  5. 移液器提示
  6. 15ml锥形聚丙烯Greiner离心管(Greiner Bio One International,目录号:188271)
  7. 50ml锥形聚丙烯Greiner离心管(Greiner Bio One International,目录号:227261)
  8. 微量离心管桥(Milian,目录号:045427)
  9. 手套
  10. R上。茄科菌株GMI1000(Salanoubat等人,2002)。该菌株可从CIRM生物资源中心检索( http://www6.inra.fr/cirm_eng/CFBP-Plant-Associated-Bacteria )在CIRM-BP收集(代码CFBP6924)
  11. 丙酮> 99.8%AnalaR NORMAPUR ®(VWR,目录号:20066.296)
  12. 在25℃电阻率为18.2MOhm cm下获得的Milli-Q水
  13. 乙酰丙酮> 99%AnalaR NORMAPUR ®(VWR,目录号:20092.230)
  14. 乙醇,99.8%(Sigma-Aldrich,目录号:02851)
  15. 4-(二甲基氨基)苯甲醛,98%(Erlich's试剂)(Sigma-Aldrich,目录号:109762)
  16. L-谷氨酸一水合物钠(Sigma-Aldrich,目录号:49621)
  17. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(Sigma-Aldrich,目录号:F8263)
  18. 硫酸铵(NH 4)2 SO 4(Sigma-Aldrich,目录号:A4418)
  19. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:M5921)
  20. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  21. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60377)
  22. 氯化钠(NaCl)> 99.5%AnalaR NORMAPUR ®(VWR,目录号:27810.295)
  23. N-乙酰半乳糖胺,98%(Sigma-Aldrich,目录号:A2795)
  24. (HCl),37%,AnalaR NORMAPUR (VWR,目录号:20252.290)
  25. 碳酸钠(Na 2 CO 3)> 99.9 AnalaR NOR MAPUR ®(VWR,目录号:27771.290)
  26. 最小介质(见配方)
  27. 5 M NaCl溶液(参见食谱)
  28. 标准样品的N-乙酰半乳糖胺(参见食谱)
  29. N-乙酰半乳糖胺标准储备溶液(参见食谱)
  30. 2%乙酰丙酮,1.5M Na 2 CO 3溶液(参见食谱)
  31. Erlich的试剂(见食谱)

设备

  1. 干式加热器具有双块Corning ®(Corning,型号:Corning ® LSE TM数字干浴加热器,目录号:6786-DB)
  2. 通风柜
  3. Eppendorf ®微量离心机5415 R(Eppendorf,型号:5415 R)
  4. 迷你微型离心机Corning ®(Corning,型号:Corning ® LSE TM 迷你微型离心机,目录号:6766)
  5. 涡旋用于微量离心管
  6. Ultrospec TM TM/pro> 2100 pro UV/Visible spectrophotometer(GE Healthcare,型号:Ultrospec TM TM/sup> 2100 pro UV/Visible Spectrophotometer,目录号:80211221)
  7. 10ml容量瓶

软件

  1. 适当的软件(SciDavis,Excel,等等)

程序

  1. 培养和样品制备
    1. 运行液体。 solanacearum 文化。培养在含有以600nm的0.01光密度接种的100ml液体基本培养基的Erlenmeyer中进行,在28℃的温度下以180rpm搅拌。
    2. 以指数或稳定期收集1 ml培养液。细胞培养物必须至少高于600nm或5×10 8细胞ml -1的光密度(理想地约0.6),以确保通过群体感测阈值,从而产生外多糖
    3. 用0.22μm过滤器过滤细胞培养液,并将上清液收集在2.0 ml微量离心管中
    4. 将样品储存在-20°C或立即运行提取。

  2. 外多糖提取
    1. 为了沉淀外多糖,在2.0ml微量离心管中混合0.2ml上清+ 0.004ml 5M NaCl溶液+0.8ml丙酮。与涡旋混合10秒,并将样品置于4℃过夜(约12小时)。
    2. 以16,000 x g(13,000 rpm)和4℃离心样品10分钟,然后弃去上清液。将管子倒在纸巾上,在15分钟内排出沉淀物。然后加入0.2毫升Milli-Q水溶解颗粒
    3. 样品可以在4°C储存几天(可选)直到定量。
    4. 在进行测定的同一天,在65℃下在干浴加热器上加热10分钟。涡旋,然后旋转5分钟,16,000 x g(13,000 rpm),4°C。收集上清液并置于新的2.0 ml微量离心管中。该制剂现在称为EPS(外多糖)样品。

  3. 外多糖定量
    1. 打开位于通风橱中的干浴加热器。添加样品前,温度必须在115°C。
    2. 使用200μlEPS样品,或者可以收集高达450 ml的EPS样品。在数据处理步骤中将样本i的选定体积称为V i i,如下所示。
    3. 加入0.15ml浓盐酸(37%)至i)EPS样品或聚集的EPS样品至0.45ml,ii)标准样品,见表1。加入足够的Milli-Q水使总量达到0.6ml。混合涡流,并通过加入桥梁紧紧关闭管子。

      表1. N-乙酰半乳糖胺标准品的制备。 我们建议在实验的每次运行中执行标准曲线,或至少运行10μg标准样品和空白,见表1. 10μg标准样品中530 nm处的光密度应为0.2左右。


    4. 将管转移到干浴加热器。将干浴加热器的温度保持在115°C 30分钟。如有必要,用铝箔盖住管子和干浴加热器。
    5. 将管子转移到机架上并冷却至室温。然后使用迷你微型离心机以2,000 x g(6,000 rpm)离心5秒钟,使顶部的液体回落。
    6. 慢慢地使用移液管逐滴加入0.4ml 2M Na 2 CO 3溶液至每个管中。将此溶液加入样品管将导致非常气泡的反应。轻轻混合,但完全混合
    7. 在1.5M Na 2 CO 3中加入0.5ml含有2%乙酰丙酮的新制溶液。旋转每个管,然后打开它们以除去加热前释放的最后一个CO 2,以避免管的打开。通过添加桥梁来关闭管子。在100°C的干浴加热器中加热20分钟。
    8. 将管子转移到机架上并冷却至室温。然后快速离心管,使顶部的液体回落。
    9. 将液体转移到15ml管中,然后加入1.0ml 99.8%乙醇。混合好
    10. 缓慢加入0.5 ml Erlich's试剂溶液。将此溶液加入样品管将导致非常气泡的反应,如香槟。一旦气泡减缓,首先轻轻混合,然后旋转好。
    11. 让管子在黑暗中放置约30分钟或用铝折叠。然后,读取530nm处溶液的光密度。使用标准样品的水样为空白,见表1.

数据分析

  1. 使用适当的软件(SciDavis,Excel,Inc。),通过标准样品中的N-乙酰半乳糖胺质量(μg/ml)在530nm(OD 530)下测量的OD曲线>等。)
  2. 用以下公式确定回归方程,并用相关因子α:
    确定


  3. 使用以下公式确定样品i中[Ngal]的浓度为mM:



    其中,
    ω是N-乙酰半乳糖胺的质量重量,221.2090g mol -1,
    根据步骤C2,样品上清液i的体积通常为0.2ml至0.450ml。

笔记

  1. 在本文中,我们建议非常小心,因为i)协议需要使用浓HCl,ii)酸性溶液被加热超过100℃,Eppendorf管可能打开,iii)将Na 2 CO酸性溶液导致强烈的起泡,因此液体可能从管中溢出。因此,需要在通风橱下使用手套。因此,我们建议使用微量离心管桥,以避免在热浴期间打开管。
  2. 使用两个带有两个加热块的干式电加热器可以方便地避免不同协议步骤之间的温度调节,也可以运行标准样品加上复制。

食谱

  1. 最小媒体
    20mM L-谷氨酸一水合物(碳和能量源)
    盐组成:
    1.25×10 -4 -4 g/L FeSO 4·7H 2 O,
    0.5g/L(NH 4)2< 3>< 4>< 4>
    0.05g/L MgSO 4·7H 2 O·
    3.4g/L KH PO 4
    用10 N KOH调节pH至7 注意:L-谷氨酸一水合钠是Minimal培养基中碳和能量的唯一来源(Plener et al。,2010)。

  2. 5 M NaCl溶液(10ml)
    2.92克NaCl
    Milli-Q水
    使用10ml容量瓶溶解2.92g NaCl于Milli-Q水中
  3. N-乙酰半乳糖胺标准储备液,浓度为500μg/ml浓度(100ml)。 50毫克N-乙酰半乳糖胺
    100ml Milli-Q水
    使用100ml容量瓶在Milli-Q水中溶解50mg N-乙酰半乳糖胺。在2.0ml微量离心管中准备1ml等分试样,并保存在-20°C直到使用
  4. 标准样品的N-乙酰半乳糖胺(10个不同浓度的样品)
    浓度为500μg/ml的N-乙酰半乳糖胺标准储备溶液 Milli-Q水
    通过用Milli-Q水稀释各种量的具有500μg/ml浓度的N-乙酰半乳糖胺标准储备溶液以达到0.450ml终体积来制备标准样品,参见表1
  5. 2%乙酰丙酮,1.5M Na 2 CO 3溶液(20ml)
    3.18g的Na 2 CO 3
    19.6毫升Milli-Q水
    0.4ml乙酰丙酮
    将3.18g的Na 2 CO 3溶解在50ml Falcon管中的19.6ml Milli-Q水中。然后加入0.4ml乙酰丙酮
  6. Erlich的试剂(12毫升,24个样品)
    0.4g 4-(二甲基氨基)苯甲醛(Erlich's试剂)
    6.0 ml 99.8%乙醇 6.0ml HCl(37%)
    在通风橱下,将4-(二甲基氨基)苯甲醛溶解在6ml乙醇中99.8%。然后小心地加入6.0ml 37%的HCl。使新鲜,使用前保护光线

致谢

这项工作得到EMBO(长期研究金ALBF 1627-2011),Marie Curie行动(EMBOCOFUND2010,GA-2010-267146),国家农业研究所农业研究所(植物卫生部授予AAP SPE 2012)和法国实验室的支持卓越计划TULIP(ANR-10-LABX-41; ANR-11-IDEX-0002-02)。

参考

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引用:Peyraud, R., Denny, T. P. and Genin, S. (2017). Exopolysaccharide Quantification for the Plant Pathogen Ralstonia solanacearum. Bio-protocol 7(10): e2289. DOI: 10.21769/BioProtoc.2289.
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