CopR, a global regulator of transcription to maintain copper homeostasis inPyrococcus furiosus

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 analysed 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 characterise 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 microM 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 complex regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Insights into synthesis and function of KsgA/Dim1-dependent rRNA modifications in archaea

“…Marker-less in-frame deletion was performed using the pop-in/pop-out strategy as previously described ( 61 ). To avoid conflicts with homologous recombination during the disruption procedure we also deleted Pf1864 encoding for one of multiple duplicated transposases present in Pyrococcus furiosus genome.…”

“…To avoid conflicts with homologous recombination during the disruption procedure we also deleted Pf1864 encoding for one of multiple duplicated transposases present in Pyrococcus furiosus genome. Flanking upstream (us: ∼600 nt) and downstream (ds: ∼1000 nt) regions spanning the target open reading frame ( Pfu _KsgA/Dim1 = Pf1863 and Pf1864 ) were amplified and assembled together with the counter-selection cassette (cs) by fusion PCR ( 61 ). The resulting PCR fusion was cloned into Zero Blunt™ TOPO™-Vector (Invitrogen) and transformed into the recipient strain MUR37Pf ( 61 ).…”

“…Flanking upstream (us: ∼600 nt) and downstream (ds: ∼1000 nt) regions spanning the target open reading frame ( Pfu _KsgA/Dim1 = Pf1863 and Pf1864 ) were amplified and assembled together with the counter-selection cassette (cs) by fusion PCR ( 61 ). The resulting PCR fusion was cloned into Zero Blunt™ TOPO™-Vector (Invitrogen) and transformed into the recipient strain MUR37Pf ( 61 ). Positive transformants were first selected on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\frac{1}{2}$\end{document} SME medium with 40 mM pyruvate, 0.1% yeast extract and 0.1% peptone, but in the absence of agmatine sulfate, inosine, and guanine (pop-in).…”

“…Growth analyses of P. furiosus were performed as recently described ( 61 ). In brief, P. furiosus strains MURPf37 (parental strain) and MURPf81 ( Pf1863/64 deletion) were cultivated under anaerobic conditions in 40 ml \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\frac{1}{2}$\end{document} SME medium supplemented with 8 mM agmatine sulfate, 8 mM inosine, 8 mM guanine, 0.1% yeast extract, 0.1% peptone and 40 mM pyruvate at 85°C to ensure heat stability of the different chemicals used over the time of the growth experiments ( 61 ). Growth was recorded in biological triplicates during 60 h of incubation by measuring the turbidity changes in situ using a photodiode and a LED with 850 nm as light source.…”

“…Growth was recorded in biological triplicates during 60 h of incubation by measuring the turbidity changes in situ using a photodiode and a LED with 850 nm as light source. The recorded values were converted to cell/ml by using a calibration curve with known cell concentrations, calculated in a Thoma counting chamber (0.02-mm depth; Marienfeld, Lauda-Königshofen, Germany) using phase-contrast microscopy ( 61 ).…”