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Next-generation sequencing facilitates metal homeostasis and intoxication studies

ABSTRACT: Purpose: Analyze transcriptome changes in response to metal treatment in wildtype and sensitized genetic backgrounds. Methods: Zinc or manganese chloride supplementation of wildtype or mutant Drosophila S2R+ cultured cells, followed by RNA extraction. Results: Zinc chloride supplementation induces transcriptional responses in wildtype, ia-2 mutant, and ZnT63C mutant cells. Conclusions: Wildtype S2R+ cells have a transcriptional response to mild zinc chloride supplementation and the zinc response is greater (in degree and in number of genes) in genetically sensitized cell backgrounds that perturb zinc mobilization.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE99332 | GEO | 2017/06/30

SECONDARY ACCESSION(S): PRJNA388110

REPOSITORIES: GEO

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Project description:BACKGROUND: We identified that a putative CzcRS-like two-component system (TCS) in Burkholderia cenocepacia K56-2 is required for heavy metal resistance and virulence. To investigate this TCS further, we applied RNA-seq to determine the transcriptional response of wildtype and CzcRS-deficient B. cenocepacia to subinhibitory concetrations of zinc. METHODS. Wildtype B. cenocepacia K56-2 was cultured in LB broth with and without 1.5 mM zinc chloride. In parallel, a ΔczcRS mutant strain was also cultured in 1.5 mM zinc chloride. Total RNA was extracted from triplicate mid log-phase cultures for each strain/culture condition. Illumina Sequencing libraries were prepared from 50 ng ribosomal-depleted RNA using ScriptSeq v2 with indexing and 15 cycles of PCR amplification using Kapa HiFi DNA polymerase. The amplified libraries were purified using a 1:1 ratio of Ampure XP beads resulting in 45-75nM concentrations and average insert size of 350 bp. Libraries were pooled in equimolar concentrations, denatured and diluted to 6.5 pM before clustering on a cBOT (Illumina). Libraries were subjected to 100-bp paired-end sequencing on a HiSeq 2000 using TruSeq SBS v 3 reagents (Illumina). Paired-end reads were filtered using the fastq-mcf package from the ea-utils suite to remove reads with less than 90% Q20 scores or above and to trim off adaptor sequence. The reads were then aligned against the B. cenocepacia J2315 genome (NC_011001-NC_011003) using Bowtie 1 to remove reads mapping to ERCC spike-in. Abundance of ERCC spike-ins was then checked against predicted values to confirm successful library preparation. Tophat v2.0.4 was used to align reads against the reference genome. The Bedtools multicov command was used to extract per-gene counts for each gene. The DESeq suite was used to determine statistical significance of differential expression between conditions. RESULTS: Wildtype B. cenocepacia K56-2 showed a restricted transcriptional response to 1.5 mM zinc chloride, with only 54 genes exhibiting significant differential expression (≥ 1.5-fold; P < 0.05) compared to unsupplemented LB. This transcriptional response included genes encoding the CzcRS-like TCS itself and the associated efflux pump (CzcCBA). In contrast, the ΔczcRS mutant strain when cultured in 1.5 mM zinc chloride exhbited a profound transcriptional response with 767 genes differentially expressed. Many genes showing differential expression in the ΔczcRS strain are indicative of a stress response, indicating the pivotal role of the CzcRS-like TCS in maintaining cellular homeostasis in response to zinc.

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:This a model from the article: The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress Takahashi H, Oshima T, Doherty N, Clayton SR, Iqbal M, Hill PJ, Hobman JL, Tobe T, Ogasawara N, Kanaya S and Stekel DJ journal year id, Abstract: Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli. We measured the response to zinc of the main specific zinc import and export systems in the wild type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: First, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesise that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments. This model originates from BioModels Database: A Database of Annotated Published Models (http://www.ebi.ac.uk/biomodels/). It is copyright (c) 2005-2011 The BioModels.net Team. For more information see the terms of use. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le NovÃ¨re N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

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Next-generation sequencing facilitates metal homeostasis and intoxication studies

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