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Identification of zinc and Zur-regulated genes in Corynebacterium diphtheriae

ABSTRACT: We assessed changes in gene expression in response to zinc availability for the human pathogen Corynebacterium diphtheriae. Expression profiles of wild-type C. diphtheriae strain 1737 grown in semi-defined metal-poor media (mPGT) without and with 5 µM zinc chloride supplementation were compared; the expression profile of wild-type C. diphtheriae strain 1737 in zinc-replete conditions was also compared against an isogenic Δzur mutant grown in zinc-replete conditions. Three biological replicates were prepared by isolating total RNA from mid-logarithmic growth cultures and eleven genes (dip0013, dip0093, dip0173, dip0438, dip1087, dip1486, dip1724, dip2114, dip2128, dip2162, and dip2325) were assessed by real-time PCR to validate the array results. (Abstract) Corynebacterium diphtheriae is a Gram-positive bacterial pathogen and the causative agent of diphtheria, a severe disease of the upper respiratory tract of humans. Factors required for C. diphtheriae to survive in the human host are not well defined, but likely include the acquisition of essential metals such as zinc. In C. diphtheriae, zinc-responsive global gene regulation is controlled by the Zinc Uptake Regulator (Zur), a member of the Fur-family of transcriptional regulators. In this study, we use transcriptomics to identify zinc-regulated genes in C. diphtheriae by comparing gene expression of a wild-type strain grown without and with zinc supplementation. Zur-regulated genes were identified by comparing wild-type gene expression with that of an isogenic zur mutant. We observed zinc repression of several putative surface proteins, the heme efflux system hrtBA, various ABC transporters, and the non-ribosomal peptide synthetase/polyketide synthase cluster sidAB. Furthermore, increased gene expression in response to zinc was observed for the alcohol dehydrogenase, adhA. Zinc and Zur regulation were confirmed for several genes by complementing the zur deletion and subsequent qPCR analysis. We used MEME to predict Zur binding sites within the promoter regions of zinc- and Zur-regulated genes, and verified Zur binding by electrophoretic mobility shift assays. Additionally, we characterized cztA (dip1101), which encodes a putative cobalt/zinc/cadmium efflux family protein. Deletion of cztA results in increased sensitivity to zinc, but not to cobalt or cadmium. This study advances our knowledge of changes to Zur-dependent global gene expression in response to zinc in C. diphtheriae. The identification of zinc-regulated ABC transporters herein will facilitate future studies to characterize zinc transport in C. diphtheriae.

ORGANISM(S): Corynebacterium diphtheriae NCTC 13129 Corynebacterium diphtheriae

PROVIDER: GSE131485 | GEO | 2019/08/15

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.

Project description:We assessed changes in gene expression in response to manganese availability for the human pathogen Corynebacterium diphtheriae. Total RNA was harvested from wild-type C. diphtheriae strain 1737 and an isogenic ΔmntR (dip0619) grown in semi-defined metal-limited media (mPGT) without or with 5 µM manganese chloride supplementation. Three biological replicates were prepared and five genes were assessed by real-time PCR to validate the array results: dip0124, dip0169, dip0615, dip1923, and dip2261. (Abstract) Corynebacterium diphtheriae is the causative agent of a severe respiratory disease in humans. The bacterial systems required for infection are poorly understood, but the acquisition of metals such as manganese (Mn) are likely critical for host colonization. MntR is a Mn-dependent transcriptional regulator in C. diphtheriae and was previously shown to repress the expression of the mntABCD genes, which encode an ABC metal transporter. However, other targets of Mn and MntR regulation in C. diphtheriae have not been identified. In this study, we use comparisons between the gene expression profiles of wild-type C. diphtheriae strain 1737 grown without or with Mn supplementation and comparisons of gene expression between wild-type and an mntR mutant to characterize the C. diphtheriae Mn and MntR regulon. MntR was observed to both repress and induce the expression of various target genes in a Mn-dependent manner. Genes induced by MntR include the Mn-superoxide dismutase, sodA, and the putative ABC transporter locus, iutABCD. DNA binding studies showed that MntR interacts at the promoter regions for several genes identified in the expression study, and a 17-bp consensus MntR DNA binding site was identified. We found that an mntR mutant displayed increased sensitivity to Mn and Cd that could be alleviated by the additional deletion of the mntA-D transport locus, providing evidence that the MntABCD transporter functions as a Mn uptake system in C. diphtheriae. The findings in this study further our understanding of metal uptake systems and global metal regulatory networks in this important human pathogen.

Project description:The zinc uptake regulator Zur is a Zn2+-sensing metalloregulatory protein involved in the maintenance of bacterial zinc homeostasis. Up to now, regulation of zinc homeostasis by Zur is poorly understood in Y. pestis. We constructed a zur null mutant of Y. pestis biovar microtus strain 201. Microarray expression analysis disclosed a set of 154 Zur-dependent genes of Y. pestis under zinc rich condition. Real-time reverse transcription (RT)-PCR was subsequently used to validate the microarray data. Based on the 154 Zur-dependent genes, predicted regulatory Zur motifs were used to screen for potential direct Zur targets including three putative operons znuA, znuCB and ykgM-RpmJ2. The LacZ reporter fusion analysis verified that Zur greatly repressed the promoter activity of the above three operons. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified Zur protein was able to bind to the promoter regions of the above three operons. The DNase I footprinting was used to identify the Zur binding sites for the above three operons, verifying the Zur box sequence as predicted previously in Î³-Proteobacteria. The primer extension assay was further used to determine the transcription start sites for the above three operons and to localize the -10 and -35 elements. Zur binding sites overlapped the -10 sequence of its target promoters, which was consistent with the previous observation that Zur binding would block the entry of the RNA polymerase to repress the transcription of its target genes. Zur as a repressor directly controls the transcription of znuA, znuCB and ykgM-RpmJ2 in Y. pestis by employing a conserved mechanism of Zur-promoter DNA association as observed in Î³-Proteobacteria. Zur contributes to zinc homeostasis in Y. pestis likely through transcriptional repression of the high-affinity zinc uptake system ZnuACB and two alternative ribosomal proteins YkgM and RpmJ2. The wild-type (WT) Y. pestis strain 201 belongs to a newly established Y. pestis biovar, Microtus, which was thought to be avirulent to humans, but highly virulent to mice. An in-frame deletion of the zur gene was constructed by using one step inactivation method based on the lambda phage recombination in which PCR primers provide the homology to the target gene, as described previously by Datsenko and Wanner. The entire coding region of zur was replaced by a kanamycin resistance (KnR) cassette, which was verified by PCR and DNA sequencing. The resulting mutant strain was referred to as Îzur. Both the WT strain and the Zur mutant were pre-cultivated at 26 ÂºC to the middle exponential growth phase (OD620 about 1.0) in TMH medium. The cell cultures were then diluted 1:20 in fresh TMH medium and grown at 26Â°C until an OD620 of about 1.0. Finally, 5mM ZnCl2 was added into each cell culture to ensure zinc rich conditions. Growth was continued for 30 min at 26Â°C before harvested for total RNA isolation. Gene expression profiles were compared between WT and Îzur. RNA samples were isolated from four individual bacterial cultures, as biological replicates, for each strain. The dual-fluorescently (Cy3 or Cy5 dye) labeled cDNA probes, for which the incorporated dye was reversed, were synthesized from the RNA samples, and then hybridized to four separated microarray slides, respectively.

Project description:Intracellular zinc concentration needs to be maintained within strict limits due to its toxicity at high levels, and this is achieved by a finely regulated balance between uptake and efflux. Many bacteria use the Zinc Uptake Regulator Zur to orchestrate zinc homeostasis, but little is known regarding the transport of this metal across the bacterial outer membrane. In this work we determined the C. crescentus Zur regulon by global transcriptional and in silico analyses. Among the genes directly repressed by Zur are those encoding a putative high affinity ABC uptake system (znuCBA), three TonB-dependent receptors (znuD, znuE and znuF) and one new putative transporter of a family not yet characterized (zrpW). Zur is also directly involved in the activation of a RND and a P-type ATPase efflux systems, as revealed by Î²-galactosidase and site-directed mutagenesis assays. Several genes belonging to the Fur regulon were also downregulated in the zur mutant, suggesting a putative cross-talk between Zur and Fur regulatory networks. Interestingly, a phenotypic analysis of the znuD and znuE mutants has shown that these genes are essential for growth under zinc starvation, suggesting that C. crescentus uses these TonB-dependent outer membrane transporters as key zinc scavenging systems. The DNA microarray experiments were performed as described in (da Silva Neto et al., 2013). Briefly, cDNA was generated from 12.5 Âµg of total RNA and labeled with either Cy3 or Cy5 fluorescent dyes (FairPlay III Microarray Labeling System, Stratagene). Labeled cDNA samples were hybridized to a Caulobacter DNA oligo microarray (Agilent), and the arrays were scanned with an Agilent High Resolution Microarray Scanner. RNA from three independent biological cultures was used for DNA microarray analysis. We considered as differentially expressed genes those showing a minimum of 2-fold change relative to the control, considering at least three out of the four last probes for each gene (that are downstream of the translational start site) in at least two of three biological replicates. The values for the relative expression of each gene were obtained as the average of the four last probes.

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Identification of zinc and Zur-regulated genes in Corynebacterium diphtheriae

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