Regulation of heat shock response in the thermophilic Crenarchaeon Sulfolobus acidocaldarius

Onderzoeksoutput: PhD Thesis

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Temperature is a crucial environmental parameter for all living organisms. Although it is well understood how bacteria and eukaryotes sense and respond to temperature changes that could elicit stress, this is largely enigmatic for archaeal microorganisms. The thermophilic Crenarchaeon Sulfolobus acidocaldarius grows optimally at 75°C in volcanic hot springs - habitats that are typified by large temperature gradients. This imposes constant temperature stress on the cells. Heat shock response is characterized by an upregulation of heat shock proteins (HSPs), mainly chaperones. However, it is unknown how this temperature-sensing and molecular upregulation of HSPs is established. This work aims to address these questions by unravelling heat shock response of S. acidocaldarius using a system-level perspective and a focused study on the regulation of the major HSP.

Using a well-validated heat shock set-up, phenotypical assays were performed to determine the condition eliciting maximal heat shock response without affecting cellular viability. At this condition, pulse-labeling of neosynthesized RNA and protein indicated that transcriptional and translational activity was decreased. However, transcriptome (RNA-sequencing) and proteome (mass spectrometry) analyses demonstrated an extensive and fast response at the RNA level and a slower reprogramming of the protein landscape. Functional enrichment analysis indicated that nearly all biological processes are affected by heat shock, including quality control of the protein landscape, DNA replication, cell division, DNA compaction and DNA supercoiling. Our results point towards the absence of a classical transcription factor as the major regulatory mechanism of heat shock response and suggests that transcriptional regulation is established by changes in overall DNA compaction.

A direct correlation between transcriptional expression and translational production was not evident for most genes, suggesting the existence of post-transcriptional regulatory processes. One possibility includes regulation by RNA thermometers. These structured RNA elements are located at the 5’-end of transcripts (usually the 5’ untranslated region (5’UTR)) and allow for translation initiation only at elevated temperatures. This is a common regulatory mechanism for HSPs in bacteria, but no RNA thermometers have been identified in archaea thus far. For a selection of HSPs, putative RNA thermometers were investigated based on in silico RNA structure predictions, which were validated in vitro using an optimized RNA structural probing procedure called “selective 2’-hydroxyl acylation analyzed by primer extension” (SHAPE).

Heat-shock responsive regulation was further investigated for a gene encoding the major HSP, by constructing a 5’UTR deletion strain. The importance of this 5’UTR-region was confirmed as a determinant for correct HSP levels at the optimal growth temperature and for heat-shock responsive upregulation by primer extension, qRT-PCR and western blotting assays. To our knowledge, this is the first demonstration of leader-associated, temperature-responsive regulation in archaea.

With this, my research provides a comprehensive insight into heat shock responsive regulation of a model Crenarchaeon and a better understanding of the physiology of this organism in the context of its natural habitat. Given the unique phylogenetic position of archaea, this research also contributes to a better understanding of the origin and early evolution of temperature-dependent regulation, which is also relevant for the other domains of life.
Originele taal-2English
Toekennende instantie
  • Vrije Universiteit Brussel
Begeleider(s)/adviseur
  • Peeters, Eveline, Promotor
  • Vranken, Wim, Promotor
Datum van toekenning9 mrt 2023
Plaats van publicatieBrussel
Uitgever
Gedrukte ISBN's9789464443523
StatusPublished - 2023

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