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.