Functional and Biochemical Characterization of Escherichia coli Sugar Efflux Transporters

Jia Yeu Liu,Paul F Miller,Jennifer Willard,Eric R Olson

doi:10.1074/jbc.274.33.22977

Abstract

A family of bacterial transporters, the SET (sugar efflux transporter) family, has been recently reported (Liu, J. Y., Miller, P. F., Gosink, M., and Olson, E. R. (1999) Mol. Microbiol. 31, 1845-1851). In this study, the biochemical and cell biological properties of the three Escherichia coli members (SetA, SetB, and SetC) of the family are characterized. We show that both SetA and SetB can transport lactose and glucose. In addition, SetA has broad substrate specificity, with preferences for glucosides or galactosides with alkyl or aryl substituents. Consistent with the observed in vitro substrate specificities, strains that hyperexpress SetA or SetB are desensitized to lactose analogues as measured by induction of the lac operon. In addition, strains that hyperexpress SetA are resistant to the growth inhibitory sugar analogue o-nitrophenyl-beta-D-thiogalactoside. Strains disrupted for any one or all of the set genes are viable and show no defects in lactose utilization nor increased sensitivity to inducers of the lac operon and nonmetabolizable sugar analogues. The data suggest that the set genes are either poorly expressed under normal laboratory growth conditions or are redundant with other cellular gene products.

Highlights

The recently described bacterial SET family of efflux pumps shares amino acid sequence similarity with the Major Facilitator Superfamily of transport proteins (1–3)
As we show in this study, the range of sugars that are efflux substrates for the E. coli SET proteins include selective monosaccharides and disaccharides, in addition to glycoside analogues such as IPTG
Because lactose and IPTG are both substrates for Set proteincatalyzed efflux, we generated null mutations in setA, setB, and setC and used these to help define the role of the Set proteins in E. coli sugar metabolism

Summary

Introduction

The recently described bacterial SET (sugar efflux transporter) family of efflux pumps shares amino acid sequence similarity with the Major Facilitator Superfamily of transport proteins (1–3). The SET proteins were first identified in Escherichia coli, which encodes three members (SetA, SetB, and SetC) that share a high degree (at least 70%) of amino acid sequence similarity (1). Many nonmetabolizable lactose analogues such as isopropyl␤-D-thiogalactoside (IPTG) and methyl-␤-D-thiogalactoside are growth inhibitory when lactose is the only carbon source (10) These compounds enter the cell through the lactose permease, the product of the lacY gene (14). A role for the SET proteins (SetA, SetB, and SetC) in the metabolism of lactose or the detoxification of nonmetabolizable sugar analogues is investigated. Because lactose and IPTG are both substrates for Set proteincatalyzed efflux, we generated null mutations in setA, setB, and setC and used these to help define the role of the Set proteins in E. coli sugar metabolism

Methods

Results

Conclusion