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.     

The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases

Myxothiazol is synthesized by the myxobacterium Stigmatella aurantiaca DW4/3-1 via a combined polyketide synthase/polypeptide synthetase. The biosynthesis of this secondary metabolite is also dependent on the gene product of mtaA. The deduced amino acid sequence of mtaA shows similarity to 4'-phosphopantetheinyl transferases (4'-PP transferase). This points to an enzyme activity that converts inactive forms of the acyl carrier protein domains of polyketide synthetases (PKSs) and/or the peptidyl carrier protein domains of nonribosomal polypeptide synthetases (NRPSs) of the myxothiazol synthetase complex to their corresponding holo-forms. Heterologous co-expression of MtaA with an acyl carrier protein domain of the myxothiazol synthetase was performed in Escherichia coli. The proposed function as a 4'-PP transferase was confirmed and emphasizes the significance of MtaA for the formation of a catalytically active myxothiazol synthetase complex. Additionally, it is shown that MtaA has a relaxed substrate specificity: it processes an aryl carrier protein domain derived from the enterobactin synthetase of E. coli (ArCP) as well as a peptidyl carrier protein domain from a polypeptide synthetase of yet unknown function from Sorangium cellulosum. Therefore, MtaA should be a useful tool for activating heterologously expressed PKS and NRPS systems.     

Phenylalanine- and tyrosine-dependent production of enterobactin in Escherichia coli

Abstract Under low-iron conditions, Escherichia coli synthesizes the siderophore enterobactin. When compared to wild-type cells grown in iron sufficient medium, cells grown under iron limitation, in the absence of tyrosine and phenylalanine or the presence of both, increased catechol production (a measure of enterobactin and its degradation product 2,3-dihydroxybenzoic acid) 5- to 9-fold while cells supplemented with tyrosine alone produced a 10- to 20-fold increase. Mutations in fur , tyrA , pheA , or pheU generally resulted in increased enterobactin production, while a tyrR mutant was unaffected by combinations of tyrosine and phenylalanine.     

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.     

Ferric enterobactin binding and utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.     

Characterization of Cereulide Synthetase,a Toxin-Producing Macromolecular Machine

Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.     

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.     

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.     

Ferric enterobactin transport system in Escherichia coli K-12. Extraction, assay, and specificity of the outer membrane receptor.

An outer membrane preparation from cells of Escherichia coli K-12 grown in low iron medium was found to retain ferric enterobactin binding activity following solubilization in a Tris-HCl, Na2EDTA buffer containing Triton X-100. Activity was measured by means of a DEAE-cellulose column which separated free and receptor bound ferric enterobactin. The binding activity was greatly reduced in preparations obtained from cells grown in iron rich media or from cells of a colicin B resistant mutant grown in either high or low iron media. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis enabled correlation of this lack of activity to a single band missing in the outer membrane profile of the colicin B mutant. Evidence was obtained for in vitro competition between ferric enterobactin and colicin B for the extracted receptor. The binding specificity of the extracted receptor was examined by competition between ferric enterobactin and several iron chelates including a carbocyclic analogue of enterobactin, cis-1,5,9-tris(2,3-dihydroxybenzamido)cyclododecane. The ferric form of the latter compound supported growth of siderophore auxotrophs, apparently without hydrolysis to dihydroxybenzoic acid and resynthesis into enterobactin. These data may require revision of the accepted mechanism of enterobactin mediated iron utilization.     

Biosynthesis of the Escherichia coli siderophore enterobactin: sequence of the entF gene, expression and purification of EntF, and analysis of covalent phosphopantetheine.

The sequence of the entF gene which codes for the serine activating enzyme in enterobactin biosynthesis is reported. The gene encodes a protein with a calculated molecular weight of 142,006 and shares homologies with the small subunits of gramicidin S synthetase and tyrocidine synthetase. We have subcloned and overexpressed entF in a multicopy plasmid and attempted to demonstrate L-serine-dependent ATP-[32P]PPi exchange activity and its participation in enterobactin biosynthesis, but the overexpressed enzyme appears to be essentially inactive in crude extract. A partial purification of active EntF from wild-type Escherichia coli, however, has confirmed the expected activities of EntF. In a search for possible causes for the low level of activity of the overexpressed enzyme, we have discovered that EntF contains a covalently bound phosphopantetheine cofactor.     

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.     

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.     

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.     

Coordination of calcium by iron enterobactin

Iron(III) enterobactin has been shown to coordinate alkaline earth metals. By studying a range of enterobactin analogues it has been possible to propose a structure for this complex type. These observations are discussed.     

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.