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.我们开发了一种可对大肠杆菌（Escherichia coli）恒化器培养物采集的样品进行快速淬灭的方法，该方法可通过氢核磁共振波谱（1H NMR）实现胞外与胞内代谢物的可重复且可靠的检测，并将其应用于厌氧至有氧生长转变过程中主要中心代谢物的研究。通空气扰动后，几乎所有代谢物均呈现渐进式变化，这与丙酮酸甲酸裂解酶（pyruvate formate-lyase）的即时抑制、溢流代谢物的稀释以及有氧代谢酶的诱导表达相一致。令人意外的是，尽管胞内丙酮酸浓度几乎未发生变化，但其胞外浓度却出现了瞬时升高。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转位。为验证该假说，研究人员对染色体上的丙酮酸激酶（pykF）基因进行改造，使其表达PykF-绿色荧光蛋白或PykF-FLAG融合蛋白。实验检测结果表明，PykF-FLAG在曝气后5分钟内即可转位至细胞膜，随后缓慢返回细胞质，这提示在曝气条件下，PykF会与细胞膜结合以促进丙酮酸分泌，从而维持胞内丙酮酸浓度的稳定。