Mechanisms of Iron Acquisition during Bacterial Pathogenesis Phillip E. Klebba, Ph. D. and Salete M. C. Newton, Ph. D.

I. Signal and energy transduction through bacterial membranes. The thin biological membranes between a cell's interior and its environment encompass many indispensable functions. Most fundamentally, they create an inward flow of nutrients to supply metabolic precursors for the biochemical pathways that constitute life. But, transport is selective: not all molecules penetrate into cells because membranes create a permeability barrier that attunes each cell to its environment. This acquisition of desirable substances and rejection of undesirable or noxious compounds is a focal point of our research. We are biochemically characterizing active transport processes that internalize compounds against their natural concentration gradients. Electrochemical potential created by ion gradients across membrane bilayers often powers thermodynamically unfavorable uptake reactions. In these contexts the proteins of bacterial cell membranes sense the environment, identify beneficial nutrilites, and capture them by membrane transport that involves signal and energy transduction by multicomponent protein assemblies. We are researching one such complex of proteins (TonB-dependent iron transport systems) in Gram-negative cells, and another network of cell envelope proteins (sortase-dependent and independent heme/hemoglobin transporters) in Gram-positive cells. Both systems are potential targets for antibiotic discovery, which is our ultimate goal. A. Bacterial iron uptake mechanisms. We are studying the biochemical processes that prokaryotes use to obtain Fe+++ from their environments, and the function of these systems in the pathogenesis of human and animal hosts. The passage of metal ions through membranes raises questions about its mechanisms, energetics, and the thermodynamics/kinetics of the transport events (9, 10). We are characterizing iron uptake in two structurally distinct systems: the Gram-negative bacterial outer membrane (OM), and the Gram-positive bacterial cytoplasmic membrane (CM). Both systems function by active transport. The energetics underlying OM metal transport are as yet uncertain, but CM iron permeases are usually ABC-transporters, driven by ATP hydrolysis. The relationship of iron to infection is simply summarized: iron is a valuable commodity in metabolism, and therefore a key element of bacterial pathogenesis. Not just microorganisms, but virtually all organisms require iron for cellular processes, including electron transport and energy generation by hemecontaining proteins, DNA synthesis, catabolism, and detoxification of reactive oxygen species. Humans and animals sequester iron in proteins like transferrin, lactoferrin and ferritin, in large part as a means of defense against microbial infection. Some microorganisms survive in the host by directly utilizing the iron in these binding proteins, but many others synthesize and secrete small molecules called siderophores (Gr. “ironcarrier”), that chelate iron and actively remove it from the eukaryotic binding proteins. Siderophores were discovered by my doctoral advisor, J.B. Neilands, and I’ve studied the mechanistic interaction between the native E. coli siderophore, ferric enterobactin (FeEnt), and its receptor protein, FepA for most of my career. 1. Site-directed spectroscopy. One object of our investigations is a mechanistic understanding of membrane transport events. Toward this end we utilize molecular biology to introduce biophysical probes at sites of interest in transport proteins, and then observe biochemical activity during ligand uptake (5, 6, 13, 15, 16, 21). We accomplish this aim in two stages: site-directed substitution mutagenesis to insert single cysteines in a target protein, followed by maleimide-, iodoacetamide- or thiosulfonate-mediated modification of the sulfhydryl side chain with paramagnetic or fluorescent reagents. The environmental sensitivity of such probes (Fig. 1) often allows direct observation of conformational dynamics as a membrane protein internalizes its substrates. Our initial experiments with these techniques focused on FepA, which resides in the Gram-negative bacterial OM and internalizes FeEnt. The E. coli system typifies those of Salmonella typhi, Vibrio cholerae, Shigella dysenteria, Neisseria meningitidis, Bordetella pertussis and Yersinia pestis. Our findings on FepA first showed the presence of a large transmembrane, porin channel in its interior (14, 23), and then found unexpectedly dynamic conformational motion during uptake of ligands through the pore (5, 6, 15, 25). We were not the first to utilize site-directed spectroscopic techniques (1) to analyze membrane proteins, but we were the first to apply these methods in living cells to detect the sub-reactions of a transport

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Figure 1. Spectroscopic measurements of active transport through FepA. The figure summarizes experiments conducted in our lab that monitor iron transport through the bacterial OM in living bacteria. (A-D) Electron Spin Resonance measurements of FeEnt and colB uptake (6). (A and C) FeEnt– and colB–induced variations in spin labeled FepA motion. E. coli cells were grown, spin-labeled, and exposed to ligands in the EPR spectrometer. Multiple signal averaged spectra were collected from independent preparations of bacteria, and the mean and standard deviation of the ratio of weakly and strongly immobilized probes (b/a; see panel D) was calculated after the addition of FeEnt (A) (n = 4) and colB (C) (n = 6). The data showed that the siderophore mobilizes spin labels and the colicin to immobilizes them. Bars 1 through 6 are from scans at sequential times (~3 min intervals) after ligand addition ligands. (B and D) Superimposed spectra from single experiments are shown for the strongly and weakly immobilized peaks. (B) Spectra in the absence of FeEnt at 37°C (purple), and in its presence at 4°C (blue), after warming the sample to 37°C for 5 (green) and 15 (red) min, and after recooling it to 4°C (black). (D) Spectra were collected in the presence of colB at 4°C (purple), after increasing the temperature to 37°C for 5 (blue), 15 (green), and 75 (red) min, and after recooling to 4°C (black). The ESR measurements show time-dependent motion in FepA, that describes its interaction with its two ligands. For further details see reference 6. (E and F) Fluorescence spectroscopic measurements of FeEnt binding and transport (5). (E) Binding. When labeled with fluorescein maleimide (FM) at FepA residues S271C or S397C, live E. coli show FeEnt binding as quenching of fluorescence intensity (inset). The binding isotherm (1-F/F0 fitted against [FeEnt] using a “Bound versus Total” equation) produced Kd values for FepAS271C-FM and FepAS397C-FM of 0.263 nM and 0.292 nM, respectively, which are the same as Kd values from radioisotopic measurements (18). (F) Transport. The time course of fluorescence was observed in FM-labeled bacteria at 20 °C. FeEnt was added to 10 nM at t = 0, and intensity was measured in the fluorometer. The tracing shows time-dependent conformation motion during the ligand transport reaction. For details see ref. 5.

cycle through observation of conformational changes in the transporter itself (5, 6, 16). This novel technology enables us to examine dynamic motion in membrane proteins during their physiological activities, potentially superceding the “snapshots” of mechanisms that crystallography provides. Our approach and goal is to use time-resolved fluoresence intensity determinations, polarization studies, FRET, and measurements of susceptibility to external quenchers to characterize membrane transport in living cells. 2. The TonB-dependent OM iron transporters of Gram-negative bacteria: FepA and its relatives. The asymmetric OM bilayer defines the transport properties of Gram-negative cells: it permits entry of nutrients and vitamins, but excludes toxic molecules like detergents and certain antibiotics (8, 11). OM proteins called porins are the basis of this selectivity, and iron transporters like FepA form an unusual subclass of the porin superfamily: they catalyze TonB- and energy-dependent uptake of metals ( FeEnt; 9, 10), protein toxins (colicins B and D; 3), and a viruses (bacteriophage H8; 22). Unlike archaetypal porins FepA does not contain an open channel through the OM. Its C-terminus folds into a β-barrel that makes a channel, but its N-terminus 2

folds into a globular domain within the lumen of the channel and completely closes it. From a mechanistic standpoint this type of transporters is unique: it accomplishes energy-dependent uptake of small molecules in a membrane bilayer that cannot sustain an ion gradient. FepA transports FeEnt in stages. The iron complex initially binds to external loops of the OM protein, and through a series of incompletely understood reactions that involve energy and other proteins (most notably, TonB), the receptor transports the ligand (2, 4, 5, 16, 18, 19, 21, 24, 25). We are researching the physical mechanism of this transport reaction: the 150-residue Nterminus of FepA must change, either by rearranging in situ to form an opening for passage of the ligand, or by dislodging from the channel and thereby opening it for ligand passage. Besides the biophysical techniques in vivo discussed above, we are using biochemical and molecular biological approaches to elucidate the transport reaction in vitro, especially the disposition and potential motion of the N-domain (16). During uptake of its three classes of biological ligands (iron complexes, protein toxins and bacteriophage) FepA acts in concert with several other cell-envelope proteins, including TonB. The mechanistic details of these interactions are incompletely understood, but TonB is required for all OM metal transport systems (12). When metal complexes or other molecules bind to the surfaces of proteins like FepA (2, 4, 26) they compel conformational changes that begin in the outer loop regions and propagate through their transmembrane domains, until they reach the internal surface of the OM bilayer (2, 4, 5, 16, 21, 25). Thus, ligand adsorption causes conformational signal transduction through the membrane, and these structural changes expose protein domains at the N-terminus of the receptor protein that are recognized and bound by the TonB C-terminus. This conjunction of ligand recognition and ensuing uptake is prototypic of membrane signal transduction: the bacterium senses the external environment by the binding affinities of its surface proteins, identifies a desired nutrilite, and then uses protein conformational motion to activate the transport system and capture it. Figure 2. FeEnt uptake by FepA. The N-terminal 150 residues of The uptake requires energy because FepA accumulates FepA fold into a globular domain (red) that inserts into a C-terminal 575 FeEnt against a concentration gradient. Nevertheless, amino acid transmembrane β-barrel (green). Initially, the surface loops active transport is confounding in a bilayer that contains of the transporter assume an open conformation that is receptive to the open (porin) channels, because they preclude the binding of ligands. FeEnt associates with aromatic and basic residues in the surface loops, converting the loops to a closed conformation that formation of an electrochemical gradient. These holds the iron complex above the N-domain, ready for transport. One considerations may explain the need for TonB in OM possible mechanism of transport, the Ball-and-Chain theory, involves metal transport. TonB may link OM transporters to the dislodgement of the N-domain from the pore, which pulls FeEnt through energized inner membrane, and promote the opening of the membrane bilayer into the cell. Transport requires the input of energy and the actions of TonB, by currently unknown mechanisms. their closed channels by direct physical contact (9, 10, 12). We found homology in TonB to periplasmic proteins that associate with peptidoglycan (PG), and demonstrated its binding to purified E. coli PG (7a). The data suggested a membrane surveillance model, in which TonB spans the periplasm to survey the underside of the OM bilayer, locating ligand-bound receptor proteins and facilitating their transport reactions (Fig. 3). With fluorescence spectroscopic, biochemical and molecular biological approaches, we are studying the interaction of TonB with other cell envelope proteins (e.g., FepA) and with PG. This research will biochemically define the activities and role of TonB (and accessory proteins) in the facilitation of high 3

Figure 3. Prototypic TonB- and energy-dependent iron transport systems of the Gram-negative bacterial cell envelope in the “Membrane Surveillance” model of TonB action. The FhuA (green) and FepA (purple) OM transporters require the activity of TonB (blue and light green)/ExbBD (red) and the input of cellular energy to accomplish iron uptake into the periplasm. Once inside, periplasmic binding proteins (e.g., FepB, pink) adsorb the ferric siderophores and transfer them to ABC transporter permeases in the IM (e.g., FepCDG, shades of magenta). The OM transport stage is postulated to involve a physical interaction with the TonB C-terminus. (Left) In a cross-sectional view of the cell envelope, dimeric TonB (green and blue), associates with the IM by α-helices in its N-terminus that complex with ExbBD (red). TonB also contains lengths of rigid polypeptide (shown here as coiled helices) that span the periplasm, and LysM motifs in its C-terminus that associate with the PG layer (grey) underlying the OM bilayer. The TonB dimer has general affinity for PG and TonB-independent OM proteins (e.g., OmpA), which tends to localize the C-terminus at the periplasmic interface of the OM bilayer. The monomeric form of the C-terminus, on the other hand, has a specific affinity for accessible TonB boxes of (ligandbound) TonB-dependent receptors, resulting in their recruitment by its β-sheet. These affinities allow TonB to physically survey the periplasmic surface of the OM bilayer until it encounters bound LGP (FhuA, dark green and orange). This motion across the internal surface of the OM may derive from movement of the N-terminus in the fluid IM bilayer. FepA (purple and red) and the TonB-independent OM proteins OmpF (white) and TolC (white) are also shown associated with PG. The latter protein complexes with AcrAB in the IM bilayer, which provides a reference for the distance between the IM and OM bilayers. (Right) From a periplasmic view, the dimeric form of TonB (green and blue) moves among the roughly hexagonal, 50 Å cells of the PG polymer (grey), which associate with the β-barrels of OM proteins (TolC, OmpF and LamB, all shown in white). LGP in the OM bilayer may be ligand-free (FepA: purple β-barrel and red N-domain, cyan TonB-box; FhuA: dark green β-barrel and orange N-domain, cyan TonB-box) or ligand-bound (note FhuA at top and bottom right, with TonB-box relocated to the center of the channel). The TonB-C-domain remains dimeric until it encounters a ligand-bound receptor (FhuA-Fc, top right), and then dissociates into a monomeric form that recruits the TonB-box region into its β-sheet. For more information see (7a).

affinity metal uptake. These phenomena present an opportunity to elucidate transmembrane signal transduction, the bioenergetics of active transport, and a unique, multi-component metal internalization process, all in the same system. 3. Sortase-independent and dependent hemin/hemoglobin transporters of Gram-positive bacteria: the Hup and Hbp systems of Listeria monocytogenes. Gram-positive bacteria require iron in comparable amounts to Gram-negative cells. But, organisms like Staphylococcus aureus, Streptococcus pyogenes, Bacillus anthracis and Listeria monocytogenes lack an outer membrane, and instead they produce a thick layer of peptidoglycan (PG) that polymerizes outside their cytoplasmic membrane (CM). Proteins, polysaccharides and lipids associate with or anchor within the multilamellar coating of PG and extend to the cell surface. This different architecture than that of Gram-negative cells results in different solute uptake processes, that are at present comparatively obscure. Nevertheless, the rapidly expanding library of Gram-positive bacterial genomic information enabled a more facile path to the discovery and definition of their systems, which are of 4

great interest because they function by novel mechanisms. Gram (+) bacteria contain many homologous loci to the iron transporters of Gram (-) bacteria: their CM (Gram-positive) and IM (Gram-negative) systems are functionally and structurally equivalent with regard to transport of many solutes. Therefore, using genomic and proteomic analyses as starting points, we investigated the iron acquisition mechanisms of Figure 4. Listerial iron transport loci. A schematic representation of the srtB, fhu and hup regions of the L. monocytogenes chromosome shows Fur-regulated loci involved in iron acquisition. The srtB region (2.274 L. monocytogenes by Mb) contains genes for secreted, peptidoglycan-associated heme binding proteins (hbp1, hbp2), the sortase systematic chromosomal (srtB) that anchors them to the cell wall and a putative ABC-transporter (lmo2182-2184). The fhu region deletions and phenotypic (2.031 Mb) encodes a substrate binding lipoprotein (FhuD) and a putative ferric hydroxamate ABCcharacterizations. These transporter (FhuBCG). The hupDGC locus (2.499 Mb) encodes a substrate binding lipoprotein (HupD) and a putative ABC transporter for Hn/Hb. Parenthetic values refer to the distance (bp) between adjacent genes. experiments identified two For more information see refs 7, 20, 27. ABC-transporter systems for iron, one that functions to acquire ferric hydroxamates (Fhu), and another with specificity for hemin/hemoglobin (Hup) (7, 20). Our subsequent experiments characterized the biochemistry of hemin/hemoglobin uptake by L. monocytogenes, which uses sortase-independent and dependent systems to acquire the iron porphyrin in animal hosts (see below; 27). The genomic approach superceded many of the genetic, and to some extent biochemical experiments that delineated Gram-negative bacterial membrane transport systems. Once identified by deletion mutagenesis, Gram-positive bacterial iron acquisition systems often closely correlated with those of E. coli. Furthermore, although IM transporters of Gram-negative cells are shielded by the OM, CM transporters of Gram-positive cells are more accessible to chemical modification from without (by the same biophysical techniques we’ve developed for E. coli FepA; Fig. 3), because of the absence of an OM. We are genetically introducing modifiable Cys residues in the two ABC-permease transporters of L. monocytogenes noted above, and applying fluorescence spectroscopic techniques to understand their mechanisms. One target is the Hup transporter, because of its multiple ligands, ability to extract hemin or iron from hemoglobin, and its relationship to the virulence of L. monocytogenes (7), an intracellular pathogen that crosses the blood-brain barrier (see also below). Experiments on the Hup transporter complement studies on E. coli FepA in several ways. Both systems actively transport iron, in one case through the OM, in the other through the CM. The Gram-negative bacterial OM transporter utilizes proton-motive force for energization, whereas the listerial CM ABCtransporter hydrolizes ATP. Both membrane proteins undergo conformational motion, but they achieve different biochemical mechanisms of ligand uptake. B. Bacterial iron acquisition in the host environment. Iron acquisition, by both pathogens and their hosts, relates to bacterial disease. Prokaryotes and eukaryotes alike require iron for metabolic biochemistry, and iron deprivation inhibits microbial growth, reducing or eliminating virulence. The central role of iron in aerobic biochemistry makes it a focal point of pathogenesis and invasiveness: eukaryotic hosts sequester iron as a defense against infection, but successful pathogens overcome this strategy and capture the metal. 5

1. Heme and hemoglobin uptake by Gram-positive bacteria. While they lack the OM transport systems of Gram-negative bacteria, L. monocytogenes and its Gram-positive relatives contain other novel approaches to both the interaction with eukaryotic cells, and iron acquisition in animal tissues. Various proteins encoded by Fur-regulated transport operons of Gram-positive bacteria are secreted to the cell exterior, or covalently attached to PG by a class of cell envelope enzymes called sortases (17). Some of these PG-anchored protein function in bacterial adherence to animal cells, and others may act in the recognition and transport of heme by providing an initial binding site for the porphyrin and for heme-containing proteins like hemoglobin or haptoglobin. We performed thermodynamic and kinetic analyses of hemin binding and transport that revealed the role of sortase-anchored cell envelope proteins in iron uptake by L. monocytogenes (7, 20, 27). These data showed that proteins anchored to PG by sortase B, Hbp1 and Hbp2, function to bind heme. These proteins bear sequence relatedness to the IsdC protein of Staphylococcus aureus, which is also known to bind hemin. Hbp1, Hpb2 and IsdC are secreted proteins that are apparently anchored to PG by sortase B. However, the mechanism by which they transfer heme to CM hemin ABC-transporters is unknown.

Figure 5. Heme binding proteins in the Gram-positive cell envelope. (A) SauIsdC. Eight residues its single α-helix and β-strands 7 and 8 complex heme in SauIsdC (2O6P). Theses amino acids in IsdC (yellow) are identical or conservatively substituted in listerial Hbp1 and Hbp2, including Y52 and Y132, which coordinate iron in the porphyrin ring. (B, C) Sortase-dependent and independent heme transport through the Gram-positive bacterial cell envelope. The cytoplasmic membrane (phospholipids in stick format; phosphate atoms in space filling form) contains transporters for iron compounds, such as the HupDGC and FhuDBGC putative ABC-transporters. The PG polymer (wire format in panel B; space filling surfaces in panel C) surrounds the cell as conjoined hexagonal pores with approximately 70 Å diameter (coordinates through the courtesy of S. Mobashery). This architecture creates pathways for diffusion of small solutes, like ferric siderophores (Fc) or porphyrins (Hn), through the PG matrix (best viewed in E). At concentrations above 50 nM, Hn permeates the PG layer and directly adsorbs to lipoproteins of the ABC-transporters. On the other hand, sortases A and B attach haem binding proteins (for example, listerial Hbp1 and Hbp2) and other proteins to the cell wall matrix by peptide bonds to Lys or DAP in PG. The anchored proteins create a sortase-dependent pathway of Hn/Hb (Hb (PDB file 3HF4) uptake. At very low Hn concentrations Hbp1 and Hbp2 initiate transport by capturing the iron porphyrin from solution and subsequently transferring it to the underlying ABC transporters. For more information, see ref 27.

2. Host colonization. Since the early 70's biochemists and microbiologists suspected that iron acquisition by both pathogens and their hosts, relates to bacterial disease. Research on this subject confirmed what originally seemed intuitive: bacteria need iron for metabolism, they produce biosynthetic and transport systems to obtain the metal, and impairment or abrogation of these processes reduces bacterial virulence. Consequently, the characterization of systems that pathogenic bacteria employ for iron uptake in vivo, and the evaluation of their importance to bacterial infection and colonization, are central goals of our research efforts. We are studying the function of siderophore uptake systems in colonization by Gram-negative (E. coli, S. typhimurium) bacteria. E. coli is usually a commensal enteric bacterium, becomes it invasive and pathogenic when it elaborates certain siderophores. Our experiments also encompass the participation of 6

hemin/hemoglobin uptake systems in infection by Gram-positive (L. monocytogenes, S. aureus) species. L. monocytogenes, a saprophytic organism that is widespread in nature, is also a lethal intracellular pathogen that does not elaborate siderophores, but uses exogenous ferric siderophores from other organisms or directly extracts iron from hemoglobin, holotransferrin or ferritin. We are characterizing the relationship between its iron uptake and infection with experiments that evaluate site-directed chromosomal mutations on bacterial virulence (LD50 values in the murine model system). We also measure bacterial multiplication in target organs (spleen, liver, brain) by oral infection of mice with wild L. monocytogenes or its genetically engineered mutants. Finally, its intracellular mode of pathogenesis and ability to directly utilize eukaryotic iron sources makes L. monocytogenes prototypic for studies of iron and virulence. We are determining the pathogenicity of iron uptake mutants in murine macrophages and cell lines, including intracellular multiplication in primary macrophages prepared from murine bone marrow, and in human enterocytes (CACO-2 cells). C. Systems Biology: iron acquisition and microbial pathogenesis. Our experiments center on membrane transport biochemistry, mainly in the acquisition of iron by bacteria. Iron availability is a global regulator of bacterial metabolism, which makes iron uptake a focal point of prokaryotic systems biology. The success or failure of iron acquisition determines the outcome of pathogenesis, so this regulatory and transport network connects to human and animal disease. Our program seeks to understand metallo-biochemistry in microbial systems biology, and future experiments will encompass a multi-disciplinary, systems biology approach to the discovery of new pharmaceutical agents to thwart bacterial pathogenesis. We expect these research programs to lead to clinical applications in human and animal health. For additional details of our research programs and findings, please visit: http://chem.ou.edu/~pek/peklab.htm

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13. Klug, C. S., W. Su, J. Liu, P. E. Klebba, and J. B. Feix 1995. Denaturant unfolding of the ferric enterobactin receptor and ligand- induced stabilization studied by site-directed spin labeling Biochemistry 34:14230-6. 14. Liu, J., J. M. Rutz, J. B. Feix, and P. E. Klebba 1993. Permeability properties of a large gated channel within the ferric enterobactin receptor, FepA Proc Natl Acad Sci U S A. 90:10653-7. 15. Liu, J., J. M. Rutz, P. E. Klebba, and J. B. Feix 1994. A site-directed spin-labeling study of ligand-induced conformational change in the ferric enterobactin receptor, FepA Biochemistry. 33:13274-83. 16. Ma, L., W. A. Kaserer, R. Annamalai, D. C. Scott, B. Jin, X. Jiang, Q. Xiao, H. Maymani, L. M. Massia, L. C. Ferreira, S. M. Newton, and P. E. Klebba 2006. Evidence of ball-and-chain transport of ferric enterobactin through FepA J Biol Chem. 282:397-406. 17. Mazmanian, S. K., G. Liu, H. Ton-That, and O. Schneewind 1999. Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall Science. 285:760-3. 18. Newton, S. M., J. S. Allen, Z. Cao, Z. Qi, X. Jiang, C. Sprencel, J. D. Igo, S. B. Foster, M. A. Payne, and P. E. Klebba 1997. Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA Proc Natl Acad Sci U S A. 94:4560-5. 19. Newton, S. M., J. D. Igo, D. C. Scott, and P. E. Klebba 1999. Effect of loop deletions on the binding and transport of ferric enterobactin by FepA Mol Microbiol. 32:1153-1165. 20. Newton, S. M., P. E. Klebba, C. Raynaud, Y. Shao, X. Jiang, I. Dubail, C. Archer, C. Frehel, and A. Charbit 2005. The svpA-srtB locus of Listeria monocytogenes: Fur-mediated iron regulation and effect on virulence Mol Microbiol. 55:927-940. 21. Payne, M. A., J. D. Igo, Z. Cao, S. B. Foster, S. M. Newton, and P. E. Klebba 1997. Biphasic binding kinetics between FepA and its ligands J Biol Chem. 272:21950-5. 22. Rabsch, W., L. Ma, G. Wiley, F. Najar, B. Roe, W. Kaserer, B. Biel, M. Schmalley, S. M. Newton, and P. E. Klebba 2007. The T5-like bacteriophage H8: TonB-dependent infection of Escherichia coli through the ferric enterobactin receptor, FepA J Bacteriol. 189:5658-74. 23. Rutz, J. M., J. Liu, J. A. Lyons, J. Goranson, S. K. Armstrong, M. A. McIntosh, J. B. Feix, and P. E. Klebba 1992. Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane Science. 258:471-5. 24. Scott, D. C., Z. Cao, Z. Qi, M. Bauler, J. D. Igo, S. M. Newton, and P. E. Klebba 2001. Exchangeability of N termini in the ligand-gated porins of Escherichia coli J Biol Chem. 276:13025-33. 25. Scott, D. C., S. M. Newton, and P. E. Klebba 2002. Surface loop motion in FepA J Bacteriol. 184:4906-11. 26. Thulasiraman, P., S. M. Newton, J. Xu, K. N. Raymond, C. Mai, A. Hall, M. A. Montague, and P. E. Klebba 1998. Selectivity of ferric enterobactin binding and cooperativity of transport in gram-negative bacteria J Bacteriol. 180:6689-96. 27. Xiao, Q., X. Jiang, K. J. Moore, Y. Shao, H. Pi, I. Dubail, A. Charbit, S. M. Newton, and P. E. Klebba. 2011. Sortase independent and dependent systems for acquisition of haem and haemoglobin in Listeria monocytogenes. Mol Microbiol 80:1581-1597.

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