Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli

Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would b...

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Bibliographic Details
Published in:Biochemistry (Easton) 2016-05, Vol.55 (18), p.2578-2589
Main Authors: Tao, Junyi, Diaz, Rebecca K, Teixeira, César R. V, Hackmann, Timothy J
Format: Article
Language:English
Subjects:
4-Chloro-7-nitrobenzofurazan - analogs & derivatives
4-Chloro-7-nitrobenzofurazan - chemistry
4-Chloro-7-nitrobenzofurazan - metabolism
Biological Transport, Active - physiology
Deoxyglucose - analogs & derivatives
Deoxyglucose - chemistry
Deoxyglucose - metabolism
Escherichia coli - metabolism
Escherichia coli Proteins - metabolism
Isotope Labeling
Monosaccharide Transport Proteins - metabolism
Streptococcus - metabolism
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Summary:Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [14C]­glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the V max of 2-NBDG transport was 2.9-fold lower than that for [14C]­glucose, and the K m was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [14C]­mannose and [14C]­deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement.
ISSN:0006-2960
1520-4995
DOI:10.1021/acs.biochem.5b01286