|Flythe, Michael - CORNELL UNIVERSITY|
Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 1, 2004
Publication Date: May 23, 2004
Citation: Flythe, M., Russell, J.B. 2004. The effect of acidic ph on the protonmotive force and intracellular potassium of clostridium sporogenes md1. American Society for Microbiology Annual Meeting. p. 325. Technical Abstract: Background: Plant materials are preserved as silage for cattle, but clostridia that deaminate amino acids increase pH. If the pH rises, spoilage organisms proliferate, and undesirable products accumulate. Clostridium sporogenes MD1 deaminated amino acids at pH values as low as 3.5, but it did not grow at values less than 4.6. The following experiments sought to define the effect of acidic pH on C. sporogenes MD1. Methods: C. sporogenes MD1 was grown anaerobically with Casaminoacids as an energy source. Intracellular pH and membrane potential and were estimated from the uptake of 14C tetraphenylphosphonium (TPP+) and benzoate, respectively. Valinomycin and nigericin treated cells were used to correct for non-specific TPP+ binding, and membrane voltage was calculated with the Nernst equation. Cells harvested through silicon oil were digested with nitric acid and analyzed for potassium by flame photometry. ATP was determined by luciferase. Results: Cultures grown at pH 6.5 had a pH gradient across the cell membrane less than 0.2 units (-15 mV) and membrane potential only -20 mV, but the intracellular potassium concentration was 375 mM. When the culture pH was decreased to pH 4.1 with HCl, pH gradient and membrane potential both increased (-70 and -50 mV, respectively). Intracellular pH declined to 5.3, but the cells could still deaminate amino acids and generate ATP at a rapid rate. However, intracellular potassium decreased 25-fold. Cells harvested by centrifugation, washed aerobically in potassium phosphate buffer (100 mM, pH 6.8) and re-suspended in sodium phosphate buffer (100 mM, pH 6.8) maintained an intracellular potassium concentration greater than 100 mM for at least 60 min. However, if these cells were re-suspended in sodium phosphate buffer (100 mM, pH 4.0), intracellular potassium declined more than 10-fold after only 20 min. Based on these results, the inhibition of C. sporogenes MD1 by acidic pH is more closely associated with its inability to maintain a potassium gradient than a decrease in protonmotive force. Conclusions: Acidic pH decreases the intracellular potassium concentration of C. sporogenes MD1, and this decrease leads to an inhibition of growth.