Overcoming fluctuation and leakage problems in the quantification of intracellular 2-oxoglutarate levels in Escherichia coli

Abstract

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in Escherichia coli. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.

Fig. 1.

HPLC chromatograms of DMB derivatives of 2-oxo acids. Blank, HPLC-grade water (after derivatization reaction); standard, equal amounts of 2OG, 2OB, and 2OV; sample, cellular extract with the standard 2OB and 2OV. The two visible peaks in the blank overlap with those for some of other tested 2-oxo acids, possibly contamination from the reagents and/or water. Injection of water yields no fluorescence peak.

Fig. 2.

2OG leakage and steady-state pool concentration during exponential growth of E. coli. Wild-type strain NCM3722 was cultured in NH₄Cl-glycerol medium. (A) 2OG concentrations in the medium were assayed from immediately after inoculation through the late exponential phase of growth. The growth curve is shown in the inset; the doubling time was 62 min. (B) Paired filter and medium samples were collected along the exponential phase of growth. Each pool value was the result of subtraction from paired samples.

Fig. 3.

The 2OG pool is subject to a sudden decrease and increase upon nutrient shift. (A) Wild-type strain NCM3722 was inoculated in glycerol-NH₄⁺ medium. Sampling was carried out during the exponential phase of growth. Wash medium contained different concentrations of glycerol from 43 mM (0.4% [wt/vol], as in the starting medium) down to zero. (B) The wild-type, GDH⁻ (FG1113), and GOGAT⁻ (FG1195) strains were inoculated in glycerol medium with 11.5 mM NH₄⁺. Samples were collected during the exponential phase of growth, when the concentration of NH₄⁺ left in the culture was 10 ± 0.2 mM. Wash medium contained different concentrations of NH₄⁺ from 10 mM down to zero. (C) Data represented by open symbols are the same as those in panel B and plotted in a semilog scale. The closed symbols are from a washup experiment with the GOGAT⁻ mutant. Cells were inoculated in glycerol medium with 3.5 mM NH₄⁺. Samples were collected during the exponential phase of growth, when the concentration of NH₄⁺ left in the culture was 2 ± 0.2 mM. The wash medium contained different concentrations of NH₄⁺ from 2 mM up to 50 mM. All inoculations were started at an OD₆₀₀ of ∼0.05, and samplings were performed at OD₆₀₀s of between 0.35 and 0.40. For sample collection, 1 ml of cells was quickly filtered and washed twice with 2 ml medium. The time between the point when the wash medium contacted cells on the filter and the filter was immersed into acid for extraction was controlled to be 15 ± 2 s. The error bars in panels A and B are minimal and maximal values obtained in at least two independent experiments.

Fig. 4.

Temperature and time effects on 2OG pool during filtration and wash. (A) Wild-type strain NCM3722 was inoculated in glycerol medium with 11.5 mM NH₄⁺. Samples were collected during the exponential phase of growth, when the concentration of NH₄⁺ left in the culture was 10 ± 0.2 mM. Wash medium equilibrated either in a 37°C culture room or in ice water contained different concentrations of NH₄⁺. (B) Strain NCM3722 was inoculated in glycerol medium with 20 mM NH₄⁺. Samples were collected during the exponential phase of growth, when the OD₆₀₀ was between 0.3 and 0.4. Wash medium was identical to the starting medium, with 20 mM NH₄⁺ and equilibrated at 37°C. N, normal handling speed of filtration and wash that lasted ∼15 s; N + 30", cells were held in a pipette tip for 30 s after the culture was withdrawn from the vessel and before filtration; N + 60", hold for 60 s. The error bars are minimal and maximal values for two different OD points.

Fig. 5.

Steady-state pool concentration obtained by the filtration and wash strategy. Cells were grown in NH₄⁺-glycerol, NH₄⁺-glucose, or proline-glycerol medium. Samples were collected at 3 to 5 different cell densities for each culture during its exponential phase of growth. Wash medium was identical to the starting medium with 20 mM either NH₄⁺ or proline. The results were from two sets of independent experiments. Average doubling times were 63 min for the wild type (WT), 68 min for the GDH⁻ mutant, 65 min for the GOGAT⁻ mutant, 48 min for the wild type grown in NH₄⁺-glucose, and 99 min for the wild type grown in proline-glycerol.

Fig. 6.

Continuous and near linear accumulation of external 2OG without a functional KgtP. E. coli (Ec) kgtP mutant strain FG1602 (A) and Salmonella serovar Typhimurium (Se) wild-type strain SK2633 (B) were cultured in NH₄Cl-glycerol medium. 2OG concentrations in medium were assayed from immediately after inoculation through the late exponential phase of growth. Internal 2OG concentrations were also measured at different cell densities. The E. coli wild-type external 2OG data in panel A are from Fig. 2A for comparison. (C) kgtP is transcribed in E. coli but silent in Salmonella serovar Typhimurium. The three strains were cultured identically to those described in panels A and B, cells were collected at an OD₆₀₀ of ∼0.4, and RNA was extracted. cDNA was reverse transcribed and subjected to PCR detection of kgtP expression. Genomic DNA extracted from colonies served as the control template for PCR.