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RNA-seq of pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into transcriptional landscape

ABSTRACT: Background: The human pathogen Corynebacterium diphtheriae is the causative agent of the disease diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced several years ago, not much is known about the transcriptome. Our aim was to use RNA Sequencing to close this knowledge gap and gain insights into the transcriptional landscape of C. diphtheriae. Results: We applied two different RNA Sequencing techniques, one to retrieve 5' ends of primary transcripts and one to obtain full length transcripts, to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1,657 transcription start sites (TSS) of which 1,229 were assigned to genes and 428 to putative novel transcripts. By using the TSS data SigA promotor regions and their motifs could be analyzed in detail, revealing a well conserved –10 but unconserved –35 motif, respectively. Furthermore with the TSS data in hand 5' UTR lengths were explored. The observed 5' UTRs range from leaderless, which make up 20 % of all genes, up to over 450 bp long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 470 operons which are further divided into 167 sub-operon structures. In addition we discovered that the deletion of the iron sensing transcription regulator DtxR, which also controls expression of diphtheria toxin (DT), has a strong influence on general gene expression. Nearly 15 % of the genome is differentially expressed, indicating that DtxR might have other functions next to regulation of iron metabolism and DT. Furthermore we shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for an additional TSS and three tox antisense RNAs, which might reveal new ways of transcriptional regulation. Conclusions: This study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding transcriptional regulators, gene characterization and the tox gene in particular.

ORGANISM(S): Corynebacterium diphtheriae NCTC 13129

PROVIDER: GSE98202 | GEO | 2018/01/12

SECONDARY ACCESSION(S): PRJNA384241

REPOSITORIES: GEO

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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.

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RNA-seq of pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into transcriptional landscape

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