Project description:Silver-resistant Saccharomyces cerevisiae mutant was obtained by evolutionary engineering method. Briefly, genetic diversity in reference strain, CEN.PK.113-7D, was increased by ethyl methane sulfonate (EMS)-mutagenesis. The mutant population was passaged several times in gradually increasing silver stress. Several mutant individuals were selected from the final population. Among selected mutant individuals, one of them was much more resistant to silver stress than the reference strain, called as 2E. Whole-genome transcriptomic analysis was performed to identify the silver resistance mechanisms in the silver-resistant mutant strain.
Project description:The present work was devoted to a multi-level characterization of E. coli exposed to Ag+-mediated stress using for the first time an approach of integrative biology, based on the combination of physiological, biochemical and transcriptomic data sets. Bacterial growth and survival after Ag+ exposure were first quantified and related to the accumulation of intracellular silver, as detected by Nano Secondary Ion Mass Spectroscopy (NanoSIMS) at high lateral resolution. The whole transcriptomic response of E. coli cells under ionic silver-mediated stress was then characterized. Clear correlations were established between (i) cell physiology, (ii) variations in the biochemical characteristics of cell fatty acids and proteins, and (iii) regulation of gene expression. This challenging approach allowed determining key genetic markers of the E. coli response to ionic silver. In particular, we identified Ag+-mediated regulations of gene expression in correlation with growth (e.g. genes of transporters, transcriptional regulators, ribosomal proteins), necessary for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and to cope with various stress (dnaK, pspA, metA,R, oxidoreductase genes). Regulation of gene expression after Ag+ exposure was also correlated to macromolecular modifications, such as acyl chain length (e.g. fadL, lpxA, arnA), protein secondary structure (e.g. dnaJ, htpX, degP) and cell morphology (e.g. ycfS, ycbB).
Project description:Silver nanoparticles cause toxicity in exposed organisms and are an environmental health concern. The mechanisms of silver nanoparticle toxicity, however, remain unclear. We examined the effects of exposure to silver in nano-, bulk- and ionic forms on zebrafish embryos (Danio rerio) using a Next Generation Sequencing approach in an Illumina platform (High-Throughput SuperSAGE). Significant alterations in gene expression were found for all treatments and many of the gene pathways affected, most notably those associated with oxidative phosphorylation and protein synthesis, overlapped strongly between the three treatments indicating similar mechanisms of toxicity for the three forms of silver studied. Changes in oxidative phosphorylation indicated a down-regulation of this pathway at 24h of exposure, but with a recovery at 48h. This finding was consistent with a dose-dependent decrease in oxygen consumption at 24h, but not at 48h, following exposure to silver ions. Overall, our data provide support for the hypothesis that the toxicity caused by silver nanoparticles is principally associated with bioavailable silver ions in exposed zebrafish embryos. These findings are important in the evaluation of the risk that silver particles may pose to exposed vertebrate organisms. mRNA profiles of whole zebrafish embryos at 24 and 48 hours post-fertilisation (hpf) exposed to silver in nano, bulk and ionic forms were generated by deep sequencing using HT-SuperSAGE (Illumina GA2).