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PubMed1996

Role of enterobactin and intracellular iron in cell lethality during near-UV irradiation in Escherichia coli.

In Escherichia coli, fur mutants that constitutively express their native iron chelating agent, enterobactin, are significantly more sensitive to near-UV radiation (NUV) than wild type, An entA mutant, which is incapable of synthesizing enterobactin, is equal to wild type in resistance to NUV irradiation. However, the addition of Fe+3 enterobactin but not AI+3 enterobactin to entA cell suspensions just prior to irradiation results in an increased sensitivity to NUV irradiation. A fes mutant, which is unable to reduce and release iron from enterobactin, is significantly more sensitive to NUV irradiation than wild type. The addition of nontoxic levels of H2O2 (5 microM) just prior to irradiation significantly increases sensitivity of both fur and fes mutants. These results suggest that one mechanism by which NUV irradiation leads to cell lethality is by creating a transient iron overload, producing very favorable conditions for the production of highly deleterious free radicals through a variety of mechanisms that lead to oxidative stress and DNA damage including lethal and mutagenic lesions. These results are consistent with the hypothesis that enterobactin is an endogenous chromophore for NUV and contributes to cell lethality via the destruction of its ligand, releasing Fe+2 into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals and other toxic oxygen species via the Haber-Weiss reaction.

Enterobactin↗
PubMed1988

Genetics and regulation of enterobactin genes in Shigella flexneri.

Although Shigella flexneri possesses the genes for two siderophore systems, enterobactin and aerobactin, the enterobactin system is only rarely utilized. To investigate the regulation of enterobactin expression in S. flexneri, all of the genes specifically required for synthesis and transport of enterobactin were cloned from both an expressing (Ent+) and a nonexpressing (Ent-) strain. Notable differences between the cloned genes included endonuclease restriction site changes and the presence of an IS1 element in the Ent- DNA. Southern hybridization revealed that this IS1 element, present at the 3' end of the entF gene, is conserved at this location in different strains and serotypes of Ent- S. flexneri. The Ent- cloned genes were tested for their ability to complement the defect in 11 different Escherichia coli enterobactin mutants. The Ent- genes fully complemented nine mutants but failed to complement the entF mutant AN117 and only partially complemented the entE mutant AN93. Whole-cell RNA isolated from E. coli and the Shigella strains was hybridized to 32P-labeled DNA containing the entB gene or a fragment carrying a portion of the entF gene. E. coli and the Ent+ Shigella strains exhibited derepression of transcription of these genes in low-iron media. Transcription in the Ent- strain remained repressed regardless of iron concentration. Expression of the entB and entF genes was also examined in an Ent- Shigella fur mutant. Expression of entF was only partially derepressed and entB remained fully repressed at all iron concentrations, suggesting that factors other than Fur are responsible for the repression of these enterobactin genes in the Ent- Shigella strains.

Cloning, Molecular↗
PubMed1993

Cloning and characterization of the ferric enterobactin receptor gene (pfeA) of Pseudomonas aeruginosa.

Pseudomonas aeruginosa K407, a mutant lacking a high-affinity 80,000-molecular-weight ferric enterobactin receptor protein (80K protein), exhibited poor growth (small colonies) on iron-deficient succinate minimal medium containing ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and enterobactin. The gene encoding the ferric enterobactin receptor was cloned by complementation of this growth defect. The complementing DNA was subsequently localized to a 7.1-kilobase-pair (kb) SstI-HindIII fragment which was able to restore synthesis of the 80K protein in strain K407 and also to direct the synthesis of high levels of a protein of the same molecular weight in the outer membranes of Escherichia coli fepA strains MT912 and IR20. Moreover, the fragment complemented the fepA mutation in MT912, restoring both growth in EDDHA-containing medium and enterobactin-dependent uptake of 55Fe3+. Expression of the P. aeruginosa receptor in E. coli IR20 was shown to be regulated by both iron and enterobactin. The complementing DNA was further localized to a 5.3-kb SphI-SstI fragment which was then subjected to deletion analysis to obtain the smallest fragment capable of directing the synthesis of the 80K protein in the outer membrane of strain K407. A 3.2-kb DNA fragment that restored production of the receptor in strain K407 was subsequently isolated. The fragment also directed synthesis of the protein in E. coli MT912 but at levels much lower than those previously observed. Nucleotide sequencing of the fragment revealed an open reading frame (designated pfeA for Pseudomonas ferric enterobactin) of 2,241 bp capable of encoding a 746-amino-acid protein with a molecular weight of 80,967. The PfeA protein showed more than 60% homology to the E. coli FepA protein. Consistent with this, the two proteins showed significant immunological cross-reactivity.

Amino Acid Sequence↗
PubMed2000

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.

Animals↗
PubMed1970

Iron transport in Salmonella typhimurium: mutants blocked in the biosynthesis of enterobactin.

A number of mutants of Salmonella typhimurium were isolated which are blocked in the biosynthesis of enterobactin, an iron chelator that is secreted by the wild-type bacteria when they are grown on low iron media. One class of these enb mutants accumulates the enterobactin precursor 2,3-dihydroxybenzoic acid, and another class does not accumulate any detectable catechol precursor. The enb mutants grow very well on a glucose-mineral salts medium. Addition of citrate, itself an iron chelator, to the medium drastically inhibits growth unless the medium is supplemented with enterobactin or iron salts. Citrate inhibits iron uptake from the medium by enb mutants; enterobactin counteracts this inhibition and also, by itself, increases iron uptake. Thus, the apparent function of enterobactin is to promote the absorption of iron from the medium by the bacteria. Transduction experiments showed that the genes for enterobactin biosynthesis are closely linked on the S. typhimurium chromosome. It is suggested that they form an operon which is repressed by the presence of iron. S. typhimurium can utilize the iron chelate ferrichrome. (Deferriferrichrome is a cyclic hexapeptide that is produced by some fungi but not by S. typhimurium.) The enb mutants use ferrichrome as an effective growth factor.

Benzoates↗
PubMed2000

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae.

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3delta background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using 55Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.

Bacterial Outer Membrane Proteins↗
PubMed2004

The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica.

Salmochelins represent novel carbohydrate containing catecholate siderophores, which are excreted by Salmonella enterica and uropathogenic Escherichia coli strains under low-iron stress. While previous analytical data showed salmochelins to contain 2,3-dihydroxybenzoyl-L-serine and glucose, the molecular structure remained elusive. Structure elucidation with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry (ESI-FTICR-MS), GC-MS and 2D-NMR now revealed that salmochelins are enterobactin-related compounds, which are beta-C-glucosylated at the 5-position of a 2,3-dihydroxybenzoyl residue. The key compound salmochelin S4 is a twofold beta-C-glucosylated enterobactin analogue. Comparison of partial structures of salmochelin with a C-glycosylated compound previously characterized by another group strongly suggest that salmochelins represent the long sought compounds termed Salmonella resistance factors (SRF) or pacifarins. Transformation of iro-genes into enterobactin-producing E. coli K12 confers the ability to produce salmochelins. A detailed analysis proved iroB to be the sole gene with glycosyltransferase activity necessary for salmochelin production. Salmochelins compared to enterobactin are the better siderophores in the presence of serum albumin. This may indicate for salmochelins a considerably more important role for pathogenic processes in certain Escherichia coli and Salmonella infections than formerly assigned to enterobactin. This conclusion is supported by the location of the iro genes on pathogenicity islands of uropathogenic E. coli strains.

Chromatography, High Pressure Liquid↗
PubMed1995

A Bordetella pertussis fepA homologue required for utilization of exogenous ferric enterobactin.

The bfeA (Bordetella ferric enterobactin) receptor gene was cloned from a Bordetella pertussis chromosomal library by using a screen in Escherichia coli to detect iron-repressed genes encoding exported proteins translationally fused to the E. coli phoA gene. The bfeA gene encoded a protein with a molecular mass of approximately 80 kDa and about 50% amino acid sequence identity to both the fepA- and pfeA-encoded enterobactin receptors of E. coli and Pseudomonas aeruginosa, respectively. Enterobactin prepared from iron-starved E. coli cultures supported growth of B. pertussis and Bordetella bronchiseptica in the presence of the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid) (EDDA). Expression of the bfeA gene was induced by low iron availability, and iron-regulated expression appeared to be dependent upon the presence of the sequence contained within 370 bp upstream of the bfeA structural gene. An internal fragment of the bfeA structural gene and flanking regions were shown by Southern analysis to be highly conserved among Bordetella species. Insertional inactivation of bfeA in both B. pertussis and B. bronchiseptica greatly impaired their ability to grow in the presence of enterobactin and EDDA. These findings suggest that enterobactin produced by other respiratory flora could aid in the colonization of the respiratory tract by Bordetella species.

Amino Acid Sequence↗
PubMed2002

The growth response of Escherichia coli to neurotransmitters and related catecholamine drugs requires a functional enterobactin biosynthesis and uptake system.

The neurotransmitter norepinephrine (NE) stimulates the growth of low inocula of Escherichia coli in a minimal medium (SAPI) supplemented with serum (SAPI+serum) and induces the production of an "autoinducer" (AI) which, in turn, promotes E. coli growth in the absence of NE. Given the importance of NE, epinephrine, and their corresponding adrenergic agonists and antagonists in clinical medicine, we sought to investigate the molecular basis for these observations. Using a variety of NE precursors, metabolites, and therapeutic agents, we demonstrated that their ability to stimulate E. coli growth in SAPI+serum is dependent on the presence of a catechol (1,2-dihydroxybenzene) moiety with maximal activity requiring a two-carbon substituent incorporating a terminal primary amine. Serum contains the iron-binding glycoprotein, transferrin, and when SAPI+serum was supplemented with sufficient Fe(3+) to saturate transferrin, growth inhibition was relieved. Other metal cations, including Mg(2+), Ca(2+), and Zn(2+), had no effect. These data suggested that the stimulation of E. coli growth by NE in SAPI+serum may involve the catecholate siderophore, enterobactin, a cyclic triester of 2,3-dihydroxybenzoylserine. Consistent with this hypothesis, E. coli strains with mutations in ferrienterobactin transport (fepA or tonB) or enterobactin biosynthesis (entA) did not respond to NE. Furthermore, NE induced expression of the ferrienterobactin receptor, FepA, during growth in SAPI+serum. The enterobactin degradation product, 2,3-dihydroxybenzoylserine (DBS) was as effective as NE in stimulating the growth of E. coli and mutations in fepA or tonB abolished the DBS-dependent growth stimulation. In contrast to NE, however, DBS stimulated the growth of the entA mutant. Moreover, after growth in an iron-limited M9 medium in the absence of NE, ethyl acetate extracts of the E. coli entA(+) parent but not of the entA mutant contained AI, i.e., stimulated the growth of E. coli in SAPI+serum. Taken together, these data show that when low numbers of E. coli are inoculated into SAPI+serum, NE, DBS, and related catecholamines induce the enterobactin iron uptake system. This, in turn, facilitates iron sequestration from transferrin and indicates that the AI present in NE-conditioned SAPI+serum medium is enterobactin and its DBS breakdown products.

Catecholamines↗
PubMed2003

Role of receptor proteins for enterobactin and 2,3-dihydroxybenzoylserine in virulence of Salmonella enterica.

Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.

Animals↗
PubMed1991

Genetic analysis of the enterobactin gene cluster in Shigella flexneri.

The genes for transport and synthesis of the phenolate siderophore enterobactin are present on the chromosomes of both Ent+ and Ent- clinical isolates of Shigella flexneri. To determine why Ent- S. flexneri isolates fail to express a functional enterobactin system, the structure and expression of enterobactin genes were examined. Several alterations may be responsible for the inability of S. flexneri to express enterobactin. (i) The mRNA levels produced from the entC and fepB genes were not derepressed in low-iron media. (ii) DNA sequence analysis of the entC-fepB intergenic region revealed an 83-bp noncontiguous deletion in the putative fepB leader sequence. The deleted sequences are in a region which would be capable of forming extensive stem-and-loop structures. (iii) An amber codon in the 5' portion of the entC gene was also detected. (iv) An IS1 element, previously mapped to the Ent- S. flexneri enterobactin gene cluster, was found to lie within a potential transcriptional termination sequence in the entF-fepE intergenic region. (v) A mutation responsible for the inactivation of the entF gene was mapped to the entF coding region by using entF hybrid gene fusions. (vi) A comparison of outer membrane profiles from an E. coli strain harboring the cloned fepA gene from either an Ent+ or Ent- Shigella isolate revealed that the Ent- FepA protein is present in the outer membrane but at greatly reduced levels than that of the Ent+ FepA protein. This observation, along with additional studies, suggests that the Ent- FepA may be defective in translation and/or translocation.

Base Sequence↗
PubMed2001

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. Mössbauer 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.

Amino Acid Sequence↗
PubMed1993

Expression of the ferric enterobactin receptor (PfeA) of Pseudomonas aeruginosa: involvement of a two-component regulatory system.

Expression of the ferric enterobactin receptor in Pseudomonas aeruginosa is inducible by enterobactin and requires sequences upstream of the structural gene (pfeA). Nucleotide sequencing of a 2.5 kilobase pair (kb) region of DNA immediately upstream of pfeA revealed two open reading frames (ORFs), pfeR and pfeS, which appeared to comprise an operon. The predicted products of pfeR and pfeS (molecular weight 26,796 and 50,597, respectively) exhibited a high degree of homology to response-regulator and sensor components, respectively, of the superfamily of prokaryotic environmentally responsive protein pairs. Consistent with an apparent role in regulating expression of pfeA in response to enterobactin, introduction of pfeR/pfeS into P. aeruginosa on a high-copy-number vector enhanced enterobactin-dependent expression of pfeA. Furthermore, a pfeR mutant obtained by in vitro mutagenesis and gene replacement failed to express PfeA despite the presence of enterobactin in the culture medium. Analysis of the hydropathy profiles of PfeR and PfeS supported a cytoplasmic location for PfeR and a cytoplasmic membrane location for PfeS.

Amino Acid Sequence↗
PubMed2003

Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli.

Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.

Enterobactin↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2000

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.

Alanine↗
PubMed1997

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.

Amino Acid Sequence↗
PubMed2004

Involvement of enterobactin synthesis pathway in production of microcin H47.

Microcin H47 (MccH47) is a gene-encoded peptide antibiotic produced by an Escherichia coli clinical isolate which is active on strains of gram-negative bacteria. Its uptake by E. coli K-12-susceptible cells depends on the presence of any of the outer membrane proteins Cir, Fiu, and FepA, the three catechol receptors of this organism. The nucleotide sequence of a portion of the MccH47 genetic system that had not yet been studied was elucidated. Five open reading frames were identified, three of which corresponded to genes encoding functions related to catechol-type siderophores: mchA and mchS1 are iroB and iroD homologues, respectively, and mchS4 was found to promote the production of the catecholate siderophore enterobactin. The possible relationship between enterobactin synthesis and MccH47 production was studied. Enterobactin-deficient strains failed to produce MccH47 when transformed with the antibiotic genetic determinants and upon introduction of the ent genetic cluster, the production of both the siderophore and MccH47 was restored. Further studies demonstrated that at least the enterobactin nonribosomal peptide synthase EntF is necessary for MccH47 synthesis. The relationship found between MccH47 and catecholate siderophore production is discussed, and a model outlining MccH47 synthesis is proposed.

Anti-Bacterial Agents↗