Abstract
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.
Highlights
Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate
The acetyl CoA produced anaerobically is converted into coenzyme A and acetate via acetylphosphate, allowing energy conservation by substratelevel phosphorylation of adenosine diphosphate (ADP), and to ethanol in a process that reoxidizes the NADH generated during glycolysis
The results suggest that a significant fraction of the cytoplasmic PykF relocated to the cell membrane on introduction of oxygen, possibly as part of a glycolytic metabolon regulated by aerobic stress
Summary
Homeostasis of metabolites in Escherichia coli on transition from anaerobic to aerobic conditions and the transient secretion of pyruvate. It can completely oxidize it via glycolysis to pyruvate, which is taken on to the citric acid cycle and electron transport chain by the pyruvate dehydrogenase complex (PDHC). This mode of growth is by far the most energy efficient [1]. If there is no such electron acceptor, it can grow by fermentation This is the least energy efficient of the three modes, and in order to achieve redox balance it produces overflow metabolites (acetate, ethanol, formate, lactate and succinate) which it secretes into the growth medium. Its ability to switch rapidly from one growth mode to another is crucial for its success
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