The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane ␤-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy-and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the ␤-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal ␤-barrel domain confirmed the feasibility of this mechanism.FepA is a Gram-negative bacterial outer membrane (OM) 6 protein that transports ferric enterobactin (FeEnt) (1-3). The crystal structures of FepA (4) and other bacterial metal transporters (FhuA, BtuB,57)), contain a C-terminal, 22-stranded ␤-barrel, placing them in the porin superfamily (5). Their ϳ150-residue globular N termini (N-domain; see Fig. 1) reside within their ␤-barrels. This architecture is potentially consistent with the "ball-and-chain" mechanism of membrane transport, whereby the globule controls solute (ligand) uptake by moving in and out of the channel. This process was postulated for nervous system channels (6), but no demonstrated examples of ball-and-chain transport are known.FepA and its relatives are unlike other porins (7, 8), because they selectively adsorb metal chelates with high affinity (3, 9 -14). Ligand binding causes small conformational changes that activate them to transport competency (15-17), hence their designation "ligand-gated porin" (LGP). The requirements for metabolic energy (18 -20) and another cell envelope protein, TonB (21-24), in LGP mediated transport are well known but unaccounted for: the OM has no source of energy and cannot sustain an ion gradient because of its open porin channels (7); TonB is a minor cell envelope protein whose functions are not yet understood.In live cells, FepA binds and transports FeEnt via sub-reactions with different dependences on energy and TonB. (i) In the absence of ligand the receptor opens, and its flexible surface loops extend outward (25). (ii) FeEnt binds to FepA in a biphasic reaction (26) that begins with adsorption to aromatic amino acids in the loop extremities (27, 28). Multiple determinants in multiple loops, including L7 (25), converge on the iron complex, creating a closed conformation that associates the negatively charged (Ϫ3), catecholate iron center with basic and aromatic residues in the r...

SummaryListeria monocytogenes is a Gram-positive bacterium that causes severe opportunistic infections in humans and animals. We biochemically characterized, for the first time, the iron uptake processes of this facultative intracellular pathogen, and identified the genetic loci encoding two of its membrane iron transporters. Strain EGD-e used iron complexes of hydroxamates (ferrichrome and ferrichrome A, ferrioxamine B), catecholates (ferric enterobactin, ferric corynebactin) and eukaryotic binding proteins (transferrin, lactoferrin, ferritin, haemoglobin). Quantitative determinations showed 10-100-fold lower affinity for ferric siderophores ( K m ª 1-10 nM) than Gramnegative bacteria, and generally lower uptake rates.

SummaryIn Listeria monocytogenes the promoter region of the svpA-srtB locus contains a well-conserved Fur box. We characterized the iron-regulation of this locus: real-time polymerase chain reaction analyses and anti-SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80-fold and 10-fold respectively), when initiated by either the addition of the iron chelator 2,2 ¢ ¢ ¢ ¢ -bipyridyl to BHI media, or by growth in iron-restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA-srtB promoter in Escherichia coli ( 37-fold) and in L. monocytogenes (two-to threefold) when the bacteria were grown in iron-deficient conditions. A D D D D fur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA-srtB promoter. Cellular fractionation data revealed that in iron-rich media wild-type SvpA was exclusively secreted to the culture supernatant. However, both the D D D D fur derivative and wild-type L. monocytogenes grown in iron-deficient media anchored a fraction of the SvpA proteins ( ~ 5%) to peptidoglycan, and produced a lower-molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA-srtB locus (through Fur-mediated regulation), and attachment of SvpA to the cell wall (through SrtB-mediated covalent linkage). SvpA bears homology to IsdC, a haemin-binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site-directed deletions of four structural genes and the promoter of the svpA-srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA-srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus . Furthermore, the svpA-srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB ؉ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepA⌬3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.Iron is one target of gram-negative bacterial cell envelope transport systems, and microbes elaborate high-affinity siderophores that complex extracellular iron (70). However, ferric siderophores, like ferric enterobactin (FeEnt), are too large (716 Da) to pass through general porins in the outer membrane (OM), necessitating a different type of transporter to acquire them. On the basis of their 22-stranded transmembrane ␤-barrels, OM metal transporters like FepA belong to the porin superfamily (89). Ligand binding to such receptors initiates the transport reaction through their transmembrane channels, which led to their designation as ligand-gated porins (LGP) (88), by analogy to the family of eukaryotic ligand-gated ion channels. It is noteworthy that LGP are mechanistically distinct from general, diffusive porins because they bind metal complexes with high affinity and actively transport them against a concentration gradient into the cell. Once in the periplasm, binding proteins adsorb ferric siderophores and del...

The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/ H + symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (k on ) of up to ∼50-fold (reaching 10 7 M −1 ·s −1 ). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).membrane transport proteins | fluorescence | major facilitator superfamily T ypical of many transport proteins, from organisms as widely separated evolutionarily as Archaea and Homo sapiens, the lactose permease of Escherichia coli (LacY), a paradigm for the Major Facilitator Superfamily (1), catalyzes the coupled, stoichiometric translocation of a galactopyranoside and an H + (galactoside/H + symport) across the cytoplasmic membrane (reviewed in refs. 2 and 3). Although it is now generally accepted that membrane transport proteins operate by an alternating access mechanism, this has been documented almost exclusively for LacY (reviewed in refs. 4 and 5). By this means, galactoside-and H + -binding sites become alternatively accessible to either side of the membrane as the result of reciprocal opening/closing of cavities on the periplasmic and cytoplasmic sides of the molecule. LacY is highly dynamic, and alternates between different conformations (6, 7).Until recently, six X-ray structures of LacY have exhibited the same inward-facing conformation with an aqueous cavity open to the cytoplasmic side, a tightly sealed periplasmic side, and sugarand H + -binding sites in the middle of the molecule (8-11). Numerous studies confirm that this conformation prevails in the absence of sugar (12-16). Recently, however, the X-ray structure of double-Trp mutant G46W/G262W with bound sugar reveals a conformation with a narrowly open periplasmic pathway and a tightly sealed cytoplasmic side (PDB ID code 4OAA) (17), thereby providing structural evidence that an intermediate occluded conformation occurs between the outward-and inwardfacing conformations in ...

Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of Gram-negative bacteria through a group of 22-stranded ␤-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the ␤-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ionconducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellularfacing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.BtuB ͉ Cir ͉ FhuA ͉ urea-denaturation

Summary We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb), respectively. Deletion of lmo2185 in the srtB region reduced listerial [59Fe]-Hn transport, and purified Lmo2185 bound [59Fe]-Hn (KD = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a hemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [59Fe]-Hn transport at external concentrations less than 10 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (KD = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (KD = 123 nM). Deletions of Hup permease components hupD, hupG, or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC+ and hupG+ alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [59Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall KM of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (Vmax = 23 pMol/109 cells/min) to those of ferric siderophore transporters. In the ΔhupDBGC strain uptake occurred at a 3-fold lower rate (Vmax = 7 pMol/109 cells/min). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g., Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD50 of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence.

Fifty-nine single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.