Identification of a DtxR-Regulated Operon That Is Essential for Siderophore-Dependent Iron Uptake inCorynebacterium diphtheriae

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Abstract
The diphtheria toxin repressor (DtxR) uses Fe2+as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) inCorynebacterium diphtheriae. A new IRP, designated IRP6, was cloned fromC. diphtheriaeby a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in anEscherichia colisystem was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-typeC. diphtheriaeC7(β), but they were expressed constitutively in C7(β) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-typeirp6operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-typeirp1operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles ofirp6A,irp6C, andirp6Bwere responsible for the phenotypes of HC1, HC4, and HC5, respectively. Theirp6Aallele in HC1 and theirp6Ballele in HC5 encoded single amino acid substitutions in their predicted protein products, and theirp6Callele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation indtxRin addition to a missense mutation in itsirp6Ballele. These findings demonstrated that theirp6operon inC. diphtheriaeencodes a putative ABC transporter, that specific mutant alleles ofirp6A,irp6B, andirp6Care associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by theirp6operon.