Dataset Information

Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae.

ABSTRACT: Although the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of tox expression is controlled, to a large extent, by its bacterial host Corynebacterium diphtheriae. Optimal yields of tox gene products are obtained only when iron becomes the growth-rate-limiting substrate. Previous studies suggest that regulation of tox expression is mediated through an iron-binding aporepressor. To facilitate molecular cloning of the tox regulatory element from genomic libraries of C. diphtheriae, we constructed a tox promoter/operator (toxPO)-lacZ transcriptional fusion in Escherichia coli strain DH5 alpha. We report the molecular cloning and nucleic acid sequence of a diphtheria tox iron-dependent regulatory element, dtxR, and demonstrate that expression of beta-galactosidase from the toxPO-lacZ fusion is regulated by dtxR-encoded protein in an iron-sensitive manner. In addition, we show that expression of the toxPO-lacZ fusion is not affected by the E. coli iron-regulatory protein Fur and that the dtxR protein does not inhibit expression of fur-regulated outer-membrane proteins.

SUBMITTER: Boyd J

PROVIDER: S-EPMC54451 | biostudies-other | 1990 Aug

REPOSITORIES: biostudies-other

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Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae.

Boyd J J Oza M N MN Murphy J R JR

Proceedings of the National Academy of Sciences of the United States of America 19900801 15

Although the structural gene for diphtheria toxin, tox, is carried by a family of closely related corynebacteriophages, the regulation of tox expression is controlled, to a large extent, by its bacterial host Corynebacterium diphtheriae. Optimal yields of tox gene products are obtained only when iron becomes the growth-rate-limiting substrate. Previous studies suggest that regulation of tox expression is mediated through an iron-binding aporepressor. To facilitate molecular cloning of the tox re ...[more]

PMID: 2116013

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Project description:We assessed changes in gene expression in response to iron availability for the human pathogen Corynebacterium diphtheriae. Expression profiles of wild-type C. diphtheriae strain 1737 grown in semi-defined metal-poor media (mPGT) in iron-limiting (0.5 µM iron chloride supplementation) and iron-replete (10 µM supplementation) conditions were compared; the expression profiles of wild-type C. diphtheriae strain 1737 during growth in iron-replete conditions was also compared against an isogenic ΔdtxR mutant grown in iron-replete conditions. Three biological replicates were prepared by isolating total RNA from mid-logarithmic growth cultures and ten genes (dip0169, dip0415, dip1061, dip1062, dip2330, dip1486, dip0173, dip1252, dip1866, and dip0281) were quantified by real-time PCR to validate the array results. Corynebacterium diphtheriae is the causative agent of the severe respiratory disease, diphtheria. Diphtheria Toxin (DT), encoded by the tox gene, is the potent exotoxin secreted by C. diphtheriae responsible for much of the morbidity and mortality of diphtheria. Expression of the tox gene is regulated by the Diphtheria Toxin Repressor (DtxR) and iron. In addition to the regulation of toxin expression, DtxR functions as a global iron-dependent regulatory factor that mediates iron homeostasis in C. diphtheriae. While numerous genes regulated by DtxR and iron are known, a genome-wide study of both the iron and DtxR regulons is lacking in C. diphtheriae. Here, we report novel iron- and DtxR-regulated genes revealed by a comprehensive transcriptomic analysis. Not all identified genes appear to be repressed by iron and DtxR; some genes were found to be induced by iron in a DtxR-dependent manner, a mechanism of regulation not previously described in C. diphtheriae. Using a prediction algorithm (MEME) and electrophoretic mobility shift assays, we verified DtxR binding to sequences upstream of several newly identified genes. Furthermore, we characterized expression of ferritin (ftn) and catalase (cat), which are both induced by iron, but differentially affected by DtxR. We identified three DtxR binding sites in the ftn promoter, while analysis of the cat promoter establishes a role for DtxR in cat expression and suggests complex regulation by additional regulators. Collectively, these results expand our knowledge on the function of DtxR and the diverse roles of this regulatory protein in controlling gene expression.

Project description:The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by 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 an Escherichia coli system 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-type C. diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are 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 the irp6 operon.

Dataset Information

Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae.

Publications

Molecular cloning and DNA sequence analysis of a diphtheria tox iron-dependent regulatory element (dtxR) from Corynebacterium diphtheriae.

Similar Datasets

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