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Choline Uptake Assay in Bacterial Cells    

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In this protocol

Original research article

A brief version of this protocol appeared in:
Journal of Bacteriology
Feb 2013

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, Uptake assay, Choline transporter, Brucella, Brucellosis

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.
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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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