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.     

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.     

Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli.

The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains.     

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.     

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.     

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.     

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.     

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.     

Absence of ferric enterobactin receptor modification activity in mutants of Escherichia coli K-12 lacking protein a.

The modification activity for the ferric enterobactin receptor in the Triton X-100 solubilized outer membrane of $\text{Escherichia}\text{coli}$ K-12 was adsorbed to a column of $\text{p}$-aminobenzamidine-//-sepharose and eluted with free benzamidine. Recombination of the dialyzed eluate with the filtrate from the column reinstituted conversion of the receptor from 81K to 81K¹, the latter exhibiting an apparent molecular weight of 74,000 daltons in sodium dodecyl sulfate polyacrylamide gel analysis. The eluate from the $\text{p}$-aminobenzamidine column was shown to contain a component, coincident on gels with both protein and modification activity, which by mutational and other analyses appears to be identical with protein $\text{a}$ of the outer membrane.     

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.     

Docking of the periplasmic FecB binding protein to the FecCD transmembrane proteins in the ferric citrate transport system of Escherichia coli

Citrate-mediated iron transport across the cytoplasmic membrane is catalyzed by an ABC transporter that consists of the periplasmic binding protein FecB, the transmembrane proteins FecC and FecD, and the ATPase FecE. Salt bridges between glutamate residues of the binding protein and arginine residues of the transmembrane proteins are predicted to mediate the positioning of the substrate-loaded binding protein on the transmembrane protein, based on the crystal structures of the ABC transporter for vitamin B(12), consisting of the BtuF binding protein and the BtuCD transmembrane proteins (E. L. Borths et al., Proc. Natl. Acad. Sci. USA 99:16642-16647, 2002). Here, we examined the role of the residues predicted to be involved in salt-bridge formation between FecB and FecCD by substituting these residues with alanine, cysteine, arginine, and glutamate and by analyzing the citrate-mediated iron transport of the mutants. Replacement of E93 in FecB with alanine [FecB(E93A)], cysteine, or arginine nearly abolished citrate-mediated iron transport. Mutation FecB(E222R) nearly eliminated transport, and FecB(E222A) and FecB(E222C) strongly reduced transport. FecD(R54C) and FecD(R51E) abolished transport, whereas other R-to-C mutations in putative interaction sites between FecCD and FecB substantially reduced transport. The introduced cysteine residues in FecB and FecCD also served to examine the formation of disulfide bridges in place of salt bridges between the binding protein and the transmembrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggest cross-linking of FecB(E93C) to FecD(R54C) and FecB(E222C) to FecC(R60C). The data are consistent with the proposal that FecB(E93) is contained in the region that binds to FecD and FecB(E222) in the region that binds to FecC.     

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.     

Interactions between TonB from Escherichia coli and the periplasmic protein FhuD

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

Engineered Escherichia coli silver-binding periplasmic protein that promotes silver tolerance

Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo.     

EntG activity of Escherichia coli enterobactin synthetase.

The last steps in the biosynthesis of the Escherichia coli siderophore enterobactin (Ent) are carried out by Ent synthetase, a multienzyme complex believed to be composed of the entD, -E, -F, and -G products (EntD to -G). However, sequencing data showed that there is no separate entG gene and, unlike EntD to -F, no distinct EntG polypeptide has been identified. In this study, genetic, biochemical, and immunological approaches were used to study the anomalies associated with EntG activity. Two plasmids, pJS43 and pJS100, were isolated that had mutations resulting in truncated EntB proteins; both had the phenotype EntB+ EntG-. PJS43 had a Tn5 inserted 198 bp from the entB termination codon, and pJS100 had the last 25 codons of entB deleted. Plasmids isolated with Tn5 insertions in the 5' half of entB had the phenotype EntB- EntG+. These latter Tn5 mutations were EntB- EntG- when moved to the bacterial chromosome. Polyclonal antiserum was prepared and shown to react only with intact EntB in Western immunoblots. Addition of anti-EntB antiserum to Ent synthetase assays resulted in complete inhibition of enzyme activity, whereas preimmune serum had no effect. Lastly, AN462, the type strain for entG which was derived by Mu insertion and which has the phenotype EntB-G-A-, was characterized. Southern blot data showed a Mu insertion, presumably with polar effects, in the vicinity of the 5' end of entB. In summary, EntG activity was found to be encoded by the entB 3' terminus. The evidence, while not rigorously eliminating the possibility that a separate EntG polypeptide exists, strongly supports the idea that EntB is a bifunctional protein.     

Release of the periplasmic penicillinases from Escherichia coli by toluene

Summary More than 80% of the chromosomally and R factor mediated, periplasmic penicillinases of Escherichia coli K-12, strains G11a1 and D1-R1, are released into the medium upon treatment with the membrane damaging agents toluene (50 l/ml) or polymyxin B (10 g/ml) within 10 min of incubation at 37°C. A concomitant release of protein (27 to 34%), but not of the cytoplasmic -galactosidase (<5%) occurs.     

Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi. Functional study of a homolog of the Escherichia coli ferric enterobactin esterase.

Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor. It can also use enterobactin as a xenosiderophore. We began this work by sequencing the 5'-upstream region of the fct-cbsCEBA operon, which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety. We identified a new iron-regulated gene (cbsH) transcribed divergently relative to the fct gene, the translated sequence of which is 45.6% identical to that of Escherichia coli ferric enterobactin esterase. Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E. coli enterobactin synthase gene. These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E. chrysanthemi. The cbsH-negative mutants were less able to utilize ferric chrysobactin, and this effect was not caused by a defect in transport per se. In a nonpolar cbsH-negative mutant, chrysobactin accumulated intracellularly. These defects were rescued by the cbsH gene supplied on a plasmid. The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family. Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain that was inhibited by diisopropyl fluorophosphate. These data are consistent with the fact that chrysobactin is a d-lysyl-l-serine derivative. M?ssbauer spectroscopy of whole cells at various states of (57)Fe-labeled chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo. The CbsH protein may therefore be regarded as a peptidase that prevents the bacterial cells from being intracellularly iron-depleted by chrysobactin.     

A novel sec-independent periplasmic protein translocation pathway in Escherichia coli.

The trimethylamine N-oxide (TMAO) reductase of Escherichia coli is a soluble periplasmic molybdoenzyme. The precursor of this enzyme possesses a cleavable N-terminal signal sequence which contains a twin-arginine motif. By using various moa, mob and mod mutants defective in different steps of molybdocofactor biosynthesis, we demonstrate that acquisition of the molybdocofactor in the cytoplasm is a prerequisite for the translocation of the TMAO reductase. The activation and translocation of the TMAO reductase precursor are post-translational processes, and activation is dissociable from translocation. The export of the TMAO reductase is driven mainly by the proton motive force, whereas sodium azide exhibits a limited effect on the export. The most intriguing observation is that translocation of the TMAO reductase across the cytoplasmic membrane is independent of the SecY, SecE, SecA and SecB proteins. Depletion of Ffh, a core component of the signal recognition particle of E. coli, appears to have a slight effect on the export of the TMAO reductase. These results strongly suggest that the translocation of the molybdoenzyme TMAO reductase into the periplasm uses a mechanism fundamentally different from general protein translocation.