Effect of loop deletions on the binding and transport of ferric enterobactin by FepA

The siderophore ferric enterobactin enters Escherichia coli through the outer membrane (OM) porin FepA, which contains an aqueous transmembrane channel that is normally occluded by other parts of the protein. After binding the siderophore at a site within the surface loops, FepA undergoes conformational changes that promote ligand internalization. We assessed the participation of different loops in ligand recognition and uptake by creating and analysing a series of deletions. We genetically engineered 26 mutations that removed 9-75 amino acids from nine loops and two buried regions of the OM protein. The mutations had various effects on the uptake reaction, which we discerned by comparing the substrate concentrations of half-maximal binding (Kd) and uptake (Km): every loop deletion affected siderophore transport kinetics, decreasing or eliminating binding affinity and transport efficiency. We classified the mutations in three groups on the basis of their slight, strong or complete inhibition of the rate of ferric enterobactin transport across the OM. Finally, characterization of the FepA mutants revealed that prior experiments underestimated the affinity of FepA for ferric enterobactin: the interaction between the protein and the ferric siderophore is so avid (Kd < 0.2 nM) that FepA tolerated the large reductions in affinity that some loop deletions caused without loss of uptake functionality. That is, like other porins, many of the loops of FepA are superficially dispensable: ferric enterobactin transport occurred without them, at levels that allowed bacterial growth.     

Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA

In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D. To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined. In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane. One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function. Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane. Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function. Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels. The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.     

Concerted loop motion triggers induced fit of FepA to ferric enterobactin

Spectroscopic analyses of fluorophore-labeled Escherichia coli FepA described dynamic actions of its surface loops during binding and transport of ferric enterobactin (FeEnt). When FeEnt bound to fluoresceinated FepA, in living cells or outer membrane fragments, quenching of fluorophore emissions reflected conformational motion of the external vestibular loops. We reacted Cys sulfhydryls in seven surface loops (L2, L3, L4, L5, L7 L8, and L11) with fluorophore maleimides. The target residues had different accessibilities, and the labeled loops themselves showed variable extents of quenching and rates of motion during ligand binding. The vestibular loops closed around FeEnt in about a second, in the order L3 > L11 > L7 > L2 > L5 > L8 > L4. This sequence suggested that the loops bind the metal complex like the fingers of two hands closing on an object, by individually adsorbing to the iron chelate. Fluorescence from L3 followed a biphasic exponential decay as FeEnt bound, but fluorescence from all the other loops followed single exponential decay processes. After binding, the restoration of fluorescence intensity (from any of the labeled loops) mirrored cellular uptake that depleted FeEnt from solution. Fluorescence microscopic images also showed FeEnt transport, and demonstrated that ferric siderophore uptake uniformly occurs throughout outer membrane, including at the poles of the cells, despite the fact that TonB, its inner membrane transport partner, was not detectable at the poles.     

Aromatic components of two ferric enterobactin binding sites in Escherichia coli FepA

Ferric enterobactin is a catecholate siderophore that binds with high affinity (Kd approximately 10-10 M) to the Escherichia coli outer membrane protein FepA. We studied the involvement of aromatic amino acids in its uptake by determining the binding affinities, kinetics and transport properties of site-directed mutants. We replaced seven aromatic residues (Y260, Y272, Y285, Y289, W297, Y309 and F329) in the central part of FepA primary structure with alanine, individually and in double combinations, and determined the ability of the mutant proteins to interact with ferric enterobactin and the protein toxins colicins B and D. All the constructs showed normal expression and localization. Among single mutants, Y260A and F329A were most detrimental, reducing the affinity between FepA and ferric enterobactin 100- and 10-fold respectively. Double substitutions involving Y260, Y272 and F329 impaired (100- to 2500-fold) adsorption of the iron chelate more strongly. For Y260A and Y272A, the drop in adsorption affinity caused commensurate decreases in transport efficiency, suggesting that the target residues primarily act in ligand binding. F329A, like R316A, showed greater impairment of transport than binding, intimating mechanistic involvement during ligand internalization. Furthermore, immunochemical studies localized F329 in the FepA ligand binding site. The mutagenesis results suggested the existence of dual ligand binding sites in the FepA vestibule, and measurements of the rate of ferric enterobactin adsorption to fluoresceinated FepA mutant proteins confirmed this conclusion. The initial, outermost site contains aromatic residues and probably functions through hydrophobic interactions, whereas the secondary site exists deeper in the vestibule, contains both charged and aromatic residues and probably acts through hydrophobic and electrostatic bonds.     

Spectroscopic observations of ferric enterobactin transport

We characterized the uptake of ferric enterobactin (FeEnt), the native Escherichia coli ferric siderophore, through its cognate outer membrane receptor protein, FepA, using a site-directed fluorescence methodology. The experiments first defined locations in FepA that were accessible to covalent modification with fluorescein maleimide (FM) in vivo; among 10 sites that we tested by substituting single Cys residues, FM labeled W101C, S271C, F329C, and S397C, and all these exist within surface-exposed loops of the outer membrane protein. FeEnt normally adsorbed to the fluoresceinated S271C and S397C mutant FepA proteins in vivo, which we observed as quenching of fluorescence intensity, but the ferric siderophore did not bind to the FM-modified derivatives of W101C or F329C. These in vivo fluorescence determinations showed, for the first time, consistency with radioisotopic measurements of the affinity of the FeEnt-FepA interaction; K(d) was 0.2 nm by both methods. Analysis of the FepA mutants with AlexaFluor(680), a fluorescein derivative with red-shifted absorption and emission spectra that do not overlap the absorbance spectrum of FeEnt, refuted the possibility that the fluorescence quenching resulted from resonance energy transfer. These and other data instead indicated that the quenching originated from changes in the environment of the fluor as a result of loop conformational changes during ligand binding and transport. We used the fluorescence system to monitor FeEnt uptake by live bacteria and determined its dependence on ligand concentration, temperature, pH, and carbon sources and its susceptibility to inhibition by the metabolic poisons. Unlike cyanocobalamin transport through the outer membrane, FeEnt uptake was sensitive to inhibitors of electron transport and phosphorylation, in addition to its sensitivity to proton motive force depletion.     

Recognition and transport of ferric enterobactin in Escherichia coli.

The specificity of the outer membrane protein receptor for ferric enterobactin transport in Escherichia coli and the mechanism of enterobactin-mediated transport of ferric ions across the outer membrane have been studied. Transport kinetic and inhibition studies with ferric enterobactin and synthetic structural analogs have mapped the parts of the molecule important for receptor binding. The ferric complex of the synthetic structural analog of enterobactin, 1,3,5-N,N',N'-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was transported with the same maximum velocity as was ferric enterobactin. A double-label transport assay with [59Fe, 3H]MECAM showed that the ligand and the metal are transported across the outer membrane at an identical rate. Under the growth conditions used, large fractions of the transported complexes were available for exchange across the outer membrane when a large excess of extracellular complex was added to the cell suspension; at least 60% of the internalized [59Fe]enterobactin exchanged with extracellular [55Fe]enterobactin. Internalized [59Fe, 3H]MECAM was released from the cell as the intact complex when either unlabeled Fe-MECAM or Fe-enterobactin was added extracellularly. The results suggest a mechanism of active transport of unmodified coordination complex across the outer membrane with possible accumulation in the periplasm.     

Recognition of ferric catecholates by FepA

Escherichia coli FepA transports certain catecholate ferric siderophores, but not others, nor any noncatecholate compounds. Direct binding and competition experiments demonstrated that this selectivity originates during the adsorption stage. The synthetic tricatecholate Fe-TRENCAM bound to FepA with 50- to 100-fold-lower affinity than Fe-enterobactin (FeEnt), despite an identical metal center, and Fe-corynebactin only bound at much higher concentrations. Neither Fe-agrobactin nor ferrichrome bound at all, even at concentrations 10(6)-fold above the Kd. Thus, FepA only adsorbs catecholate iron complexes, and it selects FeEnt among even its close homologs. We used alanine scanning mutagenesis to study the contributions of surface aromatic residues to FeEnt recognition. Although not apparent from crystallography, aromatic residues in L3, L5, L7, L8, and L10 affected FepA's interaction with FeEnt. Among 10 substitutions that eliminated aromatic residues, Kd increased as much as 20-fold (Y481A and Y638A) and Km increased as much as 400-fold (Y478), showing the importance of aromaticity around the pore entrance. Although many mutations equally reduced binding and transport, others caused greater deficiencies in the latter. Y638A and Y478A increased Km 10- and 200-fold more, respectively, than Kd. N-domain loop deletions created the same phenotype: Delta60-67 (in NL1) and Delta98-105 (in NL2) increased Kd 10- to 20-fold but raised Km 500- to 700-fold. W101A (in NL2) had little effect on Kd but increased Km 1,000-fold. These data suggested that the primary role of the N terminus is in ligand uptake. Fluorescence and radioisotopic experiments showed biphasic release of FeEnt from FepA. In spectroscopic determinations, k(off1) was 0.03/s and k(off2) was 0.003/s. However, FepAY272AF329A did not manifest the rapid dissociation phase, corroborating the role of aromatic residues in the initial binding of FeEnt. Thus, the beta-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport.     

Purification and crystallization of ferric enterobactin receptor protein, FepA, from the outer membranes of Escherichia coli UT5600/pBB2

The ferric enterobactin receptor protein, FepA, was isolated and purified from the outer membranes of a genetically transformed strain of Escherichia coli (UT5600/pBB2) using anion-exchange chromatography, chromatofocusing and gel filtration. The purified protein was found to crystallize from 25 mM sodium phosphate buffer in the presence of 0.8% beta-D-octylglucoside under a range of conditions. The protein formed mostly small rods and needle-shaped crystals in the hanging drop method.     

Evolution of the ferric enterobactin receptor in gram-negative bacteria.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera: Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia.     

Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12

Synthetic enterobactin and enantioenterobactin (D-seryl enterobactin) have been examined for the ability to transport iron in Escherichia coli. Failure of the unnatural, D-serine-derived material to support growth of E. coli mutants indicates outer membrane receptor specificity for the naturally occurring complex having an L-seryl backbone and the delta-cis configuration of the Fe(III).catecholate center. Enantioenterobactin was markedly less effective in protecting cells against colicin B compared to synthetic or natural enterobactin.     

Binding of ferric enterobactin by the Escherichia coli periplasmic protein FepB

The periplasmic protein FepB of Escherichia coli is a component of the ferric enterobactin transport system. We overexpressed and purified the binding protein 23-fold from periplasmic extracts by ammonium sulfate precipitation and chromatographic methods, with a yield of 20%, to a final specific activity of 15,500 pmol of ferric enterobactin bound/mg. Periplasmic fluid from cells overexpressing the binding protein adsorbed catecholate ferric siderophores with high affinity: in a gel filtration chromatography assay the K(d) of the ferric enterobactin-FepB binding reaction was approximately 135 nM. Intrinsic fluorescence measurements of binding by the purified protein, which were more accurate, showed higher affinity for both ferric enterobactin (K(d) = 30 nM) and ferric enantioenterobactin (K(d) = 15 nM), the left-handed stereoisomer of the natural E. coli siderophore. Purified FepB also adsorbed the apo-siderophore, enterobactin, with comparable affinity (K(d) = 60 nM) but did not bind ferric agrobactin. Polyclonal rabbit antisera and mouse monoclonal antibodies raised against nearly homogeneous preparations of FepB specifically recognized it in solid-phase immunoassays. These sera enabled the measurement of the FepB concentration in vivo when expressed from the chromosome (4,000 copies/cell) or from multicopy plasmids (>100,000 copies/cell). Overexpression of the binding protein did not enhance the overall affinity or rate of ferric enterobactin transport, supporting the conclusion that the rate-limiting step of ferric siderophore uptake through the cell envelope is passage through the outer membrane.     

Surface topology of the Escherichia coli K-12 ferric enterobactin receptor.

Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.     

Antigenic and molecular homology of the ferric enterobactin receptor protein of Escherichia coli

The ferric enterobactin receptor protein (81 kDal) of Escherichia coli O111 was purified by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and used to raise polyclonal antiserum in rabbits. This antiserum was used in conjunction with the immunoblot technique to examine the degree of antigenic homology of the ferric enterobactin receptor protein among 17 pathogenic and laboratory strains of E. coli. Both the molecular weight and the antigenic properties of the enterobactin receptor were highly conserved. However, the laboratory strain C and a pathogenic enteroinvasive strain, E. coli O164, were unusual in not producing the 81 kDal protein. The antiserum also recognized an 81 kDal protein from iron-restricted Salmonella typhimurium and an 83 kDal protein from iron-restricted Klebsiella pneumoniae.     

Overexpression and purification of ferric enterobactin esterase from Escherichia coli. Demonstration of enzymatic hydrolysis of enterobactin and its iron complex.

The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein. The ferric enterobactin esterase (Fes) enzyme was purified as a 43-kDa monomer by gel filtration chromatography. Purified Fes preparations were examined for esterase activity on enterobactin and its metal complexes and for iron reduction from ferric complexes of enterobactin and 1,3,5-tris(N,N',N"-2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM), a structural analog lacking ester linkages. Fes effectively catalyzed the hydrolysis of both enterobactin and its ferric complex, exhibiting a 4-fold greater activity on the free ligand. It also cleaved the aluminum (III) complex at a rate similar to the ferric complex, suggesting that ester hydrolysis of the ligand backbone is independent of any reductive process associated with the bound metal. Ferrous iron was released from the enterobactin complex at a rate similar to ligand cleavage indicating that hydrolysis and iron reduction are tightly associated. However, no detectable release of ferrous iron from the MECAM complex implies that, with these in vitro preparations, metal reduction depends upon, and is subsequent to, the esterase activity of Fes. These observations are discussed in relation to studies which show that such enterobactin analogs can supply growth-promoting iron concentrations to E. coli.     

Modification of a ferric enterobactin receptor protein from the outer membrane of Escherichia coli.

Modification of a ferric enterobactin receptor protein of Escherichia coli was observed upon incubation of either whole membranes or Triton X-100 solubilized outer membrane at 37°C. The modification was characterized by a change in mobility of the receptor band on SDS polyacrylamide gel electrophoresis and by a decreased binding capacity for ferric enterobactin. The rate of modification was affected by temperature and trypsin inhibitor, benzamidine. Ferric enterobactin inhibited the reaction in whole membrane. The modification affected the limited chymotrypsin digestion pattern of the receptor. The activity may represent a specific modification of the receptor, one possibly mediated by a membran-associated enzyme.     

PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa.

PfeR (Regulator) and PfeS (Sensor), members of the superfamily of so-called two-component regulatory protein pairs, are required for the enterobactin-inducible production of the ferric enterobactin receptor (PfeA) in Pseudomonas aeruginosa. A pfeR knockout mutant failed to demonstrate enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that PfeR acts at the level of pfeA gene expression. Consistent with this, PfeR overexpressed in P. aeruginosa bound, in bandshift assays, the promoter region of pfeA. Such binding was enhanced when PfeR-containing extracts were prepared from cells cultured in the presence of enterobactin, consistent with a model of PfeR as an enterobactin-responsive activator of pfeA expression. A region showing homology to the consensus binding sequence for the global iron repressor Fur was identified upstream of pfeR, suggesting that the pfeRS operon is iron regulated. As expected, expression of a pfeR-lacZ fusion in P. aeruginosa was increased under conditions of iron limitation. Enterobactin failed, however, to provide any enhancement of pfeR-lacZ expression under iron-limiting conditions, indicating that PfeR does not positively regulate pfeRS expression. A pfeA knockout mutant demonstrated enterobactin-inducible expression of a pfeA-lacZ fusion, indicating that the receptor is not required for the enterobactin inducibility of pfeA gene expression. Such mutants show growth, albeit reduced, in enterobactin-supplemented iron-limiting minimal medium, indicating that a second route of uptake across the outer membrane exists for ferric enterobactin in P. aeruginosa and may be important for the initial induction of pfeA in response to enterobactin.