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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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Publications

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.

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.

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