Characterization of pyruvate uptake in Escherichia coli K-12

Abstract

The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.

Figure 1. Transport-activity, apparent K_m and V_max values for pyruvate uptake in E.coli LJ110.

(A) Transport-activity of [1-¹⁴C] pyruvate uptake determined with four different substrate concentrations: Δ 44 µM, ▴ 26 µM, □ 13 µM, ▪ 5 µM. (n = 3) (B) Double reciprocal plot to determine the apparent K_m value and the corresponding V_max value for LJ110. The initial uptake rates were used to calculate the K_m and V_max at substrate concentrations ranging from 5 µM to 66 µM.

Figure 2. Influence of FP on growth and pyruvate transport-activity on E. coli LJ110

(A) Cells were grown in minimal medium plus 0.2% pryruvate. The transport activity was determined with 26 µM [1-¹⁴C] pyruvate with (▪) and without (□) the addition of 2 mM FP. (n = 3) (B) The growth inhibitory effect of different FP concentrations was determined with cells in the early log phase grown on minimal medium plus 0.2% glucitol. □ 0 mM FP, ▪ 0.1 mM FP, Δ 0.25 mM FP, ◊ 0.5 mM FP, ▴ 1 mM FP. (n = 3)

Figure 3. Influence of FP on cell viability of E. coli LJ110.

The cells were grown in minimal medium plus glycerol (0.2%). Addition of FP (1 mM) is indicated by the arrow. Cell-viability was determined by plating cells at indicated time-points on LB plates. □ A₄₂₀, ▪ viable cells [%].(n = 2)

Figure 4. Transport-activity and growth on pyruvate.

(A) Pyruvate uptake of E. coli LJ110 and LJK3 was measured with [1-¹⁴C] pyruvate at a final concentration of 6 µM. Cells were grown in minimal medium plus glycerol (0.2%). (n = 3) (B) Cells pre-grown on minimal medium plus glycerol (0.2%) were washed and inoculated in minimal medium plus pyruvate (0.2%) and growth was followed by measurement of absorption at 420 nm. □ LJ110, ▪ LJK3. (n = 3)

Figure 5. Concentration of extracellular pyruvate during growth of E. coli LJK3 under potassium limiting conditions.

(A) Pyruvate concentration in µM; □ LJ110, ▪ LJK3. (n = 3) (B) Pyruvate concentration as a function of cell-density. Cells were pre-grown in minimal medium plus glucose and 115 mM potassium (0.2%) and inoculated in medium plus glucose and no potassium (0.4%). Extracellular pyruvate was measured enzymatically as described in materials and methods. A₄₂₀ = □ LJ110, ▪ LJK3; µM pyruvate/A₄₂₀ = Δ LJ110, ▴ LJK3.