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Choline Uptake Assay in Bacterial Cells
细菌细胞中的胆碱摄取分析   

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Abstract

Choline is a methylated nitrogen compound that is widespread in nature. It is a precursor of several metabolites that perform numerous biological functions and it is predominantly used for the synthesis of essential lipid components of the cell membranes. Since there is no evidence that prokariotes can synthesize choline de novo and because choline uptake from exogenous sources is energetically more favorable than de novo synthesis, bacteria have evolved different uptake mechanisms for choline transport across the bacterial membrane. This protocol describes an easy and high sensitive method to assess choline uptake in bacteria using as tracer [3H]-choline chloride. The protocol was originally intended for Brucella abortus but could be applied for any bacteria with the corresponding modifications depending on the bacteria growth requirements (composition of the culture medium, temperature for growth, etc.). It can be useful to determine the choline uptake ability of several bacterial species under different growth conditions.

Keywords: Choline(胆碱), Uptake assay(吸收法), Choline transporter(胆碱转运), Brucella(布鲁氏菌), Brucellosis(布氏杆菌病)

Materials and Reagents

  1. Brucella abortus or the bacteria species you want to test
  2. Minimal medium (MM) such as M9 or equivalent
    In the case of Brucella abortus we use Gerhardt-Wilson (GW) minimum medium (Gerhardt, 1958)
  3. Choline Chloride ([Methyl-3H]-, 250 μCi (9.25 MBq)) (PerkingElmer, catalog number: NET109250UC )
  4. [3H]-choline/cold choline chloride (1:100)
  5. Liquid Scintillation (liquid Optiphase HISAFE 3) (PerkingElmer, catalog number: 1200-437 )

Equipment

  1. Microplate reader or Spectrophotometer
  2. Liquid scintillation spectrometer (Gemini BV, model: WinSpectralTM 1414)

Procedure

  1. For radioactive choline uptake analyses, cultures of bacteria grown in an appropriate minimal medium (MM) at mid log phase were harvested, washed three times with ice-chilled MM and adjusted to an optical density at 600 nm (OD600) of 0.5 with fresh MM.
    Note: Bacterial culture is harvested at OD600= 1-1.2 that correspond to a mid log phase for Brucella under this growth conditions.
  2. Reactions were initiated by addition of [3H]-choline chloride (80.6 Ci/mmol) to a final concentration of 3.3 μM, incubated at 37 °C and aliquots (around 300 μl) were taken at different time points (0 to 30 min).
  3. Samples were immediately chilled on ice, washed five times with the same volume of ice-chilled MM, centrifuged at 10,000 x g, 15 min at 4 °C and cell pellets were resuspended in 500 μl of scintillation liquid.
  4. The radioactivity in the cell pellet was determined with a liquid scintillation spectrometer.
  5. To assess uptake kinetics at different choline concentrations, bacteria were incubated 7 min at 37 °C in MM with a mix of [3H]-choline/cold choline (1:100) ranging from 6.25 x 10-2 μM to 64 μM (total choline concentration) and incorporated radioactivity in the cell pellet was determined as describe above.

Acknowledgments

This protocol was adapted from Herrmann et al. (2013).

References

  1. Gerhardt, P. (1958). The nutrition of brucellae. Bacteriol Rev 22(2):81-98.
  2. Herrmann, C. K., Bukata, L., Melli, L., Marchesini, M. I., Caramelo, J. J. and Comerci, D. J. (2013). Identification and characterization of a high-affinity choline uptake system of Brucella abortus. J Bacteriol 195(3): 493-501.

简介

胆碱是一种在自然界广泛存在的甲基化氮化合物。它是几种代谢物的前体,其执行许多生物功能,并且其主要用于合成细胞膜的必需脂质组分。因为没有证据表明原核生物可以从头合成胆碱,并且因为来自外源的胆碱吸收在能量上比新合成更有利,所以细菌已经对胆碱发展了不同的吸收机制运输通过细菌膜。该方案描述了使用示踪物[3 H] - 氯化胆碱来评估细菌中胆碱吸收的容易和高灵敏度的方法。该方案最初用于
流感布鲁氏菌,但可应用于具有相应修饰的任何细菌,这取决于细菌生长需要(培养基的组成,生长温度, em>)。它可用于确定在不同生长条件下几种细菌物种的胆碱吸收能力。

关键字:胆碱, 吸收法, 胆碱转运, 布鲁氏菌, 布氏杆菌病

材料和试剂

  1. 布鲁氏菌或您要测试的菌种
  2. 最小介质(MM),如M9或等效
    在 Brucella abortus 的情况下,我们使用Gerhardt-Wilson(GW)最小培养基(Gerhardt,1958)
  3. 氯化胆碱([甲基 - 3 H] - ,250μCi(9.25MBq))(PerkingElmer,目录号:NET109250UC)
  4. [1:3 H] - 胆碱/冷胆碱氯化物(1:100)
  5. 液体闪烁(液相Optiphase HISAFE 3)(PerkingElmer,目录号:1200-437)

设备

  1. 酶标仪或分光光度计
  2. 液体闪烁光谱仪(Gemini BV,型号:WinSpectral TM 1414)

程序

  1. 对于放射性胆碱摄取分析,收获在对数中期在合适的基本培养基(MM)中生长的细菌培养物,用冰冷的MM洗涤三次,并调节至600的光密度 nm(OD 600)为0.5,用新鲜MM 注意:细菌培养物在OD 600 = 1-1.2下收获,对应于在该生长条件下的布鲁氏菌。
  2. 通过加入[3 H] - 胆碱氯化物(80.6Ci/mmol)至终浓度为3.3μM,在37℃下孵育开始反应,在不同的时间取出等分试样(约300μl)时间点(0至30分钟)。
  3. 将样品立即在冰上冷却,用相同体积的冰冷的MM洗涤5次,在10,000xg下离心,在4℃下15分钟,将细胞沉淀重悬于500μl闪烁液中。
  4. 用液体闪烁光谱仪测定细胞沉淀中的放射性。
  5. 为了评估在不同胆碱浓度下的摄取动力学,将细菌在37℃下在MM中与[6. 3 H] - 胆碱/冷胆碱(1:100)的混合物温育7分钟,范围为6.25×10 6如上所述测定细胞沉淀中的 -2 μM至64μM(总胆碱浓度)和掺入的放射性。

致谢

该方案改编自Herrmann等人(2013)。

参考文献

  1. Gerhardt,P。(1958)。 布鲁氏菌的营养。 Bacteriol Rev 22(2 ):81-98。
  2. Herrmann,C.K.,Bukata,L.,Melli,L.,Marchesini,M.I.,Caramelo,J.J.and Comerci,D.J。(2013)。 鉴定和表征布鲁氏菌abortus的高亲和性胆碱吸收系统 。 195(3):493-501。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bukata, L., Herrmann, C. K. and Comerci, D. J. (2013). Choline Uptake Assay in Bacterial Cells . Bio-protocol 3(18): e902. DOI: 10.21769/BioProtoc.902.
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