Purification and structural characterization of siderophore (corynebactin) from Corynebacterium diphtheriae.

During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(ß) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.

Enhanced iron availability by protein glycation may explain higher infection rates in diabetics.

Serum proteins exist in a state of higher glycation among individuals with poor glycemic control, notably diabetics. These non-enzymatic modifications via the Maillard reaction have far reaching effects on metabolism and regulation, and may be responsible for increased infection rates within this population. Here we explore the effects of glycation on iron metabolism and innate immunity by investigating the interaction between siderophores and bovine serum albumin (BSA). Using a quartz crystal microbalance with dissipation monitoring to quantify association rates, glycated BSA exhibited a significantly reduced affinity for apo and holo enterobactin compared to a non-glycated BSA standard. Bacterial growth assays in the presence of BSA and under iron-limited conditions indicated the growth rate of enterobactin-producing E. coli increased significantly when the BSA was in a glycated form. The results, in addition to data in the literature, support the hypothesis that glycation of serum proteins may effectively increase the available free iron pool for bacteria in blood serum and weaken our innate immunity. This phenomenon may be partially responsible for higher infection rates in some diabetics, especially those with poor glycemic control.

Insight into siderophore-carrying peptide biosynthesis: enterobactin is a precursor for microcin E492 posttranslational modification.

Microcin E492-producing bacteria secrete both unmodified and posttranslationally modified microcins. The modification consists of a C-glucosylated linear trimer of N-(2,3-dihydroxybenzoyl)-l-serine, a catecholate siderophore related to salmochelins and enterobactin. We show here that repression of enterobactin biosynthesis inhibits the acquisition of microcin E492 posttranslational modification, as monitored by high-performance liquid chromatography and mass spectrometry. Furthermore, exogenous enterobactin restored the production of posttranslationally modified microcin in a bacterial strain deficient in enterobactin synthesis. We thus concluded that enterobactin serves as a precursor for the synthesis of the posttranslationally modified microcin and that the unmodified microcin is an incompletely processed form of mature microcin E492. Gene disruption experiments showed that MceC and MceD, two enzymes encoded by the mceABCDEFGHIJ gene cluster, are involved in the synthesis of the microcin E492 posttranslational modification, as followed by mass spectrometry. Genes homologous to iroB and iroD, required for the conversion (linearization and C-glycosylation) of enterobactin into salmochelins, efficiently complemented mceC and mceD, respectively. Based on our results, a model is proposed for the biosynthesis of the mature siderophore-carrying peptide.

Environmental factors influence the production of enterobactin, salmochelin, aerobactin, and yersiniabactin in Escherichia coli strain Nissle 1917.

The probiotic Escherichia coli strain Nissle 1917 produces four siderophores: the catecholates enterobactin and salmochelin, the hydroxamate aerobactin, and the mixed-type siderophore yersiniabactin. We studied the influence of pH, temperature, and carbon source on the production of these four siderophores. Yersiniabactin and salmochelin were maximally produced under neutral to alkaline conditions (pH 7.0 and 7.6, respectively), whereas aerobactin was maximally produced at a more acidic pH (pH 5.6), which agrees with the slightly higher complex stability of hydroxamates at acidic pH values compared to the catecholates. Under nearly all conditions studied, catecholate siderophore production was higher with glycerol than with glucose as the carbon source. Yersiniabactin production was also higher with glycerol as the carbon source at pH 7.0. At 42 degrees C, strain Nissle 1917 grew poorly or not at all because of the iron-limiting conditions. In a competition experiment between wild-type strain Nissle 1917 and a mutant of this strain with a deletion in the yersiniabactin operon, the wild-type overgrew the mutant at pH 7.0 and 7.6 and not at pH 5.6. These results agree with yersiniabactin production being of greater advantage at neutral and slightly alkaline pH values. The production of four siderophores may help the probiotic E. coli Nissle 1917 to compete with other E. coli strains in the colon. The probiotic strain Nissle 1917 used in our experiments has many characteristics in common with uropathogenic E. coli and other pathogenic strains which also secrete these siderophores. Uropathogenic E. coli strains may need the multitude of siderophores to adapt to the pH of urine, which varies between pH 4.6 and 8.0.

HPLC separation of enterobactin and linear 2,3-dihydroxybenzoylserine derivatives: a study on mutants of Escherichia coli defective in regulation (fur), esterase (fes) and transport (fepA).

Reversed-phase HPLC separation of enterobactin and its 2,3-dihydroxybenzoylserine derivatives was used for a comparative analysis of mutants of Escherichia coli, defective in the regulation of enterobactin biosynthesis (fur), enterobactin transport (fepA) and enterobactin esterase (fes). A complete separation of all 2,3-dihydroxybenzoylserine compounds was achieved: the monomer (DHBS), the linear dimer (DHBS)2 and trimer (DHBS)3, the cyclic trimer, enterobactin, as well as 2,3-dihydroxybenzoic acid. The production of all these compounds was followed after ethylacetate extraction from acidified culture fluids. Enterobactin was found to be the predominant product in all mutant strains. The mutant strains behaved differently with regard to the breakdown products. All degradation products, such as DHBS, (DHBS)2 and (DHBS)3, were detected in the overproducing fur mutant where both transport and esterase are still functioning, while only the monomer, DHBS, was detected in the fepA mutant and no degradation was found in the esterase-deficient fes mutant. From the pattern of breakdown products it may be inferred that the esterase acts in two different ways, depending on whether transport is functioning or not. Thus, esterolytic cleavage of ferric enterobactin after entering the cells results in a mixture of all three hydrolysis products, i.e. DHBS, (DHBS)2 and (DHBS)3, while cleavage of iron-free enterobactin subsequent to its biosynthesis yields only the monomer. Thus, the results of quantitative HPLC analysis of enterobactin and its breakdown products show that different enterobactin esterase products arise, depending on whether iron is bound to enterobactin or not.

Preparation and use of double-labelled enterobactin.

Double-labelled 3H/14C-enterobactin was isolated from bacterial cultures, and evaluated as a potential tool for studying the mammalian metabolism of this iron chelator. Microbial yields were low, but adequate, with a final 3H/14C ratio of 2.95 to 1. Studies conducted with mice indicated that considerable metabolism and rapid elimination of an intraperitoneally injected sample had occurred in 24 hours.

Nucleotide sequence of a cluster of Escherichia coli enterobactin biosynthesis genes: identification of entA and purification of its product 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase.

The nucleotide sequence of a region of the Escherichia coli chromosome encoding part of a cluster of genes involved in the biosynthesis of the iron chelator enterobactin has been determined. Four closely linked open reading frames, corresponding to the coding regions of entE (carboxy-terminal 144 amino acids), entB (32,554 daltons), entA (26,249 daltons), and an unidentified gene (P15) encoding a 14,970-dalton protein, were found. The lack of intergenic sequences and promoterlike elements suggests that these genes form part of the same transcription unit. We report the purification to homogeneity of the entA product, 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase. It is an octamer of native molecular weight 210,000; the amino-terminal amino acid sequence confirmed the entA coding region. No isochorismate synthase activity was associated with this polypeptide. This finding leads to the conclusion that the recent suggestion (M. S. Nahlik, T. P. Fleming, and M. A. McIntosh, J. Bacteriol. 169:4163-4170, 1987) that 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase and isochorismate synthase activities reside on a single 26,000-dalton bifunctional enzyme is incorrect, even though the entA and entC mutations map to the same genetic locus.

[Iron chelation. Biological significance and medical application]. / Eisenchelierung. Biologische Bedeutung und medizinische Anwendungen.

Iron, an essential element for all aerobic organisms, exists in a very insoluble form under physiological conditions. Therefore, most microorganisms secrete iron chelating compounds called siderophores which are able to sequester ferric ions from the environment. A vast number of such compounds has been isolated from cultures of microorganisms and tested for enhancement of iron excretion in experimental animals. Only one compound, deferrioxamine B, has been shown to be clinically effective and well tolerated in humans suffering from chronic iron overload. However, this drug can only be administered successfully by injection or slow infusion. In spite of considerable research it has not been possible to overcome this drawback by developing suitable formulations or derivatives which are orally active. Deferri-ferrithiocin, a novel type of siderophore, has recently been isolated from a streptomyces culture. This substance is well absorbed orally and has been shown to enhance the excretion of ferric ion in iron loaded rats. Further investigations are now necessary to establish acute toxicity levels and longterm tolerability before efficacy tests in man can be planned. Other recent developments in the field of metal chelation include experimental studies using deferrioxamine for the treatment of conditions resulting from toxic levels of iron or aluminium in chronically dialyzed patients. In addition, attempts are being made to administer chelation therapy in the treatment of various infections and chronic inflammation, as well as other conditions linked with disorders of iron metabolism.

Isolation of enterochelin from the peritoneal washings of guinea pigs lethally infected with Escherichia coli.

Escherichia coli secretes enterochelin while infecting normal guinea pigs. Since production of enterochelin is a well-characterized response to an iron-restricted environment, this work establishes that host iron-binding proteins do indeed influence the metabolism of the invading organism.

Siderophore production by Vibrio cholerae.

Vibrio cholerae produces a phenolate-type siderophore that stimulates growth of the organism in low-iron medium. This compound is similar, but not identical, to enterochelin, the siderophore produced by Salmonella and Escherichia coli.

Preparation of enterochelin from Escherichia coli.

A convenient method has been developed for the preparation of enterochelin, the natural iron carrier produced by Escherichia coli. The method employs a mutant strain which is unable to transport the ferric-enterochelin complex into the cell and which excretes large quantities of enterochelin into the culture medium. The addition of excess iron to the medium allows the enterochelin to accumulate as the ferric-enterochelin complex which is purified by ion-exchange chromatography and then dissociated and the free enterochelin further purified by differential extraction and crystallization. The enterochelin is isolated in good yield and appears to be of high purity as judged by a number of criteria.