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

Abstract

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.

The transcriptional regulator EarA and intergenic terminator sequences modulate archaellation inPyrococcus furiosus

“…This analysis confirmed the observed coverage profiles and highlighted the presence of poly(T)-sequences after arlB0, arlB2, and arlD. Given that poly(T) sequences have been shown to lead to intrinsic termination, as well as leading to the recruitment of the recently identified termination factors aCPSF1, these sequences likely serve as intergenic transcriptional terminators fine-tuning the expression originating from arlB0 (Maier and Marchfelder, 2019;Sanders et al, 2020;Yue et al, 2020;Li et al, 2021;Grünberger et al, 2023). A, Annotated operons in the DOOR2 database and as suggested by ANNOgesic using short-read RNA sequencing of mixed growth conditions (Mao et al, 2014;Grünberger et al, 2019).…”

“…In contrast, prediction based on short-read RNAseq data from mixed conditions suggests that the two genes are transcribed as an extra single unit (Figure 2A) (Mao et al, 2014;Grünberger et al, 2019). This was further supported by coverage re-analysis of short-read RNA-seq recently obtained from cells grown to exponential phase and cells recovered at the optimal temperature of 95°C following an extended cold shock at 4°C (Figure 2C,D) (Grünberger et al, 2023). Cells recovered from such conditions have been demonstrated to respond swiftly by swimming, which was confirmed by the induction of the arl gene cluster (Mora et al, 2014).…”

“…To explore the potential hierarchic nature of EarAinduced transcriptional regulation, we analyzed the transcriptome landscape of the arl gene cluster in Pyrococcus furiosus. Therefore, we re-analyzed both short-and long-read RNA-seq data in addition to other database information, including transcription start and termination sites (Figure 2) (Grünberger et al, 2019(Grünberger et al, , 2023. The Arl1-type cluster is characterized by three archaellin (arlB0, arlB1, arlB2) genes followed by the arl accessory genes arlC, arlD, arlF, arlG, arlH, arlI, arlJ and a hypothetical gene (Näther-Schindler et al, 2014;Jarrell et al, 2021).…”

“…To achieve a higher resolution of the transcriptional output, we re-analyzed long-read single-molecule data from samples cultivated under optimal growth conditions (Grünberger et al, 2023). Previous transcriptional start site mapping using differential RNA sequencing of Terminator-Exonuclease (TEX) treated RNA revealed that the primary start site is situated upstream of arlB0, the essential major archaellin protein (Grünberger et al, 2019).…”

“…B, Gene annotation of the arl cluster, including the transcriptional regulator EarA and a postulated methyltransferase upstream. C, Log2 fold changes obtained from differential gene expression analysis of samples recovered at 95°C after prolonged cold shock at 4°C (24 h) compared to cells in exponential growth phase (Grünberger et al, 2023). D, Mean coverage profiles normalized by CPM per position of these conditions are shown in grey (exponential phase) and blue (recovery) (Grünberger et al, 2023).…”