Commentary on: Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

RecA ATPases are a family of proteins that catalyzes the exchange of complementary DNA regions via homologous recombination. They are conserved from bacteria to humans and are crucial for DNA damage repair and genetic diversity. In this work, Knadler et al. examine how ATP hydrolysis and divalent cations impact the recombinase activity of Saccharolobus solfataricus RadA protein (ssoRadA). They find that the ssoRadA-mediated strand exchange depends on ATPase activity. The presence of Manganese reduces ATPase activity and enhances strand exchange, while calcium inhibits ATPase activity by preventing ATP binding to the protein, yet destabilizes the nucleoprotein ssoRadA filaments, allowing strand exchange regardless of the ATPase activity. Although RecA ATPases are highly conserved, this research offers intriguing new evidence that each member of the family requires individual evaluation.

Biotransformation of High Concentrations of Ginsenoside Substrate into Compound K by ß-glycosidase from Sulfolobus solfataricus.

The rare ginsenoside Compound K (CK) is an attractive ingredient in traditional medicines, cosmetics, and the food industry because of its various biological activities. However, it does not exist in nature. The commonly used method for the production of CK is enzymatic conversion. In order to further improve the catalytic efficiency and increase the CK content, a thermostable ß-glycosidase from Sulfolobus solfataricus was successfully expressed in Pichia pastoris and secreted into fermentation broth. The recombinant SS-bgly in the supernatant showed enzyme activity of 93.96 U/mg at 120 h when using pNPG as substrate. The biotransformation conditions were optimized at pH 6.0 and 80 °C, and its activity was significantly enhanced in the presence of 3 mM Li+. When the substrate concentration was 10 mg/mL, the recombinant SS-bgly completely converted the ginsenoside substrate to CK with a productivity of 507.06 µM/h. Moreover, the recombinant SS-bgly exhibited extraordinary tolerance against high substrate concentrations. When the ginsenoside substrate concentration was increased to 30 mg/mL, the conversion could still reach 82.5% with a productivity of 314.07 µM/h. Thus, the high temperature tolerance, resistance to a variety of metals, and strong substrate tolerance make the recombinant SS-bgly expressed in P. pastoris a potential candidate for the industrial production of the rare ginsenoside CK.

Interplay between transcriptional regulators and VapBC toxin-antitoxin loci during thermal stress response in extremely thermoacidophilic archaea.

Thermoacidophilic archaea lack sigma factors and the large inventory of heat shock proteins (HSPs) widespread in bacterial genomes, suggesting other strategies for handling thermal stress are involved. Heat shock transcriptomes for the thermoacidophilic archaeon Saccharolobus (f. Sulfolobus) solfataricus 98/2 revealed genes that were highly responsive to thermal stress, including transcriptional regulators YtrASs (Ssol_2420) and FadRSs (Ssol_0314), as well as type II toxin-antitoxin (TA) loci VapBC6 (Ssol_2337, Ssol_2338) and VapBC22 (Ssol_0819, Ssol_0818). The role, if any, of type II TA loci during stress response in microorganisms, such as Escherichia coli, is controversial. But, when genes encoding YtrASs , FadRSs , VapC22, VapB6, and VapC6 were systematically mutated in Sa. solfataricus 98/2, significant up-regulation of the other genes within this set was observed, implicating an interconnected regulatory network during thermal stress response. VapBC6 and VapBC22 have close homologues in other Sulfolobales, as well as in other archaea (e.g. Pyrococcus furiosus and Archaeoglobus fulgidus), and their corresponding genes were also heat shock responsive. The interplay between VapBC TA loci and heat shock regulators in Sa solfataricus 98/2 not only indicates a cellular mechanism for heat shock response that differs from bacteria but one that could have common features within the thermophilic archaea.

Divalent metal cofactors differentially modulate RadA-mediated strand invasion and exchange in Saccharolobus solfataricus.

Central to the universal process of recombination, RecA family proteins form nucleoprotein filaments to catalyze production of heteroduplex DNA between substrate ssDNAs and template dsDNAs. ATP binding assists the filament in assuming the necessary conformation for forming heteroduplex DNA, but hydrolysis is not required. ATP hydrolysis has two identified roles which are not universally conserved: promotion of filament dissociation and enhancing flexibility of the filament. In this work, we examine ATP utilization of the RecA family recombinase SsoRadA from Saccharolobus solfataricus to determine its function in recombinase-mediated heteroduplex DNA formation. Wild-type SsoRadA protein and two ATPase mutant proteins were evaluated for the effects of three divalent metal cofactors. We found that unlike other archaeal RadA proteins, SsoRadA-mediated strand exchange is not enhanced by Ca2+. Instead, the S. solfataricus recombinase can utilize Mn2+ to stimulate strand invasion and reduce ADP-binding stability. Additionally, reduction of SsoRadA ATPase activity by Walker Box mutation or cofactor alteration resulted in a loss of large, complete strand exchange products. Depletion of ADP was found to improve initial strand invasion but also led to a similar loss of large strand exchange events. Our results indicate that overall, SsoRadA is distinct in its use of divalent cofactors but its activity with Mn2+ shows similarity to human RAD51 protein with Ca2+.

Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation.

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.

Structural-Functional Relationship of the Ribonucleolytic Activity of aIF5A from Sulfolobus solfataricus.

The translation factor IF5A is a highly conserved protein playing a well-recognized and well-characterized role in protein synthesis; nevertheless, some of its features as well as its abundance in the cell suggest that it may perform additional functions related to RNA metabolism. Here, we have undertaken a structural and functional characterization of aIF5A from the crenarchaeal Sulfolobus solfataricus model organism. We confirm the association of aIF5A with several RNA molecules in vivo and demonstrate that the protein is endowed with a ribonuclease activity which is specific for long and structured RNA. By means of biochemical and structural approaches we show that aIF5A can exist in both monomeric and dimeric conformations and the monomer formation is favored by the association with RNA. Finally, modelling of the three-dimensional structure of S. solfataricus aIF5A shows an extended positively charged surface which may explain its strong tendency to associate to RNA in vivo.

Engineering of a thermophilic dihydroxy-acid dehydratase toward glycerate dehydration for in vitro biosystems.

Dihydroxy-acid dehydratase (DHAD) plays an important role in the utilization of glycerol or glucose for the production of value-added chemicals in the in vitro synthetic enzymatic biosystem. The low activity of DHAD in the dehydration of glycerate to pyruvate hampers its applications in biosystems. Protein engineering of a thermophilic DHAD from Sulfolobus solfataricus (SsDHAD) was performed to increase its dehydration activity. A triple mutant (I161M/Y145S/G205K) with a 10-fold higher activity on glycerate dehydration was obtained after three rounds of iterative saturation mutagenesis (ISM) based on computational analysis. The shrunken substrate-binding pocket and newly formed hydrogen bonds were the reason for the activity improvement of the mutant. For the in vitro synthetic enzymatic biosystems of converting glucose or glycerol to L-lactate, the biosystems with the mutant SsDHAD showed 3.32- and 2.34-fold higher reaction rates than the wild type, respectively. This study demonstrates the potential of protein engineering to improve the efficiency of in vitro synthetic enzymatic biosystems by enhancing the enzyme activity of rate-limited enzymes. KEY POINTS: • A screening method was established for the protein engineering of SsDHAD. • A R3 mutant of SsDHAD with 10-fold higher activity was obtained. • The R3 mutant exhibits higher productivity in the in vitro biosystems.

Interplay between Alba and Cren7 Regulates Chromatin Compaction in Sulfolobus solfataricus.

Chromatin compaction and regulation are essential processes for the normal function of all organisms, yet knowledge on how archaeal chromosomes are packed into higher-order structures inside the cell remains elusive. In this study, we investigated the role of archaeal architectural proteins Alba and Cren7 in chromatin folding and dynamics. Atomic force microscopy revealed that Sulfolobus solfataricus chromatin is composed of 28 nm fibers and 60 nm globular structures. In vitro reconstitution showed that Alba can mediate the formation of folded DNA structures in a concentration-dependent manner. Notably, it was demonstrated that Alba on its own can form higher-order structures with DNA. Meanwhile, Cren7 was observed to affect the formation of Alba-mediated higher-order chromatin structures. Overall, the results suggest an interplay between Alba and Cren7 in regulating chromatin compaction in archaea.

Structural basis of cyclic oligoadenylate degradation by ancillary Type III CRISPR-Cas ring nucleases.

Type III CRISPR-Cas effector systems detect foreign RNA triggering DNA and RNA cleavage and synthesizing cyclic oligoadenylate molecules (cA) in their Cas10 subunit. cAs act as a second messenger activating auxiliary nucleases, leading to an indiscriminate RNA degradation that can end in cell dormancy or death. Standalone ring nucleases are CRISPR ancillary proteins which downregulate the strong immune response of Type III systems by degrading cA. These enzymes contain a CRISPR-associated Rossman-fold (CARF) domain, which binds and cleaves the cA molecule. Here, we present the structures of the standalone ring nuclease from Sulfolobus islandicus (Sis) 0811 in its apo and post-catalytic states. This enzyme is composed by a N-terminal CARF and a C-terminal wHTH domain. Sis0811 presents a phosphodiester hydrolysis metal-independent mechanism, which cleaves cA4 rings to generate linear adenylate species, thus reducing the levels of the second messenger and switching off the cell antiviral state. The structural and biochemical analysis revealed the coupling of a cork-screw conformational change with the positioning of key catalytic residues to proceed with cA4 phosphodiester hydrolysis in a non-concerted manner.

Chromosome segregation in Archaea: SegA- and SegB-DNA complex structures provide insights into segrosome assembly.

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA-DNA and SegB-DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon-helix-helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA-SegB-DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation.

Promoter-proximal elongation regulates transcription in archaea.

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.

Insights into the mismatch discrimination mechanism of Y-family DNA polymerase Dpo4.

Nucleobases within DNA are attacked by reactive oxygen species to produce 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as major oxidative lesions. The high mutagenicity of oxoG is attributed to the lesion's ability to adopt syn-oxoG:anti-dA with Watson-Crick-like geometry. Recent studies have revealed that Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) inserts nucleotide opposite oxoA in an error-prone manner and accommodates syn-oxoA:anti-dGTP with Watson-Crick-like geometry, highlighting a promutagenic nature of oxoA. To gain further insights into the bypass of oxoA by Dpo4, we have conducted kinetic and structural studies of Dpo4 extending oxoA:dT and oxoA:dG by incorporating dATP opposite templating dT. The extension past oxoA:dG was ∼5-fold less efficient than that past oxoA:dT. Structural studies revealed that Dpo4 accommodated dT:dATP base pair past anti-oxoA:dT with little structural distortion. In the Dpo4-oxoA:dG extension structure, oxoA was in an anti conformation and did not form hydrogen bonds with the primer terminus base. Unexpectedely, the dG opposite oxoA exited the primer terminus site and resided in an extrahelical site, where it engaged in minor groove contacts to the two immediate upstream bases. The extrahelical dG conformation appears to be induced by the stabilization of anti-oxoA conformation via bifurcated hydrogen bonds with Arg332. This unprecedented structure suggests that Dpo4 may use Arg332 to sense 8-oxopurines at the primer terminus site and slow the extension from the mismatch by promoting anti conformation of 8-oxopurines.

Multi-scale architecture of archaeal chromosomes.

Chromosome conformation capture (3C) technologies have identified topologically associating domains (TADs) and larger A/B compartments as two salient structural features of eukaryotic chromosomes. These structures are sculpted by the combined actions of transcription and structural maintenance of chromosomes (SMC) superfamily proteins. Bacterial chromosomes fold into TAD-like chromosomal interaction domains (CIDs) but do not display A/B compartment-type organization. We reveal that chromosomes of Sulfolobus archaea are organized into CID-like topological domains in addition to previously described larger A/B compartment-type structures. We uncover local rules governing the identity of the topological domains and their boundaries. We also identify long-range loop structures and provide evidence of a hub-like structure that colocalizes genes involved in ribosome biogenesis. In addition to providing high-resolution descriptions of archaeal chromosome architectures, our data provide evidence of multiple modes of organization in prokaryotic chromosomes and yield insights into the evolution of eukaryotic chromosome conformation.

iCLIP analysis of RNA substrates of the archaeal exosome.

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.

Overexpression of a multifunctional ß-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter.

The ß-glucosidase, which hydrolyzes the ß(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of ß-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including ß-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80 °C, and pH 2-4. We overexpressed the ß-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with ß-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of ß-glucosidase in plants could further increase. The plant-expressed ß-glucosidase had optimal activity at 80 °C and pH 5-6. In addition, the plant-expressed ß-glucosidase showed high thermostability; on heat pre-treatment at 80 °C for 2 h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with ß-glucosidase activity of about 80% still remaining after 1 week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial ß-glucosidase gene as a reporter in plants based on alternative ß-galactosidase activity.

Promutagenic bypass of 7,8-dihydro-8-oxoadenine by translesion synthesis DNA polymerase Dpo4.

Reactive oxygen species induced by ionizing radiation and metabolic pathways generate 7,8-dihydro-8-oxoguanine (oxoG) and 7,8-dihydro-8-oxoadenine (oxoA) as two major forms of oxidative damage. The mutagenicity of oxoG, which promotes G to T transversions, is attributed to the lesion's conformational flexibility that enables Hoogsteen base pairing with dATP in the confines of DNA polymerases. The mutagenesis mechanism of oxoA, which preferentially causes A to C transversions, remains poorly characterized. While structures for oxoA bypass by human DNA polymerases are available, that of prokaryotic DNA polymerases have not been reported. Herein, we report kinetic and structural characterizations of Sulfolobus solfataricus Dpo4 incorporating a nucleotide opposite oxoA. Our kinetic studies show oxoA at the templating position reduces the replication fidelity by ∼560-fold. The catalytic efficiency of the oxoA:dGTP insertion is ∼300-fold greater than that of the dA:dGTP insertion, highlighting the promutagenic nature of oxoA. The relative efficiency of the oxoA:dGTP misincorporation is ∼5-fold greater than that of the oxoG:dATP misincorporation, suggesting the mutagenicity of oxoA is comparable to that of oxoG. In the Dpo4 replicating base pair site, oxoA in the anti-conformation forms a Watson-Crick base pair with an incoming dTTP, while oxoA in the syn-conformation assumes Hoogsteen base pairing with an incoming dGTP, displaying the dual coding potential of the lesion. Within the Dpo4 active site, the oxoA:dGTP base pair adopts a Watson-Crick-like geometry, indicating Dpo4 influences the oxoA:dGTP base pair conformation. Overall, the results reported here provide insights into the miscoding properties of the major oxidative adenine lesion during translesion synthesis.

Characterization of an archaeal recombinase paralog that exhibits novel anti-recombinase activity.

The process of homologous recombination is heavily dependent on the RecA family of recombinases for repair of DNA double-strand breaks. These recombinases are responsible for identifying homologies and forming heteroduplex DNA between substrate ssDNA and dsDNA templates, activities that are modified by various accessory factors. In this work we describe the biochemical functions of the SsoRal2 recombinase paralog from the crenarchaeon Sulfolobus solfataricus. We found that the SsoRal2 protein is a DNA-independent ATPase that, unlike the other S. solfataricus paralogs, does not bind either ss- or dsDNA. Instead, SsoRal2 alters the ssDNA binding activity of the SsoRadA recombinase in conjunction with another paralog, SsoRal1. In the presence of SsoRal1, SsoRal2 has a modest effect on strand invasion but effectively abrogates strand exchange activity. Taken together, these results indicate that SsoRal2 assists in nucleoprotein filament modulation and control of strand exchange in S. solfataricus.

The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling.

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an 'off-switch' for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

Development of an Improved Peroxidase-Based High-Throughput Screening for the Optimization of D-Glycerate Dehydratase Activity.

Successful directed evolution examples span a broad range of improved enzyme properties. Nevertheless, the most challenging step for each single directed evolution approach is an efficient identification of improved variants from a large genetic library. Thus, the development and choice of a proper high-throughput screening is a central key for the optimization of enzymes. The detection of low enzymatic activities is especially complicated when they lead to products that are present in the metabolism of the utilized genetic host. Coupled enzymatic assays based on colorimetric products have enabled the optimization of many of such enzymes, but are susceptible to problems when applied on cell extract samples. The purpose of this study was the development of a high-throughput screening for D-glycerate dehydratase activity in cell lysates. With the aid of an automated liquid handling system, we developed a high-throughput assay that relied on a pre-treatment step of cell extract prior to performing the enzymatic and assay reactions. We could successfully apply our method, which should also be transferable to other cell extract-based peroxidase assays, to identify an improved enzyme for the dehydration of D-glycerate.

The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus.

Maintaining an appropriate DNA topology with DNA-based processes (DNA replication, transcription and recombination) is crucial for all three domains of life. In bacteria, the homeostatic regulation for controlling DNA supercoiling relies on antagonistic activities of two DNA topoisomerases, TopoI and gyrase. In hyperthermophilic crenarchaea, the presence of such a regulatory system is suggested as two DNA topoisomerases, TopoVI and reverse gyrase, catalyze antagonistic activities. To test this hypothesis, we estimated and compared the number of the TopoVI with that of the two reverse gyrases, TopR1 and TopR2, in Sulfolobus solfataricus cells maintained either at 80 or at 88°C, or reciprocally shifted from one temperature to the other. From the three DNA topoisomerases, TopR1 is the only one exhibiting significant quantitative variations in response to the up- and down-shifts. In addition, the corresponding intrinsic activities of these three DNA topoisomerases were tested in vitro at both temperatures. Although temperature modulates the three DNA topoisomerases activities, TopR1 is the sole topoisomerase able to function at high temperature. Altogether, results presented in this study demonstrate, for the first time, that the DNA topological state of a crenarchaeon is regulated via a homeostatic control, which is mainly mediated by the fine-tuning of TopR1.