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 be transported with the same kinetics and transporters as [(14)C]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 Vmax of 2-NBDG transport was 2.9-fold lower than that for [(14)C]glucose, and the Km 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 [(14)C]mannose and [(14)C]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.
Department of Animal Sciences, University of Florida , P.O. Box 110910, Gainesville, Florida 32611, United States.
Junyi
J
Tao
Department of Animal Sciences, University of Florida , P.O. Box 110910, Gainesville, Florida 32611, United States.
Rebecca K
RK
Diaz
Departamento de Zootecnia, Universidade Federal de Viçosa , Viçosa, Minas Gerais 36570-000, Brazil.
César R V
CR
Teixeira
Department of Animal Sciences, University of Florida , P.O. Box 110910, Gainesville, Florida 32611, United States.
Timothy J
TJ
Hackmann
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001693:Biological Transport, Active / Q000502:physiology; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007553:Isotope Labeling; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D013291:Streptococcus / Q000378:metabolism*
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D003847:Deoxyglucose; D009327:4-Chloro-7-nitrobenzofurazan; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose