The Transcriptional Regulator TFB-RF1 Activates Transcription of a Putative ABC Transporter in Pyrococcus furiosus

Reichelt, Robert; Ruperti, Katharina M. A.; Kreuzer, Martina; Dexl, Stefan Albin; Thomm, Michael; Hausner, Winfried

doi:10.3389/fmicb.2018.00838

Cited by 9 publications

(15 citation statements)

References 51 publications

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“…RNAP, TBP, TFB: For in vitro transcription assays and EMSA analysis, we used RNAP purified from P. furiosus cells and recombinant TBP and TFB as described previously (33,35,36).…”

Section: Methodsmentioning

confidence: 99%

“…RT-qPCR reactions were performed similar as described previously (36). In short, total isolated RNA was reverse transcribed using the ProtoScript® II First Strand cDNA Synthesis Kit (NEB), according to the manufacturer´s instructions and using a random primer mix (Promega).…”

Section: Confirmation Of Data Using Rt-qpcrmentioning

confidence: 99%

“…Relative expression levels were calculated using the delta-delta Ct method (2 -∆∆Ct ), by comparing the Ct values from biological triplicates of the gene of interest to a house-keeping gene pf0256. The applicability of pf0256 as a calibrator was evaluated before (36).…”

Section: Confirmation Of Data Using Rt-qpcrmentioning

confidence: 99%

“…In vitro transcription assay: In vitro transcription assays were performed similar as described previously (35,36). To this end, template DNAs containing the promoter regions of the respective gene were amplified from genomic DNA or plasmid pUC19/gdh by PCR amplification (see Supplementary Table 1).…”

Section: In Vitro Assaysmentioning

confidence: 99%

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

Grünberger

Reichelt

Waege

et al. 2020

Preprint

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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.

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“…RNAP, TBP, TFB: For in vitro transcription assays and EMSA analysis, we used RNAP purified from P. furiosus cells and recombinant TBP and TFB as described previously (33,35,36).…”

Section: Methodsmentioning

confidence: 99%

Section: Confirmation Of Data Using Rt-qpcrmentioning

confidence: 99%

Section: Confirmation Of Data Using Rt-qpcrmentioning

confidence: 99%

Section: In Vitro Assaysmentioning

confidence: 99%

See 2 more Smart Citations

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

Grünberger

Reichelt

Waege

et al. 2020

Preprint

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“…Phr ( Pyrococcus heat shock regulator) is also a regulator in Thermococcales that responds to heat shock (19, 20). TFB-RF1 from Pyrococcus furiosus activates transcription of a putative ABC transporter by recruiting TFB to the promoter (21). In the Crenarchaeota , BarR, an Lrp-type transcription factor in Sulfolobus acidocaldarius, regulates an aminotransferase gene in response to the presence of β-alanine (22).…”

Section: Introductionmentioning

confidence: 99%

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Yamamoto

Kanai

Kaneseki

et al. 2019

mBio

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TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar (Thermococcales aromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensis. IMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.

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Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics

Grünberger

Schmid

El-Ahmad

et al. 2023

Preprint

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Extremophiles, such as the hyperthermophilic archaeon Pyrococcus furiosus, thrive under extreme conditions and must rapidly adapt to changes in the physical parameters of their natural environment for short-term and long-term survival. When inhabiting hydrothermal vents, these organisms face substantial temperature gradients, necessitating the evolution of adaptive thermal stress mechanisms. However, the dynamics and coordination of cellular responses at the transcriptome and proteome levels remain underexplored. This study presents an integrated analysis of RNA-sequencing and mass spectrometry data to elucidate the transcriptomic and proteomic responses to heat and cold shock stress and recovery in P. furiosus. Our results reveal surprisingly rapid and dynamic changes in gene and protein expression patterns associated with these stress responses. Heat shock triggers extensive transcriptome reprogramming, orchestrated by the transcriptional regulator Phr, which targets a broader gene repertoire than previously demonstrated. For heat shock signature genes, RNA levels swiftly return to baseline upon recovery, while protein levels remain persistently upregulated, reflecting a rapid but more sustained response. Intriguingly, cold shock at 4 °C elicits distinct short-term and long-term responses at both RNA and protein levels. By conducting a cluster analysis, we identified gene sets with either congruent or contrasting trends in RNA and protein changes. Notably, these clusters represent well-separated arCOG groups and appear to be tailored to their individual cellular responses. Our study provides a comprehensive overview of the cellular response to temperature stress, advancing our understanding of stress response mechanisms in hyperthermophilic archaea and provide valuable insights into the molecular adaptations that facilitate life in extreme environments.

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The Transcriptional Regulator TFB-RF1 Activates Transcription of a Putative ABC Transporter in Pyrococcus furiosus

Cited by 9 publications

References 51 publications

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

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

The TK0271 Protein Activates Transcription of Aromatic Amino Acid Biosynthesis Genes in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics

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