The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energycoupling factor transporters for the new class of membrane transporters.Transport proteins residing in the cytoplasmic membrane allow the selective uptake and efflux of solutes and are essential for cellular growth and metabolism (20). Reflecting the importance of transporters, between 3% and 16% of the genes in prokaryote genomes are predicted to encode transporter proteins (26). These transporters form numerous families that are diverse in structure, energy-coupling mechanisms, and substrate specificities (25). As only a small fraction of predicted transporter proteins have known substrates, the functional prediction and annotation of the specificities of transporter proteins in the rapidly growing number of sequenced genomes represent a substantial challenge (25, 36). For example, the uptake of many cofactors and their precursors is essential for the growth of various pathogenic bacteria whose genomes are sequenced, but the transport proteins involved have not yet been identified. The use of computational comparative genomic techniques including gene colocalization, cooccurrence, and coregulation analyses combined with experimental assays is a powerful approach to identify novel transporters and to uncover their cellular role (for a recent review, see reference 11).The starting point for the present analysis was our recent discovery of multicomponent transport systems for the vitamin biotin (BioYNM) and the transition metals nickel (NikMNQO) and cobalt (CbiMNQO) (14,30). These transporters all have substrate-specific components (S components), which...

Energy coupling factor (ECF) transporters are used for the uptake of vitamins in Prokarya. They consist of an integral membrane protein that confers substrate specificity (the S-component) and an energizing module that is related to ATP-binding cassette (ABC) transporters. S-components for different substrates often do not share detectable sequence similarity but interact with the same energizing module. Here we present the crystal structure of the thiamine-specific S-component ThiT from Lactococcus lactis at 2.0 Å. Extensive protein-substrate interactions explain its high binding affinity for thiamine (K(d) ~10(-10) M). ThiT has a fold similar to that of the riboflavin-specific S-component RibU, with which it shares only 14% sequence identity. Two alanines in a conserved motif (AxxxA) located on the membrane-embedded surface of the S-components mediate the interaction with the energizing module. Based on these findings, we propose a general transport mechanism for ECF transporters.

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Excitatory amino acid transporters (EAATs) are secondary transport proteins that mediate the uptake of glutamate and other amino acids. EAATs fulfil an important role in neuronal signal transmission by clearing the excitatory neurotransmitters from the synaptic cleft after depolarization of the postsynaptic neuron. An intensively studied model system for understanding the transport mechanism of EAATs is the archaeal aspartate transporter GltPh. Each subunit in the homotrimeric GltPh supports the coupled translocation of one aspartate molecule and three Na(+) ions as well as an uncoupled flux of Cl(-) ions. Recent crystal structures of GltPh revealed three possible conformations for the subunits, but it is unclear whether the motions of individual subunits are coordinated to support transport. Here, we report the direct observation of conformational dynamics in individual GltPh trimers embedded in the membrane by applying single-molecule fluorescence resonance energy transfer (FRET). By analysing the transporters in a lipid bilayer instead of commonly used detergent micelles, we achieve conditions that approximate the physiologically relevant ones. From the kinetics of FRET level transitions we conclude that the three GltPh subunits undergo conformational changes stochastically and independently of each other.

The putative thiamin transporter ThiT from Lactococcus lactis was overproduced in the membrane of lactococcal cells. In vivo transport assays using radiolabeled thiamin demonstrated that ThiT indeed was involved in thiamin transport. The protein was solubilized from the membranes and purified in detergent solution. Size exclusion chromatography coupled to static light scattering, refractive index, and UV absorbance measurements (SEC-MALLS) showed that ThiT is a monomer of 22.7 kDa in detergent solution. When the cells overexpressing ThiT had been cultivated in complex growth medium, all binding sites of the purified protein were occupied with substrate, which had copurified with the protein. MALDI-TOF mass spectrometry analysis confirmed that the copurified substance was thiamin. Substrate-depleted ThiT was obtained by expressing the protein in cells that were cultivated in chemically defined growth medium without thiamin. The intrinsic tryptophan fluorescence of substrate-depleted ThiT was strongly quenched upon thiamin binding. The quenching of the fluorescence was used to determine dissociation constants for thiamin and related compounds. ThiT had an unusually high affinity for thiamin (K(D) = 122 +/- 13 pM) and bound the substrate with a 1:1 (protein:ligand) stoichiometry. TPP, TMP, and pyrithiamin bound to ThiT with nanomolar affinity. A multiple sequence alignment of ThiT homologues revealed that well-conserved residues were clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanning segments 5 and 6. Mutational analysis of the conserved residues in this region combined with binding assays of thiamin and related compounds was used to build a model of the high-affinity binding site. The model was compared with thiamin binding sites of other proteins and interpreted in terms of the transport mechanism.