Physiological Functions of Bacterial "Multidrug" Efflux Pumps.

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.

Short-chain diamines are the physiological substrates of PACE family efflux pumps.

Acinetobacter baumannii has rapidly emerged as a major cause of gram-negative hospital infections worldwide. A. baumannii encodes for the transport protein AceI, which confers resistance to chlorhexidine, a widely used antiseptic. AceI is also the prototype for the recently discovered proteobacterial antimicrobial compound efflux (PACE) family of transport proteins that confer resistance to a range of antibiotics and antiseptics in many gram-negative bacteria, including pathogens. The gene encoding AceI is conserved in the core genome of A. baumannii, suggesting that it has an important primordial function. This is incongruous with the sole characterized substrate of AceI, chlorhexidine, an entirely synthetic biocide produced only during the last century. Here we investigated a potential primordial function of AceI and other members of the PACE family in the transport of naturally occurring polyamines. Polyamines are abundant in living cells, where they have physiologically important functions and play multifaceted roles in bacterial infection. Gene expression studies revealed that the aceI gene is induced in A. baumannii by the short-chain diamines cadaverine and putrescine. Membrane transport experiments conducted in whole cells of A. baumannii and Escherichia coli and also in proteoliposomes showed that AceI mediates the efflux of these short-chain diamines when energized by an electrochemical gradient. Assays conducted using 8 additional diverse PACE family proteins identified 3 that also catalyze cadaverine transport. Taken together, these results demonstrate that short-chain diamines are common substrates for the PACE family of transport proteins, adding to their broad significance as a novel family of efflux pumps.

Microbial expression systems for membrane proteins.

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.

Molecular mechanism of ligand recognition by membrane transport protein, Mhp1.

The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.

Production of membrane proteins for characterisation of their pheromone-sensing and antimicrobial resistance functions.

Despite the importance of membrane proteins in cellular processes, studies of these hydrophobic proteins present major technical challenges, including expression and purification for structural and biophysical studies. A modified strategy of that proposed previously by Saidijam et al. (2005) and others, for the routine expression of bacterial membrane proteins involved in environmental sensing and antimicrobial resistance (AMR), is proposed which results in purification of sufficient proteins for biophysical experiments. We report expression successes amongst a collection of enterococcal vancomycin resistance membrane proteins: VanTG, VanTG-M transporter domain, VanZ and the previously characterised VanS (A-type) histidine protein kinase (HPK). Using the same strategy, we report on the successful amplification and purification of intact BlpH and ComD2 HPKs of Streptococcus pneumoniae. Near-UV circular dichroism revealed both recombinant proteins bound their pheromone ligands BlpC and CSP2. Interestingly, CSP1 also interacted with ComD. Finally, we evaluate the alternative strategy for studying sensory HPKs involving isolated soluble sensory domain fragments, exemplified by successful production of VicKESD of Enterococcus faecalis VicK. Purified VicKESD possessed secondary structure post-purification. Thermal denaturation experiments using far-UV CD, a technique which can be revealing regarding ligand binding, revealed that: (a) VicKESD denaturation occurs between 15 and 50 °C; and (b) reducing conditions did not detectably affect denaturation profiles suggesting reducing conditions per se are not directly sensed by VicKESD. Our findings provide information on a modified strategy for the successful expression, production and/or storage of bacterial membrane HPKs, AMR proteins and sensory domains for their future crystallisation, and ligand binding studies.

Topological Dissection of the Membrane Transport Protein Mhp1 Derived from Cysteine Accessibility and Mass Spectrometry.

Cys accessibility and quantitative intact mass spectrometry (MS) analyses have been devised to study the topological transitions of Mhp1, the membrane protein for sodium-linked transport of hydantoins from Microbacterium liquefaciens. Mhp1 has been crystallized in three forms (outward-facing open, outward-facing occluded with substrate bound, and inward-facing open). We show that one natural cysteine residue, Cys327, out of three, has an enhanced solvent accessibility in the inward-facing (relative to the outward-facing) form. Reaction of the purified protein, in detergent, with the thiol-reactive N-ethylmalemide (NEM), results in modification of Cys327, suggesting that Mhp1 adopts predominantly inward-facing conformations. Addition of either sodium ions or the substrate 5-benzyl-l-hydantoin (L-BH) does not shift this conformational equilibrium, but systematic co-addition of the two results in an attenuation of labeling, indicating a shift toward outward-facing conformations that can be interpreted using conventional enzyme kinetic analyses. Such measurements can afford the Km for each ligand as well as the stoichiometry of ion-substrate-coupled conformational changes. Mutations that perturb the substrate binding site either result in the protein being unable to adopt outward-facing conformations or in a global destabilization of structure. The methodology combines covalent labeling, mass spectrometry, and kinetic analyses in a straightforward workflow applicable to a range of systems, enabling the interrogation of changes in a protein's conformation required for function at varied concentrations of substrates, and the consequences of mutations on these conformational transitions.

Allantoin transport protein, PucI, from Bacillus subtilis: evolutionary relationships, amplified expression, activity and specificity.

This work reports the evolutionary relationships, amplified expression, functional characterization and purification of the putative allantoin transport protein, PucI, from Bacillus subtilis. Sequence alignments and phylogenetic analysis confirmed close evolutionary relationships between PucI and membrane proteins of the nucleobase-cation-symport-1 family of secondary active transporters. These include the sodium-coupled hydantoin transport protein, Mhp1, from Microbacterium liquefaciens, and related proteins from bacteria, fungi and plants. Membrane topology predictions for PucI were consistent with 12 putative transmembrane-spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. The pucI gene was cloned into the IPTG-inducible plasmid pTTQ18 upstream from an in-frame hexahistidine tag and conditions determined for optimal amplified expression of the PucI(His6) protein in Escherichia coli to a level of about 5 % in inner membranes. Initial rates of inducible PucI-mediated uptake of 14C-allantoin into energized E. coli whole cells conformed to Michaelis-Menten kinetics with an apparent affinity (Kmapp) of 24 ± 3 µM, therefore confirming that PucI is a medium-affinity transporter of allantoin. Dependence of allantoin transport on sodium was not apparent. Competitive uptake experiments showed that PucI recognizes some additional hydantoin compounds, including hydantoin itself, and to a lesser extent a range of nucleobases and nucleosides. PucI(His6) was solubilized from inner membranes using n-dodecyl-ß-d-maltoside and purified. The isolated protein contained a substantial proportion of α-helix secondary structure, consistent with the predictions, and a 3D model was therefore constructed on a template of the Mhp1 structure, which aided localization of the potential ligand binding site in PucI.

Purification of bacterial membrane sensor kinases and biophysical methods for determination of their ligand and inhibitor interactions.

This article reviews current methods for the reliable heterologous overexpression in Escherichia coli and purification of milligram quantities of bacterial membrane sensor kinase (MSK) proteins belonging to the two-component signal transduction family of integral membrane proteins. Many of these methods were developed at Leeds alongside Professor Steve Baldwin to whom this review is dedicated. It also reviews two biophysical methods that we have adapted successfully for studies of purified MSKs and other membrane proteins-synchrotron radiation circular dichroism (SRCD) spectroscopy and analytical ultracentrifugation (AUC), both of which are non-immobilization and matrix-free methods that require no labelling strategies. Other techniques such as isothermal titration calorimetry (ITC) also share these features but generally require high concentrations of material. In common with many other biophysical techniques, both of these biophysical methods provide information regarding membrane protein conformation, oligomerization state and ligand binding, but they possess the additional advantage of providing direct assessments of whether ligand binding interactions are accompanied by conformational changes. Therefore, both methods provide a powerful means by which to identify and characterize inhibitor binding and any associated protein conformational changes, thereby contributing valuable information for future drug intervention strategies directed towards bacterial MSKs.

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins.

Chlorhexidine is widely used as an antiseptic or disinfectant in both hospital and community settings. A number of bacterial species display resistance to this membrane-active biocide. We examined the transcriptomic response of a representative nosocomial human pathogen, Acinetobacter baumannii, to chlorhexidine to identify the primary chlorhexidine resistance elements. The most highly up-regulated genes encoded components of a major multidrug efflux system, AdeAB. The next most highly overexpressed gene under chlorhexidine stress was annotated as encoding a hypothetical protein, named here as AceI. Orthologs of the aceI gene are conserved within the genomes of a broad range of proteobacterial species. Expression of aceI or its orthologs from several other γ- or ß-proteobacterial species in Escherichia coli resulted in significant increases in resistance to chlorhexidine. Additionally, disruption of the aceI ortholog in Acinetobacter baylyi rendered it more susceptible to chlorhexidine. The AceI protein was localized to the membrane after overexpression in E. coli. This protein was purified, and binding assays demonstrated direct and specific interactions between AceI and chlorhexidine. Transport assays using [(14)C]-chlorhexidine determined that AceI was able to mediate the energy-dependent efflux of chlorhexidine. An E15Q AceI mutant with a mutation in a conserved acidic residue, although unable to mediate chlorhexidine resistance and transport, was still able to bind chlorhexidine. Taken together, these data are consistent with AceI being an active chlorhexidine efflux protein and the founding member of a family of bacterial drug efflux transporters.

Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase.

Noncovalent mass spectrometry (MS) is emerging as an invaluable technique to probe the structure, interactions, and dynamics of membrane proteins (MPs). However, maintaining native-like MP conformations in the gas phase using detergent solubilized proteins is often challenging and may limit structural analysis. Amphipols, such as the well characterized A8-35, are alternative reagents able to maintain the solubility of MPs in detergent-free solution. In this work, the ability of A8-35 to retain the structural integrity of MPs for interrogation by electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is compared systematically with the commonly used detergent dodecylmaltoside. MPs from the two major structural classes were selected for analysis, including two ß-barrel outer MPs, PagP and OmpT (20.2 and 33.5 kDa, respectively), and two α-helical proteins, Mhp1 and GalP (54.6 and 51.7 kDa, respectively). Evaluation of the rotationally averaged collision cross sections of the observed ions revealed that the native structures of detergent solubilized MPs were not always retained in the gas phase, with both collapsed and unfolded species being detected. In contrast, ESI-IMS-MS analysis of the amphipol solubilized MPs studied resulted in charge state distributions consistent with less gas phase induced unfolding, and the presence of lowly charged ions which exhibit collision cross sections comparable with those calculated from high resolution structural data. The data demonstrate that A8-35 can be more effective than dodecylmaltoside at maintaining native MP structure and interactions in the gas phase, permitting noncovalent ESI-IMS-MS analysis of MPs from the two major structural classes, while gas phase dissociation from dodecylmaltoside micelles leads to significant gas phase unfolding, especially for the α-helical MPs studied.

TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor.

Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [(15)N-(1)H]- and [(13)C-(1)H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan (15)N backbone positions and also resolved signals for (15)N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [(13)C-(1)H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

Probing the contacts of a low-affinity substrate with a membrane-embedded transport protein using 1H-13C cross-polarisation magic-angle spinning solid-state NMR.

Solid-state NMR combined with sample deuteration was used to probe the proximity of the low-affinity substrate D-glucose to its binding site within the Escherichia coli sugar transport protein GalP. Samples of E. coli inner membranes with amplified expression of GalP were incubated in D(2)O with D-[(13)C(6)]glucose and (13)C NMR signals from the substrate were assigned in two-dimensional dipolar-assisted rotational resonance (DARR) spectra. The signals were confirmed as representing D-glucose bound to GalP as the peaks were abolished after the substrate was displaced from the specific site with the inhibitor forskolin. The (13)C chemical shift values for D-[(13)C(6)]glucose in solution revealed some differences compared to those for ligand bound to GalP, the differences being most pronounced for positions C1 and C2, and especially for C1 in the α-anomer. (13)C cross-polarization build-up was measured for C1 and C2 of D-[(13)C(6)]glucose and D-[(2)H(7), (13)C(6)]glucose in GalP membranes suspended in D(2)O. The build-up curves for the deuterated substrate reflect intermolecular (1)H-(13)C interactions between the protein and the fully deuterated substrate; the signal build-up suggests that the α-anomer is situated closer to the protein binding site than is the ß-anomer, consistent with its relatively high signal intensities and more pronounced chemical shift changes in the 2D-correlation spectra. These results demonstrate the utility of solid-state NMR combined with sample deuteration for mapping the binding interface of low affinity ligands with membrane proteins.

A systematic approach to the amplified expression, functional characterization and purification of inositol transporters from Bacillus subtilis.

Abstract A systematic approach was used for the cloning and amplified expression in Escherichia coli of the genes for each of three inositol transport proteins (IolF, IolT, YfiG) from Bacillus subtilis that are evolutionarily-related to human transporters. Inducible amplified expression of each was achieved to levels of ∼ 10-15% of total protein in E. coli inner membrane preparations. The functional integrity of each heterologously-expressed protein was demonstrated by measuring the kinetics of (3)H-myo-inositol transport into energized whole cells; this confirmed that IolT is the major inositol transporter, IolF is an inefficient transporter of this substrate and demonstrated that YfiG is an inositol transport protein for the first time. Competition for (3)H-myo-inositol transport by 17 unlabelled compounds revealed all three proteins to be highly specific in recognizing inositols over sugars. IolT was confirmed to be highly specific for both myo- and D-chiro-inositol and IolF was confirmed to prefer D-chiro-inositol over myo-inositol. YfiG selectively recognized myo-inositol, D-chiro-inositol and, uniquely, L-chiro-inositol. All three proteins were successfully solubilized and purified in milligram quantities from inner membrane preparations and their suitability for inclusion in crystallization trials was assessed by analysis of structural integrity and thermal stability using circular dichroism spectroscopy followed by examination for monodispersity using gel filtration chromatography.

Synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM) for solubilization of membrane proteins in TROSY NMR experiments.

This work reports the first synthesis of uniformly deuterated n-dodecyl-ß-D-maltoside (d39-DDM). DDM is a mild non-ionic detergent often used in the extraction and purification of membrane proteins and for solubilizing them in experimental studies of their structure, dynamics and binding of ligands. We required d39-DDM for solubilizing large α-helical membrane proteins in samples for [(15)N-(1)H]TROSY (transverse relaxation-optimized spectroscopy) NMR experiments to achieve the highest sensitivity and best resolved spectra possible. Our synthesis of d39-DDM used d7-D-glucose and d25-n-dodecanol to introduce deuterium labelling into both the maltoside and dodecyl moieties, respectively. Two glucose molecules, one converted to a glycosyl acceptor with a free C4 hydroxyl group and one converted to a glycosyl donor substituted at C1 with a bromine in the α-configuration, were coupled together with an α(1 → 4) glycosidic bond to give maltose, which was then coupled with n-dodecanol by its substitution of a C1 bromine in the α-configuration to give DDM. (1)H NMR spectra were used to confirm a high level of deuteration in the synthesized d39-DDM and to demonstrate its use in eliminating interfering signals from TROSY NMR spectra of a 52-kDa sugar transport protein solubilized in DDM.

Unfolding free energy of a two-domain transmembrane sugar transport protein.

Understanding how an amino acid sequence folds into a functional, three-dimensional structure has proved to be a formidable challenge in biological research, especially for transmembrane proteins with multiple alpha helical domains. Mechanistic folding studies on helical membrane proteins have been limited to unusually stable, single domain proteins such as bacteriorhodopsin. Here, we extend such work to flexible, multidomain proteins and one of the most widespread membrane transporter families, the major facilitator superfamily, thus showing that more complex membrane proteins can be successfully refolded to recover native substrate binding. We determine the unfolding free energy of the two-domain, Escherichia coli galactose transporter, GalP; a bacterial homologue of human glucose transporters. GalP is reversibly unfolded by urea. Urea causes loss of substrate binding and a significant reduction in alpha helical content. Full recovery of helical structure and substrate binding occurs in dodecylmaltoside micelles, and the unfolding free energy can be determined. A linear dependence of this free energy on urea concentration allows the free energy of unfolding in the absence of urea to be determined as +2.5 kcal·mol(-1). Urea has often been found to be a poor denaturant for transmembrane helical structures. We attribute the denaturation of GalP helices by urea to the dynamic nature of the transporter structure allowing denaturant access via the substrate binding pocket, as well as to helical structure that extends beyond the membrane. This study gives insight into the final, critical folding step involving recovery of ligand binding for a multidomain membrane transporter.

Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site.

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.

The alternating access mechanism of transport as observed in the sodium-hydantoin transporter Mhp1.

Secondary active transporters move molecules across cell membranes by coupling this process to the energetically favourable downhill movement of ions or protons along an electrochemical gradient. They function by the alternating access model of transport in which, through conformational changes, the substrate binding site alternately faces either side of the membrane. Owing to the difficulties in obtaining the crystal structure of a single transporter in different conformational states, relatively little structural information is known to explain how this process occurs. Here, the structure of the sodium-benzylhydantoin transporter, Mhp1, from Microbacterium liquefaciens, has been determined in three conformational states; from this a mechanism is proposed for switching from the outward-facing open conformation through an occluded structure to the inward-facing open state.

'Carbon-Monoxide-Releasing Molecule-2 (CORM-2)' Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects.

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological 'gasotransmitter', in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 µM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 µM; His, Kd 130 µM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 µM; GSSG, Kd 25,000 µM). The toxicity of low, but potent, levels (15 µM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

The sodium-dependent D-glucose transport protein of Helicobacter pylori.

Helicobacter pylori is a gram-negative pathogenic microaerophile with a particular tropism for the mucosal surface of the gastric epithelium. Despite its obligatory microaerophilic character, it can metabolize D-glucose and/or D-galactose in both oxidative and fermentative pathways via a Na(+)-dependent secondary active transport, a glucokinase and enzymes of the pentose phosphate pathway. We have assigned the Na(+)-dependent transport of glucose to the protein product of the H. pylori 1174 gene. The gene was heterologously expressed in a glucose transport-deficient Escherichia coli strain, where transport activities of radiolabelled D-glucose, D-galactose and 2-deoxy-D-glucose were restored, consistent with the expected specificity of the hexose uptake system in H. pylori. D-mannose was also identified as a substrate. The HP1174 transport protein was purified and reconstituted into proteoliposomes, where sodium dependence of sugar transport activity was demonstrated. Additionally the tryptophan/tyrosine fluorescence of the purified protein showed quenching by 2-deoxy-D-glucose, D-mannose, D-glucose or D-galactose in the presence of sodium ions. This is the first reported purification and characterization of an active glucose transport protein member of the TC 2.1.7 subgroup of the Major Facilitator Superfamily, constituting the route for entry of sugar nutrients into H. pylori. A model is derived of its three-dimensional structure as a paradigm of the family.