Abstract
Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.
Highlights
The archaeal transcription system combines strategies and regulatory mechanisms known from eukaryotic as well as from bacterial species (Werner and Grohmann, 2011; Peeters et al, 2013)
This cop cluster consists of a copper-exporting P1B-ATPase CopA (Ferroplasma acidarmanus Fer1: CopB), a transcriptional regulator CopR (Saccharolobus solfataricus P2: CopT, F. acidarmanus Fer1: CopY) and occasionally the metallochaperone CopT (S. solfataricus P2: CopM, F. acidarmanus Fer1: CopZ) (Figure 1A; Baker-Austin et al, 2005; Ettema et al, 2006; Villafane et al, 2009; Hong et al, 2019)
In Pyrococcus furiosus, the transcriptional regulator CopR is encoded by the gene pf0739, which is in divergent orientation to copA
Summary
The archaeal transcription system combines strategies and regulatory mechanisms known from eukaryotic as well as from bacterial species (Werner and Grohmann, 2011; Peeters et al, 2013). CopR-Mediated Copper Homeostasis in Pyrococcus expression is mainly achieved by bacterial-like transcriptional regulators (Lemmens et al, 2019). Positive or negative regulation is mediated by the binding of these transcription factors (TFs) to promoter regions of specific genes. The genome of the hyperthermophilic euryarchaeon Pyrococcus furiosus contains a total number of 86 putative DNA-binding TFs. the exact function of most of these factors, which represent about 4 % of all open reading frames (ORFs), is unknown (Denis et al, 2018). While the regulation of sugar or sulfur metabolism and other changing environmental conditions have been studied in detail, the underlying mechanisms to maintain metal homeostasis are only poorly understood (Vierke et al, 2003; Lipscomb et al, 2009; Yang et al, 2010; Gindner et al, 2014; Karr, 2014)
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