Identification of protein targets and quantification of biochemical and toxicogenomic effects of inorganic and organic mercury exposure in e. coli
Lavoie, Stephen Patrick
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Mercury is a ubiquitous environmental toxin that poses a risk to human health in all of its chemical forms, but toxicity mechanisms at the molecular level remain poorly understood. The ecological and health associated roles of microorganisms are increasingly being recognized, but there remains limited knowledge of how bacteria respond to mercury exposure. This dissertation details advancements in methodologies for studying mercury exposure and expands knowledge of toxicity mechanisms using the model prokaryotic organism, Escherichia coli. The combined results from three studies provide a system wide comparative view of the biochemical effects, in vivo protein targets, and transcriptional changes in response to varying concentrations of inorganic and organic mercury exposure. In one study, several biochemical methods were used to study the effects of acute exposure to different mercurials. Inorganic mercury was more effective in binding total and protein thiols than PMA or merthiolate and non-thiol ligands were observed as inorganic mercury depleted the cellular thiol pool. All mercurials disrupted the electrolyte balance of the cell and iron homeostasis with varying effectiveness. In another study, methodologies were developed to identify in vivo mercury binding proteins within a global proteome. There were 307 proteins observed with mercury modifications distributed across all functional roles, but translation and cysteine rich metabolism categories were predominately affected. In addition, twenty-two proteins with highly conserved domains were identified that could serve as biomarkers. These results greatly expand current knowledge of mercury targets and methods could be implemented for studies in other organisms. The final study compares the transcriptional response over time for sub-acute exposure to inorganic and organic mercury using the RNA-Seq method for the first time. Exposure to either mercurial resulted in over 40% of all genes being differentially expressed ten minutes after exposure and subsequent time points reveal gene expression changes throughout recovery of growth. Inorganic and organic mercury displayed distinct transcriptional profiles, which suggests different toxicity mechanisms. The down-regulated gene response was highly conserved, while the up-regulated response was more unique to each compound. These findings provide the most comprehensive view of the effects of mercury exposure in any organism to date.