Choline Uptake Assay in Bacterial Cells

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: WinSpectral^TM 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.