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Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

ABSTRACT: Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of these genes have similarity to genes involved in zinc uptake. Transcription of zrg and cmrA was also shown to be regulated in response to iron and manganese, respectively, by mechanisms that are independent of Zur. We demonstrate that the activity of the zur promoter is slightly decreased under low zinc conditions in a process that is dependent on Zur itself. This regulation of zur transcription is distinctive and has not yet been described for any other zur. An adjacent gene, predicted to encode a metal-dependent transcriptional regulator in the ArsR/SmtB family, is transcribed from a separate promoter whose activity is unaffected by Zur. A C. diphtheriae zur mutant was more sensitive to peroxide stress, which suggests that zur has a role in protecting the bacterium from oxidative damage. Our studies provide the first evidence of a zinc specific transcriptional regulator in C. diphtheriae and give new insights into the intricate regulatory network responsible for regulating metal ion concentrations in this toxigenic human pathogen.

SUBMITTER: Smith KF

PROVIDER: S-EPMC2648206 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

Smith Kelsy F KF Bibb Lori A LA Schmitt Michael P MP Oram Diana M DM

Journal of bacteriology 20081212 5

Regulation of metal ion homeostasis is essential to bacterial cell survival, and in most species it is controlled by metal-dependent transcriptional regulators. In this study, we describe a Corynebacterium diphtheriae ferric uptake regulator-family protein, Zur, that controls expression of genes involved in zinc uptake. By measuring promoter activities and mRNA levels, we demonstrate that Zur represses transcription of three genes (zrg, cmrA, and troA) in zinc-replete conditions. All three of th ...[more]

PMID: 19074382

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Project description: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.

Project description:Zinc is an essential trace element for all living organisms and plays pivotal roles in various cellular processes. However, an excess of zinc is extremely deleterious to cells. Bacteria have evolved complex machineries (such as efflux/influx systems) to control the concentration at levels appropriate for the maintenance of zinc homeostasis in cells and adaptation to the environment. The Zur (zinc uptake regulator) protein is one of these functional members involved in the precise control of zinc homeostasis. Here we identified a zur homologue designated 310 from Streptococcus suis serotype 2, strain 05ZYH33, a highly invasive isolate causing streptococcal toxic shock syndrome. Biochemical analysis revealed that the protein product of gene 310 exists as a dimer form and carries zinc ions. An isogenic gene replacement mutant of gene 310, the Delta310 mutant, was obtained by homologous recombination. Physiological tests demonstrated that the Delta310 mutant is specifically sensitive to Zn(2+), while functional complementation of the Delta310 mutant can restore its duration capability, suggesting that 310 is a functional member of the Zur family. Two-dimensional electrophoresis indicated that nine proteins in the Delta310 mutant are overexpressed in comparison with those in the wild type. DNA microarray analyses suggested that 121 genes in the Delta310 mutant are affected, of which 72 genes are upregulated and 49 are downregulated. The transcriptome of S. suis serotype 2 with high Zn(2+) concentrations also showed 117 differentially expressed genes, with 71 upregulated and 46 downregulated. Surprisingly, more than 70% of the genes differentially expressed in the Delta310 mutant were the same as those in S. suis serotype 2 that were differentially expressed in response to high Zn(2+) concentration, consistent with the notion that 310 is involved in zinc homeostasis. We thus report for the first time a novel zinc-responsive regulator, Zur, from Streptococcus suis serotype 2.

Project description:Corynebacterium diphtheriae is the etiological agent of diphtheria, a disease caused by the presence of the diphtheria toxin. However, an increasing number of records report non-toxigenic C. diphtheriae infections. Here, a C. diphtheriae strain was recovered from a patient with a past history of bronchiectasis who developed a severe tracheo-bronchitis with multiple whitish lesions of the distal trachea and the mainstem bronchi. Whole-genome sequencing (WGS), performed in parallel with PCR targeting the toxin gene and the Elek test, provided clinically relevant results in a short turnaround time, showing that the isolate was non-toxigenic. A comparative genomic analysis of the new strain (CHUV2995) with 56 other publicly available genomes of C. diphtheriae revealed that the strains CHUV2995, CCUG 5865 and CMCNS703 share a lower average nucleotide identity (ANI) (95.24 to 95.39%) with the C. diphtheriae NCTC 11397T reference genome than all other C. diphtheriae genomes (>98.15%). Core genome phylogeny confirmed the presence of two monophyletic clades. Based on these findings, we propose here two new C. diphtheriae subspecies to replace the lineage denomination used in previous multilocus sequence typing studies: C. diphtheriae subsp. lausannense subsp. nov. (instead of lineage-2), regrouping strains CHUV2995, CCUG 5865, and CMCNS703, and C. diphtheriae subsp. diphtheriae subsp. nov, regrouping all other C. diphtheriae in the dataset (instead of lineage-1). Interestingly, members of subspecies lausannense displayed a larger genome size than subspecies diphtheriae and were enriched in COG categories related to transport and metabolism of lipids (I) and inorganic ion (P). Conversely, they lacked all genes involved in the synthesis of pili (SpaA-type, SpaD-type and SpaH-type), molybdenum cofactor and of the nitrate reductase. Finally, the CHUV2995 genome is particularly enriched in mobility genes and harbors several prophages. The genome encodes a type II-C CRISPR-Cas locus with 2 spacers that lacks csn2 or cas4, which could hamper the acquisition of new spacers and render strain CHUV2995 more susceptible to bacteriophage infections and gene acquisition through various mechanisms of horizontal gene transfer.

Project description:Zinc has an abounding occurrence in the prokaryotes and plays paramount roles including catalytic, structural, and regulatory. Zinc uptake regulator (Zur), a Fur family transcriptional regulator, is connoted in maintaining zinc homeostasis in the pathogenic bacteria by binding to zinc and regulating the genes involved in zinc uptake and mobilization. Zinc homeostasis has been marginally scrutinized in Bacillus anthracis, the top-rated bio-terror agent, with no decipherment of the role of Zur. Of the three Fur family regulators in B. anthracis, BAS4181 is annotated as a zinc-specific transcriptional regulator. This annotation was further substantiated by our stringent computational and experimental analyses. The residues critical for zinc and DNA binding were delineated by homology modeling and sequence/structure analysis. ba zur existed as a part of a three-gene operon. Purified BaZur prodigiously existed in the dimeric form, indicated by size exclusion chromatography and blue native-polyacrylamide gel electrophoresis (PAGE). Computational and manual strategies were employed to decipher the putative regulon of ba zur, comprising of 11 genes, controlled by six promoters, each harboring at least one Zur box. The DNA binding capability of the purified BaZur to the upstream regions of the ba zur operon, yciC, rpmG, znuA, and genes encoding a GTPase cobalamine synthesis protein and a permease was ascertained by electrophoretic mobility shift assays. The regulon genes, implicated in zinc uptake and mobilization, were mostly negatively regulated by BaZur. The ba zur expression was downregulated upon exposure of cells to an excess of zinc. Conversely, it exhibited a marked upregulation under N, N, N', N'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) mediated zinc-depleted environment, adding credence to its negative autoregulation. Moreover, an increase in the transcript levels of the regulon genes znuA, rpmG, and yciC upon exposure of cells to TPEN connoted their role in combating hypo-zincemic conditions by bringing about zinc uptake and mobilization. Thus, this study functionally characterizes Zur of B. anthracis and elucidates its role in maintaining zinc homeostasis.

Project description:Corynebacterium diphtheriae, a Gram-positive, aerobic bacterium, is the causative agent of diphtheria and cutaneous infections. While mechanisms required for heme iron acquisition are well known in C. diphtheriae, systems involved in the acquisition of other metals such as zinc and manganese remain poorly characterized. In this study, we identified a genetic region that encodes an ABC-type transporter (iutBCD) and that is flanked by two genes (iutA and iutE) encoding putative substrate binding proteins of the cluster 9 family, a related group of transporters associated primarily with the import of Mn and Zn. We showed that IutA and IutE are both membrane proteins with comparable Mn and Zn binding abilities. We demonstrated that the iutABCD genes are cotranscribed and repressed in response to iron by the iron-responsive repressor DtxR. Transcription of iutE was positively regulated in response to iron availability in a DtxR-dependent manner and was repressed in response to Zn by the Zn-dependent repressor Zur. Electrophoretic mobility shift assays showed that DtxR does not bind to the iutE upstream region, which indicates that DtxR regulation of iutE is indirect and that other regulatory factors controlled by DtxR are likely responsible for the iron-responsive regulation. Analysis of the iutE promoter region identified a 50-bp sequence at the 3' end of the iutD gene that is required for the DtxR-dependent and iron-responsive activation of the iutE gene. These findings indicate that transcription of iutE is controlled by a complex mechanism that involves multiple regulatory factors whose activity is impacted by both Zn and Fe.IMPORTANCE Vaccination against diphtheria prevents toxin-related symptoms but does not inhibit bacterial colonization of the human host by the bacterium. Thus, Corynebacterium diphtheriae remains an important human pathogen that poses a significant health risk to unvaccinated individuals. The ability to acquire iron, zinc, and manganese is critical to the pathogenesis of many disease-causing organisms. Here, we describe a gene cluster in C. diphtheriae that encodes a metal importer that is homologous to broadly distributed metal transport systems, some with important roles in virulence in other bacterial pathogens. Two metal binding components of the gene cluster encode surface exposed proteins, and studies of such proteins may guide the development of second-generation vaccines for C. diphtheriae.

Dataset Information

Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

Publications

Regulation and activity of a zinc uptake regulator, Zur, in Corynebacterium diphtheriae.

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