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.

HtaA Is an Iron-Regulated Hemin Binding Protein Involved in the Utilization of Heme Iron in Corynebacterium diphtheriae

“…PCR was used to confirm the mutations in all of the deletion mutants (not shown). An R47H point mutation was introduced into the C7 dtxR gene using a previously described procedure (15).…”

“…Siderophore transport systems are ubiquitous in bacteria, and it has been known for many years that C. diphtheriae secretes a siderophore and contains a siderophore-specific uptake system (10,30). The structure of the C. diphtheriae siderophore has not been determined; however, a gene required for siderophore biosynthesis and genes encoding an ABC-type siderophore transporter were recently identified in C. diphtheriae and were shown to be regulated by DtxR and iron (15).…”

“…Expression of diphtheria toxin is repressed by iron, and transcription of the structural gene, tox, is regulated by the diphtheria toxin repressor protein, DtxR, in association with iron (4,36). DtxR is a global iron-dependent repressor in C. diphtheriae that controls the expression of at least 50 genes at more than 20 different promoters (7,14,15,29,34,38). While iron-dependent regulation of bacterial virulence factors has been well studied, it is also known that acquisition of iron from the extracellular environment is often critical for microbial pathogens to cause disease (5,6).…”

Novel Hemin Binding Domains in the Corynebacterium diphtheriae HtaA Protein Interact with Hemoglobin and Are Critical for Heme Iron Utilization by HtaA

“…As with Gram-negative species, Gram-positive organisms utilize siderophores and binding protein-dependent uptake systems for iron transport. In the absence of an outer membrane, siderophore uptake in Gram-positive bacteria involves fewer components than are required by gram-negative bacteria; only a membraneanchored substrate binding lipoprotein and its cognate ABC transporter appear to be required for mobilization of the ferric-siderophore complex into the cell (8,19,35). Analysis of hemin acquisition systems in Gram-positive species, however, has revealed transport mechanisms of greater complexity than those observed with siderophore uptake.…”

The ChrA-ChrS and HrrA-HrrS Signal Transduction Systems Are Required for Activation of the hmuO Promoter and Repression of the hemA Promoter in Corynebacterium diphtheriae

“…The dearth of free iron within an organism presents a challenge to pathogenic bacteria in obtaining the necessary iron from the host; thus, pathogens have developed a number of mechanisms for acquiring iron from host molecules such as heme. Corynebacterium diphtheriae, the causative agent of the severe upper respiratory infection diphtheria, has transport systems that are specific for various iron sources including heme (12,21,22,35,37,38). In C. diphtheriae, an ATP-binding cassette transporter for heme, encoded by hmuTUV, and a heme oxygenase (HmuO), which releases iron from the protoporphyrin ring of heme, have been described previously (12,40,60).…”