Abstract
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(β) 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.
Highlights
Iron is essential for growth of most bacteria
Preliminary experiments showed that most of the corynebactin detected by the Chrome Azurol S (CAS) assay was present in the 6% ammonium acetate eluate and that an additional step-wise elution with 8.5% ammonium acetate yielded little additional siderophore
Our findings show that corynebactin, after purification to apparent homogeneity from low-iron culture supernatants of C. diphtheriae C7(b) DdtxR, exhibits the biological activities previously attributed to C. diphtheriae siderophore based on studies with low-iron culture supernatants or partially purified preparations of siderophore
Summary
Iron is essential for growth of most bacteria. Iron is plentiful in animals and humans, most of it is complexed with ironbinding proteins such as transferrin, lactoferrin, or ferritin, or incorporated into compounds such as heme, or hemoglobin. To overcome the limited bioavailability of iron from such sources, most pathogenic bacteria, including Corynebacterium diphtheriae, use energy-dependent iron acquisition systems to assimilate iron from their hosts [1,2,3,4]. One class of iron acquisition systems uses siderophores, low-molecular-weight iron-chelating compounds that are secreted by bacteria under low-iron growth conditions, to scavenge host iron. Siderophores can be classified by their metal binding groups as catecholates, hydroxamates, or complexones Ferrichrome is typical of hydroxamate siderophores, which contain either N6-acyl-N6-
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