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Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli
大肠杆菌脂多糖中酮基-脱氧-d-甘露-8辛酸(KDO)的测定   

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Abstract

2-Keto-3-deoxy-octonate (KDO) is an essential constituent of lipopolysaccharide (LPS) that forms the outermost leaflet of Gram-negative bacterial outer membrane. LPS is mainly composed of lipid A, O-antigen and a core oligosaccharide. Two molecules of KDO are present per one molecule of LPS. A proper level of LPS is required to maintain the outer membrane integrity and either high or low levels of LPS are toxic to the cell. Various methods are available for quantification of LPS; of these, determination of KDO is a simple and accurate method and it can be estimated either directly from crude bacterial cell lysates or from purified LPS by a simple colorimetric assay. Although this procedure can be theoretically used for any Gram-negative bacterium, we used it routinely to measure KDO from cell lysates of Escherichia coli (E. coli) K12 strains.
Method: The protocol is taken from Karkhanis et al. (1978). It is a simple, sensitive and reliable method to measure KDO. The assay is performed after complete acid hydrolysis of cell lysates or LPS to release the various components of LPS. Further, reaction with periodate, arsenite and thiobarbituric acid gives a pink to red color chromophore, which is measured at 548 nm after stabilizing with DMSO.

Keywords: KDO(KDO), Lipopolysaccharide(内毒素), Outer membrane(外层膜), Escherichia coli(大肠杆菌)

Materials and Reagents

  1. Bacterial strain(s) MG1655 (wild-type) (Escherichia coli K12 strain)
  2. Periodate (H5IO6) (Merck Millipore Corporation, catalog number: 10450-60-9 )
  3. Sodium Arsenite (NaAsO2) (Sigma-Aldrich, catalog number: S7400 )
  4. 2-Thiobarbituric acid (Sigma-Aldrich, catalog number: T5500 )
  5. Sulphuric acid (H2SO4) (Thermo Fisher Scientific, catalog number: 7664-93-9 )
  6. Dimethyl sulfoxide (DMSO) (Merck Millipore Corporation, catalog number: 802912 )
  7. HEPES (Merck Millipore Corporation, CalBiochem, catalog number: 7365-45-T )
  8. Quick Start-Bradford Reagent (Bio-Rad Laboratories, catalog number: 5000205 )
  9. 2-Keto-3-deoxyoctonate ammonium salt (KDO) (Sigma-Aldrich, catalog number: K2755 )
  10. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
  11. Tryptone (Difco Laboratories, catalog number: 211705 )
    Note: Currently, it is “BD, Bacto, catalog number: 211705”.
  12. Yeast extract (Difco Laboratories, catalog number: 212750 )
    Note: Currently, it is “BD, Bacto, catalog number: 212750”.
  13. Sodium chloride (NaCl) (Sisco Research Laboratories, catalog number: 7647-14-5 )
  14. Hydrochloric acid (HCl) (Thermo Fisher Scientific, catalog number: 7647-01-0 )
  15. Ultra Pure water (Milli Q water) Millipore Progard TL1Cl2, MilliQ system (Merck Millipore Corporation, catalog number: PROGTLCS1 )
  16. LB broth (see Recipes)
  17. 0.1 M periodate reagent (see Recipes)
  18. 4% sodium arsenite reagent (see Recipes)
  19. 0.6% thiobarbituric acid reagent (see Recipes)
  20. 0.5 N sulphuric acid (see Recipes)
  21. 10 mM HEPES buffer (see Recipes)
  22. 0.5 N HCl (see Recipes)

Equipment

  1. Sonicator (Probe size, 3 mm) (Sonics Vibra, model: VLB 3X )
  2. Centrifuge (Thermo Fisher Scientific, Eppendorf, model: 5417R )
  3. UV-Visible Spectrophotometer (PerkinElmer, model: LAMBDA-35 )
  4. Thermo heating bath (Labnet International, Accu BlockTM Digital Dry bath)
  5. Pipettes (Gilson)

Procedure

  1. Standard
    Make KDO standard 200 μg/ml stock (in water) and use 0, 10, 20, 30, 40 μl for standards (Figure 1). Use ultra pure water to make up the volume to 50 μl.


    Figure 1. Standard curve for KDO estimation. Standard graph showing OD548 values obtained with various amounts of KDO. Approximately 36 μg of KDO corresponds to OD548 of 1.0. Y is the slope and R2 is the correlation coefficient of this standard curve.

  2. Sample preparation
    Preparation of crude cell lysates:
    1. Grow bacterial strain(s) of interest in 2 ml LB (Luria-Bertani broth) overnight at the desired temperature.
    2. Next morning, subculture 1:100 into 25 ml LB and grow until appropriate optical density is reached.
      For example: We have grown MG1655 (wild-type) and a mutant derivative known to make low LPS (MG1655 lpxC1272) to measure KDO. They were grown in 2 ml LB at 37 °C overnight. Next day subcultured 1:100 dilution into 25 ml LB and grown at 37 °C till an OD600 of 1.0.
    3. Pellet down cells by centrifuging at 10,000 rpm/9,391 rcf for 10 min at 4 °C.
    4. Wash the pellet with 1 ml of 10 mM HEPES buffer (pH 7.4) and again resuspend in 1 ml of same buffer.
    5. Sonicate cells on ice for 5 min with 10 sec on and 10 sec off at 40% amplitude.
    6. Spin at 12,000 rpm/13,362 rcf for 10 min at 4 °C to remove unbroken cells. Transfer supernatant to a fresh microcentrifuge tube and store on ice. Quantify the concentration of protein.
      Note: Although the cell lysates are stable on ice for 24 h, in most of our experiments we used them within 6-8 h of preparation. We used Quick start Bradford reagent for estimating the protein concentration of the cell lysates. The protein concentration is used to normalize KDO. Samples with protein concentration range from 8 to 20 mg/ml work well. Normally, with these volumes, we have obtained approximately 10 mg/ml protein.

  3. Assay
    1. To 50 µl of the above cell lysate (sample), add 50 µl of 0.5 N H2SO4 and vortex.
    2. Heat at 100 °C for 15 min for total hydrolysis of LPS. Cool to room temperature.
      Note: Heating is not required for KDO standards; for different Gram-negative bacteria, the time taken for hydrolysis should be standardized.
    3. Add 50 µl periodate reagent (H5IO6), vortex and let it stand for 10 min at room temperature.
    4. Add 200 µl sodium arsenite (NaAsO2) reagent and vortex until the brown color disappears (Figure 2 A).
    5. Add 800 µl thiobarbituric acid reagent and vortex.
    6. Heat at 100 °C for 15 min.
    7. Add 1 ml DMSO to each sample (Figure 2B) when it is warm to make the sample clear and also to stabilize the chromophore.
    8. Cool and measure absorbance at 548 nm on a spectrophotometer (Figure 2C).
    9. Calculate the KDO values based on the standard graph (Figure 1). Normalize the values with protein concentrations.


      Figure 2. Depiction of steps B4, 7-8 in the KDO determination assay. A. Step 4: The assay mixture turns brown immediately after the addition of sodium arsenite (tube 1) and the color disappears after vortexing (tube 2). B. Step 7: After heating at 100 °C for 15 min, the samples turn pink/red depending on the amount of KDO. Tubes 1 and 2 are blank duplicates; 3 and 4 are duplicates each containing approximately 500 μg of total protein (50 μl from the cell lysate having a concentration of 10 mg/ml protein); 5 and 6 are duplicates each containing 250 μg of total protein and; 7 and 8 are duplicates each containing 100 μg of total protein. C. Samples after addition of DMSO. The numbering on tubes is as described earlier in B.

Representative data

  1. KDO values of various E. coli strains
    Strains
    µg of KDO per mg of protein
    Wild type MG1655
    9.0 ± 0.5
    MG1655 lpxC1272
    6.8 ± 0.4
    The value of KDO obtained for wild type in our laboratory was more or less equal to that of the KDO obtained by Ogura et al. (1999).

Notes

  1. The experimental values should be determined from three independent experiments to check the variability and reproducibility of the results.
  2. Procedure should be standardized when determining KDO from other Gram-negative bacteria.

Recipes

  1. LB broth
    1% Tryptone
    0.5% yeast extract
    1% NaCl
  2. 0.1 M periodate reagent
    Dissolve 2.28 g of periodate in 100 ml of ultra pure water
  3. 4% sodium arsenite reagent
    Dissolve 2.0 g of sodium arsenite in 50 ml of 0.5 N HCl
  4. 0.6% thiobarbituric acid reagent
    Dissolve 150 mg of thiobarbituric acid in 25 ml ultra pure water
    Note: Use warm water to dissolve, make it fresh before use.
  5. 0.5 N sulphuric acid
    1.38 ml of concentrated sulphuric acid in 98.62 ml ultra pure water
  6. 10 mM HEPES buffer
    Dissolve 0.238 gm HEPES free acid in 100 ml ultra pure water and adjust pH to 7.4 with NaOH
  7. 0.5 N HCl
    Add 4.16 ml of concentrated HCl to 95.84 ml of ultra pure water

Acknowledgments

KDO estimation protocol was adapted from Karkhanis et al. (1978). Cell lysates were prepared as described in Ogura et al. (1999). This work was supported by funds from Department of Biotechnology (DBT), and Council of Scientific and Industrial Research (CSIR), Government of India.

References

  1. Karkhanis, Y. D., Zeltner, J. Y., Jackson, J. J. and Carlo, D. J. (1978). A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal Biochem 85(2): 595-601.
  2. Mahalakshmi, S., Sunayana, M. R., SaiSree, L. and Reddy, M. (2014). yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli. Mol Microbiol 91(1): 145-157.
  3. Ogura, T., Inoue, K., Tatsuta, T., Suzaki, T., Karata, K., Young, K., Su, L. H., Fierke, C. A., Jackman, J. E., Raetz, C. R., Coleman, J., Tomoyasu, T. and Matsuzawa, H. (1999). Balanced biosynthesis of major membrane components through regulated degradation of the committed enzyme of lipid A biosynthesis by the AAA protease FtsH (HflB) in Escherichia coli. Mol Microbiol 31(3): 833-844.
  4. cmdr.ubc.ca/bobh/methods/KDOASSAY.html.

简介

2-酮-3-脱氧 - 辛酸(KDO)是形成革兰氏阴性细菌外膜的最外侧叶的脂多糖(LPS)的必需成分。 LPS主要由脂质A,O-抗原和核心寡糖组成。每一分子LPS存在两个KDO分子。需要适当水平的LPS以维持外膜完整性,并且高或低水平的LPS对细胞有毒性。各种方法可用于定量LPS;其中,KDO的测定是简单和准确的方法,并且可以通过简单的比色测定直接从粗制细菌细胞裂解物或从纯化的LPS估计。尽管该方法可以理论上用于任何革兰氏阴性菌,我们常规地使用它来测量来自大肠杆菌(大肠杆菌)K12菌株的细胞裂解物的KDO。 br /> 方法:协议取自Karkhanis等人(1978)。它是一种测量KDO的简单,灵敏和可靠的方法。该测定在细胞裂解物或LPS完全酸水解后释放LPS的各种组分进行。此外,与高碘酸盐,亚砷酸盐和硫代巴比妥酸的反应产生粉红色至红色的发色团,其在用DMSO稳定后在548nm测量。

关键字:KDO, 内毒素, 外层膜, 大肠杆菌

材料和试剂

  1. 细菌菌株MG1655(野生型)(大肠杆菌K12菌株)
  2. 高碘酸盐(H 5 IO 6)(Merck Millipore Corporation,目录号:10450-60-9)
  3. 亚砷酸钠(NaAsO 2)(Sigma-Aldrich,目录号:S7400)
  4. 2-硫代巴比妥酸(Sigma-Aldrich,目录号:T5500)
  5. 硫酸(H 2 SO 4)(Thermo Fisher Scientific,目录号:7664-93-9)
  6. 二甲基亚砜(DMSO)(Merck Millipore Corporation,目录号:802912)
  7. HEPES(Merck Millipore Corporation,CalBiochem,目录号:7365-45-T)
  8. Quick Start-Bradford试剂(Bio-Rad Laboratories,目录号:5000205)
  9. 2-酮-3-脱氧辛酸铵盐(KDO)(Sigma-Aldrich,目录号:K2755)
  10. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
  11. 胰蛋白胨(Difco Laboratories,目录号:211705)
    注意:目前为"BD,Bacto,目录号:211705"。
  12. 酵母提取物(Difco Laboratories,目录号:212750)
    注意:目前为"BD,Bacto,目录号:212750"。
  13. 氯化钠(NaCl)(Sisco Research Laboratories,目录号:7647-14-5)
  14. 盐酸(HCl)(Thermo Fisher Scientific,目录号:7647-01-0)
  15. 超纯水(Milli Q水)Millipore Progard TL1Cl2,MilliQ系统(Merck Millipore Corporation,目录号:PROGTLCS1)
  16. LB肉汤(见配方)
  17. 0.1 M高碘酸盐试剂(见配方)
  18. 4%亚砷酸钠试剂(见配方)
  19. 0.6%硫代巴比妥酸试剂(见配方)
  20. 0.5 N硫酸(见配方)
  21. 10 mM HEPES缓冲液(参见配方)
  22. 0.5 N HCl(参见配方)

设备

  1. 超声波仪(探头尺寸,3mm)(Sonics Vibra,型号:VLB 3X)
  2. 离心机(Thermo Fisher Scientific,Eppendorf,型号:5417R)
  3. 紫外可见分光光度计(PerkinElmer,型号:LAMBDA-35)
  4. 热热浴(Labnet International,Accu Block TM Digital Dry bath)
  5. 移液器(Gilson)

程序

  1. 标准
    使KDO标准品200μg/ml(在水中),并使用0,10,20,30,40μl标准品(图1)。使用超纯水补足体积至50μl。


    图1.显示用不同量的KDO获得的OD <548个值的标准图。约36μg的KDO对应于1.0的OD 548。 Y是斜率,R 2 是该标准曲线的相关系数
  2. 样品准备
    粗细胞裂解物的制备:
    1. 在所需温度下在2ml LB(Luria-Bertani肉汤)中生长目的细菌菌株过夜。
    2. 第二天早上,以1:100传代培养至25ml LB中,并生长直到达到适当的光密度。
      例如:我们已经生长了MG1655(野生型)和突变衍生物 已知使低LPS(MG1655 lpxC1272)测量KDO。他们长大了 在2ml LB中在37℃过夜。次日培养1:100稀释 ?25ml LB中并在37℃下生长直到OD <1.0/a> 为1.0。
    3. 通过在4℃下以10,000rpm/9,391rcf离心10分钟沉淀细胞
    4. 用1ml 10mM HEPES缓冲液(pH 7.4)洗涤沉淀,并再次悬浮于1ml相同的缓冲液中。
    5. 超声处理细胞在冰上5分钟,10秒开和10秒关闭在40%幅度。
    6. 以12,000rpm/13,362rcf在4℃下旋转10分钟以除去未破碎的 细胞。将上清转移到新鲜的微量离心管中并储存 ?冰。量化蛋白质的浓度。
      注意:虽然 细胞裂解物在冰上稳定24小时,在我们的大多数实验中 使用它们在6-8小时的准备。我们使用快速启动Bradford 用于评估细胞裂解物的蛋白质浓度的试剂。 蛋白质浓度用于标准化KDO。蛋白质样品 ?浓度范围从8到20 mg/ml工作良好。通常,用这些 体积,我们已经获得约10mg/ml蛋白质。

  3. 测试
    1. 向50μl上述细胞裂解物(样品)中加入50μl0.5N H 2 SO 4 4并涡旋。
    2. 在100℃加热15分钟以使LPS完全水解。冷却至室温。
      注意:KDO标准不需要加热;为不同 革兰氏阴性菌,水解所需的时间应该是 标准化。
    3. 加入50μl高碘酸盐试剂(H 5 IO IO 6),涡旋,并在室温下静置10分钟。
    4. 加入200μl亚砷酸钠(NaAsO 2)试剂,并涡旋,直到棕色消失(图2A)。
    5. 加入800μl硫代巴比妥酸试剂和涡旋。
    6. 在100℃加热15分钟。
    7. 当温度升高时,向每个样品(图2B)中加入1ml DMSO以使样品澄清,并稳定发色团。
    8. 冷却并在分光光度计上测量548nm处的吸光度(图2C)
    9. 基于标准图计算KDO值(图1)。用蛋白质浓度标准化值。


      F igure 2.在KDO确定分析中描述步骤B4,7-8 。 A。 ?步骤4:测定混合物在加入后立即变成棕色 ?亚砷酸钠(管1),并且涡旋后颜色消失(管 ?2)。 B.步骤7:在100℃加热15分钟后,样品转向 粉红色/红色,取决于KDO的量。管1和2为空白 重复; 3和4是重复的,每个含有约500μg 的总蛋白(50μl,来自细胞裂解物, 10 mg/ml蛋白); 5和6是重复的,每个含有250μg 总蛋白; 7和8是重复的,每个含有100μg 总蛋白。 C.加入DMSO后的样品。管上的编号 是如前面的B.

代表数据

  1. 各种E的KDO值。大肠杆菌菌株
    菌株
    μg的KDO/mg蛋白质
    野生型 MG1655
    9.0±0.5
    MG1655 lpxC1272
    6.8±0.4
    在我们的实验室中获得的野生型KDO的值或多或少等于Ogura等人(1999)获得的KDO的值。

笔记

  1. 实验值应由三个独立实验确定,以检查结果的可变性和重现性
  2. 当从其他革兰氏阴性菌确定KDO时,应当对程序进行标准化

食谱

  1. LB肉汤
    1%胰蛋白胨
    0.5%酵母提取物
    1%NaCl
  2. 0.1 M高碘酸试剂
    将2.28g高碘酸盐溶于100ml超纯水中
  3. 4%亚砷酸钠试剂
    将2.0g亚砷酸钠溶于50ml 0.5N HCl中
  4. 0.6%硫代巴比妥酸试剂
    将150mg硫代巴比妥酸溶于25ml超纯水中
    注意:使用温水溶解,在使用前清新。
  5. 0.5 N硫酸
    1.38ml浓硫酸在98.62ml超纯水中的溶液
  6. 10mM HEPES缓冲液
    将0.238gm HEPES游离酸溶于100ml超纯水中,用NaOH调节pH至7.4。
  7. 0.5 N HCl
    将4.16ml浓HCl加入到95.84ml超纯水中

致谢

KDO估计方案改编自Karkhanis等人(1978)。如Ogura等人(1999)所述制备细胞裂解物。这项工作得到生物技术部(DBT)和印度政府科学和工业研究理事会(CSIR)的资助。

参考文献

  1. Karkhanis,Y.D.,Zeltner,J.Y.,Jackson,J.J.and Carlo,D.J。(1978)。 一种新的改进的微测定法,用于测定革兰氏阴性菌脂多糖中的2-酮-3-脱氧辛酸盐。 Anal Biochem 85(2):595-601
  2. Mahalakshmi,S.,Sunayana,M. R.,SaiSree,L.和Reddy,M.(2014)。 yciM是调节大肠杆菌中脂多糖合成所需的必需基因。 。 Mol Microbiol 91(1):145-157。
  3. Ogura,T.,Inoue,K.,Tatsuta,T.,Suzaki,T.,Karata,K.,Young,K.,Su,LH,Fierke,CA,Jackman,JE,Raetz,CR,Coleman, ,Tomoyasu,T。和Matsuzawa,H。(1999)。 通过由AAA蛋白酶调节脂质A生物合成的承诺酶的降解来平衡主要膜组分的生物合成FtsH(HflB)in大肠杆菌。 Mol Microbiol 31(3):833-844。
  4. cmdr.ubc.ca/bobh/methods/KDOASSAY.html />
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sunayana, M. R. and Reddy, M. (2015). Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli. Bio-protocol 5(24): e1688. DOI: 10.21769/BioProtoc.1688.
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