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DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

ABSTRACT: The structural gene encoding DtxR, an iron-dependent diphtheria tox regulatory element, has recently been cloned and sequenced from the C7(-) strain of Corynebacterium diphtheriae (J. M. Boyd, M. Oza, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 87:5972, 1990). We report here the molecular cloning, DNA sequence analysis, and characterization of DtxR from the PW8(-), 1030(-), and C7hm723 strains of C. diphtheriae. While the sequence of dtxR from PW8(-) is identical to that of the C7(-) allele, the sequence of dtxR from the 1030(-) strain is only 91.4% identical; however, the deduced amino acid sequence of DtxR from 1030(-) differs by only 6 of 678 amino acids. Moreover, DtxR from all three strains is shown to regulate expression of beta-galactosidase from a tox promoter-operator (toxPO)-lacZ transcriptional fusion. In contrast, the dtxR allele from the iron-insensitive tox constitutive mutant C7hm723 was found to have a single G----A transition, resulting in a substitution of Arg-47 to His and the loss of tox regulatory activity in recombinant Escherichia coli.

SUBMITTER: Boyd JM

PROVIDER: S-EPMC206420 | biostudies-other | 1992 Feb

REPOSITORIES: biostudies-other

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DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

Boyd J M JM Hall K C KC Murphy J R JR

Journal of bacteriology 19920201 4

The structural gene encoding DtxR, an iron-dependent diphtheria tox regulatory element, has recently been cloned and sequenced from the C7(-) strain of Corynebacterium diphtheriae (J. M. Boyd, M. Oza, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 87:5972, 1990). We report here the molecular cloning, DNA sequence analysis, and characterization of DtxR from the PW8(-), 1030(-), and C7hm723 strains of C. diphtheriae. While the sequence of dtxR from PW8(-) is identical to that of the C7(-) allele, th ...[more]

PMID: 1735717

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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

DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

Publications

DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

Similar Datasets

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