Analysis of a DtxR-regulated iron transport and siderophore biosynthesis gene cluster in Corynebacterium diphtheriae

Abstract

This report describes a genetic locus associated with siderophore biosynthesis and transport in Corynebacterium diphtheriae. A BLAST search of the C. diphtheriae genome identified a seven-gene cluster that included four genes, designated ciuA, ciuB, ciuC, and ciuD, whose predicted products are related to ABC-type iron transporters. Downstream from ciuD is the ciuE gene, whose predicted product is similar to the aerobactin biosynthetic enzymes IucA and IucC. The CiuE protein, which has a predicted mass of 121,582 Da and is approximately twice the size of either IucC or IucA, is homologous to each of these proteins in both its N- and C-terminal regions. C. diphtheriae ciuE deletion mutants exhibited a defect in siderophore production, iron uptake, and growth in low-iron medium. Mutations in the ciuA gene, whose predicted product is a lipoprotein component of an iron transport system, resulted in a severe defect in iron uptake and reduced ability to use the C. diphtheriae siderophore as an iron source. Site-directed mutations in irp6A, a gene previously reported to be associated with siderophore transport, had no effect on iron uptake or the utilization of the C. diphtheriae siderophore as an iron source. Transcriptional analysis demonstrated that expression of ciuA and ciuE is DtxR and iron regulated, and DNase I protection experiments confirmed the presence of DtxR binding sites upstream from each of these genes. Thus, this iron- and DtxR-regulated gene cluster is involved in the synthesis and transport of the C. diphtheriae siderophore.

FIG. 1.

(A) Genetic map of the ciu gene cluster (DIP0582 to DIP0588). The predicted sizes of the gene products are indicated in parentheses (in kilodaltons) below the map. P, location of putative promoter; DBS, DtxR binding site; arrow, direction of transcription. Stem and loop indicates a putative transcriptional terminator. (B) Alignment of CiuE with an approximately 400-amino-acid internal region from IucA/IucC. IucC and IucA share similarity to CiuE in both the N-terminal region and C-terminal region. Homologies are indicated below the alignment (percent identity/percent similarity).

FIG. 2.

Growth analysis of C. diphtheriae strains. C. diphtheriae strains were grown in PGTH medium containing various supplements as described in Materials and Methods, and the OD₆₅₀ of cultures was determined at the indicated times. Strains were grown in PGTH medium that contained 5 μM FeSO₄ (+Fe), no supplement (PGTH), or 1 μM EDDA (PGTH-E). (A) 1737 strains. Closed triangle, 1737 wild type (wt) (+Fe); X, 1737 wt (PGTH); open triangle, 1737 wt (PGTH-E); closed diamond, 1737E/pKE (+Fe); open diamond, 1737E/pKE (PGTH-E); closed square, 1737E (ciuE) (+Fe); open square, 1737E (ciuE) (PGTH-E); closed circle, 1737A (ciuA) (+Fe); open circle, 1737A (ciuA) (PGTH-E). (B) C7(-) strains. Closed triangle, C7(-) wt (+Fe); X, C7(-) wt (PGTH); open triangle, C7(-) wt (PGTH-E); closed square, C7E (ciuE) (+Fe); open square, C7E (ciuE) (PGTH-E); closed circle, C7A (ciuA) (+Fe); open circle, C7A (ciuA) (PGTH-E). (C) Closed triangle, C7(-) wt (+Fe); X, C7(-) wt (PGTH); open triangle, C7(-) wt (PGTH-E); closed square, C7A-1 (irp6A pt.) (+Fe); open square, C7A-1 (PGTH-E); closed circle, C7p6A (irp6A Δ) (+Fe); open circle, C7p6A (PGTH-E); closed diamond, HC1 (+Fe); +, HC1 (PGTH); open diamond, HC1 (PGTH-E). Results represent the means of three independent experiments, and values varied by less than 15% from the mean.

FIG. 3.

⁵⁵Fe uptake in C. diphtheriae. ⁵⁵Fe uptake assays were done as described in Materials and Methods. Strains were passaged into 1 ml of PGTH medium in the presence or absence of EDDA, and the assays were initiated by the addition of 1 μl of 0.1-μCi/μl ⁵⁵FeCl₃ to cultures that had attained an OD₆₅₀ of 0.5 to 0.8. (A) C7(-) strains assayed in unsupplemented PGTH medium. Open circle, C7(-) wild type (wt); closed triangle, C7E (ciuE); closed circle, HC1; open triangle, C7A (ciuA); open diamond, C7p6A (irp6AΔ). (B) C7(-) strains assayed in PGTH medium that contained 1 μM EDDA. Strain designations are the same as those indicated for panel A. Open square, C7E/pKE. (C) 1737 strains assayed in unsupplemented PGTH medium. Open circle, 1737 wt; closed triangle, 1737E; closed circle, 1737E/pKE; open triangle, 1737A; open diamond, 1737p6A (irp6AΔ). (D) 1737 strains assayed in PGTH medium that contained 1 μM EDDA. Strain designations are the same as those indicated for panel C. (E) Stimulation of ⁵⁵Fe uptake by culture supernatants. Experiments were done as described in Materials and Methods. C7E was grown in 3 μM EDDA, and culture supernatants from C7(-) wt (open diamond) or C7E (closed triangle) were added to washed cells prior to initiation of the assay. C7A (open triangle) and HC1 (closed circle) were assayed after growth in unsupplemented PGTH medium to which culture supernatants from C7(-) wt were added prior to initiation of the assay. Results are the means of at least three independent experiments, and values for each time point varied by less than 10% from the mean.

FIG. 4.

(A) DNA sequences of ciuA and ciuE upstream regions, including the DtxR binding sites. DtxR binding sites are in bold and underlined with a bar. Brackets indicate regions protected by DtxR from DNase I digestion. Underlined nucleotides indicate putative start codons, and the line above the sequence indicates the ciuD stop codon. (B) DNase I protection at the ciuA and ciuE upstream sequences. DNase I footprinting was done as described in Materials and Methods. Brackets indicate sequences protected from DNase I digestion by DtxR. DtxR was included at the concentrations indicated above the autoradiogram (micromolar concentrations). M, DNA sequencing ladder used to identify the protected sequences; G, ladder for ciuE; C, ladder for ciuA. (Several independent footprint experiments, including the gels shown, were used to define the protected region.)