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.     

Sideromycins: tools and antibiotics

Sideromycins are antibiotics covalently linked to siderophores. They are actively transported into gram-positive and gram-negative bacteria. Energy-coupled transport across the outer membrane and the cytoplasmic membrane strongly increases their antibiotic efficiency; their minimal inhibitory concentration is at least 100-fold lower than that of antibiotics that enter cells by diffusion. This is particularly relevant for gram-negative bacteria because the outer membrane, which usually forms a permeability barrier, in this case actively contributes to the uptake of sideromycins. Sideromycin-resistant mutants can be used to identify siderophore transport systems since the mutations are usually in transport genes. Two sideromycins, albomycin and salmycin, are discussed here. Albomycin, a derivative of ferrichrome with a bound thioribosyl-pyrimidine moiety, inhibts seryl-t-RNA synthetase. Salmycin, a ferrioxamine derivative with a bound aminodisaccharide, presumably inhibts protein synthesis. Crystal structures of albomycin bound to the outer membrane transporter FhuA and the periplasmic binding protein FhuD have been determined. Albomycin and salmycin have been used to characterize the transport systems of Escherichia coli and Streptococcus pneumoniae and of Staphylococcus aureus, respectively. The in vivo efficacy of albomycin and salmycin has been examined in a mouse model using Yersinia enterocolitica, S. pneumoniae, and S. aureus infections. Albomycin is effective in clearing infections, whereas salmycin is too unstable to lead to a large reduction in bacterial numbers. The recovery rate of albomycin-resistant mutants is lower than that of the wild-type, which suggests a reduced fitness of the mutants. Albomycin could be a useful antibiotic provided sufficient quantities can be isolated from streptomycetes or synthesized chemically.     

FhuD1, a ferric hydroxamate-binding lipoprotein in Staphylococcus aureus: a case of gene duplication and lateral transfer

Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.     

Identification of genes involved in siderophore transport in Streptomyces coelicolor A3(2)

The potential iron siderophore transporter genes have been determined from the genome sequence of Streptomyces coelicolor A3(2). One of these gene clusters, cdtABC, was disrupted and characterized to determine its role in the uptake of the siderophores produced by S. coelicolor. Resistance to the siderophore-like antibiotics, salmycin and albomycin, was tested in the parent and cdtABC mutant, showing that the parent, but not the mutant, was sensitive to salmycin, while both were resistant to albomycin. Ferrioxamine competition assays against salmycin suggest that the uptake of salmycin is via a ferrioxamine transport system. However, Fe-55 ferrioxamine B uptake experiments did not reveal any difference between the parent and mutant. This suggests that CdtABC specifically transports salmycin, while ferrioxamine uptake maybe substituted by another transport system.     

Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein

Due to its extreme insolubility, Fe3+ is not transported as a monoatomic ion. In microbes, iron is bound to low molecular weight carriers, designated siderophores. For uptake into cells of Escherichia coli Fe3+ siderophores have to be translocated across two membranes. Transport across the outer membrane is receptor-dependent and energy-coupled; transport across the cytoplasmic membrane seems to follow a periplasmic binding protein-dependent transport mechanism. In support of this notion we demonstrate specific binding of the Fe3+ hydroxamate compounds ferrichrome, aerobactin, and coprogen, which are transported via the Fhu system, to the periplasmic FhuD protein, and no binding of the transport inactive ferrichrome A, ferric citrate, and iron sulfate. About 10(4) ferrichrome molecules were bound to the FhuD protein of cells which overproduced plasmid-encoded FhuD. Binding depended on transport across the outer membrane mediated by the FhuA receptor and the TonB protein. Binding to FhuD was supported by the exclusive resistance of FhuD to proteinase K in the presence of the transport active hydroxamates. The overproduced precursor form of the FhuD protein was not protected by the Fe3+ hydroxamates indicating a conformation different to the mature form. The FhuD protein apparently serves as a periplasmic carrier for Fe3+ hydroxamates with widely different structures.     

FhuF, part of a siderophore-reductase system

FhuF is a cytoplasmic 2Fe-2S protein of Escherichia coli loosely associated with the cytoplasmic membrane. E. coli fhuF mutants showed reduced growth on plates with ferrioxamine B as the sole iron source, although siderophore uptake was not defective in transport experiments. Removal of iron from coprogen, ferrichrome, and ferrioxamine B was significantly lower in fhuF mutants compared to the corresponding parental strains, which suggested that FhuF is involved in iron removal from these hydroxamate-type siderophores. A redox potential E(1/2) of -310 +/- 25 mV relative to the normal hydrogen electrode was determined for FhuF by EPR redox titration; this redox potential is sufficient to reduce the siderophores coprogen and ferrichrome. M?ssbauer spectra revealed that FhuF in its [Fe(2+)-Fe(3+)] state is also capable of direct reduction of ferrioxamine B-bound ferric iron, thus proving its reductase function. This is the first report on a bacterial siderophore-iron reductase which in vivo seems to be specific for a certain group of hydroxamates.     

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.     

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 characterization of the iron-hydroxamate uptake system in Staphylococcus aureus

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.     

Iron acquisition systems for ferric hydroxamates, haemin and haemoglobin in Listeria monocytogenes

Listeria 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 (Km approximately 1-10 nM) than Gram-negative bacteria, and generally lower uptake rates. Vmax for [59Fe]-enterobactin (0.15 pMol per 10(9) cells per minute) was 400-fold lower than that of Escherichia coli. For [59Fe]-corynebactin, Vmax was also low (1.2 pMol per 10(9) cells per minute), but EGD-e transported [59Fe]-apoferrichrome similarly to E. coli (Vmax=24 pMol per 10(9) cells per minute). L. monocytogenes encodes potential Fur-regulated iron transporters at 2.031 Mb (the fur-fhu region), 2.184 Mb (the feo region), 2.27 Mb (the srtB region) and 2.499 Mb (designated hupDGC region). Chromosomal deletions in the fur-fhu and hupDGC regions diminished iron uptake from ferric hydroxamates and haemin/haemoglobin respectively. In the former locus, deletion of fhuD (lmo1959) or fhuC (lmo1960) strongly reduced [59Fe]-apoferrichrome uptake. Deletion of hupC (lmo2429) eliminated the uptake of haemin and haemoglobin, and decreased the virulence of L. monocytogenes 50-fold in mice. Elimination of srtB region genes (Deltalmo2185, Deltalmo2186, Deltalmo2183), both sortase structural genes (DeltasrtB, DeltasrtA, DeltasrtAB), fur and feoB did not impair iron transport. However, deletion of bacterioferritin (Deltafri, lmo943; 0.97 Mb) decreased growth and altered iron uptake: Vmax of [59Fe]-corynebactin transport tripled in this strain, whereas that of [59Fe]-apoferrichrome decreased 20-fold.     

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.     

Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.     

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.     

Iron-hydroxamate uptake systems in Bacillus subtilis: identification of a lipoprotein as part of a binding protein-dependent transport system

Bacillus subtilis was shown to utilize three types of hydroxamate siderophores, ferrichromes, ferrioxamines and shizokinen, each of which is taken up by different transport systems. Mutants deficient in the uptake of ferrichrome and/or ferrioxamine B were isolated. The gene fhuD, which was able to complement a mutant defective in ferrichrome uptake, was cloned. The deduced sequence of FhuD showed low but significant homology to the binding proteins FepB, FecB and FhuD of Escherichia coli, which are all components of binding protein-dependent, ferric siderophore transport systems. The first 23 amino acids of FhuD of B. subtilis possessed all characteristics of a lipoprotein signal sequence. The processing of FhuD in E. coli was inhibited by globomycin. Inhibition by globomycin indicated a lipid modification at the N-terminal cysteine in E. coli. It is highly likely that this step may also take place in B. subtilis. As in other binding protein-dependent transport systems of Gram-positive organisms it is proposed that the lack of a periplasm is compensated for by the lipid through which the binding protein is anchored to the cytoplasmic membrane.     

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.     

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.     

Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus

Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis. DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG, all of which show significant similarities to ferric hydroxamate uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus. This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.