Characterization of the Zymomonas mobilis glucose facilitator gene product (glf) in recombinant Escherichia coli: examination of transport mechanism, kinetics and the role of glucokinase in glucose transport

Abstract

Zymomonas mobilis is known to transport glucose by a facilitated diffusion process. A putative glucose facilitator gene (glf), closely related to a large family of glucose transporters, is located in a cluster of genes that code for enzymes of glucose metabolism. The Z. mobilis glf gene is able to complement glucose transport in an Escherichia coli strain that is defective in native glucose transport and glucokinase. In this study, the recombinant E. coli was shown to be capable of influx counterflow when preloaded with glucose and had an apparent Km for glucose of approximately 1.1-2.9 mM, consistent with the function of Glf as a low-affinity glucose facilitator. The ability of glucokinase mutants expressing glf to transport glucose made it clear that glucokinase activity was not required for Glf-dependent glucose transport. The possibility that glucokinase can interact with Glf to improve the affinity for glucose was not supported since expression of the Z. mobilis glucokinase gene, in addition to glf, did not affect the Km of Glf for glucose in recombinant E. coli. The inability of various sugars to compete with glucose during glucose transport by recombinant E. coli expressing glf indicated that Glf is specific for glucose. While the results of fructose transport assays did not completely rule out the possibility of very low affinity for fructose, the apparent specificity of Glf for glucose makes it possible that Z. mobilis utilizes a different transporter(s) for fructose.

Active efflux of bile salts by Escherichia coli

“…We found that the use of ice-cold dilution-and-wash buffer, containing an excess of unlabeled TOC, was sufficient to give us reproducible results. The presence of unlabeled TOC was apparently crucial in lowering the binding of TOC to the filters, and the use of a wash solution cooled to subzero temperature (20) was not necessary in our system. The time course of accumulation of TOC by everted membrane vesicles of CS1253, measured by this method, showed that the accumulation process was nearly linear with time during the first 30 s and essentially reached the steady state in about 1 min (Fig.…”

“…However, it did not allow us to measure the initial rates of uptake. For this purpose, we developed a modified filtration method, based on the rapid quench filtration method described for the measurement of facilitated glucose transport in Zymomonas mobilis (20). We found that the use of ice-cold dilution-and-wash buffer, containing an excess of unlabeled TOC, was sufficient to give us reproducible results.…”

“…For determination of kinetics of TOC transport, a quick-quench filtration method, modified from that of Parker et al (20), was used. Assay buffer (50 mM K phosphate [pH 7.5] containing 1 mM MgSO 4 ); (45-l total volume) containing thawed everted vesicles (50 g of protein), with or without an energy source (20 mM Li D-lactate), was incubated at 30ЊC.…”

The gntP gene of Escherichia coli involved in gluconate uptake

“…Semiquantitative measurement of gluconate transport was conducted as described by Istúriz et al (15). The method for kinetic measurements of GntP-mediated gluconate transport was a slight modification of the procedure of Walsh et al (31), as described previously (23). For determination of the substrate specificity of GntP, uptake of 50 M radioactive gluconate was assayed in the presence of a fourfold excess of unlabelled sugars.…”

“…For determination of the substrate specificity of GntP, uptake of 50 M radioactive gluconate was assayed in the presence of a fourfold excess of unlabelled sugars. The assay was conducted as described previously (23). The data were analyzed by using Enzfitter software (Biosoft, Cambridge, United Kingdom).…”