Data from: Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate

We have developed a method for rapid quenching of samples taken from chemostat cultures of Escherichia coli that gives reproducible and reliable measurements of extracellular and intracellular metabolites by 1H NMR and have applied it to study the major central metabolites during the transition from anaerobic to aerobic growth. Almost all metabolites showed a gradual change after perturbation with air, consistent with immediate inhibition of pyruvate formate-lyase, dilution of overflow metabolites and induction of aerobic enzymes. Surprisingly, although pyruvate showed almost no change in intracellular concentration, the extracellular concentration transiently increased. The absence of intracellular accumulation of pyruvate suggested that one or more glycolytic enzymes might relocate to the cell membrane. To test this hypothesis, chromosomal pyruvate kinase (pykF) was modified to express either PykF-green fluorescent protein or PykF-FLAG fusion proteins. Measurements showed that PykF-FLAG relocates to the cell membrane within 5 min of aeration and then slowly returns to the cytoplasm, suggesting that on aeration, PykF associates with the membrane to facilitate secretion of pyruvate to maintain constant intracellular levels.

我们开发了一种可快速淬灭大肠杆菌（Escherichia coli）恒化培养样本的方法，该方法能够通过1H核磁共振波谱法（1H NMR）对胞外与胞内代谢物进行可重复且可靠的检测，并将其应用于探究从厌氧生长向有氧生长转变过程中的主要中心代谢物变化。几乎所有代谢物在受空气扰动后均呈现渐进式变化，这与丙酮酸甲酸裂解酶（pyruvate formate-lyase）被快速抑制、溢出代谢物被稀释以及有氧代谢相关酶被诱导表达的现象一致。令人意外的是，尽管胞内丙酮酸浓度几乎未发生改变，但其胞外浓度却出现了短暂升高。胞内未出现丙酮酸积累的现象，提示一种或多种糖酵解酶可能发生了向细胞膜的移位。为验证这一假说，我们对染色体上的丙酮酸激酶（pyruvate kinase, pykF）进行改造，使其表达PykF-绿色荧光蛋白或PykF-FLAG融合蛋白。检测结果显示，PykF-FLAG在通气后5分钟内即可移位至细胞膜，随后缓慢返回细胞质中，这表明通气时PykF会与细胞膜结合，以促进丙酮酸的分泌，从而维持胞内丙酮酸浓度的稳定。