Discussion of siderophore-mediated iron transport will be divided between the two structural classes of bacteria (Figure 7). Novel uptake systems will also be briefly covered. This review will not be able to cover the details of such a large and well-studied field, which has been covered in many good reviews.19–21,23,36,66,93
8.6.4.1 Gram-negative Iron Transport 8.6.4.1.1 Outer membrane
Although passive diffusion, via nonspecific porins, mediates passage across the outer membrane (OM) in Gram-negative bacteria, the low concentration and large size of ferric siderophore complexes require an energized active transport process in order to fulfill the iron requirements of the cell. Recognition and incorporation of the ferric siderophore complex begins at an OM receptor, and this is the rate-limiting step for the entire siderophore-mediated iron-transport process.21,94 Under periods of iron deprivation, organisms express highly specific receptors cap- able of binding a very narrow subset of ferric siderophores.23,95However, several bacteriophages (T1, T5,80, UC-1) and bacteriocins (colicin M and microcin 25) opportunistically utilize these OM receptors for entry into the cell.24 Once the ferric siderophore complex is recognized, transport across the membrane is accomplished through transduction of the cytoplasmic proton motive force by the cytoplasmic membrane (CM) TonB-ExbB-ExbD complex.20,93 Thus, ferric siderophore receptors are referred to as the TonB-dependent class of OM receptor proteins.96The crystal structure of the C-terminal domain of TonB has been reported.97 Binding of the ferric siderophore to the OM receptor induces a conformational change in the receptor protein. Most notably, the unwinding of the switch helix in the receptor protein yields a flexible extended conformation. This unwinding of the switch helix allows facile distinction between receptors with and without bound siderophore complex, facilitating contact between TonB and the activated receptor. TonB transduces the electrochemical potential of the cytoplasmic membrane, resulting in a second conformational change in the receptor protein, and lowering the affinity of TonB for the receptor. The ferric siderophore is then released into the periplasmic space and TonB is retrieved and recycled by the cytoplasmic proteins ExbB and ExbD.20,93
The crystal structures of three OM proteins of Escherichia coli, each responsible for the recognition of a different type of ferric siderophore, have been described. FhuA98,99 binds ferrichrome; FepA100 recognizes ferric enterobactin, and similar tris-catecholate analogs. In 2002, the structure of the ferric citrate receptor, FecA (Figure 8),101appeared. Each of these receptors consists of approximately 700 residues organized into a C-terminal, 22-stranded -barrel and an N-terminal plug domain located within the barrel channel. This N-terminal plug serves to block passage through the barrel. The unfolded plug domain of FepA binds ferric enterobactin, though
Outer membrane Periplasm
FhuA
FhuD FhuD
FhuB FhuB
FhuC FhuC Cytoplasmic
membrane
Cytoplasm Cytoplasm
ExbD
ExbB
Gram-postive Gram-negative
ATP ADP +Pi ATP ADP +Pi
Ton B
Figure 7 Gram-negative (left) and Gram-positive (right) siderophore-mediated iron uptake. In Gram-negative bacteria: the proteins are specific for hydroxamate siderophore transport. The OM receptor protein (FhuA) binds the ferric siderophore and transports it through the OM via energy transduced by the TonB-ExbBD system. The BP FhuD shuttles the ferric siderophore complex from the OM to the CM protein FhuB. ATPase FhuC hydrolyzes ATP, transporting the ferric siderophore complex into the cytoplasm. In Gram-positive bacteria: the lipoprotein FhuD binds the ferric siderophore and directs it to the CM protein FhuB. Hydrolysis
of ATP by FhuC permits passage into the cytoplasm through the transmembrane protein FhuB.
at a hundredfold lower affinity than the intact protein, suggesting that the plug domain rearranges to allow the ferric enterobactin to pass through the channel, rather than moving out as the intact plug.94 Interestingly, FecA contains a third domain residing completely within the periplasm, termed the N-terminal extension. This periplasmic domain is responsible for signaling the presence of substrate bound to FecA. Binding of ferric citrate to FecA results in the interaction of the N-terminal extension with the transmembrane protein FecR, which in turn controls the activity of FecI and directs the transcription of genes required for the uptake of ferric citrate.101
8.6.4.1.2 ATP-binding cassette (ABC) transport system
Following passage through the gated channel formed by the OM protein, the ferric siderophore complex binds to a substrate-binding protein (BP), a part of the ATP-binding cassette (ABC) transport system responsible for the translocation of the complex across the periplasm CM.102 While the interaction of the BP with the OM receptor protein has been postulated, experimental evidence for such an interaction has not yet been documented. The specificity and affinity of the BP for the ferric siderophore complex is far lower than their OM counterparts. While three separate OM receptors are required to transport ferric complexes of ferrichrome (FhuA), aerobactin (Iut), and coprogen (FhuE), the same FhuBCD ABC transporter can transport all of these complexes. The generalized mechanism for transport of ferric siderophores via the widely conserved ABC transport system includes: (i) binding of the substrate by the periplasmic-binding protein (BP); (ii) interaction of the BP with the CM spanning protein; and (iii) hydrolysis of ATP via a membrane-bound protein to transport ferric siderophore across the CM.
8.6.4.1.3 Periplasm
The crystal structure of the BP FhuD receptor protein with gallichrome (the gallium complex of ferrichrome),103 albomycin,104 coprogen,104 and Desferal (ferrioxamine B,Figure 2)104reveals the greater flexibility of this protein as compared to the OM counterparts. A long-helix connects the two dissimilar lobes of FhuD, forming a shallow-binding pocket lined with hydrophobic residues.
The ‘‘classic’’ BP fold (causing a ‘‘Venus flytrap’’ closing upon substrate binding) is absent,102 Figure 8 The bacterial ferric citrate uptake protein, FecA, without ferric citrate bound (left) and with ferric citrate (right). The 22-stranded barrel (in blue) forms the transmembrane channel; the plug domain (in green) blocks passage of non-ferric citrate substrates; the switch helix (in orange, not seen in the ligated
structure) unwinds to signal binding of ferric citrate.101
resulting in a smaller structural difference between the FhuD holo and apo forms. Most of the substrate–protein interactions center on the iron core, leaving the majority of the complex exposed.104 Increased hydrogen-bond contacts seem to correlate with increased substrate affinity (e.g., FhuD has more contacts and a higher affinity for coprogen (0.3mM) than for ferrichrome (1.0mM)).104Accommodation of a large variety of structural types is accomplished through recog- nition of only the central iron hydroxamate core of the ferric siderophore complex, and then adjusting BP around the rest of the substrate. This follows a prediction made almost 20 years ago regarding this hydroxamate uptake system and the establishment of its stereospecific recog- nition of the metal center: ‘‘. . .we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake.’’219
Transport of catecholate siderophores also employs the same type of transport system. Although the structure of BP FepB has not yet been determined, binding studies suggest that this protein is far more specific for enterobactin (Kd=30 nM)105 than the similar catecholate siderophores vibriobactin106 and agrobactin.105 Perhaps the tertiary structure of FepB contains a deeper and more shielded-binding pocket for the ferric complex, allowing more hydrogen-bonding contacts to the complex. Less solvent exposure would protect the ferric siderophore from dissociation from the BP, resulting in a higher-binding affinity for ferric siderophore complexes with the BP.
8.6.4.1.4 Cytoplasmic membrane
The periplasmic-binding protein (FhuD, FecB, or FepB) delivers the substrate (Fe-hydroxamate, Fe-citrate, or Fe-enterobactin), to the cytoplasmic transmembrane protein (FhuB, FecCD, or FepDG). Transport across the CM is driven by hydrolysis of ATP, achieved via ATP-binding proteins (FhuC, FecE, or FepC), attached to the CM. Interaction between FhuD and FhuB is evident from several studies.107,108Based upon these experimental results, a mechanism emerges whereby FhuD interacts with both the periplasmic loops and transmembrane segments of FhuB, perhaps forming a channel for FhuD to deposit the substrate into the cytoplasm. Direct contact between FhuD and FhuC within this channel, or a conformational change of the FhuB upon FhuD binding, could trigger the ATPase activity of FhuC, which allows transport of the ferric siderophore complex across the CM.18
8.6.4.2 Gram-positive Transport
8.6.4.2.1 Comparison to Gram-negative iron transport
Gram-positive bacteria lack the lipopolysaccharide layer encasing the cell, and therefore do not require the OM receptors such as FepA or FhuA to transport their siderophores into the cell (Figure 4).21,95,109 Passage via diffusion is possible through the thick but porous peptidoglycan cell wall, eliminating the need for the energy-transducing TonB-ExbBD proteins. Transport across the CM likely occurs via the ABC transport system, as described for Gram-negative bacteria. One modification to the transport system between Gram-negative and Gram-positive bacteria is the probable use of a membrane-bound lipoprotein for transporting the ferric siderophore complex to the transmembrane component of the ABC system, rather than utilizing a soluble periplasmic-binding protein as in Gram-negative bacteria. The lipoprotein would provide the same high affinity for the ferric siderophore complex, but tethering it to the CM would prevent loss to the surrounding medium.
8.6.4.2.2 Cytoplasmic membrane receptors
Gram-positive siderophore receptor proteins are not as well documented as the Gram-negative receptor proteins. In 1993, Hantke reported a binding protein anchored via a glyceryl–cysteine–
lipid residue to the CM of a Gram-positive bacterium.110This lipoprotein had sequence homology with both FepB (22.9%) and FhuD (16.2%) and was proposed to transport a wide variety of hydroxamate siderophores. More recently, at least three ABC transport systems have been reported for the Gram-positive Staphylococcus aureus. S. aureus produces two carboxylate
siderophores, staphyloferrin A111 and B,112 and the incompletely characterized aureochelin,113 but is also able to utilize exogenous siderophores such as the hydroxamates ferrichrome and aerobactin, as well as catecholates such as enterobactin and 2,3-dihydroxybenzoic acid. Although each of the three transport systems appears to transport a different type of siderophore, all three posses a lipid-anchored binding protein, a transmembrane protein, and an ATPase protein. The ferric hydroxamate transporter, FhuCBG114,115 (or FhuCBD116), indicated that S. aureus FhuC (29.5 kDa) was an ATPase similar to FhuC from B. subtilis. The S. aureus transmembrane permease proteins FhuB (36 kDa) and FhuG (36.1 kDa) are also very similar to their B. subtilis protein counterparts.115 The more recently identified FhuD2 is a lipoprotein responsible for shuttling ferric hydroxamate complexes to the transmembrane protein.114 Two other putative ABC transport systems, SirABC117 and SstABCD118, have been reported, although their sider- phore substrates, in early 2003, have yet to be identified.
8.6.4.3 Iron Regulation
Both Gram-negative and Gram-positive bacteria produce siderophores, and their corresponding receptor proteins, only in response to iron-poor environments. Hantke recently reviewed this area,95and only a brief description will be included here. Fur (ferric uptake regulator), and Fur- like proteins, regulate iron uptake in many bacterial species. Some Gram-positive bacteria, such as streptomyces, corynebacteria, and mycobacteria, use the DtxR (diphtheria toxin regulator) protein. The two-domain Fur protein regulates iron metabolism in E. coli; similar proteins have been found in other bacteria. The protein contains two metal-binding sites: Zn2þ and Fe2þ (however, other divalent metals such as Mn2þand Co2þcan also bind in the Fe2þsites). Binding of Fe2þ to Fur suppresses the transcription of genes regulating siderophore production. The sequences for DtxR and Fur are different, but some structural similarity exists, including the presence of two metal-binding sites. However, in the DtxR protein, both sites are occupied by iron.
8.6.4.4 Novel Transport Mechanisms 8.6.4.4.1 Siderophore shuttle
In 2000, Stintziet al. published a novel iron-transport mechanism (the ‘‘siderophore shuttle’’) that employs ligand exchange to provide the organismAeromonas hydrophilawith a flexible mechan- ism to satisfy its iron requirement (Figure 9).119 An iron-free siderophore first binds the OM receptors. When a ferric siderophore complex subsequently binds, the iron-free siderophore is pushed into the receptor channel. The iron-bound siderophore transfers its iron to the iron-free ligand, prompting the release of this new ferric siderophore complex into the periplasmic space. The second siderophore, now without iron, remains bound to the receptor, waiting to accept iron from the next ferric siderophore complex in the same manner. This siderophore recognition and transport mechanism provides Aeromonas hydrophila with great flexibility, since a wide variety of siderophore types can be bound to the receptor protein instead of selectively recognizing only one. This ability to utilize iron from a wide variety of ferric complexes makes it a fierce competitor for iron. A similar mechanism involving the initial binding of an iron-free siderophore to the OM receptor has been reported for Pseudomonas aeruginosa.120 In this system, however, the iron- bound siderophore displaces the iron-free siderophore, rather than transferring its iron as found in the siderophore shuttle.
8.6.4.4.2 Photodecarboxylation
Transition-metal complexes of -hydroxy acids can be photolabile. The oil-degrading marine bacterium Marinobacter hydrocarbonoclasticusproduces a siderophore, which appears to exploit photodecarboxylation to facilitate iron release. Petrobactin (Figure 4) forms a stable ferric complex through iron chelation by two catecholate moieties and a citryl group. Decarboxylation of the citryl moiety via photolysis of ferric petrobactin yields a less stable ferric complex than the
parent complex.55Photodecomposition of siderophores incorporating -hydroxy acids may pro- vide marine microorganisms with an effective iron-release pathway.