Tripartite ATP-independent Periplasmic Transporter Research Articles

Sialic Acid Catabolism and Transport Gene Clusters Are Lineage Specific in Vibrio vulnificus

Sialic or nonulosonic acids are nine-carbon alpha ketosugars that are present in all vertebrate mucous membranes. Among bacteria, the ability to catabolize sialic acid as a carbon source is present mainly in pathogenic and commensal species of animals. Previously, it was shown that several Vibrio species carry homologues of the genes required for sialic acid transport and catabolism, which are genetically linked. In Vibrio cholerae on chromosome I, these genes are carried on the Vibrio pathogenicity island-2 region, which is confined to pathogenic isolates. We found that among the three sequenced Vibrio vulnificus clinical strains, these genes are present on chromosome II and are not associated with a pathogenicity island. To determine whether the sialic acid transport (SAT) and catabolism (SAC) region is universally present within V. vulnificus, we examined 67 natural isolates whose phylogenetic relationships are known. We found that the region was present predominantly among lineage I of V. vulnificus, which is comprised mainly of clinical isolates. We demonstrate that the isolates that contain this region can catabolize sialic acid as a sole carbon source. Two putative transporters are genetically linked to the region in V. vulnificus, the tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM and a component of an ATP-binding cassette (ABC) transporter. We constructed an in-frame deletion mutation in siaM, a component of the TRAP transporter, and demonstrate that this transporter is essential for sialic acid uptake in this species. Expression analysis of the SAT and SAC genes indicates that sialic acid is an inducer of expression. Overall, our study demonstrates that the ability to catabolize and transport sialic acid is predominately lineage specific in V. vulnificus and that the TRAP transporter is essential for sialic acid uptake.

Structural, Bioinformatic, and In Vivo Analyses of Two Treponema pallidum Lipoproteins Reveal a Unique TRAP Transporter

Treponema pallidum, the bacterial agent of syphilis, is predicted to encode one tripartite ATP-independent periplasmic transporter (TRAP-T). TRAP-Ts typically employ a periplasmic substrate-binding protein (SBP) to deliver the cognate ligand to the transmembrane symporter. Herein, we demonstrate that the genes encoding the putative TRAP-T components from T. pallidum, tp0957 (the SBP), and tp0958 (the symporter), are in an operon with an uncharacterized third gene, tp0956. We determined the crystal structure of recombinant Tp0956; the protein is trimeric and perforated by a pore. Part of Tp0956 forms an assembly similar to those of “tetratricopeptide repeat” (TPR) motifs. The crystal structure of recombinant Tp0957 was also determined; like the SBPs of other TRAP-Ts, there are two lobes separated by a cleft. In these other SBPs, the cleft binds a negatively charged ligand. However, the cleft of Tp0957 has a strikingly hydrophobic chemical composition, indicating that its ligand may be substantially different and likely hydrophobic. Analytical ultracentrifugation of the recombinant versions of Tp0956 and Tp0957 established that these proteins associate avidly. This unprecedented interaction was confirmed for the native molecules using in vivo cross-linking experiments. Finally, bioinformatic analyses suggested that this transporter exemplifies a new subfamily of TPATs (TPR-protein-associated TRAP-Ts) that require the action of a TPR-containing accessory protein for the periplasmic transport of a potentially hydrophobic ligand(s).

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.

Benzoate Mediates Repression of C 4 -Dicarboxylate Utilization in “Aromatoleum aromaticum” EbN1

Diauxic growth was observed in anaerobic C(4)-dicarboxylate-adapted cells of "Aromatoleum aromaticum" EbN1 due to preferred benzoate utilization from a substrate mixture of a C(4)-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C(4)-dicarboxylate uptake. In the first active growth phase, benzoate utilization was paralleled by maximal abundance of proteins involved in anaerobic benzoate degradation (e.g., benzoyl-coenzyme A [CoA] reductase) and minimal abundance of DctP (EbA4158), the periplasmic binding protein of a predicted C(4)-dicarboxylate tripartite ATP-independent periplasmic (TRAP) transporter (DctPQM). The opposite was observed during subsequent succinate utilization in the second active growth phase. The increased dctP (respectively, dctPQM) transcript and DctP protein abundance following benzoate depletion suggests that repression of C(4)-dicarboxylate uptake seems to be a main determinant for the observed diauxie.

Enhanced Substrate Release from a TRAP Transporter Binding Protein by Remote Modulation of its Intrinsic Conformational Dynamics

The tripartite ATP-independent periplasmic (TRAP) transporter TeaABC enables Halomonas elongata to uptake ectoine, a compatible solute protective under osmotic stress. TeaABC consists of three proteins: the Na+-coupled transporter TeaC; the transmembrane protein TeaB; and the periplasmic binding-protein TeaA. Like the binding proteins in ABC transporters, TeaA contains two globular domains, in-between which the substrate binds with high affinity. This notwithstanding, TeaA must somehow release ectoine upon interaction with TeaC. By analogy with similar transport systems, it is plausible that release is facilitated by a conformational change in TeaA, induced by TeaC. However, direct evidence and structural detail of such a mechanism are still unknown.Here, we use experimental and theoretical methods to demonstrate that modulation of the conformational dynamics of TeaA, as may occur by association with TeaC, results in an enhanced propensity for ectoine release. First, a novel crystal structure of apo TeaA reveals a pronounced domain rearrangement and opening of the binding cleft, relative to the known substrate-bound structure. Extensive molecular simulations and free-energy calculations are then employed to elucidate the mechanism by which the conformational dynamics of TeaA is modulated not only by ectoine binding and release, but also by structural elements remote from the binding cleft. This insight allows us to design a triple mutation whose influence on the dynamics of the protein may resemble that of the transporter domain TeaC, while not directly affecting the binding site. Calorimetric studies of mutant and wild-type TeaA demonstrate how this conformational modulation results in a reduction of its substrate affinity. Altogether, this data provides compelling support for the view that ectoine release from TeaA onto TeaC may be induced by the latter through a mechanism of conformational modulation of the former.

Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea

The tripartite ATP-independent periplasmic (TRAP) transporters are the best-studied family of substrate-binding protein (SBP)-dependent secondary transporters and are ubiquitous in prokaryotes, but absent from eukaryotes. They are comprised of an SBP of the DctP or TAXI families and two integral membrane proteins of unequal sizes that form the DctQ and DctM protein families, respectively. The SBP component has a structure comprised of two domains connected by a hinge that closes upon substrate binding. In DctP-TRAP transporters, substrate binding is mediated through a conserved and specific arginine/carboxylate interaction in the SBP. While the SBP component has now been relatively well characterized, the membrane components of TRAP transporters are still poorly understood both in terms of their structure and function. We review the expanding repertoire of substrates and physiological roles for experimentally characterized TRAP transporters in bacteria and discuss mechanistic aspects of these transporters using data primarily from the sialic acid-specific TRAP transporter SiaPQM from Haemophilus influenzae, which suggest that TRAP transporters are high-affinity, Na(+)-dependent unidirectional secondary transporters.

A TRAP Transporter for Pyruvate and Other Monocarboxylate 2-Oxoacids in the Cyanobacterium Anabaena sp. Strain PCC 7120

In the cyanobacterium Anabaena sp. strain PCC 7120, open reading frames (ORFs) alr3026, alr3027, and all3028 encode a tripartite ATP-independent periplasmic transporter (TRAP-T). Wild-type filaments showed significant uptake of [(14)C]pyruvate, which was impaired in the alr3027 and all3028 mutants and was inhibited by several monocarboxylate 2-oxoacids, identifying this TRAP-T system as a pyruvate/monocarboxylate 2-oxoacid transporter.

Identification of a TRAP transporter for malonate transport and its expression regulated by GtrA from Sinorhizobium meliloti

Sinorhizobium meliloti can live as a saprophyte in soil or as a nitrogen-fixing symbiont inside the root nodule cells of alfalfa and related legumes by utilizing different organic compounds as its carbon source. Here we have identified the matPQMAB operon in S. meliloti 1021. Within this operon, matP, matQ and the M region of the fused gene matMA encode an extracytoplasmic solute receptor, a small transmembrane protein and a large transmembrane protein, consisting of three components of the tripartite ATP-independent periplasmic (TRAP) transporter for malonate transport. The A region of the fused gene matMA and matB encode malonate-metabolizing enzymes, malonyl-CoA decarboxylase and malonyl-CoA synthetase. The null mutant of each matPQMAB gene is unable to grow on M9 minimal medium containing malonate as the sole carbon source. However, these mutants can induce the formation of efficient nitrogen-fixing root nodules on alfalfa. The matPQMAB operon is expressed in free-living bacterial cells and symbiotic bacterial cells from infection threads and root nodules. The GntR family transcriptional regulator, GtrA, specifically binds the promoter of the matPQMAB operon, positively regulating its expression. Moreover, the matPQMAB can be transcriptionally induced by malonate. These results suggested that a C 3-dicarboxylic acid TRAP transporter is responsible for malonate transport in S. meliloti.

The Crystal Structure of UehA in Complex with Ectoine—A Comparison with Other TRAP-T Binding Proteins

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.

The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter

Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophilus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP.

Control of Gluconate Utilization in Sinorhizobium meliloti

The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.

Development and use of genetic system to identify genes required for efficient low-temperature growth of Psychrobacter arcticus 273-4

We describe the development of genetic tools (electroporation, conjugation, vector for targeted gene replacement) for use in the psychrophile Psychrobacter arcticus 273-4 to test hypotheses about cold adaptation. Successful electroporation only occurred with nonstandard parameters, such as: electrocompetent cells freshly prepared from stationary-phase cultures, high field strengths (25 kV cm(-1)), long recovery times (16-24 h), and selection with low concentrations of antibiotics. Transformation frequencies were greatly affected by a methylation-dependent restriction barrier homologous to DpnI. The vector pJK100 (which was self-transmissible and contained a Pir-dependent R6K origin of replication) proved effective as a suicide plasmid that could be used to recombine mutations into the P. arcticus 273-4 genome. We used this vector for targeted replacement of dctT, the substrate-binding periplasmic subunit of a TRAP (tripartite ATP-independent periplasmic) transporter (which we have named dctTUF), as it was more highly expressed at cold temperatures. The replacement of dctT (with kan) decreased the rate of growth at low temperatures in mineral medium with glutamate, acetate, butyrate, and fumarate, but not with pyruvate suggesting that DctTUF participates in the transport of glutamate, acetate, butyrate, and fumarate at cold temperatures. This is the first report to demonstrate the creation of site-specific mutants in the genus Psychrobacter, their affect on low-temperature growth, and a substrate range for TAXI proteins of TRAP transporters.

1.55 Å Structure of the Ectoine Binding Protein TeaA of the Osmoregulated TRAP-Transporter TeaABC from Halomonas elongata

TeaABC from the moderate halophilic bacterium Halomonas elongata belongs to the tripartite ATP-independent periplasmic transporters (TRAP-T), a family of secondary transporters functioning in conjunction with periplasmic substrate binding proteins. TeaABC facilitates the uptake of the compatible solutes ectoine and hydroxyectoine that are accumulated in the cytoplasm under hyperosmotic stress to protect the cell from dehydration. TeaABC is the only known TRAP-T activated by osmotic stress. Currently, our knowledge on the osmoregulated compatible solute transporter is limited to ABC transporters or conventional secondary transporters. Therefore, this study presents the first detailed analysis of the molecular mechanisms underlying substrate recognition of the substrate binding protein of an osmoregulated TRAP-T. In the present study we were able to demonstrate by isothermal titration calorimetry measurements that TeaA is a high-affinity ectoine binding protein ( K d = 0.19 microM) that also has a significant but somewhat lower affinity to hydroxyectoine ( K d = 3.8 microM). Furthermore, we present the structure of TeaA in complex with ectoine at a resolution of 1.55 A and hydroxyectoine at a resolution of 1.80 A. Analysis of the TeaA binding pocket and comparison of its structure to other compatible solute binding proteins from ABC transporters reveal common principles in compatible solute binding but also significant differences like the solvent-mediated specific binding of ectoine to TeaA.

Identification of a Gene Encoding a Transporter Essential for Utilization of C 4 Dicarboxylates in Corynebacterium glutamicum

The Corynebacterium glutamicum R genome contains a total of eight genes encoding proteins with sequence similarity to C4-dicarboxylate transporters identified from other bacteria. Three of the genes encode proteins within the dicarboxylate/amino acid:cation symporter (DAACS) family, another three encode proteins within the tripartite ATP-independent periplasmic transporter family, and two encode proteins within the divalent anion:Na+ symporter (DASS) family. We observed that a mutant strain deficient in one of these genes, designated dcsT, of the DASS family did not aerobically grow on the C4 dicarboxylates succinate, fumarate, and malate as the sole carbon sources. Mutant strains deficient in each of the other seven genes grew as well as the wild-type strain under the same conditions, although one of these genes is a homologue of dctA of the DAACS family, involved in aerobic growth on C4 dicarboxylates in various bacteria. The utilization of C4 dicarboxylates was markedly enhanced by overexpression of the dcsT gene. We confirmed that the uptake of [13C]labeled succinate observed for the wild-type cells was hardly detected in the dcsT-deficient mutant but was markedly enhanced in a dcsT-overexpressing strain. These results suggested that in C. glutamicum, the uptake of C4 dicarboxylates for aerobic growth was mainly mediated by the DASS transporter encoded by dcsT. The expression level of the dcsT gene transiently increased in the early exponential phase during growth on nutrient-rich medium. This expression was enhanced by the addition of succinate in the mid-exponential phase and was repressed by the addition of glucose in the early exponential phase.

Characterization of the N-Acetyl-5-neuraminic Acid-binding Site of the Extracytoplasmic Solute Receptor (SiaP) of Nontypeable Haemophilus influenzae Strain 2019

Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4A resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.

Crystal Structures of two Bordetella pertussis Periplasmic Receptors Contribute to Defining a Novel Pyroglutamic Acid Binding DctP Subfamily

Gram-negative bacteria have developed several different transport systems for solute uptake. One of these, the tripartite ATP independent periplasmic transport system (TRAP-T), makes use of an extracytoplasmic solute receptor (ESR) which captures specific solutes with high affinity and transfers them to their partner permease complex located in the bacterial inner membrane. We hereby report the structures of DctP6 and DctP7, two such ESRs from Bordetella pertussis. These two proteins display a high degree of sequence and structural similarity and possess the “Venus flytrap” fold characteristic of ESRs, comprising two globular α/β domains hinged together to form a ligand binding cleft. DctP6 and DctP7 both show a closed conformation due to the presence of one pyroglutamic acid molecule bound by highly conserved residues in their respective ligand binding sites. BLAST analyses have revealed that the DctP6 and DctP7 residues involved in ligand binding are strictly present in a number of predicted TRAP-T ESRs from other bacteria. In most cases, the genes encoding these TRAP-T systems are located in the vicinity of a gene coding for a pyroglutamic acid metabolising enzyme. Both the high degree of conservation of these ligand binding residues and the genomic context of these TRAP-T-coding operons in a number of bacterial species, suggest that DctP6 and DctP7 constitute the prototypes of a novel TRAP-T DctP subfamily involved in pyroglutamic acid transport.

Tripartite ATP-Independent Periplasmic Transporters: Application of a Relational Database for Genome-Wide Analysis of Transporter Gene Frequency and Organization

Tripartite ATP-independent periplasmic (TRAP) transporters are a family of extracytoplasmic solute receptor-dependent secondary transporters that are widespread in the prokaryotic world but which have not been extensively studied. Here, we present results of a genome-wide analysis of TRAP sequences and genome organization from application of TRAPDb, a relational database created for the collection, curation and analysis of TRAP sequences. This has revealed a specific enrichment in the number of TRAP transporters in several bacteria which is consistent with increased use of TRAP transporters in saline environments. Additionally, we report a number of new organizations of TRAP transporter genes and proteins which suggest the recruitment of TRAP transporter components for use in other biological contexts.

Mapping the Sinorhizobium meliloti 1021 solute-binding protein-dependent transportome

The number of solute-binding protein-dependent transporters in rhizobia is dramatically increased compared with the majority of other bacteria so far sequenced. This increase may be due to the high affinity of solute-binding proteins for solutes, permitting the acquisition of a broad range of growth-limiting nutrients from soil and the rhizosphere. The transcriptional induction of these transporters was studied by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. In total, specific inducers were identified for 76 transport systems, amounting to approximately 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 are previously uncharacterized in Rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This study provides a global expression map of one of the largest transporter families (transportome) and an invaluable tool to both understand their solute specificity and the relationships between members of large paralogous families.

Conservation of Structure and Mechanism in Primary and Secondary Transporters Exemplified by SiaP, a Sialic Acid Binding Virulence Factor from Haemophilus influenzae

Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.

Novel ligands for the extracellular solute receptors of two bacterial TRAP transporters

Tripartite ATP-independent periplasmic (TRAP) transporters are relatively common prokaryotic secondary transporters which comprise an extracytoplasmic solute receptor (ESR) protein and two dissimilar membrane proteins or domains, yet the substrates and physiological functions of only a few of these systems are so far known. In this study, a biophysical approach was used to identify the ligands for the purified Rhodobacter capsulatus RRC01191 and Escherichia coli YiaO proteins, which are members of two phylogenetically distinct families of TRAP-ESRs found in diverse bacteria. In contrast to previous indirect evidence pointing to RRC01191 orthologues being involved in polyol uptake, it was shown that RRC01191 binds pyruvate, 2-oxobutyrate and a broad range of aliphatic monocarboxylic 2-oxoacid anions with varying affinities (K(d) values 0.08-3 muM), consistent with a predicted role in monocarboxylate transport related to branched-chain amino-acid biosynthesis. The E. coli YiaMNO TRAP transporter has previously been proposed to be an l-xylulose uptake system [Plantinga et al. (2004) Mol Membr Biol 21, 51-57], but purified YiaO did not bind l- or d-xylulose as judged by fluorescence spectroscopy, circular dichroism or mass spectrometry. Instead, these techniques showed that a breakdown product of l-ascorbate, 2,3-diketo-l-gulonate (2,3-DKG), binds by a simple one-step mechanism with sub-micromolar affinity. The data provide the first evidence for the existence of ESR-dependent transporters for 2-oxoacids and 2,3-DKG, homologues of which appear to be widespread amongst prokaryotes. The results also underline the utility of direct ESR ligand-binding studies for TRAP transporter characterization.