Article pubs.acs.org/biochemistry
Transport of a Fluorescent Analogue of Glucose (2-NBDG) versus Radiolabeled Sugars by Rumen Bacteria and Escherichia coli Junyi Tao,† Rebecca K. Diaz,† César R. V. Teixeira,‡ and Timothy J. Hackmann*,† †
Department of Animal Sciences, University of Florida, P.O. Box 110910, Gainesville, Florida 32611, United States Departamento de Zootecnia, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-000, Brazil
‡
S Supporting Information *
ABSTRACT: 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 Vmax of 2-NBDG transport was 2.9-fold lower than that for [14C]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 [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 2NBDG 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.
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Despite their potential as a substitute for radioisotopes, fluorescent tracers such as 2-NBDG have never been evaluated by direct comparison to their radiolabeled counterparts. Comparisons, when made, have been indirect; for example, one study reported the Km for 2-NBDG transport broadly agreed with those for transport of radiolabeled glucose analogues in other studies,18 but different cell types and measurement conditions were used in these studies. Transport of 2-NBDG has been measured by several techniques based on microscopy,3,18 fluorimetry,3 and flow cytometry,19 but these techniques have not been adapted to accommodate radiolabeled tracers. Further, measurements reported often have been only qualitative. Electrophysiological techniques have been used to compare transport of fluorescent and nonfluorescent compounds,14 which is an attractive approach but compatible only with electrogenic transport. Here we compared transport of 2-NBDG versus that of [14C]glucose and other radiolabeled sugars, facilitated by a technique commonly used for radiolabeled tracers that we adapted to accommodate fluorescent tracers. Unlike previous
inetics of solute transport traditionally have been measured with radiolabeled tracers.1,2 Over the past two decades, transport has also been measured with fluorescently labeled tracers, including analogues of glucose,3−6 other monosaccharides,7 trehalose,8 amino acids,9,10 peptides,11 toluene,12 and polyamines.13 Some fluorescent tracers are not labeled analogues but are naturally fluorescent compounds that fortuitously are transported by a transporter system of interest; for example, esculin is transported by type I plant sucrose transporters.14 These fluorescent tracers offer certain advantages over their radiolabeled counterparts, such as (i) being compatible with fluorescent techniques (e.g., microscopy and flow cytometry), (ii) being adaptable to single, living cells, and (iii) not requiring use of radioactive facilities. Of all fluorescent tracers, the glucose analogue 2-NBDG {2[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose} has been most broadly applied and studied. 2-NBDG can be transported by several types of bacterial,3,9 yeast,15,16 plant,17 and mammalian cells.18 As expected for a glucose tracer, transport is competitively inhibited by glucose and its analogues.3,16,18,19 Transport by GLUTs in mammalian cells has been demonstrated by overexpressing the transporter and by inhibition with cytochalasin B.18 © 2016 American Chemical Society
Received: November 30, 2015 Revised: April 19, 2016 Published: April 20, 2016 2578
DOI: 10.1021/acs.biochem.5b01286 Biochemistry 2016, 55, 2578−2589
Article
Biochemistry
centrifuge; Hettich), washed twice with ice-cold modified Simplex buffer (composition described below), and resuspended to give a concentration of ∼0.1 g of protein L−1. For S. equinus JB1, the duration of centrifugation steps was 5 min (not 10 min). For E. coli, all steps were done aerobically and with M9 buffer [M9 medium with glucose omitted (pH 6.8)]; Simplex buffer could not be used because its bicarbonate buffering system requires a CO2 headspace. Modified Simplex buffer (pH 6.8) contained 6.35 g of K2HPO4, 5 g of KH2PO4, 650 mg of NaC1, 90 mg of MgSO4· 7H2O, 60 mg of CaC12·2H2O, 7.5 g of NaCHO3, and 0.5 g of cysteine hydrochloride per liter. Following earlier practice in our lab,23,24 it was modified from its original composition25 to include a higher proportion of (i) a Coleman-type salt solution (to increase buffering capacity) and (ii) a cysteine hydrochloride solution (to make the sample more resistant to oxidation). Transport Experiments. Washed cells (0.25 mL aliquots) were transferred anaerobically to culture tubes and prewarmed for 10 min. Transport was then initiated by adding 5 μL of 2NBDG or radiolabeled sugar ([U-14C]-D-glucose, [U-14C]-2deoxy-D-glucose, or [2-3H]-D-mannose; PerkinElmer). Cells were gassed with O2-free CO2 and maintained at 39 °C. Transport was terminated by adding 2 mL of a stop solution (composition described below). The reaction mixture was then transferred with a pipet to a white polycarbonate membrane filter (0.4 μm; Nuclepore; Whatman). The tube, pipet, and filter were washed with 2 mL of a stop solution. Filtration was always completed within
