title: Homeostasis of metabolites inEscherichia colion transition from anaerobic to aerobic conditions and the transient secretion of pyruvate.
abstract: We have developed a method for rapid quenching of samples taken from chemostat cultures ofEscherichia colithat gives reproducible and reliable measurements of extracellular and intracellular metabolites by1H 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.
authors.affiliation: Department of Molecular Biology and Biotechnology , University of Sheffield , Firth Court, Western Bank, Sheffield S10 2TN , UK.
authors.forename: Nur Adeela
authors.initials: NA
authors.lastname: Yasid
authors.affiliation: Department of Molecular Biology and Biotechnology , University of Sheffield , Firth Court, Western Bank, Sheffield S10 2TN , UK.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Rolfe
authors.affiliation: Department of Molecular Biology and Biotechnology , University of Sheffield , Firth Court, Western Bank, Sheffield S10 2TN , UK.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Green
authors.affiliation: Department of Molecular Biology and Biotechnology , University of Sheffield , Firth Court, Western Bank, Sheffield S10 2TN , UK.
authors.forename: Mike P
authors.identifier: 0000-0001-5572-1903
authors.initials: MP
authors.lastname: Williamson
keywords: Escherichia coli; glycolysis; metabolomics; metabolon; nuclear magnetic resonance