X-ray crystallographic structures of the Escherichia coli periplasmic protein FhuD bound to hydroxamate-type siderophores and the antibiotic albomycin.

Siderophore-binding proteins play an essential role in the uptake of iron in many Gram-positive and Gram-negative bacteria. FhuD is an ATP-binding cassette-type (ABC-type) binding protein involved in the uptake of hydroxamate-type siderophores in Escherichia coli. Structures of FhuD complexed with the antibiotic albomycin, the fungal siderophore coprogen and the drug Desferal have been determined at high resolution by x-ray crystallography. FhuD has an unusual bilobal structure for a periplasmic ligand binding protein, with two mixed beta/alpha domains connected by a long alpha-helix. The binding site for hydroxamate-type ligands is composed of a shallow pocket that lies between these two domains. Recognition of siderophores primarily occurs through interactions between the iron-hydroxamate centers of each siderophore and the side chains of several key residues in the binding pocket. Rearrangements of side chains within the binding pocket accommodate the unique structural features of each siderophore. The backbones of the siderophores are not involved in any direct interactions with the protein, demonstrating how siderophores with considerable chemical and structural diversity can be bound by FhuD. For albomycin, which consists of an antibiotic group attached to a hydroxamate siderophore, electron density for the antibiotic portion was not observed. Therefore, this study provides a basis for the rational design of novel bacteriostatic agents, in the form of siderophore-antibiotic conjugates that can act as "Trojan horses," using the hydroxamate-type siderophore uptake system to actively deliver antibiotics directly into targeted pathogens.     

Iron(III) hydroxamate transport across the cytoplasmic membrane ofEscherichia coli

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm ofEscherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. AfhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [⁵⁵Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N-and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately infhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems. The FhuC 30 kDa protein, most likely involved in ATP binding, contains two domains representing consensus sequences among all peripheral cytoplasmic membrane proteins of these systems. Amino acid replacements in domain I (LysGlu and Gln) and domain II (AspAsn and Glu) resulted in a transport-deficient phenotype.     

The Solution Structure,Binding Properties,and Dynamics of the Bacterial Siderophore-binding Protein FepB

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs.     

Demonstration of the functional role of conserved Glu-Arg residues in the Staphylococcus aureus ferrichrome transporter

The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B₁₂ transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by “charge swapping” experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.     

ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20–24 residue peptides of FhuB by determining the activity of β-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD. Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane. FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB. The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport. The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA…G (in FhuB DTA…G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase. Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7. Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.     

Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions

Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long -helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B₁₂. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.     

In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence

Transport of iron(III) hydroxamates across the inner membrane ofEscherichia coli depends on a binding protein-dependent transport system composed of the FhuB,C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in afhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.     

Evidence for siderophore-dependent iron acquisition in group B streptococcus

Mutagenesis of group B streptococcus (GBS) with TnphoZ, a transposon designed to identify secreted protein genes, identified the gene homologues fhuD and fhuG. The encoded proteins participate in siderophore (hydroxamate)-dependent iron(III) transport in other bacterial species. Sequence analysis of the genome determined that fhuD and fhuG are members of a polycistronic operon comprised of four genes, fhuCDBG, that encode a putative ATPase, cell surface receptor and two transmembrane proteins respectively. We hypothesized that FhuD was a siderophore receptor. Western analysis of cell extracts localized FhuD to the bacterial cell membrane. Fluorescence quenching experiments determined that purified FhuD bound hydroxamate-type siderophores. FhuD displayed highest affinity for iron(III)-desferroxamine, with a K(D) (microM) = 0.05, identical to that described for FhuD2 from Staphylococcus aureus. The role of Fhu in siderophore-iron transport was also characterized. A fhu mutant, ACFhu1, was equally sensitive to the iron-dependent antibiotic streptonigrin as the wild-type strain, suggesting that ACFhu1 was not reduced for intracellular iron concentrations in the absence of exogenous siderophore. However, ACFhu1 transported significantly less siderophore-bound iron in (55)Fe accumulation assays. These data provide the first evidence of siderophore-mediated iron acquisition by GBS.     

Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB.

FhuD is the periplasmic binding protein of the ferric hydroxamate transport system of Escherichia coli. FhuD was isolated and purified as a His-tag-labeled derivative on a Ni-chelate resin. The dissociation constants for ferric hydroxamates were estimated from the concentration-dependent decrease in the intrinsic fluorescence intensity of His-tag-FhuD and were found to be 0.4 microM for ferric aerobactin, 1.0 microM for ferrichrome, 0.3 microM for ferric coprogen, and 5.4 microM for the antibiotic albomycin. Ferrichrome A, ferrioxamine B, and ferrioxamine E, which are poorly taken up via the Fhu system, displayed dissociation constants of 79, 36, and 42 microM, respectively. These are the first estimated dissociation constants reported for a binding protein of a microbial iron transport system. Mutants impaired in the interaction of ferric hydroxamates with FhuD were isolated. One mutated FhuD, with a W-to-L mutation at position 68 [FhuD(W68L)], differed from wild-type FhuD in transport activity in that ferric coprogen supported promotion of growth of the mutant on iron-limited medium, while ferrichrome was nearly inactive. The dissociation constants of ferric hydroxamates were higher for FhuD(W68L) than for wild-type FhuD and lower for ferric coprogen (2.2 microM) than for ferrichrome (156 microM). Another mutated FhuD, FhuD(A150S, P175L), showed a weak response to ferrichrome and albomycin and exhibited dissociation constants two- to threefold higher than that of wild-type FhuD. Interaction of FhuD with the cytoplasmic membrane transport protein FhuB was studied by determining protection of FhuB degradation by trypsin and proteinase K and by cross-linking experiments. His-tag-FhuD and His-tag-FhuD loaded with aerobactin specifically prevented degradation of FhuB and were cross-linked to FhuB. FhuD loaded with substrate and also FhuD free of substrate were able to interact with FhuB.     

Interactions between TonB from Escherichia coli and the periplasmic protein FhuD

For uptake of ferrichrome into bacterial cells, FhuA, a TonB-dependent outer membrane receptor of Escherichia coli, is required. The periplasmic protein FhuD binds and transfers ferrichrome to the cytoplasmic membrane-associated permease FhuB/C. We exploited phage display to map protein-protein interactions in the E. coli cell envelope that contribute to ferrichrome transport. By panning random phage libraries against TonB and against FhuD, we identified interaction surfaces on each of these two proteins. Their interactions were detected in vitro by dynamic light scattering and indicated a 1:1 TonB-FhuD complex. FhuD residue Thr-181, located within the siderophorebinding site and mapping to a predicted TonB-interaction surface, was mutated to cysteine. FhuD T181C was reacted with two thiol-specific fluorescent probes; addition of the siderophore ferricrocin quenched fluorescence emissions of these conjugates. Similarly, quenching of fluorescence from both probes confirmed binding of TonB and established an apparent KD of approximately 300 nM. Prior saturation of the siderophorebinding site of FhuD with ferricrocin did not alter affinity of TonB for FhuD. Binding, further characterized with surface plasmon resonance, indicated a higher affinity complex with KD values in the low nanomolar range. Addition of FhuD to a preformed TonB-FhuA complex resulted in formation of a ternary complex. These observations led us to propose a novel mechanism in which TonB acts as a scaffold, directing FhuD to regions within the periplasm where it is poised to accept and deliver siderophore.     

Iron(III) hydroxamate transport of Escherichia coli: restoration of iron supply by coexpression of the N- and C-terminal halves of the cytoplasmic membrane protein FhuB cloned on separate plasmids

Summary Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm of Escherichia coli cells is mediated by the FhuC, FhuD and FhuB proteins. We studied the extremely hydrophobic FhuB protein (70 kDa) which is located in the cytoplasmic membrane. The N- and C-terminal halves of the protein [FhuB(N) and FhuB(C)] show homology to each other and to the equivalent polypeptides involved in uptake of ferric dicitrate and of vitamin B₂. Various plasmids carrying only one-half of the fhuB gene were expressed in fhuB ^– mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamates as sole iron source; no activity was obtained with either half of FhuB alone. These results indicate that both halves of FhuB are essential for substrate translocation and that they combine to form an active permease when expressed separately. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be partially active in transport, indicating that the extra portion did not perurb proper insertion of the active FhuB segments into the cytoplasmic membrane.     

Iron-hydroxamate transport into Escherichia coli K12: localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Summary ThefhuB, fhuC andfhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm ofEscherichia coli. ThefhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence offhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.     

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.     

A structural and functional analysis of type III periplasmic and substrate binding proteins: their role in bacterial siderophore and heme transport

In Escherichia coli the Fhu, Fep and Fec transport systems are involved in the uptake of chelated ferric iron-siderophore complexes, whereas in pathogenic strains heme can also be used as an iron source. An essential step in these pathways is the movement of the ferric-siderophore complex or heme from the outer membrane transporter across the periplasm to the cognate cytoplasmic membrane ATP-dependent transporter. This is accomplished in each case by a dedicated periplasmic binding protein (PBP). Ferric-siderophore binding PBPs belong to the PBP protein superfamily and adopt a bilobal type III structural fold in which the two independently folded amino and carboxy terminal domains are linked together by a single long α-helix of approximately 20 amino acids. Recent structural studies reveal how the PBPs of the Fhu, Fep, Fec and Chu systems are able to bind their corresponding ligands. These complex structures will be discussed and placed in the context of our current understanding of the entire type III family of Gram-negative periplasmic binding proteins and related Gram-positive substrate binding proteins.     

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus.     

Requirement of Staphylococcus aureus ATP-binding cassette-ATPase FhuC for iron-restricted growth and evidence that it functions with more than one iron transporter

In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The delta fhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the delta fhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the "genetically unlinked" ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the delta fhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.     

Molecular basis for the differential interaction of plant mitochondrial VDAC proteins with tRNAs

In plants, the voltage-dependent anion-selective channel (VDAC) is a major component of a pathway involved in transfer RNA (tRNA) translocation through the mitochondrial outer membrane. However, the way in which VDAC proteins interact with tRNAs is still unknown. Potato mitochondria contain two major mitochondrial VDAC proteins, VDAC34 and VDAC36. These two proteins, composed of a N-terminal α-helix and of 19 β-strands forming a β-barrel structure, share 75% sequence identity. Here, using both northwestern and gel shift experiments, we report that these two proteins interact differentially with nucleic acids. VDAC34 binds more efficiently with tRNAs or other nucleic acids than VDAC36. To further identify specific features and critical amino acids required for tRNA binding, 21 VDAC34 mutants were constructed and analyzed by northwestern. This allowed us to show that the β-barrel structure of VDAC34 and the first 50 amino acids that contain the α-helix are essential for RNA binding. Altogether the work shows that during evolution, plant mitochondrial VDAC proteins have diverged so as to interact differentially with nucleic acids, and this may reflect their involvement in various specialized biological functions.     

Hemolytic Lectin CEL-III Heptamerizes via a Large Structural Transition from α-Helices to a β-Barrel during the Transmembrane Pore Formation Process

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.     

Albomycin uptake via a ferric hydroxamate transport system of Streptococcus pneumoniae R6

The antibiotic albomycin is highly effective against Streptococcus pneumoniae, with an MIC of 10 ng/ml. The reason for the high efficacy was studied by measuring the uptake of albomycin into S. pneumoniae. Albomycin was transported via the system that transports the ferric hydroxamates ferrichrome and ferrioxamine B. These two ferric hydroxamates antagonized the growth inhibition by albomycin and salmycin. Cross-inhibition of the structurally different ferric hydroxamates to both antibiotics can be explained by the similar iron coordination centers of the four compounds. [(55)Fe(3+)]ferrichrome and [(55)Fe(3+)]ferrioxamine B were taken up by the same transport system into S. pneumoniae. Mutants in the adjacent fhuD, fhuB, and fhuG genes were transport inactive and resistant to the antibiotics. Albomycin, ferrichrome, ferrioxamine B, and salmycin bound to the isolated FhuD protein and prevented degradation by proteinase K. The fhu locus consisting of the fhuD, fhuB, fhuG, and fhuC genes determines a predicted ABC transporter composed of the FhuD binding lipoprotein, the FhuB and FhuG transport proteins, and the FhuC ATPase. It is concluded that active transport of albomycin mediates the high antibiotic efficacy in S. pneumoniae.