Dynamics of Glucose Uptake by Single Escherichia coli Cells

Arvind Natarajan, Friedrich Srienc

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60 Scopus citations

Abstract

The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells. When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate. The dependence upon substrate concentration could be described according to Michaelis-Menten kinetics with apparent saturation constant KM=1.75μM, and maximum 2-NBDG uptake rate=197molecules/cell-second. Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by d-glucose, 3-o-methyl glucose, and d-glucosamine, and lack of inhibition by l-glucose. Inhibition of 2-NBDG uptake by d-glucose was competitive in nature. The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of d-glucose in the culture medium (~0.2μM) is detectable. The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity. The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics. A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates. The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation

Original languageEnglish (US)
Pages (from-to)320-333
Number of pages14
JournalMetabolic Engineering
Volume1
Issue number4
DOIs
StatePublished - Oct 1999

Bibliographical note

Funding Information:
We thank H. Matsuoka for kindly providing 2-NBDG. This work was supported in part by the National Science Foundation (BES-9708146).

Keywords

  • 2-NBDG
  • Escherichia coli
  • glucose uptake
  • single-cell analysis

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