Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius

ABSTRACT High-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. We set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75°C. Pulse-labeling experiments demonstrated that a temperature shift to 86°C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by a more subtle reprogramming of the protein landscape. Functional enrichment analysis indicated that nearly all cellular processes are affected by heat shock. A limited correlation was observed in the differential expression on the RNA and protein level, suggesting a prevalence of post-transcriptional and post-translational regulation. Furthermore, promoter sequence analysis of heat shock regulon genes demonstrated the conservation of strong transcription initiation elements for highly induced genes, but an absence of a conserved protein-binding motif. It is, therefore, hypothesized that histone-lacking archaea such as Sulfolobales use an evolutionarily ancient regulatory mechanism that relies on temperature-responsive changes in DNA organization and compaction induced by the action of nucleoid-associated proteins, as well as on enhanced recruitment of initiation factors. IMPORTANCE Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius, which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism’s lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response.

S. acidocaldarius SK-1 electrocompetent cells were transformed by electroporation as described in (Wagner et al., 2012) with the exception that the template plasmid was not methylated.For growth on plates, 0.6 % Gelrite® (Duchefa Biochemie, Netherlands, Haarlem) was used as a solidifying agent of Brock basal salts medium supplemented with 0.2 % sucrose, 0.1 % NZ-Amine, but lacking uracil, and acidified to pH 3.0-3.5 with H2SO4, with addition of 3 mM CaCl2 and 10 mM MgCl2.After incubating during 5 days at 75°C, colonies were treated by spraying with a solution of 5 mg ml -1 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal), revealing "pop-in" integrants by blue color formation caused by LacS-activity.These colonies were further grown in liquid culture without uracil upon confirmation by PCR analysis.At mid/end-exponential phase (OD600 of 0.6-0.8),cells were spread on plates containing 20 µg ml -1 uracil and 200 µg ml -1 5-fluoroorotic acid (5-FOA) and incubated for 5 days at 75°C.Candidate "pop-out" transformants were inoculated in liquid medium with uracil, and presence of the desired C-terminal tag was confirmed by PCR analysis and Sanger sequencing.This procedure was performed first for the introduction of the Thβ-6xHis tag and repeated twice for introduction of the Thα-FLAG and Thγ-HA tag, respectively, finally generating a strain harboring all three tags in the genome (SK-1xThα-FLAG+Thβ-6xHis+Thγ-HA).

Analysis of cellular viability by plating
Culture samples of SK-1xThα-FLAG+Thβ-6xHis+Thγ-HA were diluted with Basic Brock medium to OD600nm 0.1 (i.e. the 10 -1 dilution), based on the OD600nm measurement at the start of the experiment.A serial dilution series was constructed to 10 -6 and 10 µL of each dilution were spotted on freshly prepared plates.For growth on plates, 0.6 % gelrite was used as a solidifying agent of the Brock medium with addition of 3 mM CaCl2 and 10 mM MgCl2.Plates were incubated for 5 days at 75°C and analyzed as previously described (Baes et al., 2020).

RNA sequencing
Total RNA was extracted from MW001-stabilized cell pellets using the RNeasy Mini Kit (Qiagen, USA, Maryland) and on-column DNase treatment (Qiagen, USA, Maryland).Cell pellets were resuspended in 600 µL RLT TM lysis buffer and centrifuged for 10 minutes at 12,108 x g and 4°C.RNA was finally eluted from the column in 30 µL nuclease-free water.The total RNA quantity was determined with a Qubit RNA High Sensitivity Assay (Thermo Fisher Scientific, USA, Eugene) and RNA integrity was evaluated on a Bioanalyzer instrument with a RNA 6000 Nano chip (Agilent Technologies, USA, Santa Clara) (Supplementary Dataset S1).Ribosomal RNA depletion was established using a PAN-Archaea riboPOOL kit (siTOOLS, Germany, Planegg), followed by purification with a Zymo RNA Clean and Concentrator-5 kit (Zymo Research, USA, Irvine).Sequencing libraries were subsequently prepared with the TruSeq Stranded Total RNA Library Kit (Illumina, USA, San Diego) in combination with the RNA Unique Dual Indices (IDT for Illumina, USA, San Diego).Library enrichment PCR proceeded for 9 cycles.Library quality was verified on a Bioanalyzer instrument with a DNA High Sensitivity chip (Agilent Technologies, USA, Santa Clara) and concentrations were measured with qPCR according to 'Sequencing library quantification guide' (Illumina, USA, San Diego).Sequencing was performed on a NextSeq500 instrument (Illumina, USA, San Diego) in high output with single reads of 75 nts and 2% Phix spike-in (Supplementary Dataset S1).
Data were processed by first performing a quality control by FastQC (Andrews et al., 2010); quality trimming and filtering was performed by Trimmomatic (Bolger et al., 2014) (Supplementary Dataset S1).Sequencing reads were mapped to the S. acidocaldarius DSM639 genome (NC_007181.1)using STAR (Dobin et al., 2013) and read counts were produced by RSEM (Li et al., 2011).A minimum of 2182 genes out of 2351 coding genes were covered (= 92.8 %).Normalization and differential expression analysis was performed with the R-package EdgeR (Robinson et al., 2010).Genes were considered differentially expressed if the false discovery rate (FDR) was lower than 0.05 (Supplementary Dataset S2).

TMT-labeled Liquid Chromatography-Tandem-Mass-Spectrometry
For protein extraction, cell pellets were resuspended in 500 µL lysis buffer (PBS pH 7.5, 1% SDS, 5 mM phenylmethylsulfonyl fluoride (PMSF), cOmplete™ Protease Inhibitor Cocktail (Roche, Switzerland, Basel), lysed by ultrasonication and centrifuged for 15 minutes at 16,100 x g.Total protein concentrations were determined employing the Pierce™ Rapid Gold BCA Protein Assay Kit (Pierce Biotechnology, Inc., USA, Rockford) (Supplementary Dataset S1).Samples of 450 µL lysate were flash-frozen and stored at -80°C.Next, 100 µL lysate samples were diluted 1:10 with 30 mM triethylammonium bicarbonate (TEAB) followed by incubation at 95°C during 5 minutes.Trypsin was added at a 1:50 ratio followed by a 5-hour incubation at 37°C and a 10-minute centrifugation at 10,000 x g for the collection of peptide-containing supernatants.Digested samples were stored overnight at -20°C and peptides pelleted by speedvacevaporation.Pellets were resolved in 80 µL 30 mM TEAB using water bath sonication for 2 minutes.Remaining SDS was removed employing the Pierce™ Detergent Removal Spin Columns with a 0.5-ml volume (Pierce Biotechnology, Inc., USA, Rockford) using 30 mM TEAB as equilibration buffer.Peptides were quantified with the Pierce™ Quantitative Colorimetric Peptide kit (Pierce Biotechnology, Inc., USA, Rockford).For each peptide sample, 10 µg was Tandem Mass Tag (TMT) labeled using the TMTpro 16plex Label Reagent Set (Pierce Biotechnology, Inc., USA, Rockford).Samples were multiplexed to 120 µg, dried by speedvac centrifugation and the pellets were resuspended in 100 µl of 0.5 % trifluoroacetic acid in 5 % acetonitrile.Unincorporated TMT-labels were removed on a Pierce™ C18 Spin Column (Pierce Biotechnology, Inc., USA, Rockford) and eluted in 20 µL 70 % acetonitrile.Samples were dried by speedvac centrifugation and resuspended in 0.1 % trifluoroacetic acid 3.5% acetonitrile to a final peptide concentration of 0.5 µg/µL.One µg of peptides were directly loaded onto a reversed-phase pre-column Acclaim PepMap 100 (Thermo Fisher Scientific, USA, Eugene) and eluted in backflush mode.Peptide separation was achieved using a reversed-phase analytical column Acclaim PepMap RSLC on an Ultimate 3000 RSLN nanoHPLC system (Thermo Fisher Scientific, USA, Eugene) as described by (Ouni et al., 2022).Peptides were analyzed by mass spectrometry (MS) at an Orbitrap Fusion Lumos tribrid (Thermo Fisher Scientific, USA, Eugene) with enabled advanced peak determination (APD) and with relative quantification by MS2.Intact peptides were detected in the Orbitrap at a resolution of 120,000 with a scan range m/z from 375 to 1500 and an AGC target of 4x10 5 , maximum injection time was set to 50 ms.A data-dependent procedure of MS/MS scans was applied for the top precursor ions above a threshold ion count of 3.0 × 10 4 in the MS survey scan with 60 s dynamic exclusion.The total cycle time was set to 3 s.For MS2 quantification of the TMT reporter ions, MS/MS spectra were in the Orbitrap at a resolution of 50,000 after HCD fragmentation at 35%, with an AGC target of 1 × 10 5 ions and a maximum injection time of 120 ms.

Supplementary Methods Table 1. DNA oligonucleotides used in this work for the construction of SK-1 (derivative) strains expressing tagged thermosome subunits
. FW = forward; RV = reverse.

Methods Table 2. Plasmids used in this work for construction of SK-1 (derivative) strains expressing tagged thermosome subunits.
Nucleotide sequences can be provided upon request.