Paul Villain

Violette Da Cunha

Etienne Villain

Patrick Forterre

Jacques Oberto

Ryan Catchpole
email: ryan.catchpole@uga.edu

Tamara Basta
email: tamara.basta@i2bc.paris-saclay.fr

The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase

In all cells, DNA topoisomerases dynamically regulate DNA supercoiling allowing essential DNA processes such as transcription and replication to occur. How this complex system emerged in the course of evolution is poorly understood. Intriguingly, a single horizontal gene transfer event led to the successful establishment of bacterial gyrase in Archaea, but its emergent function remains a mystery. To better understand the challenges associated with the establishment of pervasive negative supercoiling activity, we expressed the gyrase of the bacterium Thermotoga maritima in a naïve archaeon Thermococcus kodakarensis which naturally has positively su- percoiled DNA. We found that the gyrase was catalytically active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA which was stably maintained over at least eighty generations. An increased sensitivity of gyrase-expressing T. kodakarensis to ciprofloxacin suggested that gyrase also modulated chromosomal topology. Accordingly, global transcriptome analyses revealed large scale gene expression deregulation and identified a subset of genes responding to the negative supercoiling activity of gyrase. Surprisingly, the artificially introduced dominant negative supercoiling activity did not have a measurable effect on T. kodakarensis growth rate. Our data suggest that gyrase can become established in Thermococcales archaea without critically interfering with DNA transaction processes.

List of figures

List of tables

The description and classification of living organisms plays a fundamental and historical role in Biology. This discipline reaches a turning point in the 18 th century with the binomial classification of Carl Von Linné. According to this classification, each organism's name is composed of the general name, the genus, which is shared with several organisms and a specific name, which refers to a particular species. This binomial nomenclature considerably facilitated exchange between scientists and is still used today. It paved the way to the new scientific discipline the Taxonomy, which aims to establish a general classification of living organisms. At the end of the 18 th century, Jean-Baptiste de Lamarck introduced the concept of Evolution to explain the diversification of living organisms and their adaptation to their environment. This theory was further developed by Alfred Russel Wallace and Charles Darwin who independently introduced the concept of Natural Selection [START_REF] Darwin | On the Origin of Species by Charles Darwin[END_REF]. They postulated that organisms are evolving by chance mutation and those having the most competitive characters are more likely to transmit them to their offspring. To illustrate his theory, Charles Darwin was the first to draw a phylogenetic tree (Figure 1) thus setting the stage for the appearance of the new scientific field: the molecular phylogeny.

Figure 1: Darwin first draw of phylogenetic tree "I think case must be that one generation should have as many living as now. To do this and to have as many species in same genus (as is) requires extinction. Thus between A + B the immense gap of relation. C + B the finest gradation. B+D rather greater distinction. Thus genera would be formed. Bearing relation to ancient types with several extinct forms". These notes from Charles Darwin state principles of phylogeny. Parental relations between living organisms can be represented by a tree where each branch represent a distinct lineage, connected by nodes that represent common ancestors. The distance between the end of the branches is proportional to the evolutive distance between the organisms they symbolized. Thus, distantly related lineages like A and B are very far in the tree while close cousins like B and C are just nearby. New lineages are continuously emerging from the tree by speciation at the same time that others get extinct (Darwin 1859).

1. Molecular phylogeny: a breakthrough for understanding Evolution and diversity of organisms Phylogeny assumes that as long as organisms are sharing a common origin and common features, it is theoretically possible to establish their family relationships. The more similar they are, the more closely related they are (Figure 1). This implies that the features used to establish the phylogeny were vertically inherited. Historically, first phylogenies were done by comparing easily describable macroscopic features such as morphological characteristics, thus excluding the use of molecular characters for classification of microorganisms. This limitation disappeared with the work of Emile

Zuckerkandl and Linus Pauling who showed that molecular characteristics (digestion patterns of proteins) can be used to establish evolutionary relationship between organisms [START_REF] Zuckerkandl | Molecules as Documents of Evolutionary History[END_REF]. To be useful for phylogenetic analysis, Zuckerkandl and Pauling postulated that the biomolecules must behave as molecular clocks, i.e. have a constant rate of mutation over long periods of time. By using fossil record data, it is even possible to calibrate the molecular clocks thus allowing to associate a quantity of time to each branch of the tree.

The concept of molecular clock was a major breakthrough that led to the most famous discovery of modern evolutionary biology [START_REF] Cavicchioli | Archaea -Timeline of the Third Domain[END_REF][START_REF] Albers | The Legacy of Carl Woese and Wolfram Zillig: From Phylogeny to Landmark Discoveries[END_REF]. In 1977, Carl Woese and George Edward Fox used the universally conserved small rRNA subunit (16S rRNA) to establish a first biologically meaningful classification of microorganisms based on molecular phylogeny [START_REF] Fox | Classification of Methanogenic Bacteria by 16S Ribosomal RNA Characterization[END_REF]. By comparing RNase T1 digestion patterns they noticed that methanogenic "bacteria" were distinct from other Bacteria (Figure 2). In fact, these data showed that methanogenic "bacteria" were phylogenetically as distant from bacteria, as bacteria were from eukaryotes. They proposed the same year in another article to reclassify these organisms as a third domain of life: the Archaebacteria [START_REF] Woese | Phylogenetic Structure of the Prokaryotic Domain: The Primary Kingdoms[END_REF] that was renamed Archaea a few years later to avoid any confusion with Bacteria [START_REF] Woese | Towards a Natural System of Organisms: Proposal for the Domains Archaea, Bacteria, and Eucarya[END_REF]. The existence of Archaea as a domain of life distinct from bacteria was supported by comparisons of complex cellular machineries (see I A 2. "Specific features of Archaea" for more details) such as the RNA polymerase (RNAP). Indeed, the archaeal RNAP is very different from the bacterial one in terms of subunits composition (Zillig 1979).

The work of Carl Woese deeply changed our understanding of the diversity and evolution of living beings, placing for the first time microorganisms in the tree of life.

It also helped to promote the evolutionary point of view in all biology fields. Moreover, C. Woese's classification approach has been successfully adapted to investigate uncultivable microbial diversity by studying small subunit rRNA amplified directly from the environment. It shed light on the fantastic diversity of microorganisms and their strong contribution to geochemical cycles.

Figure 2: Nucleic acids can be used to make phylogeny Autoradiography of a T1 ribonuclease digestion of small rRNA subunits resolved by two-dimensional electrophoresis (Pace et al., 2012). Annotations were written by Carl Woese in the course of the discovery of Archaea. The digestion pattern is depending on the sequence of the molecule considered and the accumulation of mutations is proportional to the evolutive distance between lineages. Thus, the comparison of digestion patterns between organisms gives an estimation of their evolutive divergence. But to follow this rule, biomolecules have to respect some conditions towards the organism dataset: (i) they have to be distributed in all organisms considered ; (ii) to have the same function in all of them ; (iii) to be enough informative (i.e. to have residues conserved and enough residues divergent to be compared) ; (iv) to evolved at a similar rate all along the dataset ; (v) to have an evolution rate adapted to the degree of divergence of the dataset (i.e. to evolved quick enough to resolved closely related organisms or slow enough to be informative on distantly related organisms). Biomolecules meeting these conditions are called molecular clocks and can be used to draw phylogenetic trees that reflect the relative degree of relationships of their owners.

Specific features of Archaea

After initial discovery of Archaea by Carl Woese, scientists soon realised that these organisms had some particular features in common thus corroborating their classification in a separate domain (Table 1). One such feature is the chemical composition of the lipid membranes surrounding archaeal cells. Archaea harbour original lipids that were initially discovered in the extreme halophilic Halobacteria [START_REF] Sehgal | Lipids of Halobacterium Cutirubrum[END_REF][START_REF] Marshall | The Membrane Lipids of Halobacterium Halobium[END_REF]. Instead of classical fatty acid ester-linked lipids with a glycerol-3-phosphate backbone, Archaea have isoprenoid ether-linked lipids with a glycerol-1-phosphate backbone. Some archaea from Euryarchaeota and Crenarchaeota phyla also harbour tetraether lipids that span the membrane and form a monolayer that contrast with the classical lipid bilayer of Bacteria and Eukarya [START_REF] Langworthy | Long-Chain Glycerol Diether and Polyol Dialkyl Glycerol Triether Lipids of Sulfolobus Acidocaldarius[END_REF][START_REF] Rosa | Structural Regularities in Tetraether Lipids of Caldariella and Their Biosynthetic and Phyletic Implications[END_REF] (Figure 3). All these lipid specificities have deep impact on membrane function, making them less permeable and more resistant to harsh conditions like acid or high temperature.

Archaea have a mixture of bacterial and eucaryotic traits. They distinguish themselves by special ether-linked lipids built on glycerol-1-phosphate, the ability to perform methanogenesis and an intriguing absence of pathogenicity (adapted from Cavicchioli 2011).

Table 1: Comparison of the characteristics from Bacteria, Archaea and Eukaryotes

Methanogenesis is another feature so far restricted exclusively to Archaea, meaning that all the biogenic methane, à strong greenhouse gas, is produced by Archaea.

Methanogenesis and its counterpart, methanotrophy, are complex enzymatic pathways (Figure 4). But they involve the same key enzyme, the methyl-coenzyme M reductase (Mcr), making it the marker of methane metabolizing organisms [START_REF] Evans | An Evolving View of Methane Metabolism in the Archaea[END_REF].

Methanogenesis was initially discovered in cultured Euryarchaea (see I.B Diversity and Ecology of Archaea) where at least, eight orders of methanogens exist. More recently,

Mcr was also detected in several uncultured lineages like Bathyarchaeota and Verstraetearchaeota from the TACK superphylum, indicating that methanogenesis is a more widely spread metabolic pathway in Archaea than initially thought (Adam et al. 2017). Another specificity of Archaea is the absence of pathogenic species [START_REF] Cavicchioli | Pathogenic Archaea: Do They Exist?[END_REF]. This is intriguing because Archaea are ubiquitous and they interact tightly with a wide range of eukaryotes. A good example are methanogens found in the gut of mammals or associated with plant roots [START_REF] Vogels | Association of Methanogenic Bacteria with Rumen Ciliates[END_REF][START_REF] Koskinen | First Insights into the Diverse Human Archaeome: Specific Detection of Archaea in the Gastrointestinal Tract, Lung, and Nose and on Skin[END_REF][START_REF] Großkopf | Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms[END_REF]. Moreover, in humans, besides intestines, the archaea were found in significant proportion also on skin, lungs, nose and oral Phylogeny of Archaea with methanogenic lineages highlighted with colours. The potential for the different lineages to perform methanogenesis is inferred from the presence of mcr and mcr-like genes in their genome. Most of the methanogenic lineages of archaea are belonging to the Euryarchaeota superphylum (adapted from [START_REF] Evans | An Evolving View of Methane Metabolism in the Archaea[END_REF].

cavity [START_REF] Koskinen | First Insights into the Diverse Human Archaeome: Specific Detection of Archaea in the Gastrointestinal Tract, Lung, and Nose and on Skin[END_REF]. From a molecular point of view, there is no evident explanation for absence of pathogens among archaea. Archaeal genomes encode toxins [START_REF] Mullis | Diversity, Ecology, and Prevalence of Antimicrobials in Nature[END_REF]) such as halocins in halophiles and sulfolobicin in Sulfolobales [START_REF] O'connor | Halocins and Sulfolobicins: The Emerging Story of Archaeal Protein and Peptide Antibiotics[END_REF]. They also encode secretion systems [START_REF] Makarova | Diversity and Evolution of Type IV Pili Systems in Archaea[END_REF]) and adhesion systems [START_REF] Kachlany | Nonspecific Adherence by Actinobacillus Actinomycetemcomitans Requires Genes Widespread in Bacteria and Archaea[END_REF] which are common virulent factors. And finally, archaeal genomes are dynamic [START_REF] Wagner | Mechanisms of Gene Flow in Archaea[END_REF], with horizontal gene transfers occurring from Bacteria and Eukarya (HGT), occurrence of different mobile elements and recombination systems [START_REF] Cossu | Flipping Chromosomes in Deep-Sea Archaea[END_REF]. Dynamic genomes are crucial for the acquisition of virulence factors and to drive quick, reversible phenotypic changes during the infection process. As Archaea apparently fulfil all the conditions to be pathogens, it is tempting to speculate that we have not discovered enough archaea to find the pathogenic ones. But even if this is true, by comparing the proportion of pathogens in Bacteria and Eukarya with the number of described archaea, there is still an obvious bias against pathogenicity in Archaea [START_REF] Cavicchioli | Pathogenic Archaea: Do They Exist?[END_REF].

Despite these specificities, Archaea share a lot of characteristics with Bacteria and Eukarya. Like Bacteria, Archaea lack intracellular compartmentation and perform simultaneous transcription and translation. They contain small circular genomes, with a high density of genes that can be organized in operons [START_REF] Koonin | Evolution of Genome Architecture[END_REF]. Along with these typical prokaryotic features, Archaea harbour a number of eukaryotic features.

Notably, the factors involved in cellular information processing (transcription, translation, DNA replication and repair) are homologous to eukaryotic equivalents. For example, the transcription in Archaea is performed by a single RNA polymerase (RNAP) homologous to the eukaryotic RNA Pol II [START_REF] Huet | Archaebacteria and Eukaryotes Possess DNA-Dependent RNA Polymerases of a Common Type[END_REF]) (Figure 5). Both enzymes present a similar crystal structure [START_REF] Hirata | The X-Ray Crystal Structure of RNA Polymerase from Archaea[END_REF] and require the same partners [START_REF] Werner | Evolution of Multisubunit RNA Polymerases in the Three Domains of Life[END_REF]. Archaeal Pol II-like holoenzyme interacts with the eukaryotic-like transcription factors TFB, TFE and TBP, to bind the TATA box and B recognition elements (BRE). Another example is the structure and composition of ribosome as shown by C. Woese's initial work on small ribosomal subunit. But archaeal ribosome is not the only translation actor with counterparts in Eukarya [START_REF] Bult | Complete Genome Sequence of the Methanogenic Archaeon, Methanococcus Jannaschii[END_REF][START_REF] Klenk | The Complete Genome Sequence of the Hyperthermophilic, Sulphate-Reducing Archaeon Archaeoglobus Fulgidus[END_REF]. Archaeal translation initiation factors (aIFs) aIF-1A, aIF-2, aIF-2B and aIF-5 are all homologous to those of Eukarya (eIFs) [START_REF] Benelli | Translation Initiation in Archaea: Conserved and Domain-Specific Features[END_REF] as well as elongation factors (aEF) aEF-1α, aEF-2 and the release factor aRF-1. Moreover, their sequential actions during translation are reminiscent of their eukaryotic homologs.

Since the initial recognition of Archaea as a separate domain of life in 1977, much has been learned about their molecular biology. We presently know that these organisms harbour their own specific molecular characteristics but also a curious mix of bacterial and eukaryotic traits. How these traits have been acquired throughout the history of archaeal lineage and how archaea contributed to the emergence of complex, eukaryotic life forms are fascinating questions for future studies.

B. Diversity and Ecology of Archaea

Inhabitants of extreme environments

Traditionally, Archaea have the image of extremophiles. Indeed, most of the initially described archaea where harbouring at least one criterion of extremophily (Figure 6).

For example, halophilic archaea thrive in highly salty environments like the dead sea, that were thought to be unsuitable for life. This ability to grow in such (from anthropocentric standpoint) a hostile environment stimulated researchers' curiosity and rapidly a lot of extreme halophilic archaea were isolated [START_REF] Oren | Molecular Ecology of Extremely Halophilic Archaea and Bacteria[END_REF], making

Halobacteria one of the best described group of archaea.

A lot of archaea are thermophiles or hyperthermophiles, meaning that they grow optimally between 50 and 80°C or above 80°C, respectively. The high diversity of archaea sampled in terrestrial hot springs and hydrothermal vents [START_REF] Stetter | History of Discovery of the First Hyperthermophiles[END_REF] challenged our vision of life boundaries, showing that even at these temperatures, often in anaerobic or sometimes acidic conditions [START_REF] Brock | Sulfolobus: A New Genus of Sulfur-Oxidizing Bacteria Living at Low PH and High Temperature[END_REF][START_REF] Zillig | Thermoproteales: A Novel Type of Extremely Thermoacidophilic Anaerobic Archaebacteria Isolated from Icelandic Solfataras[END_REF], life is possible.

To complete the collection, Archaea can also be extreme acidophiles like Sulfolobus acidocaldarius that grows optimally at pH 2 (equivalent of acidity in human stomach)

or the most acidophilic organism Picrophilus torridus that grows optimally at pH 0,7

and that can even grow at negative pH values [START_REF] Brock | Sulfolobus: A New Genus of Sulfur-Oxidizing Bacteria Living at Low PH and High Temperature[END_REF][START_REF] Fütterer | Genome Sequence of Picrophilus Torridus and Its Implications for Life around PH 0[END_REF]) ; extreme psychrophiles like Methanogenium frigidum and Methanococcoides burtonii that grows optimally at 15°C and 23°C but that can even grow at slightly negative temperatures [START_REF] Franzmann | A Methanogenic Archaeon from Ace Lake, Antarctica: Methanococcoides Burtonii Sp[END_REF]1997) ; extreme piezophiles like Thermococcus barophilus that grows optimally at 40 MPa that is 400 times the atmospheric pressure [START_REF] Marteinsson | Thermococcus Barophilus Sp. Nov., a New Barophilic and Hyperthermophilic Archaeon Isolated under High Hydrostatic Pressure from a Deep-Sea Hydrothermal Vent[END_REF] ; extreme alkaliphiles like Natronomonas pharaonis that grows up to pH 11 [START_REF] Soliman | Halobacterium Pharaonis Sp. Nov., a New, Extremely Haloalkaliphilic Archaebacterium with Low Magnesium Requirement[END_REF]. But the image of extremophiles that sticks to Archaea is actually misleading, because a lot of them live in much more "reasonable" conditions (DeLong 1998). 2. Your discrete neighbours

The huge progress in the last 20 years in DNA sequencing technologies completely changed our vision of microbial diversity. Coupled with metagenomic approaches, the microbial diversity can be easily analysed in any environment, with no restriction to cultivable organisms. These analyses revealed that archaea are present in all kinds of environments and not just in extreme ones (Adam et al. 2017). For example, a significant part of ocean microbial diversity is made of archaea [START_REF] Karner | Archaeal Dominance in the Mesopelagic Zone of the Pacific Ocean[END_REF][START_REF] Corte | Spatial Distribution of Bacteria and Archaea and Amo A Gene Copy Numbers throughout the Water Column of the Eastern Mediterranean Sea[END_REF]. While the proportion of archaea in different samples from marine water is strongly variable, it seems that their numbers increase proportionally with the depth. This indicated that Archaea may be major players in cycling of organic matter in the deep sea [START_REF] Karner | Archaeal Dominance in the Mesopelagic Zone of the Pacific Ocean[END_REF]. A good example of the ubiquity and ecological importance of Archaea is the Thaumarchaeota phylum that was discovered in sea water and initially described as "non-thermophilic Crenarchaeota" [START_REF] Delong | Archaea in Coastal Marine Environments[END_REF][START_REF] Fuhrman | Novel Major Archaebacterial Group from Marine Plankton[END_REF][START_REF] Brochier-Armanet | Mesophilic Crenarchaeota: Proposal for a Third Archaeal Phylum, the Thaumarchaeota[END_REF].

Interestingly, these organisms encoded ammonia monooxygenase an enzyme that catalyses the oxidation of ammonia, the first step of biological nitrification, a process which was believed to be exclusive to bacteria. The discovery of these ammoniaoxidising archaea at a high proportion in marine environment and soil, highlighted the fact that Archaea are important contributors to the global nitrogen cycling [START_REF] Offre | Archaea in Biogeochemical Cycles[END_REF]. More recently, Thaumarcheota have also been found to participate significantly to human microbiome. They are found principally on the skin, while Euryarchaeota are dominant in the nose and digestive tract, and Woesearchaeota in lungs [START_REF] Koskinen | First Insights into the Diverse Human Archaeome: Specific Detection of Archaea in the Gastrointestinal Tract, Lung, and Nose and on Skin[END_REF][START_REF] Borrel | The Host-Associated Archaeome[END_REF]) (Figure 7). Taxonomic distribution of archaea on human body. Archaea were sampled: from lung through bronchoalveolar lavage (BAL) (blue), from skin (yellow), from nose (red), from gastrointestinal tract (GIT) (brown). Similarities between datasets are plotted as a network (adapted from [START_REF] Koskinen | First Insights into the Diverse Human Archaeome: Specific Detection of Archaea in the Gastrointestinal Tract, Lung, and Nose and on Skin[END_REF].

Numerous other new phyla of Archaea have been discovered from metagenomics studies (Figure 8). These new proposed phyla expanded from initially 2 (Crenarchaeota and Euryarchaeota) to 27 the number of major archaeal lineages (Baker et al. 2020).

Some of the more recently discovered phyla include Bathyarchaeota methanogens which were recovered from deep aquifers [START_REF] Evans | Methane Metabolism in the Archaeal Phylum Bathyarchaeota Revealed by Genome-Centric Metagenomics[END_REF] and Vestraetearchaeota methanogens from bioreactors [START_REF] Vanwonterghem | Methylotrophic Methanogenesis Discovered in the Archaeal Phylum Verstraetearchaeota[END_REF]. Metagenomic studies have also allowed the surprising discovery of a wealth of nanosized archaea accounting for about a third of the archaeal diversity! These organisms contain atypical genomic features and thus could not be recovered initially using Archaea-specific 16S rDNA amplification. They were regrouped into the proposed DPANN superphylum, composed of: Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, Nanoarchaeota [START_REF] Rinke | Insights into the Phylogeny and Coding Potential of Microbial Dark Matter[END_REF], Woesearchaeota, Pacearchaeota, Micrarchaeota, Huberarchaeota and potentially, Altiarchaeota (Baker et al. 2020). DPANN were detected in a wide range of environments. They generally have small genomes of about 1 Mbp and they seem to have underwent intensive horizontal gene transfers. These characteristics suggested that DPANN are fast-evolving species with parasitic or symbiotic lifestyle.

Since their discovery by Carl Woese, Archaea have definitively earned a major place in the tree of life over the last 40 years. Their ubiquity, specific metabolisms, eukaryotic features and importance in geochemical cycles make them of great interest. But despite great progresses to sample their diversity and understand their ecology, the principal forces driving this diversification and adaptation of Archaea to a wide range of environments is still an interesting and largely unresolved question. C. Evolution of Archaea

Phylogenetic tree of Archaea

The fast expansion of archaeal lineages has strongly complexified the initial dichotomic archaeal tree (Figure 8), divided between Euryarchaeota and Crenarchaeota. Newly discovered uncultured lineages provide lots of information about archaeal ecology, metabolism and evolution but are challenging to place on the phylogenetic tree. For instance, it is still unclear if the monophyly of DPANN is real or just a long branch artefact (LBA) due to their propensity to evolve fast. Indeed, such fast-evolving species accumulate mutations, making them more similar for tree construction algorithms that artificially group them, often at the base of the tree, closest to the outgroup.

The profusion of new archaea is also a taxonomic problem. Taxonomic ranks assigned to archaeal lineages are not necessarily homogeneous in term of genomic divergence.

This problem, general to all microorganisms, can be explained by the early phenotypic classifications that often reflect convergence rather than evolutionary relationships and the over-splitting of extensively studied groups irrespectively of their real divergence.

The emergence of the new Genome Taxonomy DataBase (GTDB) is a recent attempt to try to standardize and equalize classification all along the tree of life [START_REF] Dh | A Standardized Bacterial Taxonomy Based on Genome Phylogeny Substantially Revises the Tree of Life[END_REF](Parks et al. , 2020)). To date four major phyla of archaea are recognize: Euryarchaeota, TACK, DPANN and the recently described Asgards. This last group is particularly important from an evolutionary point of view, as detailed below.

The position of Archaea in the tree of life and Eukaryogenesis

The position of the Archaea on the tree of life with respect to Eukaryotes, is of special interest to understand eukaryogenesis, one of the major evolutionary questions.

Indeed, two contradictory hypotheses, highly dependent on the archaeal phylogeny, have been proposed. In the first one, Eukarya are sister group of Archaea, giving the "Woese" three-domains (3D) tree of life. In the second one, Eukarya emerged from within Archaea, giving a two-domains (2D) tree of life where Archaea are not monophyletic. This hypothesis is known as the "Eocyte" one, according to the first group of archaea (current Crenarchaeota) proposed to be at the origin of Eukarya [START_REF] Lake | Eocytes: A New Ribosome Structure Indicates a Kingdom with a Close Relationship to Eukaryotes[END_REF]. The 2D and 3D tree topologies, imply distinct scenario of eukaryogenesis.

A 3D tree of life means that complex features of Eukarya, like the spliceosome, the nucleus, the ability to perform meiosis or mitosis, were acquired step by step by a protoeukaryote after the eukaryotic lineage had split from archaeal one (Figure 9).

Coupled with the crucial endosymbiosis of an alphaproteobacterium to form mitochondria, this complexification would conduct to modern eukarya [START_REF] Spang | Genomic Exploration of the Diversity, Ecology, and Evolution of the Archaeal Domain of Life[END_REF]. Conversely, in the 2D topology, many complex eukaryal features must have emerged relatively fast , in the relatively short period between splitting from archeal lineage and the last common eukaryotic ancestor. This fast evolution period could be explained by the early acquisition of mitochondria, that would dramatically A three-domains tree of life implies that Eukaryotes evolved their specific features (intracellular compartmentation, cytoskeleton, organelles) independently of Archaea, while in a two-domains tree of life all these features were evolved from an archaeon probably already very similar to modern archaea. The recent discovery of Asgards archaea, that share characteristics with Eukaryotes, has given support to the two-domains tree of life (C). However, the topology of the tree of life is still matter of intense debates.

relax evolutive constrains by providing the necessary burst of energy (Lane and Martin 2010) thus allowing the development of complex features.

The 2D topology gained in notoriety with the discovery of "eukaryote signature proteins" (ESPs) in some Thaumarchaeota [START_REF] Ettema | An Actin-Based Cytoskeleton in Archaea[END_REF][START_REF] Yutin | Archaeal Origin of Tubulin[END_REF][START_REF] Lindås | A Unique Cell Division Machinery in the Archaea[END_REF]) and, especially, in the recently discovered Asgardarchaeota superphylum [START_REF] Spang | Complex Archaea That Bridge the Gap between Prokaryotes and Eukaryotes[END_REF]. Asgards formed a monophyletic group with Eukarya that was branching in TACK archaea. Moreover, Lokiarchaeota harboured 29% of bacterial genes and about 3% of eukaryotic genes. Among these genes, a lot of ESPs were present, notably: (i) five actin homologues, (ii) a lot of small GTPases from the Ras superfamily (2% of lokiarchaea genome) known to regulate cytoskeleton dynamics (iii) a cluster of ESCRT genes and other genes involved in multivesicular endosome pathway, suggesting an active vesicle trafficking system. These findings were confirmed later with the Thorarchaeota, Heimdallarchaeota, Odinarchaeota, Helarchaeota asgards metagenomes that enriched the pantheon of Nordic gods [START_REF] Seitz | Genomic Reconstruction of a Novel, Deeply Branched Sediment Archaeal Phylum with Pathways for Acetogenesis and Sulfur Reduction[END_REF][START_REF] Zaremba-Niedzwiedzka | Asgard Archaea Illuminate the Origin of Eukaryotic Cellular Complexity[END_REF][START_REF] Seitz | Asgard Archaea Capable of Anaerobic Hydrocarbon Cycling[END_REF]) and with the isolation and sequencing of the lokiarchaeon Prometheoarchaeum syntrophicum [START_REF] Imachi | Isolation of an Archaeon at the Prokaryote-Eukaryote Interface[END_REF]).

However, the 2D topology is still controversial with plenty different opinions. Recent studies argue against 2D topology because of (i) the presence of fast-evolving species in the dataset; (ii) a subset of universal markers supports the 3D topology; (iii) asgard metagenomes are contaminated (Da Cunha et al. 2017;[START_REF] Drolet | DNA Topoisomerases: Methods and Protocols[END_REF]; (iv) phylogenetic signal is too weak in protein sequences to resolve a universal tree contrary to structural motifs (Harish and Kurland 2017a;2017b). Spang et al. rejected all these arguments [START_REF] Spang | Asgard Archaea Are the Closest Prokaryotic Relatives of Eukaryotes[END_REF], as other authors [START_REF] Williams | Phylogenomics Provides Robust Support for a Two-Domains Tree of Life[END_REF]. From another perspective, it has been claimed that only the placement of the root of the Archaea-Eukarya phylogeny can discriminate between a 2D or a 3D topology [START_REF] Fournier | A Briefly Argued Case That Asgard Archaea Are Part of the Eukaryote Tree[END_REF]. Indeed, the root is essential to orientate the sequence of evolutionary events and thus, discriminate between ancestral and recent characters. Recent studies currently propose different rooting: between Euryarchaeota and TACK, within Euryarchaeota, or between DPANN and other archaea (Petitjean et al. 2014;Raymann, Brochier-Armanet, and Gribaldo 2015;Williams et al. 2017). Further metagenomes and phylogenetic analyses will hopefully allow to distinguish between the different points of view.

II.

Three-dimensional structure of DNA and chromosomes [START_REF] Verma | Architecture of the Escherichia Coli Nucleoid[END_REF]. Knowing that E. coli measures on average 1 µm long by 0.25 µm large, resulting approximatively in a 0.05 µm 3 cylinder, it is crucial to compact DNA to fit into the cell and even more by considering that the volume of nucleoid is less than 1 µm 3 (Figure 10). This is also true in the nucleus of Eukaryotes or in every other organism (Figure 11). To fit in such a small space, DNA is compacted at least 1000-fold by supercoiling and interactions with various architectural proteins.

The genome expression is a tightly regulated process involving numerous proteins and chemical signals. DNA compaction is playing a key role in this process by modulating the accessibility of transcription machinery to different genome regions. A well-known To fit in nucleus, chromosomes from eukaryotes must be compacted more than a hundred-time in length. This compaction involves different levels: first DNA is wrapped around nucleosomes to form the bead on a string structure; then beads are packed together in a 30 nm chromatin fibre; these fibres are folded in higher-order structures by structural proteins leading to highly condensed chromosomes (adapted from Felsenfeld and Groudine 2003).

example is the heterochromatin in Eukaryotes that mostly contains non-transcribed genes [START_REF] Janssen | Heterochromatin: Guardian of the Genome[END_REF]. Functional compaction was discovered progressively by fluorescence in situ hybridization (FISH) and chromosome conformation capture methods (3C, Hi-C) [START_REF] Kempfer | Methods for Mapping 3D Chromosome Architecture[END_REF]. These findings revealed that the genomes are highly organized at different scales as detailed in the following sections.

Chromosomal domains

In Bacteria, chromosome can be divided in large Mb domains, grouping multiple topological domains, that have a higher probability of interaction with each other than with other parts of the genome. This level of genome organization in Bacteria was first brought to light in Escherichia coli, thanks to fluorescence in situ hybridization (FISH) showing a co-localization of some genomic markers in the nucleoid (H. [START_REF] Niki | Dynamic Organization of Chromosomal DNA in Escherichia Coli[END_REF][START_REF] Niki | Polar Localization of the Replication Origin and Terminus in Escherichia Coli Nucleoids during Chromosome Partitioning[END_REF]. In this way, two macrodomains were discovered and named Ori and Ter because they encompassed the origin and termination of replication of the chromosome. Two other macrodomains framing the Ter macrodomain, were discovered later on by measuring the rate of recombination of two att sites from the lambda phage inserted at different places in the chromosome [START_REF] Valens | Macrodomain Organization of the Escherichia Coli Chromosome[END_REF]). These four macrodomains are covering all the E. coli genome except two non-structured regions, called NS-left and NS-right (Figure 10 [START_REF] Dame | Chromosome Organization in Bacteria: Mechanistic Insights into Genome Structure and Function[END_REF][START_REF] Val | A Checkpoint Control Orchestrates the Replication of the Two Chromosomes of Vibrio Cholerae[END_REF].

In Eukaryotes, chromosomes occupy a precise place in the nucleus rather than being all mixed together [START_REF] Cremer | Chromosome Territories[END_REF]. Each volume occupied by a specific chromosome in the nucleus is defined as a chromosome territory, in which, intrachromosomic interactions are favoured. [START_REF] Takemata | Physical and Functional Compartmentalization of Archaeal Chromosomes[END_REF].

As in Eukaryotes, highly transcribed genes of Sulfolobus and replication origins locate in the A compartment while mobile genetic elements, poorly and conditionally expressed genes are in the B compartment (Takemata and Bell 2020) (Figure 12). The second study has been performed in Haloferax volcanii, Halobacterium salinarum and T. kodakarensis, that belong to the Euryarchaeota phylum, and shows a more bacteriallike chromosome organization with no A/B compartmentalization [START_REF] Cockram | Euryarchaeal Genomes Are Folded into SMC-Dependent Loops and Domains, but Lack Transcription-Mediated Compartmentalization[END_REF]). These discrepancies between Euryarchaeota and Crenarchaeota suggest an interesting diversity of chromosome architectures in Archaea, that is waiting to be further explored.

Topological domains or chromosomal interaction domains

To take advantage of the torsional energy stored in DNA, chromosomes supercoiling is tightly regulated and chromosomes are compartmentalized into topological domains [START_REF] Sinden | Chromosomes in Living Escherichia Coli Cells Are Segregated into Domains of Supercoiling[END_REF]. This level of chromosome organization seems to be common to every organism (Figure 13). However, it can differ in terms of domain size and importance according to the species considered. Topological domains are Chromosomes from eukaryotes are confined into nucleus where they each occupy a defined territory and are split in a highly transcribed A compartment and a less transcribed B compartment (not shown on this schema) (Ba). At a smaller scale, eukaryotic chromosomes are subdivided into topologically associating domains (TADs) (Bb). TADs are formed by loop extrusion through SMC-complex until collision with CTCF-DNA complex occurs. At the smallest scale, sub-TAD domains can be detected corresponding to one to five insulated genes (Bc).

delimited by topological barriers that block the diffusion of supercoils. This allows the coexistence of various topological states in the same chromosome. Topological barriers can be made of combinations of (i) proteins that wrap or loop DNA (histones, NAPs, CTCF, SMC) ; (ii) palindromic repeats (BIMES elements in E. coli) ; (iii) actively transcribed regions [START_REF] Verma | Architecture of the Escherichia Coli Nucleoid[END_REF]. All these elements cooperate in a complex and dynamic manner, that is not well understood for the moment.

DNA loops

Inside topological domains, DNA is organized depending on the prevalence of packaging proteins like histones, NAPs or SMC proteins. In Escherichia coli about half of the chromosome is constrained by NAPs (Bliska and Cozzarelli 1987) and the rest is forming plectonemic loops, while nearly all the DNA is constrained by histones in Eukaryotes (Sinden, Carlson, and Pettijohn 1980). This creates various architectures, but a common point is the importance of DNA loops that create a favourable environment for a lot of DNA transactions by promoting DNA contacts. These loops can for instance (i) bring together promoters and transcription terminators, thus promoting RNApolymerase recycling and increasing transcription rate (Figure 14a) (O'Sullivan 2004).

DNA loops can also (ii) bring enhancers or silencers in close proximity to a promoter to regulate its transcription (Figure 14b

Chromosome architects

Chromosome condensation and organization is dependent upon different classes of proteins that bend, wrap, link and cover DNA. They cooperate to form the chromatin, a highly packaged and organized state of the DNA that can take several different forms across the tree of life. The main proteins families involved in the formation of chromatin are described in this section. The list of most known nucleoid-associated proteins and their properties is found in Table 2.

Histones are surely the most famous chromosome packaging proteins. These highly basic proteins exhibit a positive charge at physiological pH that confers them a strong ability to bind DNA. They are sharing a general structure called the "histone-fold", made of three α-helices, two short and one long, connected by loops [START_REF] Arents | The Nucleosomal Core Histone Octamer at 3.1 A Resolution: A Tripartite Protein Assembly and a Left-Handed Superhelix[END_REF]. Ubiquitous in Eukaryotes [START_REF] Soo | Slaying the Last Unicorn: Discovery of Histones in the Microalga Nanochlorum Eucaryotum[END_REF], histones assemble in an octamer made of two copies of each core histones H2A, H2B, H3 and H4 [START_REF] Luger | Crystal Structure of the Nucleosome Core Particle at 2.8 A Resolution[END_REF]). The octamer constrains roughly 150 bp DNA by wrapping it twice in a lefthanded superhelical manner (Figure 15). Distribution of nucleosomes along the DNA molecule forms the famous beads-on-a-string structure [START_REF] Woodcock | Structural Repeating Units in Chromatin. I. Evidence for Their General Occurrence[END_REF]. The stability of the octamer is enhanced by the binding of the histone linker H1 at the entry and the exit of the nucleosome [START_REF] Fyodorov | Emerging Roles of Linker Histones in Regulating Chromatin Structure and Function[END_REF].

This H1-nucleosome structure is thought to contribute in Eukaryotes, to the further compaction of DNA in a 30 nm fibre [START_REF] Thoma | Involvement of Histone H1 in the Organization of the Nucleosome and of the Salt-Dependent Superstructures of Chromatin[END_REF][START_REF] Robinson | Structure of the "30 Nm" Chromatin Fibre: A Key Role for the Linker Histone[END_REF]. N and C-terminal tails of core histones extrude from the octamer where they can participate to interactions between nucleosomes (Shogren- [START_REF] Shogren-Knaak | Histone H4-K16 Acetylation Controls Chromatin Structure and Protein Interactions[END_REF]).

These interactions are regulated by post-translational modifications (among others: acetylation, methylation, phosphorylation, ubiquitination, and biotinylation) that have a wide variety of effect on chromatin condensation [START_REF] Tolsma | Post-Translational Modifications and Chromatin Dynamics[END_REF]. For instance, histone-tails acetylation inhibits nucleosome-nucleosome interaction, thus decondensing chromatin.

This Eukaryotes. This is especially true for transcription that is directly sensitive to the NAPsconstrained supercoiling, and the ability of NAPs to control promoters and enhancers/silencers accessibility.

Archaea harbour more diverse chromatin organizations than Bacteria and Eukaryotes, based both on histones and NAPs. Indeed, histones are not solely present in Eukaryotes, almost every phylum of archaea except Crenarchaeota possess histones [START_REF] Sandman | HMf, a DNA-Binding Protein Isolated from the Hyperthermophilic Archaeon Methanothermus Fervidus, Is Most Closely Related to Histones[END_REF][START_REF] Henneman | Structure and Function of Archaeal Histones[END_REF]Sanders, Marshall, and Santangelo 2019).

Histones from archaea are homologous to H3 and H4 eukaryotic histones and share the same core of α-helices. But in contrast to eukaryotic histones, they are devoid of tails subjected to post-translational modifications (Figure 15). Moreover, early study of Methanothermus fervidus and Methanobacterium thermoautotrophicus, suggested that DNA is wrapped around histone tetramers that constrained only about 60 bp [START_REF] Pereira | Archaeal Nucleosomes[END_REF]. This was confirmed later on by micrococcal nuclease digestion coupled with deep sequencing (MNase-seq) in H. volcanii [START_REF] Ammar | Chromatin Is an Ancient Innovation Conserved between Archaea and Eukarya[END_REF] or by structural studies of M. fervidus histones with DNA (Mattiroli et al. 2017). Remarkably, archaeal nucleosomes can constrain either negative (left-handed) or positive (righthanded) supercoils similar to what is observed in Eukaryotes [START_REF] Musgrave | DNA Binding by the Archaeal Histone HMf Results in Positive Supercoiling[END_REF][START_REF] Musgrave | Negative Constrained DNA Supercoiling in Archaeal Nucleosomes[END_REF]. Another interesting feature of archaeal histones is their ability to organize in polymers of homodimers or heterodimers from various size beyond the initially described tetramer and instead of the classic eucaryotic octamer (Maruyama et al. 2013). This peculiar structure unique to archaea, can imply up to sixteen histone dimers in T. kodakarensis, covering 480 bp by 30 bp increment (Maruyama et al. 2013). Similar histone organization, now called "hypernucleosome" (Figure 16), has also been described for M. fervidus and predicted from amino-acid sequence similarities in other species suggesting that it is a general mechanism in Archaea [START_REF] Henneman | Structure and Function of Archaeal Histones[END_REF]. This raised the possibility that modulation of hypernucleosome length could be one of the main regulators of the transcription in Archaea, reminiscent to eukaryotic heterochromatin dynamics [START_REF] Henneman | Mechanical and Structural Properties of Archaeal Hypernucleosomes[END_REF].

As in Bacteria, NAPs are numerous and highly diversified in Archaea (Zhang, Guo, and Huang 2012; Sanders, Marshall, and Santangelo 2019) (Figure 17). The most studied one is the acetylation lowers binding affinity protein (Alba) [START_REF] Bell | The Interaction of Alba, a Conserved Archaeal Chromatin Protein, with Sir2 and Its Regulation by Acetylation[END_REF]. This NAP was discovered in Sulfolobus acidocaldarius and then turned out to be widely spread in Archaea through the Sac10b family, especially in thermophiles [START_REF] Green | Histone-like Protein in the Archaebacterium Sulfolobus Acidocaldarius[END_REF][START_REF] Forterre | Identification of the Gene Encoding Archeal-Specific DNA-Binding Proteins of the Sac10b Family[END_REF]. Alba can bind both DNA and RNA, thus playing a major role in genome organization and RNA stabilization. When bound to DNA, Alba can coat it and constrain negative supercoiling in addition to bridging distant sequences by its ability to dimerize. Potential regulation of Alba function in vivo through post-translational modifications is still debated (Sanders, Marshall, and Santangelo 2019). Other highly studied archaeal NAPs include Sul7 and Cren7 specific for Crenarchaeota where they serve as substitutes for histones [START_REF] Driessen | Nucleoid-Associated Proteins in Crenarchaea[END_REF], as well as the HU homolog HTa in Thermoplasmata (Hocher et al. 2019). Some NAPs can also derive from transcription factors as the transcription regulator of the maltose-like system 2 (TrmBL2) [START_REF] Lee | Characterization of the TrmB-like Protein, PF0124, a TGM-Recognizing Global Transcriptional Regulator of the Hyperthermophilic Archaeon Pyrococcus Furiosus[END_REF][START_REF] Wierer | TrmBL2 from Pyrococcus Furiosus Interacts Both with Double-Stranded and Single-Stranded DNA[END_REF]).

This NAP is ubiquitous in Thermococcales and sporadically present in some DPANN and Bacteria. In Thermococcales, TrmBL2 is a major architect of the chromatin that competes with histones packaging by creating rigid protein-DNA filaments that cannot be transcribed (Maruyama et al. 2011;[START_REF] Efremov | Transcriptional Repressor TrmBL2 from Thermococcus Kodakarensis Forms Filamentous Nucleoprotein Structures and Competes with Histones for DNA Binding in a Salt-and DNA Supercoiling-Dependent Manner[END_REF]. [START_REF] Meselson | THE REPLICATION OF DNA IN ESCHERICHIA COLI[END_REF]. This mechanism implies the opening of the double-helix to allow synthesis of new complementary strands according to canonical base-pairing possibilities. More generally, DNA transactions like replication, transcription or recombination all require the opening of the double helix. Because of their circular state and/or the contact with proteins, DNA ends are not free to rotate during these processes. For instance, the growing RNA attached to RNA-polymerase during transcription generates a drag force thus braking DNA rotation [START_REF] Koster | Cellular Strategies for Regulating DNA Supercoiling: A Single-Molecule Perspective[END_REF]) (Figure 18). This phenomenon is even stronger in prokaryotes, where replication and transcription are coupled, creating a bulkier scaffold, or when a membrane protein is produced, anchoring the DNA to the membrane. A less obvious case is the possibility for nascent RNA to intertwine with DNA during transcription, creating a chimeric local duplex (Rloop by opposition to D-loop occurring with DNA) [START_REF] Drolet | Growth Inhibition Mediated by Excess Negative Supercoiling: The Interplay between Transcription Elongation, R-Loop Formation and DNA Topology[END_REF]) that is taking the place of the complementary strand on DNA helix. The resulting constrained DNA is put under mechanical tension anytime the double helix is opened. These tensions could be lethal if they are not maintained to a level compatible with DNA transactions. Cells dispose of plenty of actors dedicated to this purpose, that are detailed further in this manuscript (section II. C. 1.).

Topological parameters

The formalization of DNA three-dimensional structure transitions and the action of cell actors to deal with them, gave rise to a new field, the DNA Topology. While complex in many respects, topology of a constrained DNA molecule relies on a relatively simple equation: Lk = Tw + Wr. In this equation, Lk is the number of topological links that can be defined as the number of times that one strand is crossing the other one for a DNA constrained in one plane; Tw is the number of twists that only depends on the size of the molecule (N) in bp and the pitch of the double helix (h), Tw = N/h ; Wr is the number of writhes that correspond to the number of times that the axis of the double helix is crossing the DNA plane. Practically, Tw reflects the number of coils and Wr the number of supercoils. For relaxed DNA molecule Wr is null. Overwinding introduces positive supercoils (overtwisting) while underwinding introduces negative supercoils (untwisting). These supercoils can be free or constrained by proteins. This leads to two kinds of supercoiling: (i) the toroidal where DNA is wrapped around a protein, typically a histone ; (ii) the plectonemic where DNA is interwound, which is the classical state of circular supercoiled DNA in solution (Figure 19). To compare the topological state of different DNA molecules it is convenient to use a value that is not dependant of their respective size. The superhelical density (σ) is commonly used for this purpose. This value is defined by the difference of topological links between a molecule and its relaxed state (Lk0), normalized by the Lk0: σ = (Lk -Lk0) / Lk0. By doing the approximation that the Tw is constant in average for a given molecule, this equation results in a measure of its degree of supercoiling normalized by its length. The σ is positive for a positively supercoiled DNA and negative for a negatively supercoiled one.

Physiological consequences of DNA supercoiling

The sense of supercoiling is of special importance for DNA stability. The minimum energetic state for a DNA molecule is the relaxed state. In other words, every supercoil in a DNA corresponds to an excess of energy that could promote the opening of the double helix for a negative supercoiling or prevent it for a positive supercoiling, respectively, by decreasing or increasing base stacking. Thus, supercoils are a precious reserve of energy for the cell favouring or preventing DNA transactions to occur.

This means that all proteins that require DNA melting are more likely to interact with At high temperature, molecular motion is thought to replace negative supercoiling as major DNA melting factor (Forterre 2002). Thus, negative supercoiling is theoretically not essential for DNA transactions and could even be deleterious by promoting excessive DNA denaturation. On the other hand, positive supercoiling can become advantageous at high temperature by stabilizing double-stranded DNA. However, in vitro work produced contradictory results about specific DNA topology requirements for transcription at high temperature. Transcription at 78°C from Sulfolobus cell-free transcription system is not sensitive to the template DNA topology (Bell et al. 1998).

Conversely, the cell-free transcription system from Pyrococcus furiosus is more efficient on negatively supercoiled DNA at 70°C and 90°C (Hethke et al. 1999). But no comparative analysis of promoters structures was performed to determine whether they can explain the observed differences instead of transcription machineries specificities. In addition, in vivo plasmid topology studies highlighted species-specific superhelical density variations in response to growth-phase and temperatures (López-García and Forterre, 1997Forterre, , 1999) ) but underlying molecular mechanisms remain elusive.

Beyond its importance for transcription, supercoiling is also implied in the condensation of chromosome. While almost all the supercoiling is constrained by nucleosomes in Eukaryotes (toroidal supercoiling), in E. coli only about 60% of supercoiling are constrained and the 40% remaining are free (plectonemic supercoiling) (Bliska and Cozzarelli 1987). Plectonemic supercoiling is not compacting DNA efficiently (2.5 fold) compared to toroidal supercoiling around NAPs and nucleosomes and higher structures of chromatin, however both types of supercoiling (plectonemic and toroidal) should be considered for genome compaction [START_REF] Boles | Structure of Plectonemically Supercoiled DNA[END_REF].

Considering 20) (Bates and Maxwell 2005) Topoisomerases families were named from I to VIII according to the order by which they were discovered. By chance, this order is coherent with their mechanistic activities that involved the cleavage of one strand for odd topoisomerases (Type I topoisomerases) or two strands for even topoisomerases (Type II topoisomerases). This principle has been conserved for the naming of all more recently discovered topoisomerases (Table 3). In Bacteria, superhelical density is maintained around -0.06 in exponential growth phase, with about 40% (-0.025) of free supercoils. The remaining 60% (-0.035) are constrained, mainly by histone-like proteins (Bliska and Cozzarelli 1987). In Eukaryotes, DNA seems to be supercoiled in the same extent but with almost all the supercoils constrained by nucleosomes (Sinden, Carlson, and Pettijohn 1980). Unconstrained DNA is relaxed in average, with hot-spots of supercoiling near transcription start sites (TSS) of expressed genes (Kouzine et al. 2013;[START_REF] Krassovsky | Genome-Wide Profiling Reveals Functional Interplay of DNA Sequence Composition, Transcriptional Activity, and Nucleosome Positioning in Driving DNA Supercoiling and Helix Destabilization in C. Elegans[END_REF].

The negative superhelical density value of -0.06 favours DNA melting and thereby allows DNA transactions. This implies that DNA supercoiling must be tightly regulated to fit within a narrow range compatible with different DNA-templated processes. These processes, however, by themselves directly modify DNA supercoiling. For instance, transcription requires DNA strands separation for DNA decoding by the RNA polymerase (RNAP) and ribonucleotides incorporation. The nascent mRNA causes molecular crowding/drag that prevents the free rotation of the RNAP around the DNA.

Consequently, it is the DNA that is forced to rotate to compensate for the mechanical tension that is introduced by transcription. The result is the accumulation of positive supercoils ahead and of negative supercoiling behind of the RNAP, as proposed in the "twin supercoiled-domain" theory (Liu and Wang 1987). This phenomenon is enhanced by everything that can increase the drag force of the nascent mRNA, such as transcript splicing in Eukaryotes or coupling of translation and transcription in Prokaryotes. In the latter case, translation of proteins bound to the membrane, creates a strong anchor that will completely block RNAP rotation [START_REF] Koster | Cellular Strategies for Regulating DNA Supercoiling: A Single-Molecule Perspective[END_REF].

DNA replication can be cited as a second example of a process in course of which, as the replication machinery extends the new-synthesized strand, DNA template must be opened. In a similar way to transcription, because the machinery is not free to rotate along DNA, positive supercoiling accumulates ahead of the replicative fork. But in contrast to transcription, negative supercoiling can not accumulate behind the replicative fork as the newly synthesized strand has a free extremity (Postow et al. 2001).

In both cases, the resulting accumulation of supercoiling acts as a physical barrier that can stop replication, transcription, and any other DNA transactions requiring DNA melting. Thus, it is critical for the cell to constantly remove excessive supercoiling. Some topoisomerases are dedicated to this purpose. In Bacteria, topoisomerase I (topo I, also called ω or swivelase) removes negative supercoiling while DNA gyrase (hereafter gyrase) introduces negative supercoiling. Their antagonistic activities are balanced by transcriptional regulation. The promoter of topA, encoding topoisomerase I, is induced by negative supercoiling and conversely, gyrA and gyrB promoters are induced by DNA relaxation. This leads to a homeostasis of supercoiling where the two main regulators, topoisomerase I and gyrase, through their activity directly up-regulate the expression of their antagonist (Figure 21) (Menzel and Gellert 1983). III. Gyrase and reverse gyrase: two atypical topoisomerases Among all topoisomerases, gyrase and reverse gyrase distinguish themselves by their unique activities of active supercoiling. They are not removing tensions by passive DNA relaxation, instead, they are introducing supercoils into DNA through ATP-dependant processes. This makes these enzymes of special interest to quickly change genome supercoiling level in response to environmental changes and cell's requirements. A. DNA gyrase

1. An ubiquitous and critical enzyme in Bacteria

In 1976, Gellert and collaborators observed that phage λ can recombine with relaxed closed-circular substrates only in the presence of ATP and Escherichia coli cell extract.

Knowing that this reaction can only occur on negatively supercoiled DNA, they presumed that something in the E. coli cell extract can introduce negative supercoils in these relaxed substrates. They purified the responsible enzyme, that they called DNA gyrase, and demonstrated that its activity is strictly ATP and MgCl2 dependant (Gellert, Mizuuchi, et al. 1976). Following studies established that gyrase is a type IIA topoisomerase, capable of (i) relaxing positively supercoiled DNA by its negative supercoiling activity (Gellert, O'Dea, et al. 1976;[START_REF] Sugino | Energy Coupling in DNA Gyrase and the Mechanism of Action of Novobiocin[END_REF]) ; (ii) decatenating and unknotting entangled substrates [START_REF] Mizuuchi | DNA Gyrase Action Involves the Introduction of Transient Double-Strand Breaks into DNA[END_REF][START_REF] Liu | Type II DNA Topoisomerases: Enzymes That Can Unknot a Topologically Knotted DNA Molecule via a Reversible Double-Strand Break[END_REF][START_REF] Kreuzer | Formation and Resolution of DNA Catenanes by DNA Gyrase[END_REF] ; (iii) relaxing negatively supercoiled substrates in absence of ATP (Gellert et al. 1977;N. P. Higgins et al. 1978;[START_REF] Sugino | Energy Coupling in DNA Gyrase and the Mechanism of Action of Novobiocin[END_REF]. Even though gyrase was shown to have versatile in vitro activities, its main in vivo activity is the relaxation of positively supercoiled substrate by introduction of negative supercoils.

Indeed, in Bacteria, decatenation is mainly ensured by the topoisomerase IV, that is 100-fold more efficient than gyrase [START_REF] Zechiedrich | Roles of Topoisomerase IV and DNA Gyrase in DNA Unlinking during Replication in Escherichia Coli[END_REF]. Moreover, gyrase is 10-fold more efficient in removing positive supercoiling than introducing it in relaxed substrate and has a higher affinity for positively supercoiled substrates [START_REF] Ashley | Activities of Gyrase and Topoisomerase IV on Positively Supercoiled DNA[END_REF].

After its discovery in E. coli, it turned out that the gyrase is present in almost every bacterium, except the hyperthermophile Aquifex aeolicus that encodes a gyrase which lost the negative supercoiling activity (Tretter, Lerman, and Berger 2010). The ubiquitous distribution in bacteria and its essential function, makes gyrase a major antibiotic target (Bush, Evans-Roberts, and Maxwell 2015). Interestingly, gyrase is also present in some Eukaryotes, like plants and apicomplexan parasites that acquired this enzyme from chloroplast and apicoplast, respectively, [START_REF] Cho | DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning[END_REF][START_REF] Dar | Molecular Cloning of Apicoplast-Targeted Plasmodium Falciparum DNA Gyrase Genes: Unique Intrinsic ATPase Activity and ATP-Independent Dimerization of PfGyrB Subunit[END_REF] and in Archaea where its distribution will be presented further in this manuscript (see section III.A.3).

Gyrase is composed of 2 GyrA subunits and 2 GyrB subunits, that form a A2B2 heterotetramer [START_REF] Klevan | Deoxyribonucleic Acid Gyrase-Deoxyribonucleic Acid Complex Containing 140 Base Pairs of Deoxyribonucleic Acid and an Alpha 2 Beta 2 Protein Core[END_REF]. GyrA (97 kDa) wraps DNA with its C-terminal part and cleaves DNA with its N-terminal part that contains the catalytic tyrosine. GyrB Gyrase is especially efficient in removing positive supercoils by its negative supercoiling activity. Because of that, gyrase plays a critical role in removing positive supercoils introduced by replication and transcription. In association with the topoisomerase I, it is responsible of supercoiling homeostasis (see section II.C.2).

2. Quick and massive gene regulation in response to environmental changes DNA supercoiling is strongly sensitive to many environmental factors, including osmotic pressure, temperature, pH but also growth phase and aerobic versus anaerobic growth conditions [START_REF] Higgins | A Physiological Role for DNA Supercoiling in the Osmotic Regulation of Gene Expression in S. Typhimurium and E. Coli[END_REF][START_REF] Dorman | DNA Supercoiling and the Anaerobic and Growth Phase Regulation of TonB Gene Expression[END_REF][START_REF] Dorman | DNA Supercoiling and Environmental Regulation of Virulence Gene Expression in Shigella Flexneri[END_REF][START_REF] Karem | The Influence of DNA Topology on the Environmental Regulation of a PH-Regulated Locus in Salmonella Typhimurium[END_REF]Cheung et al. 2003;Rui and Tse-Dinh 2003).

Gyrase, through its ATP-dependent supercoiling activity, is directly involved in this topological response to environmental stress. Its activity is directly dependent of the ATP/ADP ratio and thereby of the cell's metabolic state (Drlica 1992). So, each variation of the environment that can modify the ATP/ADP ratio will change gyrase activity and consequently the expression of many genes sensitive to supercoiling (Dorman 2006).

This is a wide and quick way to adapt prokaryotic cells to changing environment. For instance, in E. coli, inhibition of gyrase and topoisomerase IV by drugs or temperaturesensitive mutant provokes a massive DNA relaxation. This deregulates up to 7% of E.

coli genes (Peter et al. 2004). Interestingly, genes sensitive to supercoiling in E. coli are not evenly distributed in the chromosome, they are grouped in hot spots (Figure 26).

Moreover, they can be as well up or downregulated, showing the true regulatory aspect of negative supercoiling beyond its basic propension to favour transcription. The same kind of study in Streptococcus pneumoniae also shows a massive supercoilingdependent gene regulation (de la [START_REF] Campa | The Transcriptome of Streptococcus Pneumoniae Induced by Local and Global Changes in Supercoiling[END_REF]. Chromosome relaxation by gyrase inhibition and accumulation of negative supercoiling by topoisomerase I inhibition, cause respectively the deregulation of 13% and 10% of S. pneumoniae genes, organized in hot spots as for E. coli. Loss of negative supercoiling consecutive to topoisomerase inhibition changes the expression of 7% of the genome (306 genes) located in hot spots all along the genome. Up-regulated genes are coloured in red and down-regulated are coloured in green (adapted from Peter et al. 2004).

Negative supercoiling could also be seen as a reserve of energy directly stored in the DNA (Cibot, n.d.). When the cell growth is stopped because of a nutrient lack or another kind of stress, the accumulation of negative supercoiling could be a powerful tool for the cell to restart its metabolism quickly. Negative supercoiling will lower the energy required by every DNA-melting process, boosting replication and transcription. The existence of such a mechanism for quick "emergency exit" is supported by the fact that in E. coli, gyrA and gyrB transcription increases in early stationary-phase and that the enzyme remains stable throughout long stationary-phase and is thus available to supercoil DNA as soon as ATP is produced again [START_REF] Reyes-Domínguez | Plasmid DNA Supercoiling and Gyrase Activity in Escherichia Coli Wild-Type and RpoS Stationary-Phase Cells[END_REF].

Moreover, the survival of E. coli long stationary-phase cultures, that are maintained in appropriate conditions to preserve negative supercoiling (anaerobiosis and high NaCl concentrations), is enhanced by 5 log compared to normal culture after 5 days [START_REF] Conter | Plasmid DNA Supercoiling and Survival in Long-Term Cultures of Escherichia Coli: Role of NaCl[END_REF].

In summary, gyrase is ubiquitous in Bacteria, has essential role in overcoming positive supercoiling introduced by transcription and replication, and ability to regulate simultaneously the expression of many genes according to the metabolic state of the cell. By allowing Bacteria to quickly adapt to changing environment gyrase was proposed to be one of the key evolutionary inventions explaining the predominance of Bacteria in most biotopes (Forterre and Gadelle 2009).

Gyrase in Archaea

In Archaea, contrary to Bacteria, gyrase is not ubiquitous (Table 4). The first gyrase of archaea was discovered in Halobacteria and immediately used to set up selectable marker for genetic work, suggesting it is an essential enzyme in Archaea (Holmes and Dyall-Smith 1991). Then, gyrase was found in all members of group II Euryarchaeota, some DPANN and some of the recently discovered Asgards (Forterre et al. 2007;Raymann et al. 2014;Adam et al. 2017;Garnier et al. 2021). So far, the gyrase encoding genes were not detected in representatives of TACK superphylum or group I Euryarchaeota. Considering the importance of these groups in terms of number of lineages, a considerable part of Archaea does not carry gyrase. Phylogenetic analysis revealed that acquisition of gyrase by Archaea, likely occurred through one or perhaps two early HGTs from Bacteria, followed by gene losses in TACK and group I Euryarchaeota (Raymann et al. 2014). This acquisition surely had deep effects on DNA topology and cell physiology. Consequently, it was hypothesised that the benefit of gyrase acquisition should have been strong and immediate to overcome the negative outcome of such a disturbing enzymatic activity (Forterre and Gadelle 2009). (Kikuchi and Asai 1984), reverse gyrase results from the fusion of a SF2 helicase domain and a topoisomerase I domain (Confalonieri et al. 1993). The interaction of helicase and type IA topoisomerase is common in many organisms, including humans, with the topoisomerase III family and the helicase RecQ family frequently associated [START_REF] Perugino | Reverse Gyrase and Genome Stability in Hyperthermophilic Organisms[END_REF][START_REF] Chu | RecQ Helicases: Multifunctional Genome Caretakers[END_REF]. Such hybrid enzymes are involved in genome maintenance through replication, recombination and repair processes. Most reverse gyrases are in form of a single polypeptide but some of them have the helicase and the topoisomerase domains split in subunits that interact to form the active heterodimer [START_REF] Kozyavkin | A Reverse Gyrase with an Unusual Structure. A Type I DNA Topoisomerase from the Hyperthermophile Methanopyrus Kandleri Is a Two-Subunit Protein[END_REF][START_REF] Capp | Separate and Combined Biochemical Activities of the Subunits of a Naturally Split Reverse Gyrase[END_REF]Catchpole and Forterre 2019). These split reverse gyrases are found in fast evolving species and are probably not the ancestral form of reverse gyrase (Lulchev and Klostermeier 2014). Independently of the helicase domain, the topoisomerase domain can relax supercoiled DNA in ATP-independent manner [START_REF] Perugino | Reverse Gyrase and Genome Stability in Hyperthermophilic Organisms[END_REF]). The two independent helicase and topoisomerase activities led to the early model for reverse gyrase function. The model proposed that the DNA opening by the helicase activity alone creates negative supercoils behind the reverse gyrase and positive supercoils ahead while the enzyme moves along the DNA. Then topo I activity would assume the specific relaxation of negative supercoils, thus letting only the positive supercoils remain (Confalonieri et al. 1993). However, further studies proved that both domains need to cooperate all along the process to supercoil DNA [START_REF] Déclais | Reverse Gyrase, the Two Domains Intimately Cooperate to Promote Positive Supercoiling[END_REF]. The cooperation between the helicase and the topoisomerase domains is dependent of a small insertion, called the latch [START_REF] Rodriguez | Studies of a Positive Supercoiling Machine. Nucleotide Hydrolysis and a Multifunctional "Latch" in the Mechanism of Reverse Gyrase[END_REF]. The latch is proposed to assure the communication between the two domains by opening the topoisomerase domain in

Gyrase is present in all

The only hallmark of hyperthermophily

Life at high temperature is not possible without specific adaptations. Molecular motion increases proportionally to the temperature, making membrane, proteins and nucleic acids unstable (López-García et al. 2015). To overcome these difficulties hyperthermophiles have specific lipids [START_REF] Koga | Thermal Adaptation of the Archaeal and Bacterial Lipid Membranes[END_REF][START_REF] Siliakus | Adaptations of Archaeal and Bacterial Membranes to Variations in Temperature, PH and Pressure[END_REF], GC-rich rRNAs (Boussau et al. 2008), highly stable proteins (Zeldovich, Berezovsky, and Shakhnovich 2007), specific chaperones [START_REF] Makarova | Potential Genomic Determinants of Hyperthermophily[END_REF] and specific ion transporters [START_REF] Albers | Bioenergetics and Solute Uptake under Extreme Conditions[END_REF]).

In the particular case of DNA, high temperature results in local denaturation that exposes nitrogenous bases to the solvent, increasing dramatically DNA damage such as depurinations, deaminations, oxidations, alkylations, and ultimately, double strand breaks (Lindahl 1993;Marguet and Forterre 1994). To face all these lesions, genomes of hyperthermophiles can rely on efficient DNA repair mechanisms [START_REF] Grogan | The Question of DNA Repair in Hyperthermophilic Archaea[END_REF][START_REF] Ishino | DNA Repair in Hyperthermophilic and Hyperradioresistant Microorganisms[END_REF], DNA binding proteins (Sanders, Marshall, and Santangelo 2019), high amount of ions and polyamines (Grogan 1998;[START_REF] Morimoto | Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus Kodakarensis[END_REF][START_REF] Terui | Stabilization of Nucleic Acids by Unusual Polyamines Produced by an Extreme Thermophile, Thermus Thermophilus[END_REF]) and the reverse gyrase (Forterre 2002).

From all the known actors involved in adaptation to thermophily, reverse gyrase is the only one shared by all hyperthermophiles and in most thermophiles but completely absent from mesophiles (Forterre 2002;Catchpole and Forterre 2019) (Figure 28). Thus, reverse gyrase is considered as the only hallmark of hyperthermophily. This strong association between thermophily and reverse gyrase can be reasonably explained by its activity of positive supercoiling, as overwound DNA is less sensitive to denaturation. But this simple explanation is challenged by a growing number of observations, leading to the proposition of other thermal protection mechanisms for reverse gyrase.

Uncertain in vivo activities

The different studies on the importance of reverse gyrase for life at high temperature have produced contradictory results. Reverse gyrase deletion is viable at optimal growth temperature (85°C) but lethal at 93°C for the hyperthermophilic archeon T.

kodakarensis (Atomi, Matsumi, and Imanaka 2004). It is lethal for Pyrococcus furiosus at 95°C, a suboptimal growth temperature, and for Sulfolobus islandicus at 76°C its optimal growth temperature (Lipscomb et al. 2017;Zhang et al. 2018). This suggests that reverse gyrase is essential for hyperthermophilic lifestyle, thus corroborating its phylogenetic distribution. Interestingly, though, the DNA of hyperthermophiles is not always relaxed or positively supercoiled. While most of them, like Thermococcales and Sulfolobales do follow this classical rule (López-García and Forterre 1999;Charbonnier and Forterre 1994), other ones like Thermotogae bacteria and Archaeoglobales archaea have negatively supercoiled plasmid DNA (Guipaud et al. 1997;Bouthier de la Tour et al. 1998;López-García et al. 2000). The particularity of these organisms is to possess gyrase in addition to reverse gyrase. This means that gyrase activity is the dominant one when both enzymes coexist in the cell. Moreover, this suggests that underwound DNA is compatible with hyperthermophilic lifestyle and that the positive supercoiling activity of the reverse gyrase is not critical for hyperthermophiles.

This hypothesis is corroborated by several experimental observations. For instance, reverse gyrase of Archaeoglobus fulgidus prevents DNA strand breakage at apurinic sites and keeps together broken extremities independently of its topoisomerase activity. In combination with the fact that reverse gyrase selectively binds nicked DNA, this led authors to propose that reverse gyrase acts in vitro as a DNA chaperone that stabilises DNA lesions until they are repaired (Kampmann and Stock 2004). Besides, reverse gyrase was shown to massively bind DNA in vivo after UV irradiation (Napoli et al. 2004), interact with single-strand binding protein (SSB), a common actor of DNA repair processes [START_REF] Napoli | Functional Interaction of Reverse Gyrase with Single-Strand Binding Protein of the Archaeon Sulfolobus[END_REF]) and with the translational polymerase Y (Valenti et al. 2009). Reverse gyrase also has a protective effect in vivo against double-strand breaks after methyl methanesulfonate treatment (MMS), an alkylating agent (Han, Feng, and She 2017). Taking together, these results strongly support a role of reverse gyrase in DNA repair, independently of its topoisomerase activity. Which reverse gyrase activity is really determinant for hyperthermophilic lifestyle is still an open question.

Answering this important question would require decoupling DNA repair and DNA topology activities in vivo.

IV. Investigating DNA topology in Archaea: the special case of Gyrase

The acquisition of gyrase by an ancestral archaeon had, in principle, completely disturbed its original DNA topology, causing deep impacts on its genome architecture, DNA replication and gene expression. Nevertheless, the gyrase was established in many archaeal lineages suggesting that its introduction was somehow beneficial. One, and so far the only, attractive hypothesis is that this particular event could have participated to the adaptation of Archaea to mesophilic environment. In this chapter, I will first describe this theory and then present the experimental strategy I have set up to test this hypothesis.

Gyrase evolution in Archaea

As detailed above, DNA supercoiling is inherent to the very structure of DNA making topoisomerases crucial for life. This is emphasised by the presence of at least one type I and one type II topoisomerase in every living organism (Schoeffler and Berger 2008).

Even though its accumulation is lethal, DNA supercoiling turned out to be advantageous in many situations through the high potential of genome condensation and gene expression regulation that it provides. Thus, topoisomerases are general regulators of DNA transactions by reducing the burden of excessive DNA constraints and enhancing the useful ones. Despite their importance, the evolutionary history of topoisomerases is still elusive. Many different classes of topoisomerases co-exist across species and their precise activities in the cells are often not defined. The complexity of the picture is even exacerbated by the paucity of data about global genome architecture and the importance of DNA supercoiling in most organisms.

Concerning the evolution of topoisomerases, some inter-domain transfers have been proposed, like acquisition of gyrase by Archaea or reverse gyrase by Bacteria (Raymann et al. 2014;Catchpole and Forterre 2019). Given the importance of supercoiling for genome structure and gene expression regulation, it is likely that the acquisition of a topoisomerase would have an important and global impact on the physiology of the concerned organism. This is especially true for the gyrase that dictates the overall genome topology in Bacteria by its ATP-dependent negative supercoiling activity (Forterre et al. 2007;Forterre and Gadelle 2009;Raymann et al. 2014).

Previously published phylogenetic trees suggest that gyrase was transferred once or twice from Bacteria to an ancestor of one group of Euryarchaeota and then lost in several lineages depending on the root for the archaeal tree (Forterre et al. 2007;Raymann et al. 2014). Other work, using reconstruction of ancestral sequences, suggested that the ancestral archaeon recipient of gyrase was a thermophile encoding a reverse gyrase, meaning therefore that its genome was probably relaxed or positively supercoiled (Boussau et al. 2008;Groussin and Gouy 2011;López-García et al. 2015;Catchpole and Forterre 2019). In a "normal" mesophilic context, even modest changes in DNA topology would be deleterious for the cell, as demonstrated in Streptococcus pneumoniae and E. coli (Ferrándiz et al. 2016;Pruss, Manes, and Drlica 1982), but in the context of a thermophile the change from positive to negative supercoiling would correspond to drastic alteration of DNA topology with potentially lethal consequences.

Indeed, in thermophiles positively supercoiled DNA is assumed to be important for protecting DNA from excessive melting and damage at high temperature. Thus, the acquisition of a gyrase by a naïve thermophile would potentially have dramatic consequences for genome stability. To overcome such an accumulation of negative effects, the gyrase recipient ancestor may have had disposed of strong resilience mechanisms against topological stress to maintain its DNA topology and/or had benefited of significant and immediate advantage procured by the gyrase activity (Forterre and Gadelle 2009).

Gyrase and adaptation to mesophilic lifestyle

More than 20 years ago Purification López-García emitted the hypothesis that the gyrase acquisition by a euryachaeal lineage was one of the key evolutionary events allowing these organisms to adapt to mesophilic conditions and thus conquer new ecological niches [START_REF] López-García | DNA Supercoiling and Temperature Adaptation: A Clue to Early Diversification of Life?[END_REF]López-García et al. 2015). Indeed, published phylogenetic reconstructions all agree that the Last Archaeal Common Ancestor (LACA)

was probably a thermophile (López-García et al. 2015;Catchpole and Forterre 2019).

The transition from a thermophilic lifestyle to a mesophilic one, implies that the organisms need to compensate (because of temperature drop) for lower membrane fluidity/permeability, lower protein flexibility/activity and more difficult melting of DNA. It seems that this was done through the acquisition of numerous bacterial genes, involved in aerobic respiration (electron transport chains and cytochromes), protein folding (heat-shock protein Hsp70), membrane fluidity (fatty acid biosynthesis enzymes), membrane permeability (transporters) and DNA unwinding (gyrase) (López-García et al. 2015). Gyrase, through its negative supercoiling activity, would be the key actor allowing DNA transactions to occur by lowering the energy required to open DNA helix. But gyrase is not the only topological actor that can provide negative supercoiling. The same goal can be reached with histones constraining negative supercoiling by wrapping DNA around the nucleosomes as it happens in Eukaryotes and Archaea [START_REF] Musgrave | Negative Constrained DNA Supercoiling in Archaeal Nucleosomes[END_REF].

Whereas gyrase is present in almost every mesophilic archaeon, only few thermophilic lineages are equipped with a gyrase. This raises the question of an incompatibility, between the negative supercoiling generated by gyrase and life at high temperature.

Still, thermophiles such as Archaeoglobi archaea (López-García et al. 2000) or

Thermotogae bacteria challenge this hypothesis (Guipaud et al. 1997). In these organisms endogenous plasmids are negatively supercoiled, suggesting that these organisms have negatively supercoiled genomes.

V. Objectives of the thesis and experimental strategies 1. Reconstruct the evolutionary history of gyrase in Archaea

As we have seen above, the acquisition of gyrase by Archaea was perhaps one of the key events at the origin of their expansion in mesophilic environments. The discovery of new gyrase-encoding lineages outside of Euryarchaeota, like Asgards or DPANN, challenge the current view of a unique transfer of gyrase from Bacteria to Euryarchaeota. Indeed, these lineages are distantly related to Euryarchaeota and therefore the evolutionary scenario explaining the gyrase distribution becomes more complex, possibly involving multiple inter-domain and/or intra-archaea horizontal gene transfers. The first objective of my thesis was to perform a global and updated phylogenetic analysis of currently available bacterial and archaeal gyrase sequences.

Such analysis should allow us to determine more precisely the timing and number of horizontal gene transfers throughout the evolutionary history of archaea. This analysis coupled with investigation of associated genomic adaptations could provide a general overview of the required conditions for establishment of gyrase in a naïve organism.

This, still ongoing, work is presented as draft of article I of my thesis entitled "Evolutionary history of archaeal DNA gyrases".

Describe the consequences of gyrase introduction in a naïve hyperthermophilic archaeon

Gyrase most certainly altered the original DNA topology of the ancestral thermophilic recipient archaeon. But what are the concrete consequences of introducing such an "opposite twist" in a naïve organism is not known. Is it really toxic? And if so why? Does the successful implantation of gyrase imply specific adaptations? Could it be sufficient to lower the growth temperature of this organism to compensate for gyrase activity?

Is gene expression affected, and if so, what are the affected functions? To address these questions, I decided to mimic the ancestral transfer of a bacterial gyrase in a hyperthermophilic archaeon. Although, this strategy using a modern organism is not fully extendable to the ancestral situation it still can give precious insights about the basic molecular mechanisms which allow the cells to resist such a challenge.

I chose Thermococcus kodakarensis as the recipient organism for the gyrase genes.

This marine, hyperthermophilic archaeon belongs to the Euryarchaeota superphylum.

It lives in hydrothermal vents where it feeds on organic matter such as polysaccharides and peptides and it performes anaerobic sulphur-based respiration to produce energy.

Similar to other Thermococcales, T. kodakarensis exhibits a single small circular chromosome of 2.08 Mb with 52% GC-content (Atomi et al. 2004). This organism became progressively one of the most studied model archaea because of its (i) natural competence, (ii) short generation time of about 40 min in rich medium at an optimal growth temperature of 85°C, and because of (iii) the availability of a panel of genetic tools and replicative plasmids [START_REF] Hileman | Genetics Techniques for Thermococcus Kodakarensis[END_REF]. Importantly, T.

kodakarensis encodes two histone paralogs (HTkA and HTkB), two NAPs (Alba and TrmBL2), one SMC and three topoisomerases (reverse gyrase, topoisomerase III and topoisomerase VI). This topological kit should result in a slightly positive DNA superhelical density, comparable to other Thermococcales (~0.030) (Charbonnier and Forterre 1994;López-García and Forterre 1997), and a chromatin extensively covered by histones and NAPs.

I chose Thermotoga maritima, a hyperthermophilic bacterium as a donor for the gyrase genes. This bacterium occupies the same environments as T. kodakarensis [START_REF] Huber | Thermotoga Maritima Sp. Nov. Represents a New Genus of Unique Extremely Thermophilic Eubacteria Growing up to 90°C[END_REF]). Importantly, plasmids from T. maritima are negatively supercoiled in vivo proving that the gyrase activity dominates over reverse gyrase (Guipaud et al. 1997).

Moreover, T. kodakarensis and T. maritima can be co-cultured at 85°C which is optimal growth temperature for T. kodakarensis. I therefore hypothesised that the gyrase of T.

maritima could be active in T. kodakarensis.

To test this hypothesis, I investigated gyrase activity in T. kodakarensis by superhelical density measurements on plasmids, through 2D agarose electrophoresis. To understand global impact of gyrase on T. kodakarensis cells I investigated how toxic was gyrase by (i) monitoring growth; (ii) measuring plasmid loss; (iii) measuring cell shape and size parameters using light microscopy. Finally, using RNA-seq I investigated how the gyrase expression and in particular its negative supercoiling activity affects the gene expression. The results of this work are reported in article II of my thesis entitled "The hyperthermophilic archaeon Thermococcus kodakarensis is resistant to pervasive negative supercoiling activity of DNA gyrase".

Investigate the importance of DNA supercoiling for adaptation to hyperthermophilic lifestyle in archaea

Since the discovery of DNA structure, the importance of DNA supercoiling for DNA transactions has been extensively studied in model bacteria and eukaryotes. It came out from these studies that a minimum level of negative supercoiling is required in these organisms to sustain transcription and replication. Bacteria reach such state mainly through a topoisomerase-based homeostatic regulation while Eukaryotes rely on histones to constrain DNA and provide toroidal negative supercoiling.

Comparatively, very little is known in Archaea about the importance of supercoiling for DNA transactions. The results of few studies reported so far (see chapter II.B.3

Physiological consequences of DNA supercoiling) give a partial view of the importance of supercoiling in archaea in general and in hyperthermophiles in particular. This is an important caveat for understanding the basic mechanisms governing genome stability and functionality, and the specific contribution of reverse gyrase for life at high temperature.

Slow electrophoresis for separation of plasmid topoisomers has been used for decades to investigate the topological state of DNA in vivo. While useful in the frame of my thesis work to confirm gyrase activity in T. kodakarensis, this method can not give more than a global a priori for the chromosome topology. Indeed, by its size, its chromatinization and the complexity of transactions it undergoes, chromosome harbours a multitude of topologies distinct of the plasmid one. To go beyond this limitation and truly map the supercoiling in the chromosome of T. kodakarensis, I decided to use a method that relies on preferential crosslinking of trimethyl-psoralen in negatively supercoiled DNA coupled to next-generation sequencing (Kouzine et al. 2013;Lal et al. 2016;[START_REF] Kouzine | DNA Topoisomerases: Methods and Protocols[END_REF]. This technique alone is not sufficient to meaningfully interpret the mapping data. Indeed, histone occupancy also has to be taken into account and I have therefore used a well-established method called micrococcal nuclease digestion in combination with deep sequencing (MNase-seq).

This method allows precise mapping of nucleosomes across the genome by sequencing DNA protected from MNase digestion by nucleosomes [START_REF] Schones | Dynamic Regulation of Nucleosome Positioning in the Human Genome[END_REF]). In the article III of my thesis manuscript, I described the final protocol for psoralen-crosslink in Thermococcus up to producing the sequencing samples. The oncoming analysis of sequencing data should allow me to finalise this part of my work.

I also included in this analysis T. kodakarensis deleted for reverse gyrase. those that build up during natural processed involving opening of the double helix. However, within these two classes, there are currently eight subfamilies of topoisomerases distributed unequally among the three domains of life indicating that the evolutionary history of these enzymes is complex.

Known topoisomerases are all DNA-specific enzymes and their activity strongly affects DNA-templated processes in cells. Consequently, their emergence and evolution are intimately linked with the origin of DNA and its evolution as a carrier of genetic information.

As a rule, topoisomerases have the capacity to relax supercoiled DNA but only two of them can also convert relaxed DNA into supercoiled DNA. Reverse gyrase can supercoil DNA positively and this enzyme is, as yet, the only known hallmark of thermophily (Forterre 2002). Positively supercoiled DNA is more resistant to melting and therefore the particular activity of reverse gyrase is thought to help preserve the genome integrity at high temperatures (Kikuchi and Asai 1984). DNA gyrase, an antagonist of reverse gyrase, catalyses the ATP-dependent introduction of negative supercoils in constrained DNA molecules (Gellert et al. 1976). According to the twin supercoiled-domain model, DNA gyrase removes the positive supercoils which accumulate in front of transcribing RNA polymerase or during DNA replication thus allowing these essential processes to proceed (Liu and Wang 1987;Drlica 1992;Lal et al. 2016;Sutormin et al. 2018). In tandem with the topoisomerase I (Topo I), DNA gyrase regulates global supercoiling level in bacterial cells and even small deviations from optimal DNA topology are lethal for bacteria (Pruss, Manes, and Drlica 1982) [START_REF] Corbett | The C-Terminal Domain of DNA Gyrase A Adopts a DNA-Bending β-Pinwheel Fold[END_REF][START_REF] Ward | Requirement of Topoisomerase IV ParC and ParE Genes for Cell Cycle Progression and Developmental Regulation in Caulobacter Crescentus[END_REF]. This motif is essential for chiral wrapping of DNA around gyrase enabling these enzymes to introduce negative supercoils in relaxed or positively supercoiled DNA [START_REF] Lanz | The GyrA-Box Determines the Geometry of DNA Bound to Gyrase and Couples DNA Binding to the Nucleotide Cycle[END_REF]Kramlinger and Hiasa 2006).

In contrast to bacterial orthologs which were extensively studied in vitro, only one archaeal gyrase, the one from Thermoplasma acidophylum has been biochemically characterised. This enzyme exhibited in vitro activities similar to that of bacterial homologs e.g. ATP-dependent supercoiling and ATPindependent decatenation and relaxation activities [START_REF] Yamashiro | Characterization of the DNA Gyrase from the Thermoacidophilic Archaeon Thermoplasma Acidophilum[END_REF]. Early studies showed that gyrase had negative supercoiling activity in vivo and that this activity was essential in methanogens, halophiles and thermoacidophiles (Sioud, Possot, et al. 1988). Whether, as in bacteria, the gyrase activity, and through it, the supercoiling homeostasis, is linked to chromosomal gene expression regulation in Archaea is poorly understood. The gene encoding rhodopsin in extreme halophile Haloferax was strongly induced when gyrase activity was inhibited by novobiocin drug indicating that supercoiling-sensitive promoters may exist in this archaeon. In another halophile

Halobacterium sp. the treatment with novobiocin resulted in deregulation of many genes including the stimulation of gyrase, topoisomerase VI (Topo VI) and Topo I expression indicating the involvement of these enzymes in regulating the DNA supercoiling levels in this organism (Tarasov et al. 2011).

Previous studies using the taxonomic sampling available at the time, identified the presence of gyrase encoding genes in all members of monophyletic Cluster II Euryarchaeota but not in other known archaeal phyla (Forterre et al. 2007;Raymann et al. 2014). Thus, at the time, it was proposed that gyrase was transferred from Bacteria to Archaea, presumably only once at the base of Cluster II Euryarchaeota (Raymann et al. 2014). This major group within Euryarchaeota superphylum contains 7 distinct classes of organisms with various lifestyles (methanogens, halophiles, XXX) and, with exception of Archaeoglobi, these are all mesophilic archaea. The acquisition of the gyrase by the thermophilic ancestor of Cluster II Euryarchaeota was further proposed to have played a key role in adaptation of this lineage to mesophilic lifestyle (López-García et al. 2015).

With the availability of an expanded taxonomic sampling it is now clear that the gyrase is also found in the archaeal lineages that are distantly related to the Cluster II Euryarchaeota such as DPANN and Asgard archaea (Adam et al. 2017). This raises the question of the timing and the number of inter-or intra-domain horizontal gene transfer events as well as the nature of the associated gene-specific or genome-wide adaptations. Resolving these questions is important for understanding the evolution of the Archaea, and also for testing hypotheses about the DNA topology-driven regulation of gene expression in Archaea, which is still poorly understood. To address these questions, we sampled gyrases from all available archaeal and bacterial genomes and performed phylogenetic, comparative sequence and structural analysis.

We found gyrase in all lineages of Cluster II Euryarchaeota, and in several lineages of DPANN and Asgard archaea thus expanding the gyrase dataset within these recently discovered archaea. Gyrase is also present in one lineage within Cluster I Euryarchaeota. The archaeal tree topology suggests that the gyrase was transferred from Bacteria to the base of Cluster II Euryarchaeota and then spread within other lineages via secondary horizontal gene transfers. Interestingly, we could not detect gyrase in TACK clade suggesting an incompatibility between the physiology of TACK and the activity of gyrase.

Importantly, we found that global gyrase tree exhibits a tripartite topology whereby the bacteria form two clades corresponding to Terrabacteria and Gracilicutes and Archaea are monophyletic. This suggests that gyrase was transferred before the diversification of Terrabacteria and Gracilicutes but after the diversification of Archaea. Such a scenario could imply that the diversification of Archaea predates the diversification of Terrabacteria and Gracilicutes. Remarkably, deep branches separating bacteria from Archaea are very short. This suggests that gyrase acquisition by archaea has not been accompanied by drastic changes in its sequence and, consequently, that archaeal gyrases share very similar functions with the bacterial ones.

MATERIALS AND METHODS

Generation of the DNA gyrase data set

GyrA and GyrB archaeal data sets were constructed by searching in the National Center for Biotechnology Information (NCBI) data bases, for Escherichia coli GyrA (EGT66353.1) and GyrB (AKE86808.1) homologs using the BlastP program [START_REF] Altschul | Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs[END_REF]. Additional searches were performed in Helarchaoeta and Verstraetearchaeota genomes by tBlastN [START_REF] Altschul | Gapped BLAST and PSI-BLAST: A New Generation of Protein Database Search Programs[END_REF]. Only sequences containing between 450 and 1100 amino acids and sharing at least 25 % sequence identity and more than 30 % of query sequence coverage were selected. Similar to other proteins involved in DNA metabolism, gyrase is a well-known carrier of inteins [START_REF] Novikova | Enigmatic Distribution, Evolution, and Function of Inteins[END_REF]. To detect inteins, GyrA and GyrB datsets were aligned separately and visualised using Geneious 11.0.5 (Biomatters), allowing the identification of 23 GyrA and 61 GyrB intein containing sequences which were removed from the data set. Using this approach, as of 5th of March 2020, 801 archaeal GyrA and 724 archaeal GyrB were retrieved. To complete GyrA and GyrB data sets, Annotree database was searched using KEGG accession numbers for GyrA (K02469) and GyrB (K02470) [START_REF] Mendler | AnnoTree: Visualization and Exploration of a Functionally Annotated Microbial Tree of Life[END_REF].

The 25565 results for GyrA and 25597 results for GyrB were downloaded. To avoid contamination of the GyrA data set by topoisomerase IV (TopoIV) A subunits, sequences lacking the GyrA box motif Q(R/K)RGG(R/K)G were eliminated. Dataset complexity was reduced by removing sequences with more than 90% sequence similarity across 70% of the sequence length for archaeal sequences and 70% sequence similarity across 70% of the sequence length for bacterial sequences using MMseqs2 v11.e1a1c [START_REF] Steinegger | MMseqs2 Enables Sensitive Protein Sequence Searching for the Analysis of Massive Data Sets[END_REF]. The final dataset contained 385 GyrA and 348 GyrB archaeal sequences and 571 GyrA and 633 GyrB bacterial sequences. The dataset is available upon request.

Analysis of sequence conservation

Bacterial and archaeal sequence datasets were compared using BLASTp all against all analysis (E-value <0.001). The median value for sequence identity and dispersion around this value was calculated using R 3.6.1 (https://www.r-project.org/) and plotted using ggplot2 package with geom_violin function (https://CRAN.R-project.org/package=ggplot2 ).

Phylogenetic tree construction

Sequence alignment and trimming were performed using Galaxy web platform [START_REF] Afgan | The Galaxy Platform for Accessible, Reproducible and Collaborative Biomedical Analyses: 2018 Update[END_REF].

GyrA and GyrB data sets were aligned with MAFFT v7.273.1 (Katoh and Standley 2013) using BLOSUM30 matrix. Selection of phylogenetically informative positions in alignments was done using BMGE v1.12 (Criscuolo and Gribaldo 2010) with the following parameters: BLOSUM30 matrix, sliding windows size 3, minimum block size 1. In parallel, les strict trimming was performed with Noisy [START_REF] Dress | Noisy: Identification of Problematic Columns in Multiple Sequence Alignments[END_REF]) using 0.6 cut-off value to test for artificial shortening of branch lengths [START_REF] Tan | Current Methods for Automated Filtering of Multiple Sequence Alignments Frequently Worsen Single-Gene Phylogenetic Inference[END_REF].

Trees were generated using IQ-TREE multicore v1.6.7 [START_REF] Nguyen | IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies[END_REF] on the Genotoul computing cluster (http://bioinfo.genotoul.fr/). Branch support was assessed using ultrafast bootstrap method (UFBoot, 1000 iterations) [START_REF] Hoang | UFBoot2: Improving the Ultrafast Bootstrap Approximation[END_REF]) and Shimodaira-Hasegawa approximate likelihood ratio test (SH-aLRT, 1000 iterations) [START_REF] Guindon | New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0[END_REF]. One would typically start to rely on the clade if its SH-aLRT >= 80% and UFboot >= 95%. Automatic sequence evolution model selection was performed using ModelFinder with -m MFP option [START_REF] Kalyaanamoorthy | ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates[END_REF]. Tests of tree topology were performed in IQ-TREE with 10,000 resamplings using the RELL method (Hirohisa [START_REF] Kishino | Maximum Likelihood Inference of Protein Phylogeny and the Origin of Chloroplasts[END_REF][START_REF] Hasegawa | Accuracies of the Simple Methods for Estimating the Bootstrap Probability of a Maximum-Likelihood Tree[END_REF].

Trees were visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). We systematically investigated the genomic context of highly divergent sequences (appearing as long branches in the trees) or sequences branching outside their taxonomic group to detect contamination with misannotated or outlier fast evolving sequences. Sequences from small contigs (less than 10 kb) or from contigs carrying rRNA genes not congruent with their annotation were removed from the data set. The dataset used for building final phylogenetic trees are available upon request.

For building a concatenated GyrA_GyrB tree, we first confirmed the presence of both gyrA and gyrB gene within the same genomes using Taxonomy ID. The two proteins were aligned using MAFFT, concatenated, and we performed the phylogenetic analysis using IQ-tree with the MPF option for sequence evolution model selection.

Synteny analysis

The synteny conservation analysis around the gyrAB locus was performed using the Clinker tool [START_REF] Gilchrist | Clinker & Clustermap.Js: Automatic Generation of Gene Cluster Comparison Figures[END_REF]. We selected organisms for which we detected, based on our phylogenetic analysis, putative HGT of gyrase genes. We extracted the corresponding GenBank file containing the contig encoding the gyrA and or the gyrB genes or a 30kb window of the chromosome or contig (if > 50kb). Global comparative analysis of our selected set of genbank files was done using default parameters. The gene clusters were determined based on protein similarity and the color-coded graphics was plotted.

Analysis of gyrase distribution in Archaea

The archaeal phylogenetic tree generated from 122 core proteins was retrieved from the Genome Taxonomy Database (GTDB Release 03-RS86) (Parks et al. 2020). The presence of GyrA or GyrB proteins was searched using AnnoTree [START_REF] Mendler | AnnoTree: Visualization and Exploration of a Functionally Annotated Microbial Tree of Life[END_REF] with additional manual curation to eliminate false positives (TopoIV). The presence/absence profiles were visualized using iTOL [START_REF] Letunic | Interactive Tree Of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation[END_REF].

gyrBA operon conservation analysis

To assess how conserved is the gyrBA operonic structure in Bacteria and Archaea we performed systematic search using the synteny maps option grouped by taxonomy available on the Genome Browser of the MicroScope platform [START_REF] Vallenet | MicroScope: An Integrated Platform for the Annotation and Exploration of Microbial Gene Functions through Genomic, Pangenomic and Metabolic Comparative Analysis[END_REF] (https://mage.genoscope.cns.fr/microscope.)

Each line refers to a taxon, followed by the number of species within the taxon and having the same gyrase gene organization. The taxonomic rank can be modified through the «Option» button. For each taxon, the gyrA gyrB gene organization is symbolized by a colored box. The color of the box corresponds to percentage of species which have the same organization comparted to the reference gene. This percentage is computed by dividing the number of species having the particular gene organization with the total number of species belonging to this taxon. For under-represented bacterial taxonomic ranks we used the synteny conservation tool SyntTax. The search was performed using GyrA and GyrB proteins of E. coli [START_REF] Oberto | SyntTax: A Web Server Linking Synteny to Prokaryotic Taxonomy[END_REF] .

RESULTS

Collection and analysis of gyrase sequences

The phenomenal amount of sequence data available in the public sequence databases represents a valuable asset for understanding the evolution and diversity of life using phylogenomics and comparative genomics approaches. We took advantage of this resource to establish a comprehensive comparative sequence analysis of the DNA gyrase in Archaea. As initial step in this analysis, we constituted a curated set of GyrA and GyrB sequences across entire phylogenetic diversity of Archaea and Bacteria (see materials and methods).

Briefly, GyrA and GyrB datasets were constituted by searching for E. coli GyrA or GyrB orthologs in NCBI nr database by protein BLAST and by searching Annotree (REF). Intein-containing sequences were identified using sequence alignment and removed from dataset. We also removed redundant sequences, fast evolving sequences and misassigned sequences from metagenome-assembled genomes. To avoid contamination with subunit A of TopoIV, the GyrA dataset was filtered for GyrA box Archaeal GyrA sequences contained the classical GyrA Box motif suggesting that the negative supercoiling function was conserved in archaeal gyrases (Figure 1,A). Using pairwise BLAST searches, we found that the sequence identity between archaeal and bacterial orthologs is comparable to sequence identity within archaeal or bacterial sequences (Figure 1,B). Together, the data suggest low amount of sequence evolution since introduction of DNA gyrase into archaeal domain. B) Sequence conservation analysis using BLAST.

motif. GyrA box motif (Q(R/K)RGG(R/K)G)
Sequence identity was determined using all against all BLASTp search. The statistical analysis and graphical representation were generated using R packages (see materials and methods). The analysis shows that sequence conservation within each domain or between the two domains is similar.

Distribution of gyrase in archaeal domain

Previous work showed that the gyrase is mainly present in one monophyletic group of Euryarchaea and, sporadically, in other archaeal lineages. We took advantage of the rapid increase of sequence data in public databases to update the gyrase distribution in Archaeal domain (Figure2). As one result of this survey, we confirmed the systematic presence of gyrase in Cluster II Euryarchaea and the systematic absence of gyrase in TACK superphylum including newly discovered lineages. The gyrase is also almost totally absent in Cluster I Euryarchaeota with exception of sporadic presence in Theionarchaea. We detected gyrase within DPANN superphylum in majority of Micrarchaeota, Woesearchaeota, Pacearchaeota, and in recently described (and still nameless) UBA583 order. Gyrase is only sporadically present in Altiarchaeota and is completely missing in the remaining seven DPANN orders. In asgard archaea gyrase is robustly present in Loki-and Heimdall-archaeota but is missing in Thor-and Odin-archaeota. The diversity of Asgard archaea was recently substantially expanded by proposal of six additional phyla [START_REF] Liu | Expanded Diversity of Asgard Archaea and Their Relationships with Eukaryotes[END_REF]. Using BLAST search we detected gyrase genes in all phyla within a monophyletic clade composed of Heimdall-, Kari-, Gerdr-, Hod-and Wukong-archaeota and also in distantly related Loki-and Hela-archaeota. In contrast, gyrase genes were not detected in two new lineages, Baldr-and Hermo-archaeaota which form sister group with Odin-and Thorarchaeota, respectively.

The presence/absence pattern when superposed to the archaeal phylogenetic tree makes it difficult to deduce the evolutionary history leading to present-day distribution of the gyrase in archaea. Was gyrase present already in the last common ancestor of Archaea (LACA) and was subsequently lost in many lineages, of was it introduced later on as suggested by previous phylogenetic analyses? If the latter is true, how many intra or inter horizontal gene transfers occurred? Prompted by these questions we reconstructed the phylogeny of prokaryotic gyrases.

Archaeal and Bacterial gyrases segregate in three monophyletic clades

The above described distribution could be a result of vertical inheritance of gyrase genes from the last common ancestor of Archaea (LACA) or a consequence of a more recent inter-and/or intra-domain lateral gene transfers. The former scenario would imply that gyrase was already present in LUCA or that it was acquired by early lateral transfer from Bacteria before the diversification of Archaea. The latter scenario implies that the gyrase originated in bacterial lineage and was transferred to archaea after this domain diversified. These two scenarios lead to hypotheses testable by phylogenetic analyses: if the gyrase evolved in the lineage leading to the LACA, the global gyrase phylogeny should produce a bipartite tree where archaeal and bacterial sequences are monophyletic, whereas post-LACA inter-domain lateral spreading of gyrase would likely not produce a congruent bacterial and/or archaeal tree.

In order to investigate the evolutionary history of the gyrase we first built a phylogenetic tree using the alignment of concatenated GyrAB archaeal and bacterial sequences, respectively. The initial alignment was trimmed with BMGE to remove phylogenetically unreliable columns and increase the signal to noise ratio [START_REF] Criscuolo | BMGE (Block Mapping and Gathering with Entropy): A New Software for Selection of Phylogenetic Informative Regions from Multiple Sequence Alignments[END_REF]. This filtering method removed 84 % of positions (1288 remaining positions out of 7978). In the phylogenetic tree inferred from this alignment, the bacterial sequences segregated into three separate clades while the archaeal sequences were split into two clades (Supplementary Figure 1,A). However, the ultrafast bootstrap and SH-aLRT values (36%/36%) did not support the existence of the central bipartition branch, suggesting that all archaeal sequences may fall within a single monophyletic clade. This was further suggested by the tree containing only GyrA sequences (Supplementary figure 6,A) in which archaeal sequences formed a monophyletic clade with a fairly good branch support (97%/72%). To test this hypothesis, we modified the global gyrase phylogeny such that Archaea and Bacteria form monophyletic groups, and subjected this tree to various tests of phylogenetic tree selection [START_REF] Kishino | Evaluation of the Maximum Likelihood Estimate of the Evolutionary Tree Topologies from DNA Sequence Data, and the Branching Order in Hominoidea[END_REF][START_REF] Strimmer | Inferring Confidence Sets of Possibly Misspecified Gene Trees[END_REF][START_REF] Shimodaira | An Approximately Unbiased Test of Phylogenetic Tree Selection[END_REF][START_REF] Shimodaira | Multiple Comparisons of Log-Likelihoods with Applications to Phylogenetic Inference[END_REF]. These statistical analyses ask whether the tree consistent with the monophyly of archaea has a significantly worse-likelihood score than the maximum likelihood tree. These tests soundly confirmed the monophyletic tree topology (Supplementary figure 3).

We hypothesised that the conflicting, paraphyletic or monophyletic, topologies regarding the archaeal clade could be due to insufficient phylogenetic signal in our dataset. Indeed, we consistently observed that the SH-aLRT and UFBoot values have a tendency to disagree especially for deep nodes (UFBoot < 95 but SH-aLRT > 80). Such discrepancies can occur for datasets with week phylogenetic signal, that is, with insufficiently long and/or insufficiently diverged sequences [START_REF] Guindon | New Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the Performance of PhyML 3.0[END_REF]. To test if this is the case, we used Noisy [START_REF] Dress | Noisy: Identification of Problematic Columns in Multiple Sequence Alignments[END_REF]) which has high tolerance for columns containing gaps thus allowing to potentially harvest substantial phylogenetic signal that may be contained in gap-rich regions [START_REF] Dessimoz | Phylogenetic Assessment of Alignments Reveals Neglected Tree Signal in Gaps[END_REF][START_REF] Tan | Current Methods for Automated Filtering of Multiple Sequence Alignments Frequently Worsen Single-Gene Phylogenetic Inference[END_REF]. Trimming the alignment of the complete gyrase dataset with this method resulted in removal of 60 % of positions (3217 remaining positions out of 7978) thus more than doubling the number of alignment positions used for building the tree. The inferred maximum likelihood tree showed tripartite topology with archaeal sequences forming a monophyletic clade (Figure 3, Supplementary figure 1,B). Notably, the deep branches supporting the monophyly of the three clades were now robustly supported by both UFBoot and SH-aLRT suggesting that the relaxed trimming increased the signal to noise ratio in our dataset. However, despite the significant increase in the number of analysed positions the branches dividing archaeal and bacterial clades remained short indicating low level of sequence evolution since splitting of the archaeal lineage from bacteria. Remarkably, the bacterial part of the tree shows bimodal topology in agreement with the latest rooting of the bacterial tree [START_REF] Coleman | A Rooted Phylogeny Resolves Early Bacterial Evolution[END_REF]). The tree is divided into Terrabacteria (a clade encompassing Cyanobacteria, Deinococcus/Thermus, Firmicutes, etc.) and Gracilicutes (a clade encompassing Proteobacteria, Bacteroidetes, Spirochaetes, etc.) corresponding to the deepest phylogenetic divide between these two groups of bacteria suggesting mainly vertical evolution of gyrase in the bacterial domain. Three alternative roots can be proposed based on this phylogenetic tree with different consequences for our understanding of the gyrase origin and evolution of bacteria and archaea (Figure 4). Rooting the tree in-between the archaeal clade and the two bacterial clades places the origin of gyrase before LUCA and subsequent partial loss of gyrase genes in archaeal domain (Figure 4, scenario I). Placing the root on the branch of Gracilicutes or of Terrabacteria leads to two different scenarii.

Here, the gyrase originated in the branch leading to the last bacterial common ancestor (LBCA) and was then transferred to the lineage leading to Archaea Assuming that the gyrase evolutionary history recapitulates faithfully, through mostly vertical inheritance of gyrase genes, the evolutionary history of organisms, three evolutionary scenarii can be deduced with consequences for the gyrase emergence and relative timing of emergence of major taxonomic groups.

A) The gyrase was already present in LUCA and LACA is older than LBCA. This scenario suggests that LUCA had DNA genome and that massive loss of gyrase genes occurred independently in the majority of archaeal lineages.

B) Gyrase emerged in bacterial lineage and LACA is younger than LBCA. This scenario suggests again occurrence of a massive and independent loss of gyrase genes in the majority of archaeal lineages.

C) Gyrase emerged in bacterial lineage and LACA is younger than the ancestor of Gracilicutes or Terrabacteria. In this scenario, the lineage giving rise to Gracilicutes (or Terrabacteria) is the donor of the gyrase and the recipient is the ancestor of Cluster II Euryarchaeota. The monophyly of Archaea can then be explained by spread of gyrase genes via secondary HGT within archaeal domain.

Collectively, the data show that the bacterial and archaeal clades are consistently separated by short branches indicating that low amount of evolution occurred in gyrase sequences since divergence from bacteria. This is inconsistent with the presence of the gyrase in the LUCA or its acquisition early in the lineage leading to LACA because such history would most probably result in highly divergent proteins.

Instead, our data agree with the idea that the introduction of the gyrase in the archaeal domain occurred once the tempo of evolution decreased i.e. after the diversification of this domain was initiated. Furthermore, the data suggest that the gyrase was introduced in archaeal lineage by a single horizontal gene transfer from bacteria and we propose that the ancestor of Cluster II Euryarchaeota was the recipient cell. The separation of bacterial sequences into two clades as distinct from each other as they are from archaeal clade suggests that the ancestor of Gracilicutes or Terrabacteria was the donor of the gyrase.

Phylogeny of archaeal gyrases

The global gyrase tree indicated that the archaea were monophyletic but internal topology of the archaeal clade was not robustly resolved. We reasoned that limited early HGT followed mostly by vertical inheritance would produce a phylogeny in which the majority of phylum-level taxonomic groups would be monophyletic, while frequent and recent HGT would likely not produce a typical archaeal tree. To test this hypothesis, we generated gyrase data sets containing 377 GyrA and 331 Although the use of relaxed trimming of sequence alignments recovers a tree topology more congruent with the expected archaeal tree topology, branch support at some deep nodes is systematically weak thus resulting at low resolution of the gyrase tree at inter-phylum level. These results are further difficult to interpret since there is no consensus to the rooting of the archaeal tree (Petitjean et al. 2014;Raymann, Brochier-Armanet, and Gribaldo 2015;Da Cunha et al. 2017;Williams et al. 2017;Baker et al. 2020) and thus no true reference tree to compare with. Nevertheless, the repeated recovery of monophyletic clades corresponding to major taxonomic divisions within archaeal superphyla is not consistent with spread of the gyrase through multiple temporally separated HGT events. Rather, the data suggest predominantly vertical evolution with few early transfer events occurring before the diversification of archaeal superphyla. Such evolution fits with aforementioned evolutionary scenario III (Figure 4) suggesting that the gyrase was originally acquired by the lineage leading to group II Euryarchaea (which all encode gyrase) and later on introduced in the Asgard and DPANN superphyla. In case of viruses, we detected GyrA-like sequences in 13 isolated phages infecting Bacillus or Lactococcus bacteria but the closer inspection of these sequences revealed that they do not carry GyrA box motif suggesting that these are Topo IV subunits or degenerated DNA gyrases (Supplementary figure 7,A). We also detected hundreds of GyrA-like sequences in metagenome assembled phage genomes all originating from one recent meta-analysis of human-associated viruses [START_REF] Tisza | A Catalog of Tens of Thousands of Viruses from Human Metagenomes Reveals Hidden Associations with Chronic Diseases[END_REF]. These metagenomes were classified as Microviridae, Myoviridae, Siphoviridae and Podoviridae all belonging to Caudovirales bacteriophages. The alignment of representative GyrA sequences for each of these taxa showed that Microviridae, Myoviridae and Siphoviridae contained the GyrA Box suggesting that these viruses may carry functional gyrases (Supplementary figure 7,B).

Collectively, the data show that mobile genetic elements rarely, if at all, encode gyrases except perhaps the bacteriophages from human microbiome. This suggest that the dispersion of the gyrase genes via these vehicles in not a common mechanism of transmission among organisms.

Cases of inter-domain horizontal gene transfer

To gain further insight in mode of transmission of gyrase genes we have examined the cases of interdomain HGT which were the only ones we could robustly detect. Indeed, in global gyrase phylogenies we consistently recovered the clustering of Methanobrevibacter archaea within Firmicutes bacteria while Thermotoga bacteria were positioned as a sister group to Huberarchaea from DPANN superphylum. Synteny analysis revealed that gyrA and gyrB genes are encoded on distant loci in Thermotoga bacteria (Figure 6). The synteny is supported by tree topology of Thermotogales thus suggesting vertical inheritance of gyrase genes from the common ancestor of this group. However, the genomic context is not well conserved across different genera suggesting a high degree of plasticity in this genomic region. Similar results were obtained for gyrB locus (Supplementary figure 9). We noted though that the gyrase containing 11-gene cluster assigned to Kosmotoga olearia was most similar to distantly related Pseudothermotoga thermarum indicating that this gene cluster may have been horizontally exchanged. In the most closely related archaeal donor species the gyrase genes are organised in a gyrBA operonic structure suggesting that this organisation was split into two different genomic locations in Thermotogales perhaps to facilitate the establishment of the archaeal gyrase in these bacteria. B) Phylogeny of Thermotogales species used in synteny analysis. The phylogenetic tree was automatically generated using PhyloT and the Genome Taxonomy Database and visualized using iTOL [START_REF] Letunic | Interactive Tree Of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation[END_REF]. The numbers on the branches correspond to bootstrap node support values.

The transfer of gyrase genes from Firmicutes bacteria to Methanobrevibacter archaea must have been a relatively recent one since only few species of Methanobrevibacter carry gyrase. As such, this case can inform us about how gyrase can become established in a naïve organism. Synteny analysis showed that gyrase genes were clustered in gyrBA operon with no conservation of genomic context in vicinity not even between the two closely related species M. curvatus and M. cuticularis (Figure 7). This suggested that the gyrase operon may be mobilized as a stand-alone unit. Inactivation of gyrase genes by genetic drift would be expected if the gyrase offers no selective advantage to Methanobrevibacter species. Using sequence alignment, we detected all catalytically important residues in archaeal orthologs suggesting that these may indeed be functional (Supplementary figure 10). A single mutation with potential to modify the negative supercoiling activity was detected in GyrA box motif where one of the universally conserved glycines was mutated to alanine. The 170 amino acid insertion in the metal-and DNA-binding TOPRIM domain that occurs in the GyrB subunit of many gram negative bacteria (including E. coli) is missing in Methanobrevibacter species in line with these gyrases being of gram positive origin as suggested by the tree topology and synteny analysis. B) Phylogeny of Methanobrevibacter species. The phylogenetic tree was automatically generated using PhyloT and the Genome Taxonomy Database and visualized using iTOL [START_REF] Letunic | Interactive Tree Of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation[END_REF]. The numbers on the branches correspond to bootstrap node support values.

DISCUSSION

Topoisomerases are ancient enzymes with a complex evolutionary history. These ubiquitous enzymes are important for genome stability, replication and gene expression. As such, they must have played an important but still poorly understood role in the evolution of genomes. In this study, we focused on DNA gyrase which are ubiquitous in Bacteria but have a patchy distribution in Archaea. This topoisomerase maintains a tight control of chromosomal supercoiling in Bacteria and through it exercises a topology-driven control of gene expression. From evolutionary standpoint, the gyrase is of much interest because of its capacity to trigger massive gene deregulation in response to environmental stimuli. For this reason, it was suggested that gyrase is a key enzyme allowing the bacteria to adapt and colonise a variety of ecological niches. Much less is known about the in vivo function of Archaeal gyrases and about the role this enzyme played in the evolution of this domain of life. As initial approach to close this knowledge gap, we have analysed the phylogeny of prokaryotic gyrases with special focus on evolutionary history of archaeal orthologs. We found gyrase orthologs in Euryarchaeota, Asgard and DPANN superphyla but not in TACK superphylum. The constructed phylogenetic trees revealed that Archaea acquired the gyrase from bacteria probably by a single HGT and that the gyrase genes were further spread within archaea by secondary transfers. We further show that the most parsimonious evolutionary scenario implies that the gyrase emerged in the lineage giving rise to bacteria and that the archaea diversified before one of the major groups of Bacteria. Finally, the mode of transmission of gyrase genes seems not to include mobile genetic elements.

When thinking about the evolution of the gyrase we wondered why this enzyme is not more widely spread in Archaea? Plasmids and viruses are notorious as disseminators, via horizontal gene transfer, of a large diversity of genes and this mechanism is important for adaptation and genetic diversification of Bacteria and Archaea [START_REF] Polz | Horizontal Gene Transfer and the Evolution of Bacterial and Archaeal Population Structure[END_REF][START_REF] Medini | The Microbial Pan-Genome[END_REF]. Recently, the members of the type IIB topoisomerase family (topoisomerase VIII and Mini-A) were detected encoded on free or integrated conjugative plasmids in Bacteria and Archaea and our own search shows presence of TopoIV in isolated bacteriophages indicating that these topoisomerases can be horizontally transferred by the archaeal and bacterial mobilome (Takahashi et al. 2020). On the other hand, this seems not to be the case for genes encoding gyrase orthologs thus limiting the dissemination potential for gyrase genes. In bacteria, the gyrase is important for maintaining the supercoiling levels within a range compatible with DNA transactions (Cheung et al. 2003;Dorman and Dorman 2016;Peter et al. 2004;Sutormin et al. 2018). As a part of this homeostatic mechanism, the promotors of gyrA and gyrB genes are stimulated by DNA relaxation (Menzel and Gellert 1983;[START_REF] Straney | Mutations in the -10 TATAAT Sequence of the GyrA Promoter Affect Both Promoter Strength and Sensitivity to DNA Supercoiling[END_REF][START_REF] Unniraman | Regulation of DNA Gyrase Operon in Mycobacterium Smegmatis: A Distinct Mechanism of Relaxation Stimulated Transcription[END_REF]. It seems therefore plausible to think that the introduction, via horizontal gene transfer, into this tightly controlled system of an additional functional gyrase may cause imbalance leading to fitness cost or lethality.

Still, through our phylogenetic analysis, we detected two robust cases of relatively recent transfer of gyrase genes between archaea and bacteria showing that despite potential negative consequences, the establishment of exogenous gyrases is fundamentally possible. In both detected cases, the putative donor organism encoded gyrase as gyrBA operon. This makes sense since the colocation of genes within an operon facilitates their collective translocation via horizontal gene transfer and both genes are required to form functional tetrameric A2B2 holoenzyme. In Thermotogales however, the operonic structure was broken down to two distant genomic loci and similar situation is found in many bacterial and archaeal lineages (data not shown) indicating, at least in these lineages, the absence of selective pressure for the maintenance of operon structure. Since organism bearing clustered genes are more likely to act as successful donors [START_REF] Lawrence | Selfish Operons: Horizontal Transfer May Drive the Evolution of Gene Clusters[END_REF], the separation of gyrase genes into two distant genetic loci would probably limit horizontal spread among bacterial and archaeal genomes.

We also noticed little or no conservation of genetic context even between closely related organisms such as the two Methanobrevibacter species suggesting that the gyrase operon may be transferred as a stand alone unit rather than as a part of a mobile cluster of genes but the precise mechanism remains to be determined.

Interestingly, the gyrase genes found in Methanobrevibacter species probably encode functional gyrases suggesting that these Cluster I Euryarchaea archaea which naturally do not encode gyrase have successfully adapted to the presence of a pervasive negative supercoiling activity. How come such, a priori, toxic activity with a wide ranging consequences for essential DNA transactions can be so easily accommodated and adopted? We have recently demonstrated that, unexpectedly, introduction of bacterial gyrase into Thermococcus kodakarensis is well tolerated by this hyperthermophilic archaeon (Villain et al. 2021). Notably, the T. kodakarensis cells did not overexpress endogenous topoisomerases to counteract the excess of negative supercoiling, instead we proposed that the plectonemic negative supercoils can be efficiently absorbed by wrapping DNA positively around nucleosomes. This hypothesis is also supported by the distribution of histone genes in archaea which largely overlaps with gyrase distribution pattern (Adam et al. 2017) thus indicating that histones may be a key factor facilitating the establishment of gyrase in archaeal cells. Actually, as a result of our survey we noticed that all archaeal gyrase bearers also encode histones. Gyrase-encoding Thermoplasma archaea are, at first glance, an interesting exception to this rule because they lost the original archaeal histone genes but have acquired instead a histone analog (dubbed HTa) of bacterial origin (Hocher et al. 2019). In these cells, as additional peculiarity, the Topo VI, which is the main archaeal type II topoisomerase, has equally been lost, leaving the gyrase as the sole type II topoisomerase probably in charge of chromosome decatenation and supercoiling homeostasis. As another result of our survey of gyrase gene distribution, we noticed that among ten thermophilic archaeal lineages (Catchpole and Forterre 2019), Archaeoglobi are the only ones carrying both gyrase and reverse gyrase, a topoisomerase with opposite, positive supercoiling activity and the sole known marker of hyperthermophily (Forterre 2002;Kikuchi and Asai 1984;Forterre et al. 1985). This observation indicates that the concomitant presence of these two enzymes with antagonist activities may be difficult to balance and is counterselected in Archaea. Interestingly, the only known example of a bacterium without functional gyrase is Aquifex aeolicus, one of the most extreme hyperthermophilic bacteria in which the endogenous gyrase was transformed into Topo IV enzyme (Tretter, Lerman, and Berger 2010).

What would be an evolutionary advantage for adopting a DNA gyrase? Through its negative supercoiling activity, the gyrase would be expected to change the delicate balance between the stability and melting potential of the DNA (Lopez-Garcia, 1999, Lopez-Garcia et al., 2015). In Bacteria, negative supercoiling drastically reduces the energy cost necessary for DNA melting and thus promotes transcription initiation by RNA polymerase (Martis et al 2019). In hyperthermophiles, it is presumed that the high temperature would be the energy source for melting the relaxed or positively supercoiled DNA [START_REF] Meyer | Temperature dependence of the DNA double helix at the nanoscale: Structure, elasticity, and fluctuations[END_REF]. Previous work exploiting the correlation between sequence composition and optimal growth temperatures suggested that early Archaea were (hyper)thermophiles, with mesophily arising more recently in archaeal evolution (Groussin et al., 2013, Groussin et al., 2013 ). Building upon this work, Lopez-Garcia and colleagues suggested that the acquisition of the bacterial gyrase by the ancestor of cluster II Euryarchaeota was a key evolutionary event allowing the progressive adaptation to mesophilic lifestyle (Lopez-Garcia et al., 2015). Indeed, through its negative supercoiling activity the gyrase could have facilitated DNA melting thus allowing the essential DNA transactions to proceed at suboptimal temperatures.

The evidence gathered so far indicates strongly that, as in Bacteria, gyrase has an active and essential role in regulating DNA supercoiling in Archaea (Sioud, Possot, et al. 1988;Sioud, Baldacci, et al. 1988).

Together, these observations indicate that gyrase became essential in Archaea once mechanisms dealing with DNA became dependent on negative supercoiling. Indeed, in course of co-evolution with the gyrase, proteins involved in DNA transactions may have taken advantage of negative supercoiling of DNA for recognition and activity. Hence, a certain number of gene-specific and/or genome wide adaptations may have occurred since the acquisition of gyrase by Archaea. For example, DNA gyrase and histones/nucleosomes (which occur in many Archaea), both produce negative supercoils, but their mode of action is fundamentally different. Histones constrain supercoils in a solenoidal form (like a phone cord) while the gyrase produces plectonemic supercoil conformation (like a figure eight).

Moreover, the negative supercoils produced by gyrase are ready to assist in separating the double strand helix whereas the histones need to be released from nucleosomes to harness the energy contained in the negative supercoil. It's seems therefore plausible to presume that acquisition of gyrase may have impacted the evolution of genes encoding histones and, possibly, their function. Thus, the comparison of histone borne DNA condensation mechanisms between the gyrase encoding and gyrase lacking archaea provides the unique opportunity to explore evolutionary forces underlying the structuration of genomes.

A striking observation coming from phylogeny of gyrases is that the bacterial and archaeal clades are consistently separated by short branches. Similarly, the phylogenetic reconstructions of another topoisomerase, reverse gyrase, from bacteria and archaea consistently produced trees with short branches (Catchpole and Forterre 2019). This is clearly different from what is observed in phylogenies of universal marker proteins which typically exhibit very long branch separating bacterial and archaeal clades [START_REF] Coleman | A Rooted Phylogeny Resolves Early Bacterial Evolution[END_REF]. Such long branch is compatible with a fast tempo of sequence evolution which presumably occurred between LUCA and the ancestors of bacteria and archaea resulting in very divergent proteins (Woese, 1998, Forterre 2006). Consequently, the gyrase phylogeny as constructed from our sequence dataset, is inconsistent with the presence of the gyrase in the LUCA or its acquisition early in the lineage leading to LACA. Instead, our data agree with the idea that the introduction of the gyrase in the archaeal domain occurred once the tempo of evolution decreased i.e. during the diversification of this domain. According to the most parsimonious scenario, the gyrase was transferred only once from Bacteria probably into the lineage giving rise to the cluster II Euryarchaea.

Given that the archaeal gyrase tree roughly recapitulates the established phylogeny of archaea, we suggest that the gyrase mainly evolved vertically with only few lateral gene transfers that must have occurred before the diversification of DPANN and Asgard superphyla. Recent phylogenetic analyses using outgroup-free approach allowed placing the bacterial root between Terrabacteria and Gracilicutes, two major bacterial clades [START_REF] Coleman | A Rooted Phylogeny Resolves Early Bacterial Evolution[END_REF]. In line with this proposal we recovered the deep split between these two clades thus indicating that, similar to archaeal counterparts, bacterial gyrase evolution has a major vertical component.

The analysis of ancestral horizontal gene transfers can also provide insight into the temporal sequence of events during gyrase diversification, because donor lineages must be at least as old as recipients.

The topology of the gyrase tree suggests that Cluster II euryarchaeota may have been one of the earliest, if not the first, emerging archaeal lineages. This has implications for the ongoing debate concerning the position of the archaeal root for which there is currently no consensus (Petitjean et al. 2014;Raymann et al. 2015;Da Cunha et al. 2017;Williams et al. 2017). Moreover, according to our phylogenetic reconstruction, the archaea started their diversification before one of the two major bacterial clades emerged. Importantly, the gyrase evolution is incompatible with the presence of this enzyme in LUCA thus suggesting an RNA-based LUCA cell.

SUPPLEMENTARY FIGURES AND TABLES

INTRODUCTION

With the discovery of the structure of DNA, it became apparent that the opening of the double helix generates torsional stress resulting in overwinding or underwinding of the DNA molecule (1). Paradoxically, many DNA transaction processes such as transcription and replication require strand separation and will lead naturally to DNA overwinding and strand entanglement [START_REF] Liu | Supercoiling of the DNA template during transcription[END_REF][START_REF] Postow | Topological challenges to DNA replication: Conformations at the fork[END_REF][START_REF] Gilbert | Supercoiling in DNA and chromatin[END_REF]. These topological constraints antagonise these essential cellular processes and if not resolved, are lethal. To deal with this problem, all cells rely on topoisomerases, a ubiquitous class of enzymes that introduce strand breaks to relieve unfavourable topological intermediates without damaging the genome [START_REF] Wang | Cellular roles of DNA topoisomerases: a molecular perspective[END_REF][START_REF] Schoeffler | DNA topoisomerases: harnessing and constraining energy to govern chromosome topology[END_REF][START_REF] Forterre | Introduction and Historical perspective[END_REF][START_REF] Vos | All tangled up: how cells direct, manage and exploit topoisomerase function[END_REF][START_REF] Bush | DNA topoisomerases[END_REF][START_REF] Seol | The dynamic interplay between DNA topoisomerases and DNA Topology[END_REF][START_REF] Mckie | DNA topoisomerases: Advances in understanding of cellular roles and multi-protein complexes via structure-function analysis[END_REF]. Topoisomerases are mechanistically classified as type I or type II, depending on whether they cleave one or two strands of DNA, respectively [START_REF] Schoeffler | DNA topoisomerases: harnessing and constraining energy to govern chromosome topology[END_REF]. Multiple phylogenetically unrelated subclasses of each type exist in the biosphere and such diversity has made it particularly challenging to dissect the evolutionary history of topoisomerases [START_REF] Forterre | Origin and evolution of DNA topoisomerases[END_REF][START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF]. A long-standing puzzle has been to understand why so many topoisomerases have emerged in the course of evolution and what role they played in the evolution of DNA-based cells [START_REF] Forterre | Origin and evolution of DNA topoisomerases[END_REF][START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF]. DNA gyrase (hereafter gyrase), a type II A topoisomerase, is the only known enzyme that can negatively supercoil (underwind) DNA using the free energy of ATP hydrolysis to drive the process ( 14). An important antibiotic target, gyrase is essential and ubiquitous in bacteria where it controls (together with Topo I) the supercoiling density of chromosomes by introducing negative supercoils into DNA and by relaxing positive supercoils accumulating in front of moving DNA and RNA polymerases [START_REF] Drlica | Control of bacterial DNA supercoiling[END_REF][START_REF] Zechiedrich | Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli[END_REF][START_REF] Lal | Genome scale patterns of supercoiling in a bacterial chromosome[END_REF][START_REF] Sutormin | Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome[END_REF]. The contribution of DNA gyrase in maintaining the chromosome in an underwound state in bacterial cells can profoundly impact the binding of regulatory proteins, promoter firing dynamics, DNA replication, and chromosome architecture [START_REF] Gilbert | Supercoiling in DNA and chromatin[END_REF][START_REF] Hardy | Disentangling DNA during replication: a tale of two strands[END_REF][START_REF] Dorman | DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression[END_REF].

DNA supercoiling is used in a wide range of bacteria to quickly transduce environmental signals towards the chromosome and this process is conserved in distant bacterial species [START_REF] Dorman | DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression[END_REF][START_REF] Martis | DNA supercoiling: an ancestral regulator of gene expression in pathogenic bacteria?[END_REF]. The most clearly described pathway involves the modulation of gyrase activity in response to [ATP]/[ADP] ratio in the cell. When this ratio is low, the DNA gyrase supercoiling activity is significantly reduced and the expression level of many genes is simultaneously modified [START_REF] Westerhoff | DNA supercoiling by DNA gyrase[END_REF][START_REF] Hsieh | Bacterial DNA supercoiling and [ATP]/[ADP] ratio: changes associated with salt shock[END_REF]. The inhibition of gyrase supercoiling activity by quinolone antibiotics has a similar effect; the expression of up to 48% of genes can be deregulated [START_REF] Martis | DNA supercoiling: an ancestral regulator of gene expression in pathogenic bacteria?[END_REF]. This relatively simple, quick and general mechanism was suggested to be one of the key evolutionary inventions allowing the bacteria to occupy a wide variety of environments [START_REF] Forterre | Origin and evolution of DNA topoisomerases[END_REF][START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF].

In Archaea, Gyrase is found in all members of the highly diversified monophyletic group (named Cluster II Euryarchaeota by Adam and colleagues) containing seven distinct groups with very different lifestyles (acidophiles, halophiles, methanogens among others) and, sporadically, in DPANN and Asgard superphyla [START_REF] Forterre | Origin and evolution of DNA topoisomerases[END_REF][START_REF] Adam | The growing tree of Archaea: new perspectives on their diversity, evolution and ecology[END_REF][START_REF] Garnier | Archaea: a gold mine for topoisomerase diversity[END_REF] (Figure 1). Initial phylogenetic analyses indicated that archaeal gyrase is of bacterial origin and was acquired via ancient horizontal gene transfer by a hyperthermophilic archaeon [START_REF] Forterre | Origin and evolution of DNA topoisomerases[END_REF]. Later analysis including more archaeal lineages suggested that this transfer occurred only once at the base of the aforementioned late emerging Cluster II Euryarchaeota [START_REF] Raymann | Global phylogenomic analysis disentangles the complex evolutionary history of DNA replication in archaea[END_REF]. Because negative supercoiling facilitates DNA melting, it was proposed that gyrase acquisition had a profound impact on all DNA-dependent processes with important consequences for the evolution of recipient archaea [START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF][START_REF] Ópez-García | Control of DNA topology during thermal stress in hyperthermophilic archaea: DNA topoisomerase levels, activities and induced thermotolerance during heat and cold shock in Sulfolobus[END_REF][START_REF] Ópez-García | Bacterial gene import and mesophilic adaptation in archaea[END_REF]. However, how and why DNA gyrase became fixed in archaeal lineages remains obscure.

The successful establishment of bacterial gyrase in Archaea is particularly intriguing since the introduction of an uncontrolled negative supercoiling activity could potentially interfere with all DNA-templated processes. To make things worse, the recipient archaeon was probably a thermophile [START_REF] Ópez-García | Bacterial gene import and mesophilic adaptation in archaea[END_REF][START_REF] Boussau | Parallel adaptations to high temperatures in the Archaean eon[END_REF](30)[START_REF] Catchpole | The evolution of reverse gyrase suggests a nonhyperthermophilic last universal common ancestor[END_REF] and therefore encoded a reverse gyrase with opposite, positive supercoiling activity which is essential for life at high temperature [START_REF] Kikuchi | Reverse gyrase-a topoisomerase which introduces positive superhelical turns into DNA[END_REF][START_REF] Forterre | High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius[END_REF][START_REF] Atomi | Reverse gyrase is not a prerequisite for hyperthermophilic life[END_REF][START_REF] Lipscomb | Reverse gyrase is essential for microbial growth at 95 •[END_REF]. Finally, archaea encode Topo VI as the main type II topoisomerase and its predicted in vivo role in relaxing the positive supercoils overlaps with that of gyrase [START_REF] Gadelle | Phylogenomics of type II DNA topoisomerases: review articles[END_REF].

To understand better the challenges imposed by DNA gyrase to a naïve archaeal cell we introduced a bacterial gyrase in the genetically tractable hyperthermophilic archaeon Thermococcus kodakarensis TS559 which naturally has slightly positively supercoiled DNA [START_REF] Santangelo | Thermococcus kodakarensis genetics: TK1827-encoded ␤-glycosidase, new positive-selection protocol, and targeted and repetitive deletion technology[END_REF]. This archaeon encodes histones and three topoisomerases (reverse gyrase, Topo III and Topo VI) thus mimicking the topological 'kit' present in the ancestor of the Cluster II Euryarchaeota [START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF][START_REF] Boussau | Parallel adaptations to high temperatures in the Archaean eon[END_REF]. As a source of gyrase, we selected the one from the bacterium Thermotoga maritima (TmGyrAB) since its closest relatives are archaeal gyrases (Villain et al., unpublished) increasing the chance that its activity would not be impaired by the archaeal cellular context. In addition, the optimal growth temperature of T. maritima (80 • C) and that of T. kodakarensis (85 • C) are similar, both were isolated from geothermally heated sea floors [START_REF] Huber | Thermotoga maritima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90∼C[END_REF][START_REF] Atomi | Description of Thermococcus kodakaraensis sp. nov., a well studied hyperthermophilic archaeon previously reported as Pyrococcus sp. KOD1[END_REF] and they can be co-cultured in laboratory. Finally, TmGyrAB exhibits the expected negative supercoiling activity both in vitro and in vivo [START_REF] Guipaud | Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium Thermotoga maritima[END_REF](41)[START_REF] Tretter | A naturally chimeric type IIA topoisomerase in Aquifex aeolicus highlights an evolutionary path for the emergence of functional paralogs[END_REF].

We found that the gyrase of T. maritima was active and predominant in T. kodakarensis such that the normally positively supercoiled plasmid DNA was converted to strongly negatively supercoiled DNA. Gyrase interacted with the genome of T. kodakarensis and induced differential expression of hundreds of genes, a subset of which specifically responded to negative supercoiling activity. Reverse gyrase was the only topoisomerase that reacted transcriptionally, albeit modestly, to negative supercoiling activity. Despite unnatural gyrase-enforced topological changes, the growth of T. kodakarensis was not affected. We conclude that gyrase is remarkably well tolerated by T. kodakarensis suggesting the existence of resilience mechanisms against torsional stress which may have been instrumental for the natural establishment of gyrase in the archaeal domain.

MATERIALS AND METHODS

Construction of recombinant Thermococcus kodakarensis TS559 strains

All the strains, plasmids and oligonucleotides used in this study are listed in the Supplementary table S1 andS2. Plasmids were constructed in Escherichia coli strain XL1-Blue grown at 37 • C in LB supplemented with Ampicillin (100 g/ml), Kanamycin (40 g/ml) or Chloramphenicol (20 g/ml) using standard molecular biology protocols. The gyrase-encoding genes gyrA and gyrB were PCR amplified using genomic DNA of Thermotoga maritima MSB8 as template and cloned as a bi-cistronic operon in plasmid pTNAg [START_REF] Catchpole | A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors[END_REF] under the control of the strong constitutive promoter PhmtB. Plasmid constructions were performed by Gibson Assembly using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) following the manufacturer's protocol.

T. kodakarensis TS559 was transformed using standard protocol in anaerobic conditions using the rich medium ASW-YT or the synthetic medium without tryptophan ASW-AAW [START_REF] Catchpole | A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors[END_REF][START_REF] Sato | Improved and versatile transformation system allowing multiple genetic manipulations of the hyperthermophilic archaeon Thermococcus kodakaraensis[END_REF]. Briefly, 10 ml of late exponential phase culture was pelleted and resuspended in 100 l of 0.8× ASW. At least 2 g of plasmid DNA was mixed with 100 l of cell suspension and incubated on ice for 1 h, heat shocked for 1 min at 85 Then 1 ml of non-selective medium was added and cell suspensions were incubated 90 min at 85 • C for recovery. Cells were then harvested by centrifuging 4 min at 4500 g, resuspended in 200 l of 0.8× ASW and plated onto selective solid medium containing 1% (w/v) Phytagel™ (Sigma-Aldrich). Plates were reduced with 2 ml of polysulfide solution (100 g of Na 2 S nonahydrate and 30 g of sulfur dissolved in 75 ml of water) per litre of medium and starch azure was added at 0.2% (w/v) to facilitate visualisation of colonies [START_REF] Akpan | A rapid plate culture method for screening of amylase producing micro-organisms[END_REF]. After 40 h (ASW-YT) or 64 h (ASW-AAW -) of incubation at 85 • C isolated colonies were transferred to selective liquid medium in sealed bottles under N 2 atmosphere. Na 2 S was added to a final concentration of 0.02% (w/v) and resazurin was added at 1 mg/l as an indicator of medium reduction.

To measure growth, liquid cultures of T. kodakarensis were inoculated at 1:100 dilution from fresh precultures. Cell density was monitored with phase-contrast microscopy using a Thoma cell counting chamber (0.01 mm depth) or using an optical device that measured turbidity variations directly in Hungate tubes (MicrobeMeter).

The expression of the gyrA and gyrB genes in transformants was tested by RT-PCR. Briefly, three individual clones of T. kodakarensis TS559 were each grown in 25 ml of ASW-YT without agmatine until late exponential phase.

Cells were harvested (5000 × g, 10 min) and resuspended in 500 l of resuspension buffer (NaCl 1 M, Tris HCl 0.1 M, CaCl 2 5 mM, MgSO 4 0.1 M). The RNAs were extracted using TRIzol according to the supplied protocol (Sigma-Aldrich). To eliminate residual DNA, the samples were treated for 30 min at 37 • C with TURBO DNase (Ther-moFisher) and reextracted with TRIzol. The cDNA was synthetized using Maxima First Strand cDNA Synthesis kit (ThermoFisher) following the manufacturer's protocol. The PCR reactions were performed using each cDNA or total RNA sample as template. The used oligonucleotides are listed in Supplementary table S2. The obtained PCR products were separated on 1% (w/v) agarose gel and stained with ethidium bromide (0.5 g/ml).

Wide-field microscopy and DNA staining of T. kodakarensis cells

A small volume (200 l) of exponentially growing culture was rapidly cooled down in mixture of ice and water to limit the effects of cold shock and oxygen exposure during imaging. Cells were then centrifuged and resuspended in an equal volume of 0.8× ASW containing 1 g/ml of Hoechst 33342 (excitation 361 nm / emission 486 nm) for DNA staining. Cells were incubated for 10 min in the dark and then mounted onto glass slides covered with a thin layer of 1% agarose (suspended in 0.8× ASW solution). DIC and fluorescent images were obtained at room temperature on an SP8 confocal laser scanning microscope (Leica Microsystems) equipped with hybrid detectors and a 63x oil immersion objective (HC Plan Apo, 1.4 numerical aperture [NA]; Leica). Fluorescence detection was performed by exciting the sample with a 405 nm laser and collecting fluorescence between 415 and 515 nm) at the speed of 600 Hz with a line averaging of three. Image format was adjusted to provide an XY optimal sampling (pixel size of 60 nm) and for each position z-stacks (3 m width and 0.5 m step) were acquired.

Method Yen from MicrobeJ software ( 46) was used to perform automatic cell detection and size measurements. The obtained profiles were manually curated to remove debris and aggregated cells. For cell size, circularity and area measurements at least 50 independent images were analysed per strains totalling at least 600 analysed cells.

Two-dimensional agarose gel electrophoresis

Plasmids pTNAg-Y119F and pTNAg-gyrAB are too large (12 602 bp) to be easily resolved by 2D agarose gel electrophoresis we instead used the smaller reporter plasmid pTPTK2 (5455 bp) [START_REF] Catchpole | A series of new E. coli-Thermococcus shuttle vectors compatible with previously existing vectors[END_REF]. T. kodakarensis strains were grown in Ravot medium [START_REF] Gorlas | A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms[END_REF] to increase the yield of intact supercoiled plasmids.

Ravot medium was inoculated from overnight precultures grown in ASW-AAW medium at 1:100 dilution, and growth was monitored until late exponential phase. Then cultures were rapidly chilled in precooled beaker immersed in a water-ice bath to stop topoisomerase or nuclease activities. Plasmids were extracted with the NucleoSpin ® kit (Macherey-Nagel) following the low copy manufacturer's protocol with minor modifications: (i) the lysis step was reduced to 1 min to limit plasmid nicking, (ii) two steps of lysate clarification were performed and (iii) the wash step (AW) was performed to inactivate residual nucleases.

Agarose gels were prepared by dissolving 0.8% (w/v) of ultrapure agarose (Invitrogen) in 1× TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA) and pouring in 14.5 × 20 cm tray. Electrophoresis was performed at 24 • C and minimum 3 g of plasmid DNA was used for each sample. In the first dimension, no intercalating agent or 1.5 g/ml of chloroquine (Alpha Aesar) was added in the gel and in the running buffer. Electrophoresis was run at 1.2 V/cm for 40 h. Gels were subsequently equilibrated for 1 h in 1× TBE buffer supplemented with 7.5 g/ml of chloroquine and placed in the tray after a rotation of 90 • . In the second dimension, an electric field of 2 V/cm was applied for 10 h. Gels were washed 3 × 30 min in water to remove chloroquine and then stained for 1 h with 2.5 g/ml of ethidium bromide. Gels were then rinsed with water and imaged with a Typhoon Imager (Amersham) using Cy3 channel.

Calculation of the supercoiling density

Superhelical density () of the reporter plasmid pTPTK2 (5455 bp) was calculated from the imaged 2D gels using an adaptation of the band-counting method as described by L ópez-García and Forterre [START_REF] Ópez-García | DNA topology in hyperthermophilic archaea: reference states and their variation with Nucleic Acids Research, 2021 15 growth phase, growth temperature, and temperature stresses[END_REF]. As an example, we detail the calculation for the gels presented in Figure 2. Topoisomers were first separated without chloroquine in the first dimension and the major topoisomer was identified using band intensity measurement with Fiji software [START_REF] Rueden | ImageJ2: ImageJ for the next generation of scientific image data[END_REF]. This gave the writhe for the plasmid pTPTK2 (Wr = -5) in strain TKY119F. A second gel was run with 1.5 g/ml of chloroquine in the first dimension, allowing to determine a Wr = + 14 for plasmid pTPTK2 in strain TKY119F. We thus determined that the chloroquine introduced 19 positive supercoils in pTPTK2. To account for the temperature effect on plasmid topology between the growth temperature of Thermococcus (85 • C) and the electrophoresis ( 24• C) we applied the correcting factor of -0.011 • / • C/bp [START_REF] Charbonnier | Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures[END_REF]. The temperature difference thus introduced 10 negative supercoils in pTPTK2. Taking into account the chloroquine effect in the first dimension and the unwinding induced by temperature, we determined a Wr of -5 for the TKY119F strain (Wr = 14 + 10 -19) and -17 for the TKgyrAB strain (-8 + 10 -19). Tw is equal to the length of the plasmid molecule (in base pairs) divided by the number of base pairs per helix turn (h) (h = 10.5 bp per turn under standard conditions). For pTPTK2 the twist is Tw = 5455 bp/10.5 bp. Finally, the was calculated by dividing the Wr by Tw to yield TKY119F = +0.0096 and TKgyrAB = -0.0327. The mean values were calculated from three independent 2D gel electrophoresis experiments.

Plasmid toxicity assay

T. kodakarensis strains TKY119F and TKgyrAB, carrying respectively pLCAg-Y119F and pLCAg-gyrAB, were passaged 14 times at 85 • C with 100-fold dilution in Ravot medium or in Ravot medium supplemented with 1 mM of agmatine. The culture used for the first inoculation and the 4th, 9th and 14th subcultures, corresponding respectively to 0, 24, 54 and 84 cell generations, were anaerobically sampled. These samples were 10-fold serial diluted in 1X Ravot salts solution to a 10 -7 dilution. 10 l of each dilution were spotted on solid selective (Ravot) or non-selective (Ravot with agmatine) medium. Plates were reduced with polysulfides solution and supplemented with starch azure as described above. After 2 days of incubation in anaerobic jars at 85 • C, cell viability was determined by counting colonies from spotted dilutions.

Ciprofloxacin susceptibility test

Ciprofloxacin sensitivity of TKY119F and TKgyrAB was investigated on plates using an adaptation of the inverted spot test method [START_REF] Gorlas | A simple procedure to determine the infectivity and host range of viruses infecting anaerobic and hyperthermophilic microorganisms[END_REF]. Briefly, in anaerobic conditions, 25 ml of Ravot-phytagel medium supplemented with agmatine was poured in Petri dishes as described above. On this bottom layer, a 5 ml thin layer composed of 1X Ravot medium, 0.18% (w/v) of Phytagel™, 1 mM of agmatine, 1% of colloidal sulfur and 160 l of late exponential phase T. kodakarensis culture was poured. On the solidified top layer, 5 mm Whatman paper discs were gently laid. 10 l of ciprofloxacin dilutions dissolved in 0.1 N HCl were spotted on the paper discs. On such plates the top layer initially appears milky-white because of the colloidal sulfur. Growing or TKgyrAB strains and its topoisomers were separated using 2D agarose gel electrophoresis. The corresponding cartoon is depicted below each agarose gel. (A) and (C) DNA intercalating drug chloroquine was added at 7.5 g/ml only in the second-dimension run. This allows to separate positively and negatively supercoiled topoisomers as fast-migrating right arc and slow-migrating left arc, respectively. Under these running conditions, the majority of pTPTK2 isolated from TKgyrAB strain is in extremely negatively supercoiled form. The major (the most abundant) topoisomer from the TKY119F is indicated with a white arrow and was used to calculate superhelical density. (B and D) Chloroquine was added both in the first (1.5 g/ml) and the seconddimension run (7.5 g/ml). Chloroquine introduces a slight positive torsion in DNA, thus changing the apparent superhelical density of topoisomers toward a positively supercoiled state. This allowed relaxing the extremely negatively supercoiled form of pTPTK2 during the first dimension run such that the individual topoisomers could be separated and the major (the most abundant) topoisomer determined. This topoisomer is indicated by a white arrow and was used to calculate superhelical density. (E) Plasmid superhelical densities from various organisms were plotted against the optimal growth temperature of their hosts (see also supplementary table S3). The mean and standard deviation calculated from three independent experiments are indicated for gyrase expressing strain TKgyrAB and the control strains TKY119F. The point corresponding to Archaeoglobi archaea is highlighted by an asterisk. The total change in the supercoiling density ( ) resulting from gyrase activity in T. kodakarensis or from TopoI inhibition in Streptococcus pneumonia is indicated.

T. kodakarensis consumes the sulfur and the top layer becomes transparent. After overnight incubation in anaerobic jars, growth inhibition was assessed by measuring the diameter of the remaining milky-white halos around the Whatman paper discs.

Differential gene expression analysis

To prepare samples for RNA sequencing, 25 ml of Ravot medium was inoculated at 1/100 dilution with fresh T. kodakarensis preculture. Total RNA was extracted from 20 ml of exponentially growing cultures (6 h of culture, approximately 2 × 10 9 cells/ml) using a NucleoSpin RNA set for NucleoZOL (Macherey Nagel). DNA was eliminated from samples using a TURBO DNA-free kit following the manufacturers protocol (Ambion). For each strain, biological replicates were prepared from four independent cultures. Total RNA quality was assessed on an Agilent Bioanalyzer 2100, using RNA 6000 pico kit (Agilent Technologies). Directional RNA-Seq Libraries were constructed using the TruSeq Stranded Total RNA library prep kit, with bacteria Ribo-Zero reagents (Illumina), following the manufacturer's instructions; 500 ng of total RNA were used.

After the Ribo-Zero step, the samples were checked on the Agilent Bioanalyzer for proper rRNA depletion. Final libraries quality was assessed on an Agilent Bioanalyzer 2100, using an Agilent High Sensitivity DNA Kit. Libraries were pooled in equimolar proportions and sequenced on a Paired-End 2 × 75 bp run, on an Illumina NextSeq500 instrument. Demultiplexing was performed with bcl2fastq2 v2.18.12. Adapters were trimmed with Cutadapt v1.15, and only reads longer than 10 bp were kept for further analysis. Between 32 and 56 million reads for each sample were mapped on genome of T. kodakarensis TS559 and plasmid sequences with Burrow-Wheeler Aligner for short-read alignment release 0.7.17-r1188 [START_REF] Li | Fast and accurate short read alignment with Burrows-Wheeler transform[END_REF]. Reads per gene were counted using subread featureCounts v1.5.2 and differential analyses were performed in R using DESeq2 v1.28.1 package [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF].

Adjusted P-values histograms, Principal Component Analysis, Volcano Plot, Heatmap and distribution of deregulated genes across T. kodakarensis TS559 chromosome were drawn using R with gplots and ggplot2 packages. COGs categories of T. kodakarensis genes were extracted from 'The ArCOG database' ( 53) and used to draw the deviation graph with the plotrix R package.

RESULTS

Construction of gyrase-expressing Thermococcus kodakarensis strains

We transformed T. kodakarensis with the replicative plasmid pTNAg encoding gyrA and gyrB genes from T. maritima. The genes were expressed from gyrAB operon under the control of the constitutive archaeal promoter PhmtB. Single clone transformants that we named T. kodakarensis TKgyrAB strain (hereafter TKgyrAB) were readily obtained and the expression of the gyrAB was confirmed by RT-PCR (Supplementary figure S1). We also constructed a control strain carrying the empty vector (hereafter TKAg) and the strain expressing a mutated version of the gyrase where the catalytic tyrosine 119 was replaced by phenylalanine (hereafter TKY119F). This gyrase mutant was shown to bind DNA and ATP but is unable to generate double strand breaks necessary to introduce negative supercoiling in the DNA [START_REF] Horowitz | Mapping the active site tyrosine of Escherichia coli DNA gyrase[END_REF][START_REF] Critchlow | DNA cleavage is not required for the binding of quinolone drugs to the DNA Gyrase-DNA complex[END_REF]. We could not detect protein bands (90.5 kDa for GyrA and 72.5 for GyrB) corresponding to gyrase subunits on a SDS-PAGE gel indicating that in this experimental setup the gyrase was not expressed to very high levels compared to native proteins in TKgyrAB and TKY119F strains (Supplementary figure S2).

Plasmid DNA is negatively supercoiled in gyrase-expressing T. kodakarensis

Native plasmids of Thermococcus species are positively supercoiled in vivo presumably by the action of reverse gyrase [START_REF] Ópez-García | DNA topology in hyperthermophilic archaea: reference states and their variation with Nucleic Acids Research, 2021 15 growth phase, growth temperature, and temperature stresses[END_REF]. To investigate the impact of gyrase on DNA topology in T. kodakarensis we analysed the topology of plasmids isolated from the TKGyrAB, TKAg or TKY119F cultures using agarose gel electrophoresis. To facilitate these analyses, we introduced a smaller reporter plasmid pTPTK2 (5455 bp) into each of the three recombinant Thermococcus strains.

Plasmid topoisomers can be separated as single bands by one dimensional slow electrophoresis in agarose gels containing no DNA intercalating agent. In such conditions, the most highly supercoiled plasmids migrate the fastest forming a single front-band. We observed a broad range distribution of topoisomers in the two control strains as expected for plasmids with low level of positive supercoiling and only one fast-migrating major band of highly supercoiled DNA in the TKGyrAB strain (Supplementary figure S3A). This suggested that the gyrase was active in T. kodakarensis.

To confirm that the fast-migrating major band corresponded to negatively supercoiled DNA (in 1D electrophoresis, positively and negatively supercoiled topoisomers behave identically), we performed 2D agarose gel electrophoresis to determine the orientation (either positive or negative) of the supercoiling [START_REF] Gibson | Two-Dimensional gel electrophoresis to resolve DNA topoisomers[END_REF]. Initial 2D gel electrophoresis was performed in the absence of chloroquine in the first dimension to preserve the natural plasmid topology and with 7.5 g/ml of chloroquine in the second dimension to determine the direction of supercoiling. This confirmed that the vast majority of the reporter plasmid was in an extreme negatively supercoiled state, in stark contrast to plasmid isolated from the control TKY119F strain (Figure 2A andC). The distribution of the topoisomers served as a reference to determine the number of supercoils introduced by chloroquine. Next, the plasmids were separated by adding chloroquine in both the first and the second dimension. This allowed us to determine the Lk for the major topoisomer in the gyrase expressing TKgyrAB strain ( Lk = -8) as well as in the control strain TKY119F ( Lk = -5) by taking into account the number of positive supercoils (+19) introduced by intercalation of chloroquine (Figure 2B andD). We used these values to calculate the mean supercoiling density from three independent experiments, after correction for the temperature effect (due to helix pitch increase) on the plasmid topology (-10 supercoils) we obtained a superhelical density of +0.0077 ± 0.0016 for the control strain ( TKY119F ) and -0.0327 ± 0.0016 for the strain containing the active DNA gyrase ( TKgyrAB ).

Supercoiling density is used as a standardized measure of linking difference and can be compared between different organisms regardless of plasmid size or culturing conditions. The determined mean TKY119F was identical to that of the strain TKAg (Supplementary figure S3B) and it matched well with the reported native plasmid supercoiling level of Thermococcus sp. [START_REF] Ópez-García | DNA topology in hyperthermophilic archaea: reference states and their variation with Nucleic Acids Research, 2021 15 growth phase, growth temperature, and temperature stresses[END_REF][START_REF] Gorlas | Increase of positive supercoiling in a hyperthermophilic archaeon after UV irradiation[END_REF], thus suggesting that the inactive gyrase did not introduce topological changes in the reporter plasmid DNA (Figure 2E, supplementary table S3). The supercoiling density of gyrase-expressing T. kodakarensis matched that of Archaeoglobi archaea which are the only gyrase-encoding hyperthermophilic archaea (Fig- ure 2E, asterisk, Supplementary table S3) [START_REF] Ópez-García | Plasmid pGS5 from the hyperthermophilic archaeon archaeoglobus profundus is negatively supercoiled[END_REF]. Remarkably, the increase in negative supercoiling density in TKgy-rAB strain as compared to the control strain is more than 5-fold.

Collectively, the data show that active gyrase can be expressed in Thermococcus kodakarensis and that this organism can tolerate a substantial increase in negative supercoiling of its plasmid DNA. The data also suggest that the endogenous topoisomerases of T. kodakarensis with capacity to relax negatively supercoiled DNA (principally reverse gyrase and to a lesser extent Topo III and Topo VI) were outcompeted by gyrase.

The gyrase-expressing T. kodakarensis is sensitive to ciprofloxacin

We next asked if gyrase interacted with chromosomal DNA of T. kodakarensis and catalysed the double-stranded breaks required to introduce negative supercoiling. This can be tested indirectly by measuring the sensitivity of strains to the antibiotic ciprofloxacin. Ciprofloxacin binds to gyrase and DNA [START_REF] Mustaev | Fluoroquinolone-gyrase-DNA complexes[END_REF] and kills bacteria efficiently by stabilising the double-stranded break occurring during gyrase activity [START_REF] Gellert | Nalidixic acid resistance: A second genetic character involved in DNA gyrase activity[END_REF][START_REF] Sugino | Mechanism of action of nalidixic acid: Purification of Escherichia coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme[END_REF].

In contrast to what is reported for bacteria, we had to use relatively high concentrations of the drug to observe growth inhibition on plates (Figure 3). Since less than 10% of ciprofloxacin is degraded at the incubation temperature and time-scales we used [START_REF] Pan | Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge[END_REF], the low sensitivity of strain TKGyrAB to the drug may be explained by inefficient transfer of the drug across the membrane and/or very efficient efflux pumps. Intriguingly, we also observed a significant growth retardation of our control strains when exposed to ciprofloxacin indicating that the drug interferes with essential process(es) in T. kodakarensis. The figure 3 shows the result of the antibiogram test on phytagel plates. The cells were plated on non-selective medium to make sure that we detected the toxic effect of the ciprofloxacin (gyrase-DNA covalent adducts) against the chromosome and not against the plasmid that carried the selective marker. The assay repeatedly (n = 4) showed higher sensitivity of TKGyrAB strain to the ciprofloxacin compared to controls, consistent with the formation of toxic gyrase-DNA covalent adducts on the chromosome.

Global transcriptional response to gyrase expression in Thermococcus kodakarensis

To understand better how T. kodakarensis cells cope with the presence of artificial negative supercoiling activity we investigated genome-wide transcriptional responses in the three recombinant strains.

We performed RNA-seq experiments on biological replicates (n = 4) of exponentially growing cells and quantified differential transcript abundance in TKgyrAB versus TKAg, TKY119F versus TKAg and TKgyrAB versus TKY119F (Supplementary figure S4). We reasoned that the latter analysis would, in principle, allow us to identify the subset of genes enriched for those responding specifically to the negative supercoiling activity of the gyrase while TKY119F versus TKAg comparison would yield genes responding to the burden of gyrase heterologous expression and its DNA binding activity. In agreement with this hypothesis, only few differentially expressed genes (DEGs) are shared between TKgyrAB vs TKY119F (genes responding to negative supercoiling) and TKY119F versus TKAg (genes responding to gyrase expression burden and DNA binding) or in all three comparisons (Supplementary figure S5).

We first compared the transcriptional profile of the genes encoded on the empty vector and the two plasmids carrying gyrase genes. This was particularly interesting because, in contrast to the chromosome where the local DNA topology status was not known, we knew that the plasmid encoding active gyrase was negatively supercoiled and thus we could measure how negative supercoiling affected gene transcription. Negative supercoiling facilitates DNA melting and thereby promotes transcription [START_REF] Bell | Temperature, template topology, and factor requirements of archaeal transcription[END_REF][START_REF] El Houdaigui | Bacterial genome architecture shapes global transcriptional regulation by DNA supercoiling[END_REF]. We therefore anticipated an increase in expression of plasmid-borne genes when comparing TKgyrAB vs TKY119F transcriptional profiles. Intriguingly, however, none of the genes (including the gyrAB operon) were differentially expressed (Supplementary table S4). Interestingly, the expression of inactive gyrase was not neutral, three out of five plasmid-encoded genes were significantly downregulated suggesting that gyrase might bind within or in the vicinity of these genes and interfere with their transcription. It has to be noted though that the interpretation of these data needs to be taken with precaution since the precise plasmid copy number in each strain is not known.

We next quantified differential transcript abundance for chromosomal genes. The expression of inactive gyrase alone modified significantly (Padj < 0.05) the expression of 143 genes (fold change > 1.25) most of which (80%) were downregulated in agreement with the hypothesis that the DNA binding activity of gyrase alone impedes transcription (Figure 4 A). This suggested a dominant negative effect that could be explained by the formation of a stable complex in which DNA is wrapped around the CDT domain of the gyrase heterotetramer. Without the possibility to cut the DNA, it is possible that the catalytically dead gyrase would stay bound to DNA thus forming a mechanical barrier for the passage of the RNA polymerase. When active gyrase is expressed, 410 genes were affected which corresponds to ∼18% of total number of annotated genes. Finally, by comparing TKgyrAB and TKY119F expression profiles we identified 205 DEGs which specifically responded to negative supercoiling activity of the gyrase. The comparison of the relative transcript abundances for these genes using Zscore scaling shows an opposite tendency between the gyrase expressing strain and the two control strains (Supplementary figure S6). We have therefore conducted further analyses on this set of genes which we named SRGs for supercoiling-responding genes. SRGs were distributed throughout the entire chromosome of T. kodakarensis without obvious bias with respect to GC skew or transcription direction (Figure 4,B). We did not detect SRGs-specific GC content in the promoter region of SRGs or bias in the size of the corresponding transcripts (Supplementary figure S9). We noticed however, hotspots of deregulated genes in vicinity of ribosomal RNA genes. These regions would be expected to be targeted by the gyrase because the flanking regions of heavily transcribed ribosomal RNA genes are rich in supercoiled DNA [START_REF] Dorman | DNA supercoiling and transcription in bacteria: a two-way street[END_REF].

The most highly deregulated SRGs encode the archaellum components (FC ∼ 54) and the genes involved in chemotaxis which are both highly upregulated (FC ∼ 6). An opposite downregulation trend was observed for genes assigned to energy production and conversion, nucleotide transport and metabolism and cell cycle control, cell division and chromosome partitioning functional categories (Supplementary figure S7). Among 11 DNA-repair related genes (66) only TK0784 encoding a homologue of XPD was significantly deregulated (log 2 FC = 0,33) suggesting that gyrase expression does not induce high levels of DNA damage. Intriguingly, however, the genes encoding Topo VI or Topo III were not deregulated although these enzymes are known to relax negatively supercoiled DNA in vitro (Supplementary Table S5). A notable exception was the re-verse gyrase-encoding gene showing significant upregulation (P adj = 0.007) albeit with a low fold change (FC = 1.2). We next looked into the transcript level of gyrase and reverse gyrase which, although an imperfect proxy of protein quantity, can give some insight into relative protein abundances in cells. To be able to compare the read counts within each sample we converted them into fragments per kilobase million (FPKM, Supplementary table S6). Assuming that all the transcripts are translated into functional protein, this shows that reverse gyrase, Topo VI and Mini-A (distant homolog of type IIB topoisomerases, ( 67)) are present in comparable levels in T. kodakaransis. In GyrAB strain average FPKM values are 1.5 for GyrA, 0.6 for GyrB and 0.47 for reverse gyrase suggesting that the gyrase is the most abundant topoisomerase in the GyrAB strain. From these data, we can speculate that the observed upregulation of reverse gyrase in the GyrAB strain was insufficient to counteract the gyrase negative supercoiling activity as suggested by the topology of the plasmids.

The above results demonstrated that gyrase introduction in T. kodakarensis provoked a genome-wide but, in most cases, mild deregulation of gene expression. The data also suggested that the gyrase-derived negative supercoiling was not handled by the endogenous topoisomerases.

To understand better the molecular bases underpinning the observed transcriptional response, we compared our data with previously published analyses of T. kodakarensis transcriptomes. Among six available datasets one caught our attention as, similar to our study, the fla (archaellum) and che (chemotaxis) operons were the most highly deregulated [START_REF] Sanders | Extended archaeal histone-based chromatin structure regulates global gene expression in Thermococcus kodakarensis[END_REF]. Sanders and colleagues studied the transcriptional profile of T. kodakarensis that either could not build multimeric chromatin particles (strain TS620, HTkB HTkA G17D ) or relied only on Histone B for building histone polymers (strain TS622, HTkB WT HTkA G17D ). Interestingly, out of top 30 upregulated SRGs, at least 26 were downregulated in strains TS620 and TS622 (Figure 5A). Most of these genes belonged to fla and che operons but we also identified two non-operonic chemotaxis genes (TK0156 and TK2147), one gene annotated as AAA + AT-Pase (TK1139) and a predicted FprA family A-type flavoprotein electron transfer protein (TK1605). When we extended this analysis to all SRGs (298 genes with P adj < 0.05) we did not find any correlation with transcriptomes of TS620 and TS622 (Supplementary figure S8). Together, these data suggested that only the most highly upregulated SRGs are sensitive to both gyrase induced negative supercoiling and chromatin structural defect. To further examine the molecular basis for such behaviour we mapped tran-scriptional start sites (TSS) for anti-correlated SRGs based on experimental data from Jäger and coll. [START_REF] Jäger | Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis[END_REF]. The six identified intergenic sequences were subjected to MEME analysis ( 70) which revealed the presence of a 23 bp common motif (consensus sequence TTTGTGTABSTGBTTATG-TAGGT) present in one copy or in two copies for fla operon. The motif, mostly located ∼35 bp upstream of TSS, does not resemble consensus promoter motifs of T. kodakarensis which typically have a B recognition element (BRE) followed by a TATA-box ∼33 and ∼23 bp, respectively, upstream from transcription initiation (69) (Figure 5B). A search for the motif in whole T. kodakarensis genome using FIMO [START_REF] Bailey | The MEME Suite[END_REF] retrieved the six already identified motifs but no additional high scoring hits.

This analysis thus uncovered a common sequence motif found exclusively in the promoter region of the most highly upregulated SRGs, and further suggests that the dysregulation of these genes might be the consequence of alterations of chromatin structure induced by the negative-supercoiling activity of the gyrase.

Impact of gyrase on Thermococcus kodakarensis growth

To assess the impact of gyrase on T. kodakarensis growth we first monitored the growth kinetics of the three strains in batch cultures and at optimal growth conditions by direct cell counting. The recorded growth curves exhibited a typical sigmoidal shape and were overall similar (Figure 6A). The specific growth rate at the exponential phase was lower for TKgyrAB and TKY119F as compared to TKAg control strain, however, the slopes were not significantly different (P = 0.34) indicating that the observed differences are not significant.

Next, the cell shape and size as well as DNA content were assessed using light microscopy coupled with image analysis. This revealed that the exponential phase TKgyrAB cells had the typical irregular round shape as the control cells and contained DNA. However, the measurement of cell area revealed that the gyrase expressing strains were, on average, smaller than the TKAg strain and this difference was statistically significant (P = 8.1 × 10 -70 , Figure 6B, Supplemen-tary figure S10). The phenotype was the most pronounced for TKGyrAB cells which (assuming that the cells are perfectly spherical) exhibited in average 35% less volume compared to TkAg control. Despite being smaller, the TKGy-rAB cells did not exhibit a filamentous phenotype indicating they divided at a normal rate (Supplementary Figure S10).

We next tested whether the gyrase expression becomes toxic during longer culturing. If so, we expected that the gyrase expression plasmid would be gradually lost in nonselective culture conditions. We therefore quantified the gyrase expressing plasmid in the population by counting colony forming units (CFU) under selective versus nonselective conditions over 84 generations (14 subcultures). Plasmid was stably maintained in the TKgyrAB strain while about 50% plasmid loss was observed for strain TKY119F at the final stage of the experiment (Figure 6C). To ensure that the experiment was performed with cells that contained active gyrase we monitored pTPTK2 topology using 1D agarose gel electrophoresis (Supplementary figure S11). The amount of the extreme supercoiled form of pTPTK2 remained constant throughout the experiment indicating no loss of gyrase activity during prolonged culturing of Thermococcus.

Collectively, the data show that the gyrase is remarkably well tolerated by T. kodakarensis.

DISCUSSION

In this study, we demonstrate that it is possible to introduce active bacterial gyrase in the hyperthermophilic archaeon T. kodakarensis, a cellular system that naturally lacks negative supercoiling activity. We further show that, as in bacteria, the gyrase became the dominant topoisomerase converting positively supercoiled plasmids into highly negatively supercoiled DNA. Transcriptomic analyses revealed mild deregulation of hundreds of genes including induction of stress -related flagellar (archaellum) and chemotaxis systems. The analysis of the top 30 upregulated genes (including fla and che operons) revealed the presence of a conserved 23 bp sequence motif in their promoter region. These genes were also systematically downregulated in T. kodakarensis strains carrying mutations in genes encoding histones. Re-verse gyrase was the only topoisomerase of T. kodakarensis for which the expression was altered (slightly upregulated) in response to the negative supercoiling activity of gyrase. Despite global-scale alterations of its cellular context, the T. kodakarensis growth rate was not affected, suggesting that critical DNA-templated processes were not compromised by gyrase activity. This was an unexpected result because the negative supercoiling activity of the gyrase should, in principle, interfere with essential processes such as transcription or DNA replication. In particular, gyrase relaxes positive supercoils accumulating ahead of transcribing RNA polymerase [START_REF] Sutormin | Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome[END_REF], a task predicted to be accomplished by Topo VI in archaea [START_REF] Gadelle | Inhibition of archaeal growth and DNA topoisomerase VI activities by the Hsp90 inhibitor radicicol[END_REF]. Moreover, negative supercoiling facilitates DNA melting which in turn facilitates promoter firing ( 64) but, at high temperatures, also exposes ssDNA to heat-induced damage [START_REF] Lindahl | Instability and decay of the primary structure of DNA[END_REF][START_REF] Hethke | Cell-dree transcription at 95 • : thermostability of transcriptional components and DNA topology requirements of pyrococcus transcription[END_REF]. In spite of these potential threats to genome stability and expression, T. kodakarensis expressing gyrase tolerated an approximately five-fold increase in the negative supercoiling density of plasmid DNA as compared to the natural plasmid DNA topology. Granted, plasmid DNA topology does not necessarily recapitulate chromosomal DNA topology, this observation is still quite impressive when put in perspective with the life-limiting tolerance level of bacteria. Streptococcus pneumoniae can only tolerate increases in negative supercoiling levels of up to 1.4-fold (≈41% increase for plasmid DNA) ( 74) and E. coli of up to 1.1 fold (≈14% increase for plasmid and chromosomal DNA) [START_REF] Pruss | Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes[END_REF]. Besides, the RNA-seq data do not show notable induction of genes involved in DNA repair thus suggesting that the gyrase expression does not result in massive DNA damage in T. kodakarensis.

Why is gyrase so well tolerated by an organism in which the cellular DNA and machineries did not co-evolve to accommodate pervasive negative supercoiling activity? We suggest that during evolution, and in T. kodakarensis, the wrapping of genomic DNA in nucleosome-like structures may have facilitated the establishment of the gyrase in archaeal cells. In Euryarchaea, histones have evolved as most abundant chromatin proteins [START_REF] Sandman | Archaeal histones and the origin of the histone fold[END_REF][START_REF] Peeters | The interplay between nucleoid organization and transcription in archaeal genomes[END_REF] and T. kodakarensis has particularly dense histone coverage approaching 100% [START_REF] Jäger | Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis[END_REF][START_REF] Maruyama | An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis[END_REF][START_REF] Nalabothula | Archaeal nucleosome positioning in vivo and in vitro is directed by primary sequence motifs[END_REF][START_REF] Sanders | The role of archaeal chromatin in transcription[END_REF]. Commonly, archaeal histone dimers assemble into tetramers as minimal nucleosomal units that wrap ∼ 60 bp of DNA [START_REF] Bailey | Histone stoichiometry and DNA circularization in archaeal nucleosomes[END_REF] but in some archaea, including T. kodakarensis, the tetramers can be extended via incorporation of additional dimers into particles of variable size that wrap up to 480 bp of DNA in negatively constrained supercoils [START_REF] Maruyama | An alternative beads-on-a-string chromatin architecture in Thermococcus kodakarensis[END_REF][START_REF] Yunwei | Transcription by an Archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome[END_REF][START_REF] Mattiroli | Structure of histone-based chromatin in Archaea[END_REF]. This flexible chromatin structure may participate in adaptive responses of T. kodakarensis by restructuring the existing pool of histones such that the local gyrase-induced increase of negative supercoiling would be efficiently absorbed. In line with this idea, we discovered that the most highly upregulated SRGs are almost systematically downregulated in T. kodakarensis strains carrying mutations in histone genes. Remarkably, the anti-correlated transcriptional response is always associated with a 23 bp motif occurring about 35 bp upstream of transcription start site of these SRGs. The function of this motif is currently unclear but we can speculate that its presence may render the promoter region particularly sensitive to alterations of DNA topology. Along the same line, it is interesting to note that artificial chromatinization of the E. coli genome with archaeal histones resulted in downregulation of gyrase genes as a part of the adaptive cellular response [START_REF] Rojec | Chromatinization of Escherichia coli with archaeal histones[END_REF]. Together, these observations gathered in artificial settings suggest that both organisms tolerate well the introduction of major modellers of DNA topology and react by balancing chromatin structure and DNA supercoiling to achieve a DNA geometry necessary to sustain life.

Once established, the gyrase became fixed in many archaeal lineages to the point where it has become an essential protein in present-day archaea. Indeed, early studies showed that gyrase-targeting drugs such as novobiocin and ciprofloxacin inhibited growth of different archaea including methanogens, halophiles and thermoacidophiles [START_REF] Sioud | Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme[END_REF]. These experiments also established that gyrase was responsible for introducing most, if not all of the negative supercoils in plasmid DNA molecules [START_REF] Sioud | Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme[END_REF][START_REF] Sioud | Novobiocin induces positive supercoilling of small plasmids from halophilic arctiaebacterla in vivo[END_REF]. Notably, novobiocin treatment of Halobacterium halobium cultures stopped DNA replication specifically and instantaneously indicating, albeit indirectly, that gyrase acts upon chromosomal DNA during the elongation step of DNA replication [START_REF] Sioud | Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme[END_REF]. Collectively, these findings suggest that the negative supercoiling activity of the gyrase was positively selected in the course of evolution, but the selective advantage conferred by this feature remains to be established and future studies of in vivo functions of archaeal gyrases should bring some insight. In Bacteria, gyrase-controlled negative supercoiling is instrumental for quick adaptation of the cellular protein repertoire to changing environmental conditions [START_REF] Dorman | DNA supercoiling is a fundamental regulatory principle in the control of bacterial gene expression[END_REF] but whether such mechanisms operate in gyrase-encoding Archaea is not known. Indirect evidence points to involvement of negative DNA supercoiling in gene expression control in extreme halophiles. In these organisms, a plasmid-encoded gyrB gene and chromosomally encoded bop gene (encoding bacteriorodopsin) were strongly induced (up to 20-fold) by DNA relaxation in novobiocintreated cultures, a drug that inhibits gyrase activity [START_REF] Holmes | Mutations in DNA gyrase result in novobiocin resistance in halophilic archaebacteria[END_REF][START_REF] Yang | Genetic and topological analyses of the bop promoter of Halobacterium halobium: stimulation by DNA supercoiling and non-B-DNA structure[END_REF]. More recently, a global transcriptome analysis was reported for novobiocin-treated Halobacterium species [START_REF] Tarasov | A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum[END_REF]. The expression of many genes was affected including the upregulation of gyrase, topoisomerase VI and topoisomerase I expression indicating the involvement of these enzymes in regulation of the DNA supercoiling levels in this organism [START_REF] Tarasov | A small basic protein from the brz-brb operon is involved in regulation of bop transcription in Halobacterium salinarum[END_REF]. However, to what extent the gyrase controls the chromosomal supercoiling and how its activity is coordinated with other archaeal topoisomerases, histones and NAPs is currently unknown.

Related to that, it is noteworthy that reverse gyrase was the only topoisomerase of T. kodakarensis that responded to the negative supercoiling activity of the gyrase. Reverse gyrase is a Topo I enzyme and the only topoisomerase capable of supercoiling DNA positively [START_REF] Kikuchi | Reverse gyrase-a topoisomerase which introduces positive superhelical turns into DNA[END_REF]. Through this activity, reverse gyrase can remove negative supercoiling [START_REF] Kikuchi | Reverse gyrase-a topoisomerase which introduces positive superhelical turns into DNA[END_REF][START_REF] Forterre | High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius[END_REF][START_REF] Bizard | Caractérisation des topoisomérases de type IA de l'archaeon hyperthermophile Sulfolobus solfataricus[END_REF][START_REF] Couturier | The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus[END_REF]. Remarkably, reverse gyrase is found specifically in thermophilic organisms and its deletion in T. kodakarensis and Pyrococcus furiosus is lethal at 93 and 95 • C, respectively [START_REF] Atomi | Reverse gyrase is not a prerequisite for hyperthermophilic life[END_REF][START_REF] Lipscomb | Reverse gyrase is essential for microbial growth at 95 •[END_REF]. It was initially suggested that reverse gyrase prevents thermal denaturation of the double helix by introducing positive supercoils in chromosomal DNA [START_REF] Kikuchi | Reverse gyrase-a topoisomerase which introduces positive superhelical turns into DNA[END_REF][START_REF] Forterre | High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius[END_REF][START_REF] Atomi | Reverse gyrase is not a prerequisite for hyperthermophilic life[END_REF]. However, the idea that positively supercoiled DNA, in spite of its stabilising effect, is not essential for a hyperthermophilic lifestyle was put forward some time ago based on the observation that thermophilic Thermotoga bacteria and gyrase-encoding hyperthermophilic Archaeoglobi archaea contain negatively supercoiled plasmids [START_REF] Guipaud | Both DNA gyrase and reverse gyrase are present in the hyperthermophilic bacterium Thermotoga maritima[END_REF][START_REF] Ópez-García | Plasmid pGS5 from the hyperthermophilic archaeon archaeoglobus profundus is negatively supercoiled[END_REF]. Later studies reported the involvement of this topoisomerase in DNA repair [START_REF] Kampmann | Reverse gyrase has heat-protective DNA chaperone activity independent of supercoiling[END_REF][START_REF] Napoli | Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus[END_REF](94)[START_REF] Valenti | Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus[END_REF]. Recently, however, a study of gyrase-less Saccharolobus (formerly Sulfolobus) solfataricus reported that reverse gyrase is involved in homeostatic control of DNA supercoiling mainly based on the finding that the protein abundance increased about two-fold when cells were exposed to supraoptimal temperatures and the enzyme was more active in vitro [START_REF] Couturier | The reverse gyrase TopR1 is responsible for the homeostatic control of DNA supercoiling in the hyperthermophilic archaeon Sulfolobus solfataricus[END_REF]. Our data show that 1.2-fold (at the mRNA level) upregulation of reverse gyrase in T. kodakarensis is insufficient to restore natural DNA topology and suggest that, at least in Thermococcus and in these artifi-cial conditions, this topoisomerase is not essential for homeostatic control of DNA topology.

In contrast, we were intrigued to find that the transcription profile of plasmid-encoded genes in the gyraseexpressing T. kodakarensis seems not to be modified. The most clearly identified effect of supercoiling on transcription initiation results from the requirement of RNA polymerase to open the double helix in order to gain access to the template strand. Negative supercoiling facilitates melting of the double helix and a strong regulatory effect of negative supercoiling upon gene transcription is well recorded in bacteria [START_REF] Martis | DNA supercoiling: an ancestral regulator of gene expression in pathogenic bacteria?[END_REF][START_REF] Dorman | DNA supercoiling and transcription in bacteria: a two-way street[END_REF]. In hyperthermophiles, which have relaxed or slightly positively supercoiled DNA, it is thought that the elevated growth temperature may replace negative supercoiling as source of melting energy [START_REF] Forterre | A hot story from comparative genomics: reverse gyrase is the only hyperthermophile-specific protein[END_REF][START_REF] Meyer | Temperature dependence of the DNA double helix at the nanoscale: Structure, elasticity, and fluctuations[END_REF] and we therefore expected that the gyrase-induced negative supercoiling and high temperature would synergistically activate the expression of plasmid-borne genes in T. kodakarensis. The fact that this is not so suggests that T. kodakarensis is naturally equipped to allow transcription from topologically different DNA templates. This is reminiscent of observations made by Bell and colleagues who studied the effect of temperature and template topology on expression from an archaeal ribosomal RNA promoter using a highly purified in vitro system from crenarchaeon Sulfolobus [START_REF] Bell | Temperature, template topology, and factor requirements of archaeal transcription[END_REF]. They found that, in marked contrast to characterised bacterial and eukaryal systems, DNA template topology had negligible effect on transcription levels at 78 • C, the optimal growth temperature for Sulfolobus. In another study, Hethke et al. used the cell-free transcription system of Pyrococcus furiosus, a euryarchaeon closely related to Thermococcus, to study the effect of DNA template topology on expression from the gdh promoter at 70 and 90 • C (73). They found that at both temperatures negatively supercoiled DNA was the preferred template compared with relaxed DNA and that positive supercoiling deteriorates the template activity of DNA. It is unclear whether the differences between P. furiosus and Sulfolobus transcription systems come from the type of promoter used or from the intrinsic properties of their transcription machineries [START_REF] Hethke | Cell-dree transcription at 95 • : thermostability of transcriptional components and DNA topology requirements of pyrococcus transcription[END_REF]. We can now use the gyrase-expressing T. kodakarensis to perform similar studies in vivo and thus study the relationship between supercoiling and gene expression in more natural settings.

Would global negative supercoiling be advantageous to a hyperthermophile at lower, suboptimal temperatures? The experiments described by Bell and colleagues suggest so since the Sulfolobus system was unable to transcribe relaxed or positively supercoiled templates at 48 • C. They further highlighted that, in response to cold shock, hyperthermophiles rapidly reduce their plasmid linking number [START_REF] Ópez-García | DNA topology in hyperthermophilic archaea: reference states and their variation with Nucleic Acids Research, 2021 15 growth phase, growth temperature, and temperature stresses[END_REF][START_REF] Marguet | DNA topological change in the hyperthermophilic archaeon Pyrococcus abyssi exposed to low temperature[END_REF] raising the possibility that global regulation of DNA superhelical state in vivo represents an effective mechanism for ensuring continued gene expression after drastic changes in temperature of the environment. Building upon these findings, more than 20 years ago, P. L ópez-García proposed that archaea may have improved their adaptability to mesophily by importing the gyrase from bacteria [START_REF] Ópez-García | DNA supercoiling and temperature adaptation: a clue to early diversification of life?[END_REF]. This idea was reinforced later on by the finding that the vast majority of gyrase-encoding monophyletic group II Euryarchaea are mesophiles in spite of their thermophilic origin [START_REF] Forterre | Phylogenomics of DNA topoisomerases: their origin and putative roles in the emergence of modern organisms[END_REF][START_REF] Raymann | Global phylogenomic analysis disentangles the complex evolutionary history of DNA replication in archaea[END_REF][START_REF] Ópez-García | Bacterial gene import and mesophilic adaptation in archaea[END_REF]. These archaea possess histones leading to the proposal that the acquisition of the gyrase may have had a synergistic effect on DNA-dependent processes in these organisms, with associated changes in transcriptional patterns thus contributing to bacterial-like progressive adaptation to lower temperatures [START_REF] Ópez-García | Bacterial gene import and mesophilic adaptation in archaea[END_REF]. The gyrase-expressing T. kodakarensis now offers the possibility to test this evolutionary hypothesis and ultimately understand why several archaeal lineages became addicted to gyrase.

INTRODUCTION

High temperatures are considerably challenging for cell physiology and particularly for genome stability. As the molecular motion increases proportionally to rise in temperature, molecules, membranes and DNA become unstable. Concretely, in in vitro conditions exposure of DNA to high temperature results in high rates of oxidation, alkylation, deamination, depurination and finally, strand breaks (Lindahl 1993;Marguet and Forterre 1994). To protect their genomes against these toxic DNA lesions, thermophiles and hyperthermophiles rely on specific adaptations. Among others, they harbour GC-rich rRNA, specific lipids, high proportions of thermostable amino acids in their proteins (typically I, V, Y, W R, E, L), chaperones, high salts and polyamines concentrations in their cytoplasms, specific ions transporters and a topoisomerase, the reverse gyrase (Grogan 1998;Zeldovich, Berezovsky, and Shakhnovich 2007;López-García et al. 2015).

This peculiar type IA topoisomerase was discovered in 1984 in Sulfolobus acidocaldarius and named after its unique positive supercoiling activity, that mirrored the one of DNA gyrase (Kikuchi and Asai 1984). Reverse gyrase is a fusion of a SF2-helicase and a topoisomerase domains, and consequently exhibits ATP-dependent and MgCl2-dependent activity (Figure 1) (Confalonieri et al. 1993). It has been proposed that reverse gyrase creates both negative and positive supercoiling by opening DNA and then relaxing only the negative supercoiling leading to a net increase in positive supercoiling.

The helicase and the topoisomerase domains cooperate thanks to the latch during the whole process (Lulchev and Klostermeier 2014). Remarkably, so far, reverse gyrase is the only protein shared by every hyperthermophile and most thermophiles, making it the hallmark of hyperthermophily (Forterre 2002). Its protective effect against high temperature was thought to come from the higher stability of positively supercoiled DNA, a topology that displays increased base stacking and is more resistant to double strand melting at high temperature.

The coating of DNA through chromatinization is another strategy to stabilize DNA at high temperature.

Histones from archaea are classically organized in tetrameric nucleosomes (Mattiroli et al. 2017). But some archaea like T. kodakarensis, possess histones that can extend this basic structure by several dimers of histones (Maruyama et al. 2013). This results in assembly of histones into oligomers of various size that coat the DNA in a filamentous structure. T. kodakarensis was shown to exhibit almost complete histone coverage in its chromatin (Rojec et al. 2019) This high histone coverage over the genome suggests that histones are major actors of genome architecture and gene regulation in T.

kodakarensis. Importantly, histones can titrate supercoiling both by constraining it or by releasing supercoils from opposite sense. Conversely, histones binding to DNA and histones oligomerization can be impacted by DNA supercoiling.

In most hyperthermophiles investigated so far plasmids were found in relaxed or slightly positively supercoiled state (López-García and Forterre 1999; Charbonnier and Forterre 1994). However, hyperthermophiles with negatively supercoiled DNA exist and they invariably encode DNA gyrase, indicating that when the two topoisomerases co-exist in a cell, the DNA gyrase dictates the overall DNA topology (Guipaud et al. 1997;Bouthier de la Tour et al. 1998;López-García et al. 2000). We recently demonstrated, by introducing an active DNA gyrase in the naïve hyperthermophilic archaeon

Thermococcus kodakarensis (Villain et al. 2021), that the topology of plasmids can be converted from positive to negative supercoiling without adverse effect on growth of this archaeon. This demonstration further challenges the hypothesis that positively supercoiled DNA is an important requirement for hyperthermophiles and raises questions about the physiological role of reverse gyrase.

Investigation of reverse gyrase function in hyperthermophilic archaea using mutational analyses produced ambiguous results. Deletion of the gene encoding reverse gyrase in euryarchaeon T.

kodakarensis is viable and does not cause growth defect at 85°C, the optimal growth temperature.

However, at 90°C, the growth rate is decreased by 2-fold and the strain does not grow at all at 93°C (Atomi, Matsumi, and Imanaka 2004). In closely related Pyrococcus furiosus the absence of reverse gyrase is lethal at 95°C, a suboptimal growth temperature for this archaeon which optimally grows at 100°C (Lipscomb et al. 2017). In crenarchaeon Sulfolobus islandicus each of the two genes encoding reverse gyrase can be deleted separately, but the deletion of TopR2 can only be obtained after 14-20 days of incubation that seems hard to reconcile with survival in natural growth conditions [START_REF] Zhang | Genetic Manipulation in Sulfolobus Islandicus and Functional Analysis of DNA Repair Genes[END_REF][START_REF] Drolet | DNA Topoisomerases: Methods and Protocols[END_REF].

Besides its positive supercoiling activity, several lines of evidence support direct involvement of reverse gyrase in DNA repair (reviewed in Garnier et al. 2021). Reverse gyrase of Sulfolobus solfataricus forms stable covalent complexes with UV-damaged DNA in vitro either using purified enzyme or with cell extracts (Napoli et al. 2004). At high protein/DNA ratio, the reverse gyrase of Archaeoglobus fulgidus prevents inappropriate aggregation of heat-denatured DNA regions and promotes correct annealing in vitro thus protecting DNA from heat-induced double strand breaks independently of any enzymatic activity (Kampmann and Stock 2004). Using global protein-interaction network approach the interaction was detected between reverse gyrase and the single strand DNA binding protein RPA of Pyrococcus abyssi (Pluchon et al., 2013). In Sulfolobus solfataricus reverse gyrase was found to form DNA-bridged complex with the single strand binding protein SSB [START_REF] Napoli | Functional Interaction of Reverse Gyrase with Single-Strand Binding Protein of the Archaeon Sulfolobus[END_REF]. The binding to SSB enhanced the topoisomerase activity of reverse gyrase suggesting its direct involvement in the DNA repair. In the following study the same group showed that reverse gyrase co-immunoprecipitates with translational polymerase Y (PolY) known for its essential role in DNA repair (Valenti et al. 2009).

The reverse gyrase inhibits the activity of PolY in vitro and the positive supercoiling activity is required for inhibition suggesting that reverse gyrase may regulate the PolY activity when DNA damage occurs

Other experiments examined the role of reverse gyrase when Sulfolobus islandicus cells were challenged with methyl methanesulfonate (MMS), a DNA alkylation agent which provokes DNA backbone breakage. The depletion of TopR1 in these conditions resulted in accelerated genomic DNA degradation accompanied by a higher rate of cell death (Han, Feng, and She 2017).

Since reverse gyrase was recognised as the only hallmark of hyperthermophily 20 years ago, a substantial amount of work was done to understand what makes this protein indispensable for life at high temperature. In spite of this considerable effort the physiological role of reverse gyrase and in particular, its precise contribution to maintenance of genome integrity remains unclear. One of the main unresolved questions remains to understand what is the contribution of the reverse gyrase to the overall chromosomal DNA supercoiling. To address this question, we have characterized in vivo the impact of reverse gyrase deletion on cellular DNA topology. We first demonstrated that the reverse gyrase is indeed responsible for positive supercoiling of plasmids in hyperthermophilic archaeon Thermococcus kodakarensis. Using micrococcal nuclease (MNase) digestion of chromatin, we show that reverse gyrase deletion does not lead to aberrant profile suggesting that the lack of positive supercoiling activity in T. kodakarensis cells has no strong effect on nucleosome assembly.

MATERIAL AND METHODS

Strain construction

Transformation of T. kodakarensis was performed using standard protocol as previously described (Villain et al. 2021). The T. kodakarensis DAD (ΔpyrF, ΔTK0149) and DAD∆RG (ΔpyrF, ΔTK0149, ΔTK0470) strains, are both auxotroph for uracil (ΔpyrF) and agmatine (ΔTK0149). DAD∆RG carries, in addition, the deletion of the gene encoding reverse gyrase (ΔTK0470). The two strains were kindly provided by Hiroki Higashibata (Toyo University). A subsequent ΔtrpE::pyrF replacement was made in these strains to allow tryptophan-based selection, resulting in TKDAD and TK∆RG strains. These two strains were transformed for two-dimensional gel electrophoresis with pTPTK3 replicating plasmid (Catchpole et al. 2018) resulting in TKDADp and TK∆RGp strains.

Two-dimensional agarose gel electrophoresis

To prepare samples for 2D agarose gel electrophoresis, Ravot medium was inoculated from overnight precultures of TKDADp and TK∆RGp at 1:100 dilution and incubated at 85°C without shaking until late exponential growth phase. Plasmids were gently extracted to preserve their topological integrity, as previously described (Villain et al. 2021).

Agarose gel electrophoresis was performed as previously described with minor modifications (Villain et al. 2021). Briefly, agarose gels were prepared by dissolving 0.8 % (w/v) of agarose in 1X TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA). Electrophoresis was performed at room temperature (usually 24°C) and minimum 2 µg of plasmid DNA was used for each sample. In the first dimension, no intercalating agent or 1.5 µg/ml of chloroquine was added in the gel and in the running buffer.

Electrophoresis was run at 1.2 V/cm for 22 h. Gels were subsequently equilibrated for 1 h in 1X TBE buffer supplemented with 7.5 µg/ml of chloroquine and placed in the tray after a rotation of 90°. In the second dimension, an electric field of 2 V/cm was applied for 5 h. Gels were washed 3 x 30 min in water to remove chloroquine and then stained for 1h with 2.5 µg/ml of ethidium bromide. Gels were then rinsed with water and imaged with a Typhoon Imager using Cy3 channel. The main topoisomer from each plasmid preparation was determined by quantifying the intensity of individual topoisomer bands on gel images using Fiji software [START_REF] Schindelin | Fiji: An Open-Source Platform for Biological-Image Analysis[END_REF].

Nucleosome profiling by micrococcal nuclease digestion

Chromatin isolation and MNase digestion were carried out as previously described with minor modifications (Sanders et al. 2021). Briefly, strains TKDAD and TK∆RG were cultivated from fresh overnight precultures in 500 ml of Ravot medium until late exponential phase. Cultures were rapidly chilled in precooled beaker immersed in a water-ice bath to stop cellular metabolism and thus minimize the risk of nucleosome disassembly. Cells were pelleted by centrifugation (4500 x g, 15 min, 4°C) and then washed with Ravot salt solution before storing at -80°C. Frozen pellets were resuspended in 1 ml of MNase buffer (50 mM Tris-HCl pH 8, 100 mM NaCl, and 1 mM CaCl2) and then submitted to 5 cycles of nitrogen freezing plus grinding with mortar. Cell lysate was subsequently recovered by pipetting from the mortar and clarified by centrifugation for 5 min at 1,700 x g and 4°C. 500 µl of clarified lysates were carefully collected by pipetting and digested for 1 h at 37°C with 70 U of RNase A (Qiagen). Samples were separated into 5 x 100 µl fractions and then each fraction was digested 3 min at 37°C with either 0, 1, 10, 100 or 500 U of MNase (Thermo Scientific, ≥100 U/µl). DNA from digested fractions was immediately extracted with 300 µl of 10 mM Tris-HCl pH 8 and 400 µl of phenol/chloroform/isoamyl alcohol (25:24:1). Mixtures were vortexed for about 2 min and then centrifuged for 4 min at 14,000 x g, room temperature. 200 µl of the top aqueous phase were collected for each sample. DNA was precipitated by adding 200 µl of 1 M Tris-HCl pH 8 and 1 ml of pure ethanol, followed by overnight incubation at -80°C. DNA fragments were pelleted 30 min at 20,000 x g and 4°C.

Resulting pellets were resuspended in 20 µl of TE (10 mM Tris-HCl pH 8, 1 mM EDTA) and resolved on a 4% agarose gel in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA) for 3 h at 4 V/cm.

RESULTS

In vivo measurement of reverse gyrase supercoiling activity in Thermococcus kodakarensis

The positive supercoiling of plasmids extracted from Thermococcales and other hyperthermophilic archaea is thought to come from reverse gyrase based on in vitro enzymatic assays either with purified protein or with cell extracts. However, the positive supercoiling activity of the reverse gyrase has never been tested in vivo. To this end, we used two-dimensional agarose gel electrophoresis to analyse the topology of plasmids extracted from strains TKDADp or TK∆RGp.

This allowed to determine the major topoisomers for reporter plasmid pTPTK3 isolated from TKDADp and TK∆RGp strains and calculate their superhelical density (σ) which (P. López-García and Forterre 1997). This value takes into account the size of the plasmid (4710 pb) and the effect of temperature shift from 85°C, the optimal growth temperature of T. kodakarensis, to 22°C the temperature of electrophoresis (~22°C) on plasmid topology (Figure 2,A). Superhelical density of σ = +0.0089 was determined for TKDADp strain, in good agreement with published values. In contrast, the superhelical density of pTPTK3 plasmid isolated from strain TK∆RGp drops to zero (Figure 2 Reporter plasmid pTPTK3 was isolated from TKDADp or TK∆RGp strains and the topoisomers were resolved using 2D agarose gel electrophoresis. The corresponding cartoon image showing the distribution of different topological forms of pTPTK3 is depicted below each gel. Plasmid topoisomers were resolved by their degree of supercoiling in the first dimension, independently of their sense of supercoiling. In the second dimension, the addition of DNA intercalating drug chloroquine allows to discriminate between positive and negative supercoiling. Chloroquine introduces positive supercoils in plasmid DNA, thus increasing the electrophoretic mobility of positively supercoiled DNA or decreasing the migration of negatively supercoiled DNA. This results in a typical bell-shaped distribution, with negative, slow migrating topoisomers in the left arch and positive, fast migrating topoisomers in the right arch.

(A) and (C) DNA intercalating drug chloroquine was added only in the second dimension run to separate positive and negative topoisomers. Major topoisomers are indicated with a white arrow and these were used to calculate superhelical densities indicated below each gel. The error margin indicated (±0.0022) accounts for a potential mistake of ±1 during the determination of the main topoisomer.

(B) and (D) Chloroquine was added both in the first and the second dimension runs. The addition of the chloroquine in the first run allows to resolve topoisomers from TK∆RG strain by shifting them towards a positive supercoiling. Major topoisomers strain are indicated with a white arrow and used to calculate superhelical densities indicated below the gel. The error margin indicated (±0.0022) accounts for a potential mistake of ±1 during the determination of the main topoisomer.

Histone oligomerization is not affected in T. kodakarensis TK∆RG

We used micrococcal nuclease (MNase) digestion of chromatin to assess the oligomerization state of histones in nucleosomes. Using various concentration of MNase, we observed protected fragments of [START_REF] Gellert | Nalidixic acid resistance: A second genetic character involved in DNA gyrase activity[END_REF][START_REF] Bizard | Caractérisation des topoisomérases de type IA de l'archaeon hyperthermophile Sulfolobus solfataricus[END_REF]120,150 bp corresponding to oligomers of up to four dimers (Figure 3). The most prominent band corresponds to 90 bp DNA fragments showing that trimer of histone dimers is the dominant oligomer in our experimental conditions (Figure 3). Digestion patterns of TKDAD and TK∆RG strains were very similar, suggesting that reverse gyrase deletion does not affect histone oligomerization in T.

kodakarensis.

DISCUSSION

The precise role of reverse gyrase in hyperthermophilic organisms is still an open question. While positive supercoiling activity of reverse gyrase appears as an obvious candidate to protect genome against heat-induced damages, a substantial evidence suggests that this enzyme may also function as a DNA chaperone and in DNA repair. Besides this experimental evidence, the possibility for hyperthermophiles to harbour negatively supercoiled DNA argues strongly against positively supercoiled DNA as prerequisite for thermophilic lifestyle. (Guipaud et al. 1997;López-García et al. 2000). We recently reinforced this idea by showing that T. kodakarensis can accommodate a dominant gyrase activity, that changed its DNA topology from positive to negative supercoiling, without adverse effects upon growth (Villain et al. 2021). Noteworthy, the reverse gyrase was the sole endogenous topoisomerase that was upregulated in response to gyrase activity but this dysregulation was insufficient to restore the initial, positive, DNA topology in T. kodakarensis. These results suggested that the maintenance of a narrow range of supercoiling, compatible with DNA transactions, is not prerequisite for life in Thermococcus. This is in stark contrast with what is known for model bacteria where supercoiling levels are strictly regulated by joint action of DNA gyrase and Topo I and even slight perturbation of this system is lethal (Pruss, Manes, and Drlica 1982;Ferrándiz et al. 2016). In T.

kodakarensis, reverse gyrase is an obvious candidate with the topoisomerase VI to handle the accumulation of negative supercoiling consecutive to DNA replication and transcription. In line with this hypothesis, it has been recently proposed that the TopR1reverse gyrase is important for supercoiling homeostasis in Sulfolobus solfataricus (Couturier et al. 2020). In addition, topR2 was shown to be an essential gene in S. solfataricus, as expected for a gene involved in the removing of topological constrains generated by DNA transactions [START_REF] Zhang | Genetic Manipulation in Sulfolobus Islandicus and Functional Analysis of DNA Repair Genes[END_REF][START_REF] Drolet | DNA Topoisomerases: Methods and Protocols[END_REF]. However, deletion of reverse gyrase is perfectly viable in T. kodakarensis at optimal growth temperature, indicating that replication and transcription are still possible without its positive supercoiling activity (Atomi, Matsumi, and Imanaka 2004).

In light on these data it remains unclear what is the function of reverse gyrase in controlling overall chromosomal supercoiling and whether this enzyme collaborates with topoisomerase VI in preventing stalling of DNA transaction processes because of accumulation of DNA supercoils. Thus, a comprehensive study in vivo of the exact role of reverse gyrase on DNA topology seems now essential to clarify this puzzling situation.

The work presented in this paper draft is a first attempt to initiate such a study. To the best of our knowledge, we performed the first in vivo measurement of reverse gyrase contribution to DNA topology. The results confirm that all the positive supercoiling detected by plasmid topoisomers analysis can be attributed to reverse gyrase, excluding transcription-induced supercoiling. This results corroborate plasmid topology studies performed in vitro from cells extracts (Atomi, Matsumi, and Imanaka 2004) and confirm the high tolerance of T. kodakarensis to DNA topology variations as demonstrated in a previous study (Villain et al. 2021). Two-dimensional electrophoresis of small plasmid extracted directly from the organism studied is a reliable method to directly measure the supercoiling state of DNA. This will be a valuable tool to characterize further the importance of reverse gyrase on the DNA topology of hyperthermophiles.

Chromatin digestion by MNase shows the same typical protection pattern (60, 90, 120, 150 bp) corresponding to nucleosomes of up to five histones dimers in TKDAD and TK∆RG. This indicates that the loss of positive supercoiling consecutive to reverse gyrase deletion does not impact histones oligomerization. However, we did not observe longer fragments as reported in previous studies (Maruyama et al. 2011;[START_REF] Oberto | SyntTax: A Web Server Linking Synteny to Prokaryotic Taxonomy[END_REF]Mattiroli et al. 2017;Sanders et al. 2021). This difference of histone oligomerization could be explained by a different growth stage of cells used to extract chromatin, or the different culture medium used in our study (Ravot medium instead of ASW-YT). The mapping of histone binding across the whole genome by MNase-seq should yield valuable information about the interplay between the reverse gyrase function and chromatinization in T. kodakarensis.

Together, this study and previous meticulous work accomplished to develop genetic engineering in archaea, paves the way for a more detailed investigation of the relation between histones and DNA supercoiling in hyperthermophilic archaea.

INTRODUCTION

The helical nature of DNA while providing stability to the molecule, imposes strong topological constrains every time that the two strands must be separated (Watson and Crick 1953). This means that DNA transactions like replication, transcription, recombination and repair complexify DNA topology, resulting in supercoils, knots, catenanes and non-B DNA transitions (Liu and Wang 1987;Postow et al. 2001;Bates and Maxwell 2005). The accumulation of these structures influence in return DNA transactions, interfering with normal cellular processes. To maintain topological constrains at a physiological level, cells rely on a wide set of actors dedicated to DNA topology that cooperate to release constrains or contain them in specific regions of chromosomes. The most common and important type of DNA topological structure is supercoiling, which is directly regulated by topoisomerases (Schoeffler and Berger 2008;Wang 2002;Bush, Evans-Roberts, and Maxwell 2015;Seol and Neuman 2016;McKie, Neuman, and Maxwell 2021).

DNA supercoiling has been extensively studied in model organisms by studying plasmid topology using sucrose gradient or agarose gel electrophoresis. These methods are based on the increased mobility of supercoiled plasmids (as compared to relaxed plasmids) due to their compactness. While easy to set up, these methods are limited by the fact that plasmids, due to their small size, do not faithfully represent much bigger and complex chromosomes. Genetic approaches, relying on the site-specific recombination of resolvase ɤδ or bacteriophage λ integrase (Bliska and Cozzarelli 1987;Cibot, n.d.), was used in the past to estimate supercoiling distribution across the chromosome. However, these methods are restricted to genetically tractable model bacteria and imply genetic engineering with potential side effects. Other methods based on psoralen derivatives overcome these limitations by allowing measurement of relative supercoiling directly on chromosomes without need for genetic engineering [START_REF] Corless | Investigating DNA Supercoiling in Eukaryotic Genomes[END_REF]. Psoralen is a little planar aromatic molecule that intercalates between DNA base pairs and establishes crosslink under UV light. Underwound DNA is about two fold more likely to incorporate psoralen, making it a reliable tool for mapping negative DNA supercoiling [START_REF] Sinden | Psoralen Cross-Linking as Probe of Torsional Tension and Topological Domain Size in Vivo[END_REF][START_REF] Bermúdez | A Method for Genome-Wide Analysis of DNA Helical Tension by Means of Psoralen-DNA Photobinding[END_REF]). In the last decade, psoralen has been used to investigate supercoiling at the genome scale in various model bacteria and eukaryotes [START_REF] Bermúdez | A Method for Genome-Wide Analysis of DNA Helical Tension by Means of Psoralen-DNA Photobinding[END_REF][START_REF] Teves | Transcription-Generated Torsional Stress Destabilizes Nucleosomes[END_REF]Kouzine et al. 2013;[START_REF] Naughton | Transcription Forms and Remodels Supercoiling Domains Unfolding Large-Scale Chromatin Structures[END_REF]Lal et al. 2016;[START_REF] Achar | Negative Supercoil at Gene Boundaries Modulates Gene Topology[END_REF] but not in Archaea. Consequently, nothing is known about the supercoiling state of archaeal chromosomes.

Negative supercoiling of DNA has a crucial role in the regulation of DNA transactions by its potential to decrease base stacking interactions, thus favouring double helix opening. Positive supercoiling of DNA on the other hand stabilizes double helix in a closed conformation decreasing its melting potential. In Bacteria, supercoiling is used as a quick and massive transcriptional regulator by its ability to change simultaneously the expression of large number of genes in response to environmental changes (Rui and Tse-Dinh 2003;Peter et al. 2004;Dorman andDorman 2016, Villain et al. 2021). This regulation is mainly based on the DNA gyrase activity that introduces negative supercoiling in the genome proportionally to the [ATP]/[ADP] ratio in the cell (Westerhoff et al. 1988). Consequently, bacterial genomes are in average underwound but the distribution of the negative supercoiling is not homogenous and varies with respect to origin of replication and the growth phase [START_REF] Worcel | On the Structure of the Folded Chromosome of Escherichia Coli[END_REF]Lal et al. 2016). In Escherichia coli, the only bacterium for which the supercoiling has been mapped genome wide, a decreasing gradient of negative supercoiling is measured from the terminus to the origin of replication in stationary phase while no gradient was observed when the bacterium grew exponentially. This specific pattern was shown to correlate with transcription intensity and gyrase binding sites [START_REF] Sobetzko | Gene Order and Chromosome Dynamics Coordinate Spatiotemporal Gene Expression during the Bacterial Growth Cycle[END_REF]Sutormin et al. 2018), reflecting the importance of this topoisomerase for efficient transcription and global control of supercoiling.

Eukaryotes lack DNA gyrase but they also have underwound genome (Sinden, Carlson, and Pettijohn 1980). This topology is achieved by wrapping DNA around nucleosomes which cover the majority of eukaryotic genomes. The gene expression control from such constrained loci is generated by a complex cross-talk between transcription, histones binding and topoisomerase relaxation [START_REF] Baranello | The Importance of Being Supercoiled: How DNA Mechanics Regulate Dynamic Processes[END_REF]. Importantly, in Eukaryotes, free negative supercoils are concentrated near transcriptional start sites (TSS) and transcriptional termination sites (TTS) of active genes, while free positive supercoils can be found in middle of actively transcribed genes [START_REF] Achar | Negative Supercoil at Gene Boundaries Modulates Gene Topology[END_REF]. Such an organization promotes non-B DNA structures at genes boundaries that insulate the gene from the rest of the genome, thus increasing RNA polymerase recycling from TSS to TTS and keeping gene bodies overwound to avoid nucleosome repositioning.

The studies of global DNA topology in Archaea are still in their infancy but initial studies of in vivo plasmid topology indicate that, as it is often the case, these organisms harbour idiosyncratic features not found in bacteria or eukaryotes. For example, positively supercoiled plasmid DNA is so far only found in thermophilic archaeal lineages (Charbonnier et al. 1992;Charbonnier and Forterre 1994;López-García and Forterre 1997;López-García et al. 2000). This specificity is thought to protect genome integrity at high temperatures, but it probably also affects global DNA geometry and gene expression regulation. Beyond the special case of thermophiles, Archaea possess small and dense genomes with the majority of genes structured in operons and their transcription and translation are coupled. These bacterial-like properties are counterbalanced by a transcription machinery homologous to the eukaryotic one and the presence of histones in most archaeal linages (Sanders, Marshall, and Santangelo 2019). It is probable that successful integration in course of evolution of such mosaic combination of molecular features resulted in emergence of original mechanisms for control of DNA topology in Archaea.

In this study, we used trimethylpsoralen (TMP) crosslink coupled to next-generation sequencing (NGS)

to establish the first map of DNA supercoiling in an archaeon. We focused on Thermococcus kodakarensis, a genetically tractable hyperthermophilic archaeon with a relatively simple topological kit composed of two histones (HtkA and HtkB), two NAPs (TrmBL2 and Alba), one SMC and three topoisomerases (TopoIII, TopoVI and reverse gyrase). We used four previously constructed strains: a reference TKAg strain, a control strain TKY119F expressing inactive gyrase, a gyrase expressing strain TKgyrAB and a reverse gyrase deletion mutant TK∆RG (Villain et al. 2021). By comparing relative TMP crosslink enrichment on the genome of these strains, we plan to (i) map the distribution of negative supercoiling in T. kodakarensis, giving insight on interplay between local DNA topology, gene transcription and higher-order folding of chromosome (ii) establish directly the importance of reverse gyrase for global chromosome supercoiling; (iii) describe in more detail the impact of gyrase acquisition on the chromosome topology of a naïve organism.

appropriate crosslinking conditions various amounts of the TMP solution or equivalent volume of ethanol (negative controls), were injected in sealed bottles containing 20 ml of late exponential phase T. kodakarensis strains grown at 85°C. The concentration of 1 µg/ml was chosen for preparing samples for sequencing. To avoid cold-shock, injection was performed directly in the oven. After 10 min of incubation at 85°C with the TMP, cultures were poured in the lid of a 10 cm glass Petri dish maintained at 85°C using a water-bath. Cells were immediately irradiated with ~9.6 kJ.m -2 .min -1 of 365-nm light N-lauryl sarcosine, 0.12% Triton X-100). Cell lysates were digested with 0.5 mg of RNAse A (Qiagen) for 1 h at 37°C and then incubated overnight at 55°C with 0.5 mg of proteinase K (Thermofisher Scientific).

DNA was extracted by classical phenol/chloroform method, with three rounds of phenol/chloroform/isoamyl alcohol extraction (Sigma-Aldrich, 25:24:1) and one chloroform/isoamyl alcohol extraction (VWR, Ready-Red). Aqueous phase was carefully recovered and DNA precipitated by addition of 0.8 volume of isopropanol. After 1 h of incubation at -80°C, DNA was pelleted at 20,000

x g, 4°C for 30 min. Dried pellets were resuspended in 85 µl of 5 mM Tris/HCl, pH 8.5. DNA was fragmented to an average size of 200 bp by 200 cycles of 180 s of sonication with 10% duty and 175 peak, using a S220 focused-ultrasonicator (Covaris).

Separation of TMP crosslinked and non-crosslinked DNA fragments

The sonicated DNA was separated on a 0.65% agarose gel in 0.5X TAE buffer. DNA fragments of 100-300 bp were cut out of agarose gel and the DNA was extracted with the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and eluted with 15 µl of ultrapure water pH 8.5. DNA was not exposed to UV light during the gel excision. After addition of 4 µl of 0.1 M phosphate buffer (57.7 mM Na2HPO4 and 42.3 mM NaH2PO4,pH 7), DNA samples were heat-denatured for 5 min at 100°C, then 20 µl of a mix of DMSO and glyoxal (Alfa Aesar) was immediately added (respective final concentrations of 40% and 4%) followed by 90 min of incubation at 55°C. Crosslinked and non-crosslinked DNA fragments were separated by electrophoresis at 2 V/cm for 14 h, with buffer recirculation between electrodes, on 3% agarose gels. Gels were 20 cm long and made of 1.2:1.8 mixture of UltraPure agarose and NuSieve 3:1 agarose in 200 ml of 10 mM phosphate buffer pH 7. TMP-DNA crosslinks were reversed by incubating gels 3 h at 65°C in denaturing solution (0.5M NaOH, 1.5M NaCl) followed by 2 h of incubation with agitation in neutralizing solution (1.5 M NaCl, 500 mM Tris-HCl pH 8, 1 mM EDTA).

Then gels were quickly rinsed in MilliQ water before to be equilibrated 3 times 1 h in 1X TAE and stained overnight with SYBR Gold diluted in 1X TAE. Gels were then imaged with a Typhoon Imager (Amersham) using Cy2 channel. Pieces of gels containing crosslinked and non-crosslinked DNA fragments were excised with a clean scalpel and gel-extracted using a Gel and PCR Clean-up Midi kit with NTC buffer (Macherey-Nagel). DNA was eluted with 5 mM Tris/HCl and stored at -80°C before sequencing.

Nucleosome profiling by micrococcal nuclease digestion

Chromatin isolation and MNase digestion were carried out as previously described (Sanders et al. 2021) with minor modifications. Briefly, strains TKAg, TKY119F, TKgyrAB and TK∆RG were cultivated from fresh overnight precultures in 500 ml of Ravot medium until late exponential phase. Cultures were rapidly chilled in precooled beaker immersed in a water-ice bath to stop cellular metabolism and thus minimize the risk of nucleosome disassembly. Cells were pelleted by centrifugation (4500 x g, 15 min, 4°C) and then washed with Ravot salt solution before storing at -80°C. Frozen pellets were resuspended in 1 ml of MNase buffer (50 mM Tris-HCl pH 8, 100 mM NaCl, and 1 mM CaCl2) and then submitted to 5 cycles of nitrogen freezing plus grinding with mortar. Cell lysate was subsequently recovered by pipetting from the mortar and clarified by centrifugation for 5 min at 1,700 x g and 4°C.

500 µl of clarified lysates were carefully collected by pipetting and digested for 1 h at 37°C with 70 U of RNase A (Qiagen). Samples were separated into 5 x 100 µl fractions and then each fraction was digested 3 min at 37°C with either 0, 50, 100, 200 or 400 U of MNase (Thermo Scientific, ≥100 U/µl).

DNA from digested fractions was immediately extracted with 300 µl of 10 mM Tris-HCl pH 8 and 400 µl of phenol/chloroform/isoamyl alcohol (25:24:1). Mixtures were vortexed for about 2 min and then centrifuged for 4 min at 14,000 x g, room temperature. 200 µl of the top aqueous phase were collected for each sample. DNA was precipitated by adding 200 µl of 1 M Tris-HCl pH 8 and 1 ml of pure ethanol, followed by overnight incubation at -80°C. DNA fragments were pelleted 30 min at 20,000 x g and 4°C.

Resulting pellets were resuspended in 20 µl of TE (10 mM Tris-HCl pH 8, 1 mM EDTA) and resolved on a 4% agarose gel in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA) for 2 h at 4 V/cm.

RESULTS

TMP can be crosslinked in vivo to DNA of T. kodakarensis

To get a snapshot of DNA topology across the whole genome, TMP assay must be done in vivo, preferentially at optimal culture conditions. This is particularly challenging in T. kodakarensis considering that it grows optimally at 85°C and in absence of oxygen. To test the feasibility of psoralen crosslink in this archaeon we incubated T. kodakarensis cells for 30 min with saturating concentration of TMP at 85°C in the reductive culture medium. Cells were then irradiated with UV to achieve crosslink and plasmid DNA was isolated. After digestion with restriction enzymes, plasmids were heatdenatured and analysed by electrophoresis. Only the samples treated with psoralen and irradiated with UV, exhibited the expected BglII digestion pattern (Figure 1). The presence of such pattern is only possible if plasmid DNA is covalently crosslinked to TMP and resists heat-denaturation. The data thus demonstrate that in vivo psoralen-DNA crosslink can be achieved under the conditions which preserve the native DNA topology of T. kodakarensis.

Set up of TMP crosslinking conditions for chromosomal DNA supercoiling mapping

In order to be used as a topological probe, the number of TMP-DNA crosslinks must follow linear correlation with the UV irradiation dose and the quantity of TMP used to treat the cells. The range of UV irradiation producing linear incorporation of DNA crosslinks was determined by in vivo TMPcrosslinking experiments. Cells were treated with 5 µg/ml of TMP and irradiated with increasing dose of UV. The ratio of crosslinked versus non-crosslinked DNA was then assessed by electrophoresis after glyoxal denaturation. The proportion of crosslinked DNA increased in a linear manner over the entire range of UV irradiation (from 10 s to 240 s at ~9.6 kJ.m -2 .min -1 ) (Figure 2A andB).

Next, we investigated the dose-response of the TMP concentration on the quantity of DNA-crosslinks.

Cells were treated with increasing amounts of TMP and irradiated for 180 s at ~9.6 kJ.m -2 .min -1 .

Crosslinked and non-crosslinked DNA was separated by agarose gel electrophoresis after glyoxal denaturation. The proportion of crosslinked DNA increased in a linear manner up to 5 µg/ml of TMP.

Beyond this concentration, the proportion of crosslinked DNA stagnated between 22% and 24% (Figure TMP UV-crosslinking is an efficient probe for in vivo DNA supercoiling investigation in T. kodakarensis (A) TMP crosslink incorporation is linear over 240 s of 365 nm UV exposition at 9.6 kJ.m -2 .min -1 (red line) (B) TMP crosslink incorporation is linear up to a 5 µg/ml concentration in the culture medium (red line), a saturation plateau is reached beyond this limit (blue line).

From these data we chose unsaturating TMP crosslink conditions (1 µg/ml of TMP and 3 min of UV irradiation) to prepare samples for DNA supercoiling mapping (Figure 3). For the sequenced samples, the ratio crosslinked/non crosslinked DNA was quantified by image analysis before gel extraction. Values were systematically inferior to 20% for each strain, meaning that TMP photobinding produced less than one crosslink per kb. In these low hit conditions, TMPcrosslinking is suitable to investigate DNA supercoiling with minor interference from DNA-binding proteins.

MNase digestion of chromatin

TMP intercalation into DNA can be affected by histones and nucleoid associated proteins (NAPs). To limit this bias, we used low hit conditions for TMP crosslinking. In this way, the incorporation of TMP into DNA is mainly due to helical tension [START_REF] Bermúdez | A Method for Genome-Wide Analysis of DNA Helical Tension by Means of Psoralen-DNA Photobinding[END_REF]. Still, the native chromatin organization can impact the diffusion of supercoils and the activity of topoisomerases. To account for this phenomenon we performed MNase-seq experiment. We first extracted chromatin from the four T. kodakarensis strains and digested it using MNase.

The protected DNA fragments of 60/90/120/150 bp corresponding to two, three, four or five histone dimers, respectively were visible on agarose gels and their stoichiometry remained similar for all strains (Figure 4). This suggested that oligomerization of histones into polymeric nucleosomes was not strongly affected. Based on the digestion profile, we have chosen the samples digested with 200U of MNase as suitable for sequencing of MNase protected fragments. The corresponding four DNA bands were gel extracted and sent for DNA sequencing. An attractive hypothesis is that gyrase was determinant for transition from thermophilic to mesophilic lifestyle. We conducted preliminary experiments that showed no obvious growth advantage of the gyrase expressing strain at sub-optimal temperature except perhaps at early stages of growth (the lag phase). However, over a long-term culturing experiment it would be interesting to test if synergic mutations would emerge to promote growth at low temperatures. This would be a major finding enhancing our understanding of adaptation to mesophilic lifestyle in course of evolution.

III. Importance of DNA supercoiling for adaptation to hyperthermophilic lifestyle Thermophilic lifestyle requires a complex set of adaptations to ensure proper cell functioning. Among these adaptations, reverse gyrase is the only one common to all hyperthermophiles and most thermophiles but absent from mesophiles. By its positive supercoiling activity and its involvement in DNA repair, reverse gyrase contributes to genome stability but ambiguous results about the relative importance of these two activities coupled to a lack of in vivo data, make its precise role elusive. Consequently, we initiated an in vivo characterization of reverse gyrase activities to clarify their importance in hyperthermophilic lifestyle. Moreover, histones and NAPs are surely major actors contributing to genome stability at high temperature, making them obvious partners of reverse gyrase. To better understand the relation between these topological actors, we included chromatin structure analysis in this study.

Using plasmid topology analysis, we demonstrated that the positive supercoiling observed in T. kodakarensis can be attributed to the reverse gyrase. Since deletion of reverse gyrase is not associated with growth defect at optimal growth temperature, we also conclude that the topoisomerase activity of reverse gyrase is not necessary for relaxing negative supercoil build-up during DNA transactions in T. kodakarensis.

Moreover, the preliminary characterisation of chromatin structure showed that the loss of positive supercoiling has no dramatic impact on the oligomerization and DNA binding of histones.

The in vivo demonstration that reverse gyrase accounts for all the positive supercoiling in T. kodakarensis cells, suggests that the positive supercoiling deficit cannot be compensated by DNA transactions. Together with the viability of reverse gyrase deletion mutant, it reinforces the idea that positive supercoiling has a minor role in genome stability at high temperature. However, it could be envisaged that the reverse gyrase supercoiling activity is crucial outside of the "comfortable" culture conditions we used. To explore this hypothesis it would be interesting to investigate DNA topology in both the reverse gyrase deletion mutant and the wild type strain in stressful conditions, like changing temperatures or in presence of DNA-damaging agents. We I used a psoralen derivative called trimethyl-psoralen (TMP) to map DNA supercoiling over the genome of T. kodakarensis. The method used is adapted from the protocol published by Fedor Kouzine and collaborators (Kouzine et al. 2013[START_REF] Kouzine | DNA Topoisomerases: Methods and Protocols[END_REF]. It relies on the crosslinking of supercoiled DNA by 365 nm photobound TMP and its separation from non-crosslinked DNA by DNA denaturation and agarose gel electrophoresis.

Populations of DNA are then sequenced and the crosslinked enrichment reflecting DNA supercoiling mapped on the genome.

The adaptation of this method to T. kodakarensis impose strong constrains due to the growth conditions of this archaeon. As DNA topology is extremely sensitive to environmental conditions, special attention was put to keep T. kodakarensis under anaerobiosis and at a temperature of 85°C, during the TMP treatment and the UV irradiation. Under these harsh conditions we were concerned about the ability of TMP to be stable, crosslinked by UV and specific of DNA supercoiling (absence of heatinduced non-specific crosslinks). Thus, preliminaries experiments of TMP in vivo crosslinking were conducted to set up conditions adapted to these constrains. They demonstrated that (i) TMP can enter T. kodakarensis cells, (ii) TMP is stable at 85°C

during the ~15 min of the experiment, (iii) the temperature is not causing off target TMP intercalation into DNA, (iv) TMP crosslinking can be achieved by 365 nm irradiation in the growth conditions of T. kodakarensis.

In order to be used as a probe of DNA supercoiling, the relations between TMP crosslinking into DNA and the quantity of TMP used, as well as the dose of UVs used for the crosslinking, must be linear. To check for these conditions, I titrated the incorporation of TMP at constant irradiation conditions and defined this linear range for T. kodakarensis. It showed a linear incorporation of TMP in T. kodakarensis up to 5 µg/ml, a relatively low value compared to the thresholds described in E. coli or model eukaryotes. In addition, I demonstrated that TMP incorporation into T. kodakarensis DNA is linear over 4 min of UV irradiation at ~9.6 kJ/m 2 /min.

The intercalation of TMP into DNA can be affected by the chromatin structure. This phenomenon is only revealed at saturating TMP concentrations. To limit this bias, experiments were carried using low, non-saturating concentrations of TMP to produce limited amount of crosslink (ratio crosslink / not crosslink < 1/3). A MNase-seq experiment was performed in parallel to confirm that potential variations of TMP incorporation can not be attributed to nucleosome redistribution over the genome.

The successful extraction of TMP crosslinked DNA that, a priori, fulfils all the conditions for the investigation of DNA supercoiling by TMP incorporation confirms the viability of this approach for T. kodakarensis.

The development of the psoralen photo-binding method in T. kodakarensis is a major step on the characterization of DNA topology organization and regulation in Archaea and hyperthermophiles. Coupled to the genetic editing developed for this organism, it opens the way for a global and integrative characterization of its DNA topology.

Concretely, the use of this method on T. kodakarensis should give us the first view of DNA supercoiling organization in an archaeon, allowing comparison with the known situation of E. coli or eukaryotes. Moreover, this method would help to complete the data obtained on the introduction of a pervasive gyrase activity in T. kodakarensis, by giving a detailed view of the supercoiling perturbations at the chromosome scale. In the same idea, the comparison of DNA supercoiling density over the chromosomes of wild type and reverse gyrase deletion mutant strains, could be decisive to understand the importance of this peculiar enzyme on the stability of genomes of hyperthermophiles.

Article I Les topoisomérases régulent le surenroulement de l'ADN pour permettre aux processus essentiels, comme la transcription ou la réplication, d'avoir lieu. Au cours de ma thèse je me suis intéressé à la gyrase qui est unique par sa capacité de surenrouler l'ADN négativement. Des études antérieures suggèrent qu'un transfert horizontal de gènes depuis les bactéries a permis l'implantation de la gyrase chez les Archées. L'acquisition d'une telle activité doit avoir profondément bouleversé la topologie de l'ADN d'archée receveuse. Comment cette archée « naïve » a pu intégrer une gyrase et quels ont été les avantages évolutifs associés ? Pour aborder cette question, j'ai mis en oeuvre une approche intégrative, mêlant analyses phylogénétiques, génie génétique et techniques d'analyse du génome dans sa globalité. J'ai montré que la gyrase a été acquise tardivement dans l'histoire des Archées et que cet unique événement a été suivi par une expansion limitée via de rares transferts secondaires. Pour mimer le transfert ancestral, j'ai exprimé une gyrase bactérienne dans l'archée Thermococcus kodakarensis, qui en est dépourvue et présente un surenroulement de l'ADN positif. J'ai pu démontrer que la gyrase introduit un surenroulement négatif important dans l'ADN de T. kodakarensis. Ce bouleversement topologique dérégule l'expression de nombreux gènes, mais pas les topoisomérases endogènes, et n'a pas d'impact sur la croissance de T. kodakarensis. Cela indique l'existence chez cette archée, de mécanismes permettant de résister efficacement au stress topologique. Pour tester cette hypothèse, j'ai mis au point la technique de pontage au psoralène, qui permet de cartographier le surenroulement directement sur le chromosome, chez T. kodakarensis. Couplées à un séquençage à haut débit, ces données permettront pour la première fois de décrire en détail la répartition du surenroulement à l'échelle du chromosome chez une archée.

Title :

In vivo study of DNA topology in hyperthermophilic archaeon Thermococcus kodakarensis Keywords : DNA gyrase, reverse gyrase, DNA topology, Thermococcus kodakarensis, psoralen crosslink Abstract : In all cells the DNA exists in supercoiled form. Topoisomerases regulate the DNA supercoiling allowing essential processes such as transcription or replication to occur. In course of my thesis I focused on DNA gyrase which is unique by its capacity to supercoil DNA negatively. Previous studies suggested that this enzyme was transferred from Bacteria to Archaea via a single HGT. This acquisition must have had completely changed the DNA topology of the recipient archaeon, thus impacting all DNA transactions. I therefore asked how this naïve archaeon could accommodate an active gyrase and what was the associated evolutive advantage? To tackle this question I undertook an integrative approach using phylogenetic analysis, genetic engineering and genome wide analyses. I showed that gyrase was acquired relatively late in the history of Archaea and that this event was followed by a limited expansion through rare secondary transfers. To mimic the ancestral transfer, I expressed a bacterial gyrase in the naïve archaeon Thermococcus kodakarensis, which naturally has positively supercoiled DNA. I demonstrated that the gyrase was active in T. kodakarensis leading to strong negative supercoiling of plasmid DNA. Surprisingly, the drastic change of DNA topology, while mildly deregulating the expression of hundreds of genes, did not affect growth of T. kodakarensis. Notably, the endogenous topoisomerases were not deregulated suggesting existence of uncommon resilience mechanisms against torsional stress in this archaeon. To test this hypothesis, I established psoralen crosslinking assay to directly map supercoiling on the chromosome of T. kodakarensis. Coupled with next generation sequencing, this technique will allow us to map for the first time the distribution of supercoiling at the chromosome scale in an archaeon. My thesis work now provides the necessary tools to investigate how DNA supercoiling is distributed and maintained in hyperthermophiles and in archaea in general.

Maison du doctorat de l'Université Paris-Saclay 2ème étage aile ouest, Ecole normale supérieure Paris-Saclay 4 avenue des Sciences, 91190 Gif sur Yvette, France

Figure 1 :

 1 Figure 1: Darwin first draw of phylogenetic tree ...............................................................................................................

Figure 2 :

 2 Figure 2: Nucleic acids can be used to make phylogeny ............................................................................................

Figure 3 :

 3 Figure 3: Comparison of lipids from Archaea and Bacteria........................................................................................

Figure 4 :

 4 Figure 4: Methanogenesis in Archaea ................................................................................................................................

Figure 5 :

 5 Figure 5: RNA polymerase from Archaea and Eukaryotes are orthologous ........................................................

Figure 6 :

 6 Figure 6: Many lineages of archaea are extremophiles ...............................................................................................

Figure 7 :

 7 Figure 7: Archaea are part of the human microbiome .................................................................................................

Figure 8 :

 8 Figure 8: Up to date diversity and phylogeny of Archaea ..........................................................................................

Figure 9 :

 9 Figure 9: Three-domain and two-domain trees of life imply distinct scenario of eukaryogenesis ............

Figure 10 :

 10 Figure 10: Compaction of Escherichia coli chromosome in the nucleoid ............................................................

Figure 11 :

 11 Figure 11: DNA is highly compacted in eukaryotic nucleus.......................................................................................

Figure 12 :

 12 Figure 12: Chromosome organization in Sulfolobus ....................................................................................................

Figure 13 :

 13 Figure 13: Chromosomes are hierarchically organized in bacteria and eukaryotes .........................................

Figure 14 :

 14 Figure 14: Chromatin loops and transcription regulation ..........................................................................................

Figure 15 :

 15 Figure 15: Archaeal histones are very similar to the eukaryotic ones ....................................................................

Figure 16 :

 16 Figure 16: Structure of archaeal hypernucleosome .......................................................................................................

Figure 17 :

 17 Figure 17: Distribution of NAPs and histones in Archaea ...........................................................................................

Figure 18 :

 18 Figure 18: Topological consequences of transcription and replication ................................................................

Figure 19 :

 19 Figure 19: Description of DNA topology parameters ...................................................................................................

Figure 20 :

 20 Figure 20: Reactions catalysed by topoisomerases .......................................................................................................

Figure 21 :

 21 Figure 21: Model of supercoiling homeostasis in Bacteria.........................................................................................

Figure 22 :

 22 Figure 22: Model of supercoiling homeostasis in Sulfolobus ...................................................................................

Figure 23 :

 23 Figure 23: Primary domain structure of E. coli DNA gyrase .......................................................................................

Figure 24 :

 24 Figure 24: Molecular model of DNA Gyrase.....................................................................................................................

Figure 25 :

 25 Figure 25: DNA gyrase supercoiling mechanism............................................................................................................

Figure 26 :

 26 Figure 26: Chromosomal map of E. coli supercoiling sensitive genes ...................................................................

Figure 27 :

 27 Figure 27: Structural model of Thermotoga maritima reverse gyrase ...................................................................

Figure 28 :

 28 Figure 28: Phylogenetic tree of bacterial and archaeal reverse gyrase sequences ..........................................

Table 1 :

 1 Comparison of the characteristics from Bacteria, Archaea and Eukaryotes .................... Table 2: Main characteristics of known nucleoid-associated proteins ............................................... Table 3: Main characteristics of known topoisomerases ......................................................................... Table 4: Distribution of gyrase in Archaea ....................................................................................................

Figure 3 :

 3 Figure 3: Comparison of lipids from Archaea and Bacteria

Figure 4 :

 4 Figure 4: Methanogenesis in Archaea

Figure 5 :

 5 Figure 5: RNA polymerase from Archaea and Eukaryotes are orthologous Structures of RNA polymerases from Thermus aquaticus (bacterium), Saccharolobus solfataricus (archaeon) and Saccharomyces cerevisiae (eucaryote). Orthologous subunits harbour the same colours (adapted from Jun et al. 2011).

Figure 6 :

 6 Figure 6: Many lineages of archaea are extremophiles

Figure 7 :

 7 Figure 7: Archaea are part of the human microbiome

Figure 8 :

 8 Figure 8: Up to date diversity and phylogeny of Archaea

Figure 9 :

 9 Figure 9: Three-domain and two-domain trees of life imply distinct scenario of eukaryogenesis

Figure 10 :

 10 Figure 10: Compaction of Escherichia coli chromosome in the nucleoid Circular chromosome of E. coli occupies a 523 µm3 volume at thermal equilibrium without supercoiling or any stabilizing factors. This random coil must be compacted at least 1000-fold by various chromatin architects to form a nucleoid smaller than 1 µm 3 . E. coli macrodomains are indicated by colours (adapted from Verma, Qian, et Adhya 2019).

Figure 11 :

 11 Figure 11: DNA is highly compacted in eukaryotic nucleus

  ). More recently, chromosome capture technics like 3C or Hi-C have confirmed the previous studies and detailed the structure of macrodomains (Lioy et al. 2018). These technics combine Next Generation Sequencing (NGS) and genomic DNA crosslinking to map each internal contact on the chromosome. Results allowed to precise the borders of macrodomains and, coupled with genetic experiments, to determine actors involved in their formations. It also gave an idea of the range of distance in which interactions occurred, showing that DNA-DNA interactions are restricted to 280 kb in Ter macrodomain but can reach up to 1 Mb in the rest of the chromosome. While most of the work on bacterial chromosome structure has been performed in E. coli, it appears that chromosomes of other distant model bacteria like Bacillus subtilis, Caulobacter crescentus, Streptomyces ambofaciens or Vibrio cholerae exhibit similar characteristics

Figure 12 :

 12 Figure 12: Chromosome organization in Sulfolobus Schematic representation of Sulfolobus archaea chromosome organization (adapted from Takemata, Samson, et Bell 2019). Sulfolobus chromosomes are organized into topological domains that interact to form A and B compartments in a reminiscent way of eukaryotes. A compartment concentrates replication origins and highly transcribed genes while B compartment concentrates less expressed genes. The structural maintenance protein (SMC) coalescin, specific of Sulfolobus, is involved in the structuration of B repressive compartment.

Figure 13 :

 13 Figure 13: Chromosomes are hierarchically organized in bacteria and eukaryotes

  and 14c)(Tolhuis 2002 ; Tiwari 2008). In those configurations, enhancer or silencer is bound by a transcriptional regulator that will regulate the transcription initiation complex. Another possibility is (iii) the isolation of a portion of the genome from its genomic context to prevent aberrant regulation. Such structures have been first described in the Drosophila flies(Hou 2012). This kind of loops involve insulator elements that are bound by proteins like SMCs or the CTCF in Eukarya. Insulators are present on both sides of the isolated region and form the loop by collapsing together.While apparently absent from Sulfolobus crenarchaea[START_REF] Takemata | Multi-Scale Architecture of Archaeal Chromosomes[END_REF], DNA loops have been recently observed in some archaea belonging to the Euryarchaeota superphylum[START_REF] Cockram | Euryarchaeal Genomes Are Folded into SMC-Dependent Loops and Domains, but Lack Transcription-Mediated Compartmentalization[END_REF]. Haloferax volcanii and Halobacterium salinarum exhibit in Hi-C contact maps the plaid pattern characteristic of DNA loops. However, T. kodakarensis, another euryarchaeon does not exhibit this pattern, highlighting the diversity of genome organizations in Archaea, even within the same superphylum.

Figure 14 :

 14 Figure 14: Chromatin loops and transcription regulation

Figure 15 :

 15 Figure 15: Archaeal histones are very similar to the eukaryotic ones

Figure 16 :

 16 Figure 16: Structure of archaeal hypernucleosome

Figure 17 :

 17 Figure 17: Distribution of NAPs and histones in Archaea

Figure 18 :

 18 Figure 18: Topological consequences of transcription and replication

Figure 19 :

 19 Figure 19: Description of DNA topology parameters

  negatively supercoiled DNA. But independently of DNA melting, DNA supercoiling can change distances and local conformations between genetic elements. This can be exemplified by the complex relation between supercoiling and transcription. Negative supercoiling acts as a global regulator of gene expression. In many ways, negative supercoiling can influence the transcription process. (i) The recruitment of RNAP on promoter via σ factors in Bacteria or transcription factor B (TFB/TFIIB) in Archaea andEukaryotes, rely on their interaction with -10 and -35 elements. Supercoiling can modify both the distance between these elements and their orientation on the DNA helix, thus changing their potential of interaction with the RNAP-complex. (ii) Negative supercoiling promotes DNA melting, a required step for transcription initiation and DNA decoding. This is especially important for G-C rich promoters, as those involved in the stringent response in Bacteria. (iii) In Prokaryotes as well as in Eukaryotes, transcription can be dependent upon regulatory sequences called enhancer or silencers. Negative supercoiling acts by bringing together an enhancer or a silencer and its target promoter with consequences for gene expression regulation(Dorman 2006).

Figure 20 :

 20 Figure 20: Reactions catalysed by topoisomerases

Figure 21 :

 21 Figure 21: Model of supercoiling homeostasis in Bacteria Negative supercoiling increases the transcription of topA by promoting the melting of its promoter, leading to DNA relaxation by topoisomerase I. Conversely, DNA relaxation increases the transcription of gyrA and gyrB, leading to DNA supercoiling by gyrase in presence of ATP.

Figure 22 :

 22 Figure 22: Model of supercoiling homeostasis in Sulfolobus

(

  90 kDa) binds ATP with its N-terminal part and interacts with GyrA with its C-terminal part. The C-terminal part of GyrB contains the TOPRIM domain responsible for the binding of Mg 2+ ions that are necessary for the cleavage-religation reaction, while the C-terminal part of GyrA contains the GyrA-box motif (consensus sequenceQ(R/K)RGG(R/K)G) (Figure23 and 24). This motif is a defining signature of all gyrases and it causes a positive DNA bending that is required for gyrase supercoiling activity(Kramlinger and Hiasa 2006).

Figure 23 :

 23 Figure 23: Primary domain structure of E. coli DNA gyrase Schematic representation of the primary domain structure of GyrA and GyrB from E. coli including the functionally important residues. The GHKL domain hydrolyses ATP. The TOPRIM domain chelates three Mg2+ ions required for DNA cleavage and religation reactions. The catalytic tyrosine (Y122) is in the WHD (winged-helix domain) domain. The C-terminal (CTD) part of GyrA contains the GyrA box which is essential for introducing the negative supercoiling in DNA by wrapping DNA around CTD domain.

Figure 24 :

 24 Figure 24: Molecular model of DNA Gyrase E. coli gyrase interacting with 130 bp DNA was imaged by cryo-electron microscopy, then DNA and structural model of gyrase domains were built using the electron density map (adapted from Vanden Broeck et al. 2019).

Figure 25 :

 25 Figure 25: DNA gyrase supercoiling mechanism GyrA CTD is light-blue and rest of GyrA is orange. GyrB tail is green, TOPRIM domain is red and the rest of GyrB is blue. DNA T segment is purple and G segment is black. Stars indicate the active site residues for DNA cleavage and the circle indicates the ATPbinding pocket. (1) Free state of gyrase subunits in solution. (2) Formation of the heterotetramer and wrapping of the DNA around GyrA CTD. T segment is positioned above G segment in a positive cross-over. (3) ATP-binding provokes GyrB clamp (Ngate) closing, capturing T segment while the G segment is transiently cleaved within DNA-gate. (4) Hydrolysis of one ATP allows GyrB to rotate, DNA-gate to open and the passage of the T segment through the cleaved G segment. (5) G-segment is religated, introducing two negative supercoils in the DNA molecule. Hydrolysis of a second ATP resets the enzyme before a new round of DNA supercoiling (adapted from Costenaro et al. 2007).

Figure 26 :

 26 Figure 26: Chromosomal map of E. coli supercoiling sensitive genes

  Cluster 2 Euryarchaeota and sporadically in some Asgard and DPANN archaea. Homologs of gyrA and gyrB were searched by Blast and HMM against representative genomes of archaea. Full circles represent gene presence in most or all members of the taxon, empty circles absence and partial circles presence in a few members only (adapted from Adam et al. 2017).

N

  -terminal helicase part of the reverse gyrase binds and transiently unwinds DNA in an ATP-dependent manner[START_REF] Ganguly | Reverse Gyrase Transiently Unwinds Double-Stranded DNA in an ATP-Dependent Reaction[END_REF], making the reverse gyrase the only ATP-dependent type I topoisomerase. The C-terminal part of topoisomerase I-like domain is very similar to the topoisomerase I of E. coli. It consists of (i) a domain I that binds Mg 2+ through the TOPRIM fold, (ii) a domain II that defines the central cavity of the enzyme, (iii) a domain III that contains the catalytic tyrosine and (iv) a domain IV that interacts with domains I and III to form the positively charged interface for DNA binding, and to control the access to the central cavity by forming a gate that closed the central cavity(Schoeffler and Berger 2008) (Figure27).

Figure 27 :

 27 Figure 27: Structural model of Thermotoga maritima reverse gyrase

  response to DNA binding and ATP hydrolysis. These findings led to a new model where reverse gyrase segregates positive and negative supercoiling and relaxes specifically the negative one. In a third model, alternation of high affinity for ssDNA and dsDNA by the helicase domain, coupled with strand passage mechanism would result in positive supercoiling (del Toro Duany et al. 2008). Recently, the sequence of DNA topology transitions during the catalytic cycle has been uncovered by single molecule analysis (Yang et al. 2020) Despite these results, the exact catalytic cycle of reverse gyrase remains elusive.

Figure 28 :

 28 Figure 28: Phylogenetic tree of bacterial and archaeal reverse gyrase sequences

  This should allow me to describe whether this topoisomerase functions in controlling the DNA topology at the global scale in T. kodakarensis and in hyperthermophiles more generally. In article IV of my thesis I reported the ongoing work on investigation of the DNA topology in this mutant. INTRODUCTION Topoisomerases are enzymes in charge of DNA topology regulation in every living cell. By their transient DNA cleaving activities, they maintain DNA supercoiling in a range compatible with DNA transactions such as transcription and DNA replication and ensure decatenation of chromosomes prior to cell division(Wang 2002; Schoeffler and Berger 2008;[START_REF] Pommier | Cancer Drug Discovery and Development[END_REF] Bush, Evans-Roberts, and Maxwell 2015; Seol and Neuman 2016; McKie, Neuman, and Maxwell 2021). Topoisomerases are classified in type I and type II depending whether they cleave one or two DNA strands(Schoeffler and Berger 2008). In principle, only one type I and one type II topoisomerases would be necessary and sufficient for resolving all topological problems resulting from the DNA being very long molecule and

  is the hallmark of DNA gyrases and its mutation abolishes the negative supercoiling activity (but not relaxation and decatenation activity) (REF). The final dataset contained 385 GyrA and 348 GyrB archaeal sequences and 571 GyrA and 633 GyrB bacterial sequences.

Figure 1 .

 1 Figure 1. Sequence conservation of archaeal and bacterial gyrases A) Comparison of the GyrA box motif. The motif was generated using an alignment of 477 bacterial and 299 archaeal representative sequences. The size of letters is proportional to the frequency of occurrence of each letter in the alignment. WebLogo v. 3.7.4. (REF) was used to generate the sequence logo.

Figure 2 .

 2 Figure 2. Distribution of gyrase across archaeal diversity.The depicted phylogenetic tree shows the distribution of gyrase at the taxonomic rank of order. The presence of gyrase in the majority or all representatives is indicated with full red circle. The empty circle indicates presence in a few members only. It should be noted, however, that absence of genes in uncultured taxa may be due to genomes incompleteness. Plus sign indicates lineages that were discovered or significantly enriched in new genomes since the last survey of gyrase distribution by Adam and colleagues(Adam et al. 2017).

Figure 3 :

 3 Figure 3: Global phylogeny of DNA gyrases Maximum likelihood phylogenetic tree generated using the complete dataset of concatenated GyrA and GyrB sequences containing 502 bacterial and 297 archaeal sequences. The tree was inferred from an alignment trimmed with Noisy. The legend indicates the correspondence between the colours and taxonomic affiliations of the branches. SH-aLRT support (%)/ultrafast bootstrap support (%) for major bipartitions are indicated. Detailed tree is shown in Supplementary figure 2, B.

  (scenario II) or to the lineage leading to Cluster II Euryarchaeota with the donor bacterium belonging to the lineage giving rise to Gracilicutes or Terrabacteria. The scenario II implies that last archaeal common ancestor (LACA) is younger than LBCA while the third scenario infers that the transfer occurred between the donor bacterial lineage and the ancestor of Cluster II Euryarchaeota thus placing the origin of one of the major bacterial groups after LACA.

Figure 4 .

 4 Figure 4. Evolutionary scenarii derived from the phylogeny of prokaryotic DNA gyrases

  GyrB sequences, aligned them with MAFFT and performed trimming with BMGE or with Noisy. The unrooted archaeal phylogeny resulting from this analysis resolves into well supported clades corresponding to major taxonomic groups consistent with the consensus archaeal phylogeny but the three superphyla DPANN, Asgard and Euryarchaeota are not monophyletic in most of the trees (Supplementary figure 4, 5 and 6). More relaxed sequence alignment trimming resulted in GyrA phylogeny in which the Asgard monophyly was recovered and the DPANN were distributed between three closely branching clades (Figure 5, A). These two superphyla further branched within the Cluster II Euryarchaeota such that this group was split into two clades, one more closely related to Asgard archaea and the other to DPANN. In few cases, DPANN sequences branch within Asgard or Thermoplasmata clades suggesting recent HGT events or perhaps artefactual attraction of these fastevolving DPANN sequences. Gyrases are almost completely absent in group I Euryarchaeota with exception of sporadic appearance in Theinoarchaea and Methanobrevibacter. The former organisms form a monophyletic clade branching within the group II Euryarchaeota and thus likely have acquired the gyrase by rather recent lateral transfer. The position of Methanobrevibacter within Asgard clade is most likely result of artefactual long branch attraction since Methanobrevibacter gyrases were recently acquired from bacteria (see below).

Figure 5 .

 5 Figure 5. Vertical evolution is the predominant mechanism of gyrase spreading in archaeal domain. A) Schematic representation of the maximum likelihood phylogeny of archaeal GyrA sequences. The detailed tree is shown in supplementary figure 4. The tree was inferred from the alignment of 376 GyrA sequences. The UFBoot support and SH-aLRT support are indicated for major bipartitions. Group I Euryarchaeota are highlighted with an asterisk. B) Hypothetical scenario for gyrase evolution in Archaea. The acquisition of the gyrase by single HGT from bacteria before (1) or after (2) LACA is indicated with blue arrows. Putative HGT events toward ancestors of Asgard and DPANN superphyla are indicated with black arrows. The DPANN branch is represented by a dotted line to indicate the uncertain position of this group within the archaeal tree. The filled circles indicate the systematic presence of gyrase, semi-filled circles indicate partial presence and empty circles indicate absence of gyrase. Sporadic presence of gyrase in cluster I Euryarchaeota group is indicated by an asterisk. See main text for more detail.

Figure 6 .

 6 Figure 6. Synteny analysis of the gyrA locus in Thermotogales A) The genomic context around gyrA gene is depicted with each arrow corresponding to a gene. Genes are automatically color coded based on functional annotation. The scale bar at the bottom corresponds to the percentage of identity between proteins encoded by the depicted genes. The drawing is on scale

Figure 7 .

 7 Figure 7. Synteny analysis of the gyrBA locus in Methanobrevibacter species A) The genomic context around gyrBA operon is depicted with each arrow corresponding to a gene. Genes are automatically color coded based on functional annotation. The scale bar at the bottom corresponds to the percentage of identity between proteins encoded by the depicted genes. The drawing is on scale with the scale bar representing 2.5 kbp. The two closely related Methanobrevibacter species are highlighted in blue.

Figure 1 .

 1 Figure 1. DNA gyrase distribution in Archaea. Schematic phylogeny of archaea with main phyla and superphyla indicated at the top. The presence of gyrase in a phylum is indicated by a blue dot. The model organism in this study, Thermococcus kodakarenis KOD1, belongs to Thermococci phylum within Euryarchaeota I clade. The dashed line symbolizes the uncertainty of DPANN branching within the Archaea tree.

Figure 2 .

 2 Figure 2.Plasmid DNA from Thermococcus kodakarensis TKgyrAB is negatively supercoiled. Reporter plasmid pTPTK2 was isolated from TKY119F or TKgyrAB strains and its topoisomers were separated using 2D agarose gel electrophoresis. The corresponding cartoon is depicted below each agarose gel. (A) and (C) DNA intercalating drug chloroquine was added at 7.5 g/ml only in the second-dimension run. This allows to separate positively and negatively supercoiled topoisomers as fast-migrating right arc and slow-migrating left arc, respectively. Under these running conditions, the majority of pTPTK2 isolated from TKgyrAB strain is in extremely negatively supercoiled form. The major (the most abundant) topoisomer from the TKY119F is indicated with a white arrow and was used to calculate superhelical density. (B and D) Chloroquine was added both in the first (1.5 g/ml) and the seconddimension run (7.5 g/ml). Chloroquine introduces a slight positive torsion in DNA, thus changing the apparent superhelical density of topoisomers toward a positively supercoiled state. This allowed relaxing the extremely negatively supercoiled form of pTPTK2 during the first dimension run such that the individual topoisomers could be separated and the major (the most abundant) topoisomer determined. This topoisomer is indicated by a white arrow and was used to calculate superhelical density. (E) Plasmid superhelical densities from various organisms were plotted against the optimal growth temperature of their hosts (see also supplementary tableS3). The mean and standard deviation calculated from three independent experiments are indicated for gyrase expressing strain TKgyrAB and the control strains TKY119F. The point corresponding to Archaeoglobi archaea is highlighted by an asterisk. The total change in the supercoiling density ( ) resulting from gyrase activity in T. kodakarensis or from TopoI inhibition in Streptococcus pneumonia is indicated.

Figure 3 .

 3 Figure 3. Gyrase expression induces ciprofloxacin sensitivity in T. kodakarensis Thermococcus cells from overnight cultures were mixed with 0.18% (w/v) phytagel and colloidal sulfur containing culture medium and were spread as overlay onto 1% phytagel plates. The plate cartoon indicates the position of the paper discs and the quantity of ciprofloxacin used. The cell-containing overlay becomes transparent when incubated at 85 • C due to consumption of colloidal sulphur during T. kodakarensis growth. Growth inhibition can therefore be detected as a white area on the plate.

Figure 4 .

 4 Figure 4. The impact of gyrase on transcription in T. kodakarensis (A) Volcano plots showing significantly deregulated genes. The two vertical and the horizontal dashed lines indicate the threshold values of ±1.25 fold change and Padj > 0.05, respectively. The blue and red dots correspond to downregulated and upregulated genes, respectively. Each plot corresponds to a pairwise comparison of transcriptomes from strains indicated on the top. (B) Distribution of the SRGs on the chromosome of T. kodakarensis TS599. Top panel shows the distribution of DEGs on the chromosome of T. kodakarensis. Points correspond to individual DEGs and their colour indicates the orientation of transcription whereby green indicates antisense expressed genes and violet indicates sense expressed genes. The position of ribosomal genes is indicated by arrows. The annotated function of SRGs involved in DNA transactions and cell division (COG categories L, K, D and B) as well as those of outliers is indicated. The lower panel shows the distribution of the GC content along the chromosome.

Figure 5 .

 5 Figure 5. Top upregulated SRGs carry specific sequence motif in their promoter region. (A) Differentially expressed genes reacting to gyrase supercoiling activity or chromatin defect. Rectangles correspond to the top 30 upregulated SRGs and their colour corresponds to the log 2 FC values as indicated by the scale. The log 2 FC values of the genes/operons indicated by an asterisk are systematically anti-correlated between the gyrase expressing strain and the T. kodakarensis TS620 and TS622 strains. (B) Upper panel shows that the anti-correlated genes/operons invariably contain a conserved 23 bp sequence motif, indicated as red rectangle, upstream of the transcription start site (TSS). The lower panel shows the alignment of the 23 bp sequences on the left and the corresponding sequence logo on the right.

NucleicFigure 6 .

 6 Figure 6. Expression of active DNA gyrase is not toxic for T. kodakarensis. (A) Growth of the three strains was monitored by direct cell counting using a Thoma cell counting chamber. The specific growth rates (number of divisions h -1 ) of the control (TKY119, TKAg) and gyrase expressing strain TKgyrAB) were 2.11 ± 0.27 h -1 TKAg, 1.45 ± 0.35 h -1 TKY119 and 1.81 ± 0.37 h -1 TKgyrAB. Specific growth rates were calculated from the slope of the linear portions of the curves according to the equation = dY/dt, where t is time and Y is the cell density. Mean values of Y were used for the calculation. Error bars represent the standard deviation, n = 3. The differences between the slopes are not significant, P = 0.34, ANCOVA two-tailed test. (B) Measurement of plasmid loss over 84 generations corresponding to 14 subcultures. Each point corresponds to the ratio of CFUs grown on plates under non-selective or selective medium. The experiment was done in triplicate and the bars correspond to standard deviation from the mean value. (C) Representative micrographs of T. kodakaransis cells harvested at the exponential growth stage. The cells were stained with Hoechst dye and DIC and fluorescent images were superposed. (D) Density plot of cell area determined from DIC microscopy images. The number of analysed cells is indicated below the graph.

Figure 1 :

 1 Figure 1: Structure of Thermotoga maritima reverse gyrase

  , A). Identical values of superhelical density were obtained when chloroquine was added in both dimensions (Figure2, B).

Figure 2 :

 2 Figure 2: Plasmid DNA isolated from Thermococcus kodakarensis TK∆RGp is relaxed.

Figure 3 :

 3 Figure 3: The lack of reverse gyrase has no major consequences for assembly of nucleosomes in T. kodakarensis TK∆RG

using a 45 W

 45 lamp (Vilber Lourmat model VL-315.BL). For the sequenced samples were irradiated by this way 3 min (28.8 kJ/m 2 ). The anaerobic state of cultures during irradiation was controlled through the resazurin indicator contained in the Ravot medium. No yellow to pink transition, synonym of medium oxidation, was observed for irradiation times inferior to 3 min. After irradiation, cells were immediately chilled on ice before harvesting at 4°C by 15 min of centrifugation at 5,000 x g. Total DNA extraction and fragmentation Pellets were resuspended in 250 µl of TEN (40 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl) and then lysed by the addition of 250 µl TENST (40 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl, 1.6%

Figure 1 :

 1 Figure 1: Plasmid DNA and TMP can be UV-crosslinked in cells of in T. kodakarensis

Figure 2 :

 2 Figure 2: In vivo crosslinking of TMP in DNA of T. kodakarensis.

Figure 3 :

 3 Figure 3: TMP sequencing samples

Figure 4 :

 4 Figure 4: MNase-seq samples

  initiated, in collaboration with A. Gardner's laboratory (New England Biolabs) a mirror study to map DNA damages genome-wide in reverse-gyrase deletion mutant. This should tell us if reverse gyrase protects the genome from DNA damage and if so what type of damage.IV. Mapping DNA supercoiling along chromosomes by psoralen crosslinkingThe characterization of DNA topology in vivo is hindered by the complexity, the diversity and the imbrication of the involved actors and mechanisms. In addition, DNA topology is highly dynamic and its supercoiling component can diffuse over long distances along the chromosome. For these reasons, but also because it is technically difficult to faithfully describe chromosomal DNA topology, analyses are often restricted to plasmids. By their circular shape and their reasonable sizes, plasmids appear as convenient tool to study DNA topology. However, their simplicity is also their weakness as they can not account for the complexity of chromosomes. This limitation was progressively lifted by the development of psoralen-based in vivo supercoiling measurement methods. There are different ways to proceed, but the basic principle of these methods is to exploit the propensity of psoralen derivatives to intercalate preferentially in negatively supercoiled DNA and to create inter-strand crosslinks under UV irradiation. Crosslinked and non-crosslinked DNA are then separated and sequenced to estimate the relative rate of negative supercoiling over the entire genome. While difficult to implement, these methods are actually the only currently existing allowing a direct measurement of negative supercoiling at the entire genome scale. I adapted psoralen crosslinking protocol originally developed for eukaryotes to T. kodakarensis and I used it to map DNA supercoiling on its chromosome. The sequencing data are now available and need to be analysed. The results of this analysis, when available, will be precious to understand the impact of the gyrase introduction on DNA supercoiling and gene expression as well as to decipher the exact contribution of reverse gyrase to global DNA supercoiling in T. kodakarensis. More generally, the mapping of DNA supercoiling in T. kodakarensis, opens the way for a general characterization of DNA topology regulation in this archaeon.

L

  'ADN gyrase (ci-après gyrase), une topoisomérase de type II A, est la seule enzyme connue capable de surenrouler négativement l'ADN en utilisant l'énergie de l'hydrolyse de l'ATP. Cible importante des antibiotiques, la gyrase est essentielle et ubiquitaire chez les bactéries où elle contrôle (avec la Topo I) la densité de surenroulement des chromosomes en introduisant des supertours négatifs dans l'ADN et en relâchant les supertours positifs qui s'accumulent devant les ADN et ARN polymérases en mouvement. La contribution de la gyrase au maintien du chromosome dans un état de surenroulement négatif dans les cellules bactériennes peut avoir un impact profond sur la liaison des protéines régulatrices, la dynamique d'activation des promoteurs, la réplication de l'ADN et l'architecture des chromosomes.Le surenroulement de l'ADN est utilisé dans un large éventail de bactéries pour transmettre rapidement les signaux environnementaux vers le chromosome et ce processus est conservé dans des espèces bactériennes phylogénétiquement éloignées.La voie la plus clairement décrite implique la modulation de l'activité de la gyrase en réponse au rapport [ATP]/[ADP] dans la cellule. Lorsque ce rapport est faible, l'activité de surenroulement de la gyrase est significativement réduite et le niveau d'expression de nombreux gènes est simultanément modifié. L'inhibition de l'activité de surenroulement de la gyrase par les antibiotiques quinolones a un effet similaire ; l'expression de jusqu'à 48% des gènes peut être dérégulée. Ce mécanisme relativement simple, rapide et général a été suggéré comme étant l'une des inventions évolutives clé permettant aux bactéries d'occuper une grande variété d'environnements. Chez les archées, la gyrase est présente chez tous les membres d'un groupe monophylétique très diversifié (nommé cluster II Euryarchaeota par Adam et collègues) contenant sept phyla distincts aux modes de vie très différents (acidophiles, halophiles, méthanogènes entre autres) et, sporadiquement, chez les superphyla DPANN et Asgard. Les analyses phylogénétiques initiales ont indiqué que les gyrases d'archées sont d'origine bactérienne et ont été acquises par une archée hyperthermophile grâce à un transfert horizontal de gènes. Des analyses ultérieures incluant d'autres lignées d'archées ont suggéré que ce transfert n'a eu lieu qu'une seule fois à la base des Euryarchées de groupe II. Puisque le surenroulement négatif facilite la dénaturation de l'ADN, il a été proposé que l'acquisition de la gyrase a un impact profond sur tous les processus dépendant de l'ADN, avec des conséquences importantes pour l'évolution des archées réceptrices. Cependant, comment et pourquoi la gyrase s'est fixée dans les lignées d'archées reste obscur. L'établissement réussi de la gyrase bactérienne dans les archées est particulièrement intrigant, car l'introduction d'une activité de surenroulement négatif incontrôlée pourrait potentiellement interférer avec tous les processus liés à l'ADN. Pour aggraver les choses, l'archée réceptrice était probablement thermophile et codait donc une reverse gyrase. Cette topoisomérase a une activité de surenroulement positif, opposée à celle de la gyrase, et elle est essentielle pour la vie à haute température. Enfin, les archées possèdent la Topo VI comme principale topoisomérase de type II et son rôle prédit in vivo dans la relaxation des surenroulements positifs est redondant avec celui de la gyrase. Pour mieux comprendre les défis imposés par la gyrase à une cellule archéenne naïve, nous avons introduit une gyrase bactérienne dans l'archée hyperthermophile Thermococcus kodakarensis TS559, génétiquement modifiable, qui possède naturellement un ADN surenroulé positivement. Cette archée possède des histones et trois topoisomérases (reverse gyrase, Topo III et Topo VI), imitant ainsi le "kit" topologique présent chez l'ancêtre des Euryarchaeota de groupe II. Comme donneur de gyrase, nous avons choisi celle de la bactérie Thermotoga maritima (TmGyrAB) puisque les orthologues les plus proches sont des gyrases d'archées, ce qui augmente les chances que son activité ne soit pas altérée par le contexte cellulaire archée. De plus, les températures optimales de croissance de T. maritima (80°C) et T. kodakarensis (85°C) sont similaires, les deux ayant été isolées de sources hydrothermales marines et elles peuvent être co-cultivées en laboratoire. Enfin, TmGyrAB présente l'activité de surenroulement négatif attendue à la fois in vitro et in vivo. Nous avons montré que la gyrase de T. maritima était active : l'ADN plasmidique normalement surenroulé positivement est converti en ADN fortement surenroulé négativement dans T. kodakarensis. La gyrase interagit avec le génome de T. kodakarensis et altère l'expression de centaines de gènes, dont une partie répond spécifiquement à l'activité de surenroulement négatif. La reverse gyrase est la seule topoisomérase surexprimée, bien que modestement, en réponse à l'activité de surenroulement négatif. Malgré les changements topologiques non naturels induits par la gyrase, la croissance de T. kodakarensis n'est pas affectée. Nous concluons que la gyrase est remarquablement bien tolérée par T. kodakarensis, ce qui suggère l'existence de mécanismes de résilience contre le stress topologique qui ont pu être déterminants pour l'établissement de la gyrase dans le domaine des Archées au cours de l'évolution. Article III Les hautes températures représentent un défi considérable pour la physiologie cellulaire et en particulier pour la stabilité du génome. Comme le mouvement moléculaire augmente proportionnellement à l'élévation de la température, les molécules, les membranes et l'ADN deviennent instables. Concrètement, dans des conditions in vitro, l'exposition de l'ADN à des températures élevées entraîne des taux élevés d'oxydation, d'alkylation, de désamination, de dépurination et enfin de cassures de brins. Pour protéger leurs génomes contre ces lésions toxiques de l'ADN, les thermophiles et les hyperthermophiles ont recours à des adaptations spécifiques. Entre autres, ils hébergent des ARNr riches en GC, des lipides spécifiques, des proportions élevées d'acides aminés thermostables dans leurs protéines (typiquement I, V, Y, W R, E, L), des protéines chaperones, des concentrations élevées de sels et de polyamines dans leurs cytoplasmes, des transporteurs d'ions spécifiques et une topoisomérase, la reverse gyrase. Ce type particulier de topoisomérase IA a été découvert en 1984 chez Sulfolobus acidocaldarius et nommé après son activité unique de surenroulement positif, opposé à celle de la gyrase. La reverse gyrase est une fusion des domaines SF2-hélicase et topoisomérase, et présente donc une activité ATP-dépendante et MgCl2-dépendante. Il a été proposé que la reverse gyrase crée un surenroulement négatif et positif en ouvrant l'ADN, puis en relâchant uniquement le surenroulement négatif, ce qui entraîne une augmentation nette du surenroulement positif. Les domaines hélicase et topoisomérase coopèrent grâce au « latch » au cours du processus. De façon remarquable, jusqu'à présent, la reverse gyrase est la seule protéine présente chez tous les hyperthermophiles et la plupart des thermophiles, ce qui en fait la marque de l'hyperthermophilie. On pensait que son effet protecteur contre les hautes températures provenait de la plus grande stabilité de l'ADN surenroulé positivement, une topologie qui présente un empilement accru des bases et qui est plus résistante à la dénaturation des deux brins à haute température. La compaction de l'ADN par la chromatinisation est une autre stratégie de stabilisation de l'ADN à haute température. Les histones des archées sont classiquement organisées en nucléosomes tétramériques. Mais certaines archées comme T. kodakarensis, possèdent des histones qui peuvent étendre cette structure de base par plusieurs dimères d'histones. Il en résulte un assemblage d'histones en oligomères de différentes tailles qui enrobent l'ADN dans une structure filamenteuse. Il a été démontré que la couverture en histone du génome de T. kodakarensis est presque complète. Cette couverture élevée en histones suggère que les histones sont des acteurs majeurs de l'architecture du génome et de la régulation des gènes chez T. kodakarensis. De manière importante, les histones peuvent réguler le surenroulement à la fois en le contraignant ou en libérant des supertours de sens opposé. Inversement, la liaison des histones à l'ADN et l'oligomérisation des histones peuvent être affectées par le surenroulement de l'ADN. Chez la plupart des hyperthermophiles étudiés jusqu'à présent, les plasmides ont été trouvés dans un état de surenroulement relâché ou légèrement positif. Cependant, les hyperthermophiles contenant de l'ADN négativement surenroulé existent et leurs génomes codent invariablement pour la gyrase, indiquant que lorsque les deux topoisomérases coexistent dans une cellule, la gyrase dicte la topologie globale de l'ADN. Nous avons récemment démontré, en introduisant une gyrase active dans l'archée hyperthermophile naïve Thermococcus kodakarensis, que la topologie des plasmides peut être convertie d'un surenroulement positif à un surenroulement négatif sans effet négatif sur la croissance de cette archée. Ce résultat remet en question l'hypothèse selon laquelle le surenroulement positif de l'ADN est une caractéristique importante des hyperthermophiles et soulève des questions sur le rôle physiologique de la reverse gyrase.L'étude de la fonction de la reverse gyrase chez les archées hyperthermophiles par des approches génétiques a produit des résultats ambigus. La délétion du gène codant pour la reverse gyrase chez l'euryarchée T. kodakarensis est viable et ne provoque pas de défaut de croissance à 85°C, sa température de croissance optimale. Cependant, à 90°C, le taux de croissance est divisé par deux et la souche ne croît plus du tout à 93°C.Chez Pyrococcus furiosus, une espèce étroitement apparentée, l'absence de reverse gyrase est létale à 95°C, une température de croissance sous-optimale pour cette archée qui croît de manière optimale à 100°C. Chez l'archée Sulfolobus islandicus, chacun des deux gènes codant pour la reverse gyrase peut être supprimé séparément, mais la suppression de TopR2 ne peut être obtenue qu'après 14 à 20 jours d'incubation ce qui semble difficilement compatible avec la survie dans des conditions de croissance naturelles.Outre son activité de surenroulement positif, de plus en plus d'études suggèrent l'implication directe de la reverse gyrase dans la réparation de l'ADN. La reverse gyrase de Sulfolobus solfataricus forme des complexes covalents stables avec l'ADN endommagé in vitro par irradiation aux UV, en utilisant l'enzyme purifiée ou des extraits cellulaires. À un rapport protéine/ADN élevé, la reverse gyrase d'Archaeoglobus fulgidus empêche l'agrégation inappropriée de régions d'ADN dénaturées par la chaleur et favorise un appariement correct in vitro, protégeant ainsi l'ADN des cassures double brin induites par la chaleur, indépendamment de toute activité enzymatique.La reverse gyrase interagit avec la protéine de liaison à l'ADN simple brin RPA de Pyrococcus abyssi. Chez Sulfolobus solfataricus, il a été démontré que la reverse gyrase forme un complexe avec la protéine de liaison à l'ADN simple brin SSB et l'ADN. La liaison à la SSB augmente l'activité topoisomérase de la reverse gyrase, ce qui suggère son implication directe dans la réparation de l'ADN. Dans une autre étude, le même groupe a montré que la reverse gyrase co-immunoprécipite avec la polymérase translésionnelle Y (PolY) connue pour son rôle essentiel dans la réparation de l'ADN. La reverse gyrase inhibe l'activité de PolY in vitro et l'activité de surenroulement positif est requise pour l'inhibition, ce qui suggère que la reverse gyrase peut réguler l'activité de la PolY lorsque l'ADN est endommagé. D'autres expériences ont étudié le rôle de la reverse gyrase lorsque des cellules de Sulfolobus islandicus sont exposées au méthylméthane-sulfonate (MMS), un agent alkylant de l'ADN qui provoque des cassures de l'ADN. La déplétion de TopR1 dans ces conditions a entraîné une dégradation accélérée de l'ADN génomique accompagnée d'un taux plus élevé de mort cellulaire.Depuis qu'il a été montré il y a 20 ans que la reverse gyrase est la seule protéine caractéristique de l'hyperthermophilie, un travail considérable a été réalisé pour comprendre ce qui la rend indispensable à la vie à haute température. En dépit de ces efforts, le rôle physiologique de la reverse gyrase et, en particulier, sa contribution précise au maintien de l'intégrité du génome restent obscurs. L'une des principales questions non résolues reste de comprendre quelle est la contribution de la reverse gyrase au surenroulement global de l'ADN chromosomique. Pour répondre à cette question, nous avons caractérisé in vivo l'impact de la délétion de la reverse gyrase sur la topologie de l'ADN cellulaire. Nous avons d'abord démontré que la reverse gyrase est effectivement responsable du surenroulement positif des plasmides chez l'archée hyperthermophile Thermococcus kodakarensis. En utilisant la digestion de la chromatine par la nucléase micrococale (MNase), nous avons montré que la délétion de la reverse gyrase n'entraîne pas de profil aberrant, ce qui suggère que l'absence d'activité de surenroulement positif dans les cellules de T. kodakarensis n'a pas d'effet important sur l'assemblage des nucléosomes.Article IVLa nature hélicoïdale de l'ADN, tout en assurant la stabilité de la molécule, impose de fortes contraintes topologiques chaque fois que les deux brins doivent être séparés.Cela signifie que les transactions de l'ADN comme la réplication, la transcription, la recombinaison et la réparation complexifient la topologie de l'ADN, entraînant la formation de supertours, de noeuds, de caténanes et de formes non-B de l'ADN. L'accumulation de ces structures influence en retour les transactions de l'ADN, interférant avec les processus cellulaires essentiels. Pour maintenir les contraintes topologiques à un niveau physiologique, les cellules s'appuient sur un large éventail d'acteurs dédiés à la topologie de l'ADN qui coopèrent pour relâcher les contraintes ou les contenir dans des régions spécifiques des chromosomes. La structure topologique de l'ADN la plus courante est le surenroulement, qui est directement régulé par les topoisomérases. Le surenroulement de l'ADN a été largement étudié chez les organismes modèles en étudiant la topologie des plasmides à l'aide d'électrophorèse en gradient de sucrose ou en gel d'agarose. Ces méthodes sont basées sur la mobilité accrue des plasmides surenroulés (par rapport aux plasmides relâchés) du fait de leur compaction. Bien que faciles à mettre en oeuvre, ces méthodes sont limitées par le fait que les plasmides, en raison de leur petite taille, ne représentent pas fidèlement les chromosomes beaucoup plus grands et complexes. Des approches génétiques, reposant sur la recombinaison spécifique des sites de la resolvase ɤδ ou de l'intégrase du bactériophage λ, ont été utilisées dans le passé pour estimer la distribution du surenroulement à travers le chromosome. Cependant, ces méthodes sont limitées aux bactéries modèles génétiquement modifiables et impliquent de potentiels effets polaires. D'autres méthodes basées sur des dérivés du psoralène permettent de surmonter ces limites en mesurant le surenroulement relatif directement sur les chromosomes sans avoir recours à la génétique. Le psoralène est une petite molécule aromatique plane qui s'intercale entre les paires de bases de l'ADN et établit des ponts covalents intramoléculaires sous l'effet d'une exposition aux UV. L'ADN surenroulé négativement incorpore environ deux fois plus de psoralène que l'ADN relâché, ce qui en fait un outil fiable pour cartographier le surenroulement négatif de l'ADN. Au cours de la dernière décennie, le psoralène a été utilisé pour étudier le surenroulement à l'échelle du génome chez divers modèles de bactéries et d'eucaryotes mais pas chez les Archées. Par conséquent, l'état de surenroulement des chromosomes n'est pas connu chez les Archées. Le surenroulement négatif de l'ADN a un rôle crucial dans la régulation des transactions de l'ADN par sa capacité à favoriser l'ouverture de la double hélice. D'autre part, le surenroulement positif de l'ADN stabilise la double hélice dans une conformation fermée, ce qui diminue son potentiel de dénaturation. Chez les bactéries, le surenroulement est utilisé comme un régulateur transcriptionnel rapide et massif par sa capacité à changer simultanément l'expression d'un grand nombre de gènes en réponse à des changements environnementaux. Cette régulation est principalement basée sur l'activité de la gyrase qui introduit un surenroulement négatif dans le génome proportionnellement au rapport [ATP]/[ADP] dans la cellule. Par conséquent, les génomes bactériens sont en moyenne surenroulés négativement mais la distribution du surenroulement négatif n'est pas homogène et varie en fonction de l'origine de réplication et de la phase de croissance. Chez Escherichia coli, la seule bactérie pour laquelle le surenroulement a été cartographié à l'échelle du génome, un gradient décroissant de surenroulement négatif est mesuré du terminus vers l'origine de réplication en phase stationnaire alors qu'aucun gradient n'a été observé lorsque la bactérie croît de manière exponentielle. Il a été démontré que ce schéma spécifique est corrélé à l'intensité de la transcription et aux sites de liaison de la gyrase, ce qui reflète l'importance de cette topoisomérase pour une transcription efficace et un contrôle global du surenroulement. Les eucaryotes sont dépourvus de gyrase, mais possèdent également un génome surenroulé négativement. Ce surenroulement négatif est généré par la fixation des histones sur l'ADN. Le contrôle de l'expression génétique à partir de ces loci contraints est généré par une relation complexe entre la transcription, la liaison des histones et la relaxation de l'ADN par les topoisomérases. Il est important de noter que chez les eucaryotes, les supertours négatifs libres sont concentrés près des sites de début de transcription (TSS) et des sites de fin de transcription (TTS) des gènes actifs, tandis que les supertours positifs libres sont plutôt situés au milieu des gènes activement transcrits. Une telle organisation favorise les structures d'ADN non-B aux limites des gènes ce qui les isolent du reste du génome, augmentant le recyclage de l'ARN polymérase du TSS au TTS et maintenant les corps des gènes en surenroulement positif pour éviter le repositionnement des nucléosomes. Les études de la topologie globale de l'ADN chez les archées n'en sont qu'à leurs débuts, mais les premières études de la topologie des plasmides in vivo indiquent que, comme c'est souvent le cas, ces organismes présentent des caractéristiques idiosyncrasiques que l'on ne retrouve pas chez les bactéries ou les eucaryotes. Par exemple, l'ADN plasmidique positivement surenroulé n'est jusqu'à présent trouvé que dans les lignées d'archées thermophiles. Cette spécificité est censée protéger l'intégrité du génome à haute température, mais elle affecte probablement aussi la géométrie globale de l'ADN et la régulation de l'expression des gènes. Au-delà du cas particulier des thermophiles, les Archées possèdent des génomes petits et denses avec la majorité des gènes structurés en opérons et leur transcription et traduction sont couplées. Ces propriétés bactériennes sont contrebalancées par une machinerie de transcription homologue à celle des eucaryotes et la présence d'histones dans la plupart des lignées d'archées. Il est probable que l'intégration réussie au cours de l'évolution d'une telle combinaison mosaïque de caractéristiques moléculaires ait abouti à l'émergence de mécanismes originaux de contrôle de la topologie de l'ADN chez les archées. Dans cette étude, nous avons utilisé le pontage de l'ADN par le triméthylpsoralène (TMP) couplée à un séquençage de nouvelle génération (NGS) pour établir la première carte du surenroulement de l'ADN chez une archée. Nous nous sommes concentrés sur Thermococcus kodakarensis, une archée hyperthermophile génétiquement modifiable avec un kit topologique relativement simple composé de deux histones (HtkA et HtkB), deux NAPs (TrmBL2 et Alba), une SMC et trois topoisomérases (Topo III, Topo VI et reverse gyrase). Nous avons utilisé quatre souches construites précédemment : une souche TKAg de référence, une souche témoin TKY119F exprimant une gyrase inactive, une souche exprimant la gyrase TKgyrAB et un mutant de délétion de la reverse gyrase TK∆RG. En comparant l'enrichissement relatif des pontages au TMP sur le génome de ces souches, nous prévoyons de (i) cartographier la distribution du surenroulement négatif dans T. kodakarensis, donnant un aperçu de l'interaction entre la topologie locale de l'ADN, la transcription des gènes et l'architecture du chromosome ; (ii) établir l'importance de la reverse gyrase pour le surenroulement global du chromosome ; (iii) décrire plus en détail l'impact de l'acquisition de la gyrase sur la topologie du chromosome d'un organisme naïf. Titre : Étude in vivo de la topologie de l'ADN chez l'archée hyperthermophile Thermococcus kodakarensis Mots clés : ADN gyrase, reverse gyrase, topologie de l'ADN, Thermococcus kodakarensis, pontage au psoralène Résumé : Au sein des cellules, l'ADN existe dans un état surenroulé.

  These ESPs correspond to proteins thought to be absent from prokaryotes but almost ubiquitous in Eukaryotes. Their massive discovery in Asgards suggests that Eukaryotes can emerge from Archaea instead of forming an individual domain. The first asgard metagenomes were

recovered in deep marine sediments from the Arctic Mid-Ocean Ridge, next to the Loki's Castle hydrothermal vent and, accordingly the corresponding archaea were named Lokiarchaeota. The phylogenetic analysis of 36 markers proteins suggested that

Table 2 :

 2 Main characteristics of known nucleoid-associated proteins

Table 3 :

 3 Main characteristics of known topoisomerases

Table 3 : Main characteristics of known topoisomerases

 3

	(Sur-) DNA negatively supercoiled; (Sur+) DNA positively supercoiled; (Bond formed) covalent link to the 5' or 3' extremity of DNA;
	(ND) non-determined; (+) reaction catalysed (the number of + indicates the efficiency to catalyse the reaction); (-) non-catalysed
	reaction; (*) some topoisomerases VIII are ATP-dependent, others are not (adapted from Bush, Evans-Roberts, et Maxwell 2015).

Characteristics Activities Distribution Type Family Sub-family Enzyme structure Bond formed ATP- dependent Mg2+- dependent Relaxation Supercoiling Catenation/Decatenation « Knotting/Unknotting » Archaea Bacteria Eucaryotes Sur-Sur+ Sur-Sur+ I

			2. Supercoiling homeostasis
		Topoisomerases have many different functions in the cell. By their transient cleaving
		activity, they perform decatenation during replication, unknotting of entangled DNA
		IA and relaxation of supercoiled DNA. This last activity is of special importance for all DNA I Monomer 5' No Yes +++ ---+ III Monomer 5' No Yes + ---++	+ +	Yes Yes	Yes Yes	No Yes
		Reverse Gyrase IB transactions. For example, transcription, replication or recombination can only occur if Monomer 5' Yes Yes +++ --+++ ND IB Monomer 3' No No + + ---	-+	Yes Yes	Yes Yes	No Yes
		IC	V	Monomer	3'	No	No	+	+	-	-	-	ND	Yes	No	No
		IIA the supercoiling level is compatible with DNA helix opening. II Homo-dimer 5' Yes Yes + IV Hetero-tetramer 5' Yes Yes +	+ +	--	--	+ +	+ +	No No	No Yes	Yes No
	II	IIB	Gyrase VI	Hetero-tetramer Hetero-tetramer	5' 5'	Yes Yes	Yes Yes	-+	+++ +++ ++ -	--	+ ++	+ ND	Yes Yes	Yes No	No ND
			VIII	Homo-dimer	ND	Yes / No	Yes	+	+	-	-	+	ND	Yes	Yes	No

Table 4 :

 4 Distribution of gyrase in Archaea 1. An atypical topoisomerase Among topoisomerases, gyrase is not the only one that can supercoil DNA. Another atypical topoisomerase, called by opposition reverse gyrase, has a positive supercoiling activity. But the comparison stops there. Reverse gyrase is not homologous to gyrase and is part of the type IA topoisomerases, together with the prokaryotic topoisomerase I and the topoisomerase III. Initially detected in cell extracts of Sulfolobus acidocaldarius

  . Gyrase activity is, via the intracellular ATP/ADP ratio, directly linked with global state of cellular metabolism. In stressful conditions such as lack of nutrients, the ATP levels drop, DNA gyrase is less active and, consequently, the global supercoiling level of DNA is modified. In response, the expression of numerous genes (between 7 and up to 48 % of all genes) are simultaneously altered to allow quick adaptation to such unfavourable conditions (Dorman be a key feature providing the bacteria with capacity to adapt rapidly to changing environment(Rui and Tse-Dinh 2003; Dorman 2006).Bacterial DNA gyrase A and B subunits assemble into A2B2 heterotetramer of approximately 370 kDa forming three major subunit interfaces, or gates, called N-gate, DNA-gate and C-gate. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events(Schoeffler and Berger 2008;[START_REF] Soczek | CryoEM Structures of Open Dimers of Gyrase A in Complex with DNA Illuminate Mechanism of Strand Passage[END_REF] Vanden Broeck et al. 2019). The DNA gate houses the catalytic tyrosine and cooperates with the TOPRIM fold to cleave DNA[START_REF] Mckay | Structure of Catabolite Gene Activator Protein at 2.9 A Resolution Suggests Binding to Left-Handed B-DNA[END_REF][START_REF] Harrison | DNA Recognition by Proteins with the Helix-Turn-Helix Motif[END_REF][START_REF] Berger | Structure and Mechanism of DNA Topoisomerase II[END_REF][START_REF] Aravind | Toprim--a Conserved Catalytic Domain in Type IA and II Topoisomerases, DnaG-Type Primases, OLD Family Nucleases and RecR Proteins[END_REF][START_REF] Gajiwala | Winged Helix Proteins[END_REF]. The unique C-terminal DNA binding domain (CTD) carries the conserved GyrA -box motif, Q(R/K)RGG(R/K)G, which has been identified as hallmark feature of DNA gyrases[START_REF] Reece | DNA Gyrase: Structure and Function[END_REF][START_REF] Qi | C-Terminal Domain of Gyrase A Is Predicted to Have a Beta-Propeller Structure[END_REF]

and Dorman 2016;

Martis B et al. 2019; Bush, Evans-Roberts, and Maxwell 2015)

. The capacity of the DNA gyrase to quickly translate environmental stimuli into appropriate global transcriptional response is believed to

table 1 :

 1 Dispersion of sequence identity values for prokaryotic GyrA and GyrB sequences of the alignment containing the GyrA box. The first sequence is GyrA of E. coli which serves as a reference for canonical GyrA protein. The alignment shows that viral sequences do not contain GyrA box motif.

	Min.*	1st Qu. Median Mean	3rd Qu. Max.

Supplementary

Supplementary figure 3. Global gyrase tree topologies used for tests of tree selection. A) Complete gyrase phylogeny inferred from BMGE-trimmed alignment. B) The complete gyrase phylogeny where one deep branch was moved to produce monophyletic archaeal clade. C) Complete gyrase phylogeny where the branches were shuffled but the global tree topology was preserved. Results of tree selection tests are shown on the bottom part of the figure.

Values indicate p-value for exclusion and are colored in green for tree topologies not excluded by the tests, red for tree topologies significantly excluded. KH is one-sided Kishino-Hasegawa test; SH is Shimodaira-Hasegawa test; WKH is weighted KH test; WSH is weighted SH test; ELW is Expected Likelihood Weight; AU is approximately unbiased test. Plus signs denote the 95% confidence sets. Minus signs denote significant exclusion. All tests were performed with 10000 resamplings using the RELL method. part

Table 4 :

 4 Differential expression profile of pTNAg encoded genes

			pTZ18	-0.051
	Escherichia coli	37	pBR322	-0.050
	Escherichia coli	37	M13mp19	-0.049
	Thermus sp. YS45	65	pTYS45-1	-0.057
	Rhodothermus marinus R21	65	pRM21	-0.063
	Thermus thermophilus HB8	80	pTT8	-0.059
	Halobacterium halobium GRB	37	pGRB	-0.068
	Haloferax volcanii WR11	37	pHV11	-0.068	Charbonnier F. and
					Forterre P., J Bacteriol.
	Haloferax volcanii WR12	37	pHV12	-0.060	1994
	Halobacterium volcanii DS2	37	pHV2	-0.060
	Methanococcus sp. C5	30	pURB500	-0.058
	Methanosarcina acetivorans C2A	35	pC2A	-0.048
	Methanobacterium thermoautotrophicum Marburg DSM 2133	65	pME2001	-0.013
	Sulfolobus shibatae DSM 5389	78	pSSV1	+0.015
	Desulfurolobus ambivalens DSM 3772	80	pSL10	+0.007
	Pyrococcus abyssi GE5	95	pGT5	-0.003
	Thermococcus sp. GE31	80	pGN31	+0.035
	Sulfolobus islandicus REN1H1	80	pRN1	+0.008	López-García P. and
					Forterre P., Mol.
	Sulfolobus islandicus REN1H1	80	pRN2	+0.008	Microbiol. 1997
	Sulfolobus sp. NZ 54/3	80	pTAU4	+0.017
	Archaeoglobus profundus	80	pGS5	-0.033	Lopez-Garcia P. et al., J Bacteriol. 2000
	SV40 virion	37 (host)	/	-0.051
	intracellular SV40		/	-0.050
	polyoma virion		/	-0.053	Shure M. et al., Nucleic Acids Res. 1977
	intracellular polyoma		/	-0.050
	PM2 (marine bacteriophage)	37 (host)	/	-0.017

* confers agmatine prototrophy to T. kodakarensis

Supplementary

table 5 :

 5 Expression profile of topologically relevant genes

	TKgyrAB vs TKY119	TKY119F vs TKAg	TKgyrAB vs TKAg

table 6 :

 6 Fragments per kilobase million counts for annotated topoisomerases in T. kodakarensis

	TKgyrAB (FPKM)	TKY119F	TKAg

  To complete the general view of gyrase distribution in Archaea, it would be worth identifying the key determinants preventing or promoting its successful establishment in archaeal lineages. Assuming that most organisms rely on tightly regulated DNA topology to keep their genomes functional, chromatin architecture proteins like histones, NAPs and SMC as well as topoisomerases are probably critical in the establishment of gyrase in a given lineage. For instance, histones by constraining the excess of negative supercoiling generated by gyrase, can prevent or at least attenuate its deleterious consequences. From this respect, the absence of histones in Crenarchaeota and Diaforarchaea (part of TACK superphylum) could explain why gyrase is never found in these clades[START_REF] Hocher | Growth Temperature Is the Principal Driver of Chromatinization in Archaea[END_REF]).It could be also envisaged that gyrase activity interferes with the activity of the topoisomerase VI. This type II topoisomerase, ubiquitous in Archaea, is probably in charge of positive supercoiling relaxation and chromosomes decatenation in Archaea, in a reminiscent way to gyrase and topoisomerase IV, respectively, in Bacteria. Thus,The main conclusion that we draw from the introduction of gyrase in T. kodakarensis is that, in marked contrast to what is known for bacteria, DNA supercoiling does not need to be strictly maintained within a tiny range for cell survival. This unusual resistance of T. kodakarensis to DNA supercoiling variations could be explained by a buffering effect of histones. As T. kodakarensis histones deletion mutants or mutants of histones oligomerization are viable, it would be interesting to use them as recipient of gyrase to test this hypothesis. In parallel, the introduction of gyrase in Sulfolobus crenarchaeon, a thermophile without histones, would inform us how this organism would deal with alteration of DNA topology in absence of chromatin. Another pertinent model organism would be Archaeoglobus that are the only hyperthermophilic archaea that naturally encode gyrase. Unfortunately, these peculiar archaea, while cultivable, are not genetically tractable for the moment.

	the introduction of gyrase into a naïve archaeon could give raise to conflicts with
	endogenous topoisomerase VI. Such conflicts could be attenuated by adaptation of
	gyrase and/or topoisomerase VI to new repartition of the workload and if so, this
	should be detectable at the sequence level. The phylogeny of the topoisomerase VI
	could reveal such phenomenon. In addition, comparing the activity and the structure
	of topoisomerase VI from gyrase-encoding archaea with those from gyrase-less
	archaea could give additional clues. As a particular evolutionary case, the
	Thermoplasmata, who are devoid of topoisomerase VI but encode gyrase, are of special
	interest. It is probable that their gyrase in addition to control of chromosomal
	supercoiling also ensures genome decatenation thus constituting a good model to
	understand adaptations to this specific situation.

  Les topoisomérases sont des enzymes chargées de la régulation de la topologie de l'ADN dans chaque cellule vivante. Par leurs activités de clivage transitoire de l'ADN, elles maintiennent le surenroulement de l'ADN à un niveau compatible avec les transactions de l'ADN telles que la transcription et la réplication de l'ADN et assurent la décaténation des chromosomes avant la division cellulaire. Les topoisomérases sont de type I ou de type II selon qu'elles clivent un ou deux brins d'ADN. En principe, une seule topoisomérase de type I et une seule topoisomérase de type II seraient nécessaires et suffisantes pour résoudre tous les problèmes topologiques résultant de la longueur et de l'ouverture de la double-hélice d'ADN. Cependant, au sein de ces deux classes, il existe actuellement huit sous-familles de topoisomérases réparties de gyrase régule le niveau global de surenroulement dans les cellules bactériennes et même une déviation modeste de la topologie optimale de l'ADN peut être létale chez les bactéries. L'activité de la gyrase est, via le rapport ATP/ADP intracellulaire, Halobacterium sp., le traitement à la novobiocine a entraîné la dérégulation de nombreux gènes, notamment une augmentation de l'expression de la gyrase, de la Topo VI et de la Topo I, ce qui indique l'implication de ces enzymes dans la régulation des niveaux de surenroulement de l'ADN dans cet organisme.Des études antérieures utilisant l'échantillonnage taxonomique disponible à l'époque, ont identifié la présence de gènes codant pour la gyrase chez toutes les Euryarchées de groupe II, mais pas chez d'autres phyla d'archées connus. Il a donc été proposé que la gyrase aurait été transférée des bactéries aux archées, vraisemblablement une seule fois à la base des Euryarchées de groupe II. Ce groupe majeur au sein du superphylum des Euryarchaeota contient 7 classes distinctes d'organismes aux modes de vie variés (méthanogènes, halophiles) et, à l'exception des Archaeoglobi, ce sont toutes des archées mésophiles. L'acquisition de la gyrase par l'ancêtre thermophile des Euryarchées de groupe II a été proposée comme ayant joué un rôle clé dans ADN chez les archées, qui est encore mal comprise. Pour répondre à ces questions, nous avons échantillonné les gyrases de tous les génomes d'archées et de bactéries disponibles et avons effectué une analyse phylogénétique et comparative des séquences. Nous avons trouvé la gyrase dans toutes les lignées des Euryarchées de groupe II, et dans plusieurs lignées des DPANN et des Asgard, élargissant ainsi les données sur la distribution de la gyrase au sein de ces archées récemment découvertes. La topologie de l'arbre des archées suggère que la gyrase a été transférée des bactéries à la base des Euryarchées de groupe II et qu'elle s'est ensuite répandue dans d'autres lignées par le biais de transferts horizontaux secondaires de gènes. Il est intéressant de noter que nous n'avons pas pu détecter la gyrase dans le clade des TACK, ce qui suggère une incompatibilité entre la physiologie de ces organismes et l'activité de la gyrase. Fait important, nous avons constaté que l'arbre global de la gyrase présente une topologie tripartite dans laquelle les bactéries forment deux clades correspondant aux Terrabacteria et aux Gracilicutes et les Archaea sont monophylétiques. Cela suggère que la gyrase a été transférée avant la diversification des Terrabacteria et des Gracilicutes mais après la diversification des Archaea. Un tel scénario pourrait impliquer que la diversification des Archaea est antérieure à celle des Terrabacteria et des Gracilicutes. De façon remarquable, les branches séparant les bactéries des Archées sont très courtes. Cela suggère que l'acquisition de la gyrase par les archées ne s'est pas accompagnée de changements radicaux dans sa séquence et, par conséquent, que les gyrases des archées partagent des fonctions très similaires à celles des bactéries. De multiples sous-classes de chaque type de topoisomérase, existent dans la biosphère et cette diversité a rendu particulièrement difficile l'étude de l'histoire évolutive des topoisomérases. Une énigme de longue date est de comprendre pourquoi tant de topoisomérases sont apparues au cours de l'évolution et quel rôle elles ont joué dans l'évolution des cellules.

	directement liée à l'état global du métabolisme cellulaire. Dans des conditions de stress
	telles que le manque de nutriments, les niveaux d'ATP diminuent, la gyrase est moins
	active et, par conséquent, le niveau global de surenroulement de l'ADN est modifié. En
	réponse, l'expression de nombreux gènes (de 7 à 48 % de l'ensemble des gènes) est
	simultanément modifiée pour permettre une adaptation rapide à ces conditions
	défavorables. La capacité de la gyrase à traduire rapidement les stimuli
	environnementaux en une réponse transcriptionnelle globale appropriée est
	considérée comme une caractéristique clé fournissant aux bactéries la capacité de
	s'adapter rapidement à un environnement changeant.
	Contrairement aux orthologues bactériens qui ont été largement étudiés in vitro, une
	seule gyrase archée, celle de Thermoplasma acidophylum a été caractérisée
	biochimiquement. Cette enzyme a présenté des activités in vitro similaires à celles des Article II
	homologues bactériens, par exemple des activités de surenroulement dépendant de
	l'ATP et de décaténation et relaxation indépendantes de l'ATP. Les premières études Avec la découverte de la structure de l'ADN, il est devenu évident que l'ouverture de la
	En règle générale, les topoisomérases ont la capacité de relâcher l'ADN surenroulé, ont montré que la gyrase avait une activité de surenroulement négatif in vivo et que double hélice génère des contraintes mécaniques qui provoquent un surenroulement
	mais seules deux d'entre elles peuvent également surenrouler l'ADN relâché. La reverse cette activité était essentielle chez les méthanogènes, les halophiles et les de la molécule d'ADN. Paradoxalement, de nombreuses transactions de l'ADN, tels que
	gyrase peut surenrouler positivement l'ADN et cette enzyme est, à ce jour, le seul thermoacidophiles. On ne sait pas encore si, comme chez les bactéries, l'activité gyrase, la transcription et la réplication, nécessitent la séparation des brins et conduisent
	marqueur connu de la thermophilie. L'ADN surenroulé positivement est plus résistant et par son intermédiaire, l'homéostasie du surerenroulement, est liée à la régulation de naturellement à un surenroulement et à un enchevêtrement de l'ADN. Ces contraintes
	à la dénaturation et il a donc été proposé que l'activité particulière de la reverse gyrase l'expression génétique chromosomique chez les Archées. Le gène codant pour la topologiques finissent par empêcher le déroulement de ces processus cellulaires
	contribue à préserver l'intégrité du génome à haute température. La gyrase, un rhodopsine dans l'halophile extrême Haloferax a été fortement induit quand l'activité essentiels et, si elles ne sont pas résolues, conduisent à la mort cellulaire. Pour faire
	antagoniste de la reverse gyrase, catalyse l'introduction ATP-dépendante de de gyrase a été inhibée par la novobiocine indiquant que les promoteurs sensibles au face à ce problème, toutes les cellules s'appuient sur les topoisomérases, une classe
	supertours négatifs dans l'ADN. Selon le modèle du « Twin supercoiled domain », la surenroulement peuvent exister dans cette archée. Chez une autre archée halophile, d'enzymes ubiquitaire qui introduisent des cassures transitoires dans l'ADN pour
	gyrase élimine les supertours positifs qui s'accumulent devant l'ARN polymérase en résoudre les intermédiaires topologiques défavorables sans endommager le génome.

manière inégale entre les trois domaines de la vie, ce qui indique que l'histoire évolutive de ces enzymes est complexe. Les topoisomérases connues sont toutes des enzymes spécifiques de l'ADN et leur activité affecte fortement les processus liés à l'ADN dans les cellules. Par conséquent, leur apparition et leur évolution sont intimement liées à l'origine de l'ADN et à son évolution en tant que support de l'information génétique. cours de transcription ou pendant la réplication de l'ADN, permettant ainsi le déroulement de ces processus essentiels. En tandem avec la topoisomérase I (Topo I), la l'adaptation de cette lignée à un mode de vie mésophile. Avec la disponibilité d'un échantillonnage taxonomique plus large, il est maintenant clair que la gyrase est également présente dans des lignées d'archées distantes des Euryarchées de groupe II, comme les DPANN et les Asgard. Cela soulève la question du moment et du nombre de transferts horizontaux de gènes inter-ou intra-domaine, ainsi que la nature des adaptations associées, spécifiques ou à l'échelle du génome. La résolution de ces questions est importante pour comprendre l'évolution des archées, et aussi pour tester des hypothèses sur la régulation de l'expression des gènes par la topologie de l'Les topoisomérases sont dites de type I ou type II, selon qu'elles clivent respectivement un ou deux brins d'ADN.

Acknowledgements

Un grand merci à Tamara pour la qualité de son encadrement. Tu m'as toujours traité comme un collègue et pas comme un subordonné et je sais que déjà rien que pour cela j'ai été plus chanceux que beaucoup de doctorants. Travailler avec quelqu'un d'aussi gentil, rigoureux et investi que toi a été un réel plaisir et m'a permis de progresser énormément d'un point de vue scientifique.

ACKNOWLEDGMENTS

We are grateful to the Genotoul bioinformatics platform Toulouse Midi-Pyrenees for providing computing and storage resources.

ACKNOWLEDGEMENTS

We would like to thank fellow scientists who helped us along the way. Olivier Namy, Isabelle Hatin and Pauline Franc ¸ois for help with design of RNA-seq experiments and data handling. Laura Baranello and Jan Grosser, Charles Dorman and Virginia Lioy for their feedback on our data and constructive discussions. Clémence Lauden for help with plotting under R studio. We thank Aurore Gorlas and Evelyne Marguet for advice regarding 2D electrophoresis and Augustin Degaugue for technical support with plasmid loss experiments. We acknowledge the High-throughput sequencing facility of I2BC (Centre de Recherche de Gif -http://www.i2bc. paris-saclay.fr/) for its sequencing and bioinformatics expertise with particular thanks to Yan Jaszczyszyn, Erwin van Dijk and Céline Hernandez for their technical advice and help with data analysis. The present work has benefited from Imagerie-Gif core facility, supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR-11-IDEX-0003-02/SaclayPlant Sciences. Many thanks to Romain Le Bars for helping us with microscopy data acquisition and analysis. Author contributions: P.V. performed the experiments and analysed data. VdC, E.V. and J.O. analysed data. T.B, R.C. and P.F. designed the research. J.O. funded the research. P.V. and T.B. wrote the paper. All the authors read and approved the manuscript. FUNDING P.V. received the 3 year PhD scholarship from the French Ministry of Higher Education and Research; R.C. and VdC postdoctoral fellowship was funded by the European Research Council under the European Union's Seventh Framework Program [FP/2007-2013]/Project EVO-MOBIL -ERC Grant Agreement [340440]. Funding for open access charge: CNRS.

Article II

Nucleic Acids Research, 2021 1-16 https://doi.org/10.1093/nar/gkab869

DATA AVAILABILITY

The RNA-seq data reported in this article are available in ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) database and can be accessed with E-MTAB-10799 accession number. Processed tables, which were modified to homogenize gene names, are available upon request.

Evolutionary history of archaeal DNA gyrases

Paul Villain 1 , Ryan Catchpole 1,2 , Patrick Forterre 1,3 , Jacques Oberto 1 , Violette da Cunha 1, $ & Tamara Basta, 1,$ 1. Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gifsur-Yvette, France 2. Present address : Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA 3. Archaeal Virology Unit, Institut Pasteur, Paris, France. $ Corresponding authors: E-mail: tamara.basta@i2bc.paris-saclay.fr E-mail: violette.da-cunha@i2bc.paris-saclay.fr ABSTRACT Replication, transcription and other DNA-templated processes that require opening of the double helix lead to topological constraints which if not resolved are lethal for cells. Topoisomerases are ubiquitous enzymes that solve these topological problems by introducing one (type I) or two (type II) transient strand breaks. Presence of one type I and one type II topoisomerase is a minimal requirement for life but at least eight distinct families of topoisomerases exist in the biosphere. The emergence and the evolution of this plethora of topoisomerases is poorly understood. DNA gyrase is special type II topoisomerase with the unique capacity to introduce negative supercoiling in DNA. This enzyme is a major antibacterial target because of its essential role in homeostatic regulation of DNA supercoiling in nearly all bacteria. Intriguingly, despite its potential to interfere with essential DNA transactions, bacterial gyrase was successfully established in Archaeal domain by horizontal gene transfer but its emerging function and evolutionary history in this domain of life remain unclear. Here, we established archaea-specific and a global phylogeny of gyrases in prokaryotes. We find that the gyrase was introduced only once in the archaeal domain after the diversification of this group had already started.

The archaea-centred phylogeny mostly recovers the major archaeal phyla suggesting mostly vertical evolutionary trajectory and only few early horizontal gene transfers within the archaeal domain. The global gyrase tree recapitulates the recently established deep divide between Terrabacteria and Gracilicutes suggesting that one of these superphyla was the gyrase donor. This topology further suggests that archaeal diversification started before the diversification of one of these two major bacterial groups.

Supplementary figure 1. Global phylogeny of DNA gyrases

The GyrA and GyrB (502 bacterial and 297 archaeal) sequences were concatenated, aligned using MAFFT and trimmed using BMGE (A) or Noisy (B). This operation removed 6690 out of 7978 positions from the alignment when using BMGE and 4761 out of 7978 positions when using Noisy. Maximumlikelihood analysis was used for tree construction with IQ-TREE and branch support was assessed using UFboot and SH-aLRT (1000 iterations). The consensus trees are shown where sequences belonging to Euryarchaea, DPANN and Asgard superphyla are coloured in blue, green and magenta, respectively. The tree scale corresponds to number of substitutions per site.

Supplementary figure 2. Global phylogeny of GyrA or GyrB sequences inferred with stringent sequence alignment trimming

The GyrA (571 bacterial and 385 archaeal) or GyrB (633 bacterial and 348 archaeal) sequences were aligned using MAFFT and trimmed using BMGE. This operation removed 359 out of 1085 and 528 out of 1099 positions for GyrA and GyrB alignments, respectively. Maximum-likelihood analysis was used for tree construction with IQ-TREE and branch support was assessed using UFboot and SH-aLRT (1000 iterations). The consensus trees are shown where bacterial sequences are underlined in red. The legend indicates color-codes for the archaeal superphyla. The tree scale corresponding to number of substitutions per site is indicated.

Supplementary figure 4.

Maximum likelihood phylogeny of archaeal GyrA sequences.

Dataset containing 376 GyrA sequences was aligned using MAFFT and the phylogenetically uninformative regions were removed using Noisy. 1218 positions out of 1504 were used to produce the phylogenetic tree. The consensus trees is shown where sequences belonging to Euryarchaea, DPANN and Asgard superphyla are coloured in blue, green and magenta, respectively. The tree scale corresponding to number of substitutions per site is indicated.

Supplementary figure 5. Maximum likelihood phylogeny of archaeal GyrB sequences.

Dataset containing 331 GyrB sequences was aligned using MAFFT and the phylogenetically uninformative regions were removed using Noisy. 826 positions out of 1551 were used to infer the phylogenetic tree. Detailed consensus tree is shown on the left, sequences belonging to Euryarchaea, DPANN and Asgard superphyla are coloured in blue, green and magenta, respectively. The tree scale corresponding to number of substitutions per site is indicated. Schematic representation of GyrB tree is shown on the right. The members of group I Euryarchaeota are indicated with an asterisk.The numbers correspond to SH-aLRT and UFboot support for the deep nodes. Supplementary Figure 6. Maximum likelihood phylogeny of archaeal GyrA or GyrB sequences inferred from conservatively trimmed sequence alignment.

377 GyrA and 331 GyrB sequences were aligned using MAFFT and trimmed using BMGE (see material and methods for details). The filtering removed 48 % of positions (779 from 1493 positions left) and 60% of positions (614 from 1554 left) for GyrA and GyrB alignment, respectively. Maximum-likelihood analysis was used for tree construction with IQ-TREE and branch support was assessed using UFboot B) Phylogeny of Thermotogales species used in synteny analysis. The phylogenetic tree was automatically generated using PhyloT and the Genome Taxonomy Database and visualized using iTOL [START_REF] Letunic | Interactive Tree Of Life (ITOL) v5: An Online Tool for Phylogenetic Tree Display and Annotation[END_REF]. The numbers on the branches correspond to bootstrap values for node support.

Supplementary figure 10.

Sequence alignment of GyrA and GyrB orthologs from Methanobrevibacter species.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Conflict of interest statement. None declared.

SUPPLEMENTARY FIGURES AND TABLES

Supplementary figure 1. The gyrA and gyrB from T. maritima are transcribed in T. kodakarensis

TKgyrAB

The expression of gyrA and gyrB was analysed by RT-PCR for three independent clones of strain TKgyrAB. For each clone, total RNA was isolated and then retrotranscribed to cDNA (see material and methods). The total RNA (left side of the gels) or cDNA (right side of the gels) was used as template for PCR and the obtained products were separated by agarose gel electrophoresis. The position of the used specific oligonucleotides is indicated with arrows. T is a control PCR where total RNA or cDNA from T. kodakarensis TKAg which carries the empty expression vector was used as template. TK0149 is plasmid-encoded pyruvoyl-dependent arginine decarboxylase which confers prototrophy to agmatine.

Supplementary figure 2. SDS-PAGE analysis of soluble proteins from T. kodakarensis.

TKAg, TKY119F and TKgyrAB were grown until exponential (expo) or stationary phase (stat). Cells were pelleted and resuspended in Laemmli buffer and heat-denatured. Resulting cell lysates were analysed on a 4-20% polyacrylamide gel. Protein ladder sizes are indicated on the right side of the gel.

Expected bands at 90,5 kDa and 72,5 are not visible in TKY119F and TKgyrAB lysates indicating that gyrA and gyrB are not overexpressed.

Supplementary figure 3. Topological profile of plasmids isolated from Thermococcus kodakarensis

TKgyrAB, TKAg or TKY119F strains.

A) The plasmid DNA was migrated in 0.8% (w/v) agarose gel in absence of intercalating agents for 24h at 1,6 V/cm at 4°C. The ethidium bromide gel is shown on the left and its schematic representation on the right. Different topological forms of plasmids are indicated.

B) The plasmid DNA was migrated in two dimensions in a 1% (w/v) agarose gel prepared with 1X TEP (36 mM Tris-HCl, 30 mM NaH2PO4, 1 mM EDTA, pH 7.8). Chloroquine was added at 10 µg/ml only in the second dimension. First dimension was run for 15 h at 1.2 V/cm and second dimension was run

ABSTRACT

Hyperthermophiles, organisms living at temperatures above 80°C, have evolved a plethora of protective features helping them maintain genome stability in conditions in which DNA is subjected to high rates of DNA damage. Only one of these features, a topoisomerase reverse gyrase, is known to be uniquely found in all hyperthermophiles and many thermophiles suggesting an important role of this enzyme for adaptation to thermophily. Considerable effort has been made in the past 20 years to characterise the function of reverse gyrase both in vivo and in vitro. These studies resulted in many new and sometimes contradictory findings which, in a very resumed manner, point collectively to general function of this enzyme in DNA repair but also in controlling the global DNA supercoiling level in most hyperthermophilic archaea. In the present study, we characterized in vivo the impact of reverse gyrase deletion on DNA supercoiling in hyperthermophilic archaeon Thermococcus kodakarensis. We found that plasmid DNA was converted from positively supercoiled to relaxed topology in reverse gyrase deletion mutant thus demonstrating that reverse gyrase indeed functions as topoisomerase in vivo. The micrococcal digestion of chromatin did not reveal a detectable modification in the mutant T.

kodakarensis cells suggesting that the lack of positive supercoiling activity has no effect on nucleosome assembly. We anticipate that the global scale studies using psoralen crosslink and MNase digestion coupled to next generation sequencing will allow us to determine without ambiguity how reverse gyrase affects the global supercoiling of the chromosome in hyperthermophiles. DNA due to deletion of reverse gyrase. We found that psoralen can be efficiently crosslinked to T.

kodakarensis DNA in vivo under optimal growth conditions of this organism. Under low hit conditions (ratio crosslinked/non-crosslinked DNA = 0.2) we did not see significant difference in amount of crosslinked DNA between different strains suggesting that the global amount of negative supercoiling remained similar and that differences, if they exist, may occur locally on their genomes. The MNase digestion profile of chromatin did not show significant difference between the different strains suggesting that introduced topological perturbations do not affect histone oligomerisation.

MATERIAL AND METHODS

Thermococcus kodakarensis strains and culture

T. kodakarensis strains TKAg, TKY119F, TKgyrAB and TK∆RG were cultured in anaerobic conditions in ravot medium at 85°C as described previously (Villain et al. 2021). The TKgyrAB strain encodes the gyrase of Thermotoga maritima on the pTNAg plasmid, the strain TKY119F encodes a catalytic mutant of the gyrase and the TKAg strain encodes the empty pTNAg plasmid. The TK∆RG is a reverse gyrase deletion mutant was generously supplied by Hiroki Higashibata (Tokyo university).

In vivo plasmid DNA crosslinking with psoralen

Trimethylpsoralen (Sigma-Aldrich, ref. T6137) was diluted to saturation (0.9 mg/ml) in absolute ethanol. 50 µg/ml of psoralen were injected in sealed bottles containing 20 ml of late exponential phase cultures of T. kodakarensis strains TKY119F and TKgyrAB grown at 85°C. To avoid cold-shock, injections were performed directly in the oven. After 30 min of incubation at 85°C with the TMP, cultures were poured in the lid of a 10 cm glass Petri dish maintained at 85°C using a water-bath. Cells were immediately irradiated with ~9.6 kJ.m -2 .min -1 of 365-nm light using a 45 W lamp (Vilber Lourmat model VL-315.BL). After irradiation, cells were immediately chilled on ice before to be pelleted by 15 min of centrifugation at 5,000 x g and at 4°C. Control samples not treated with TMP or treated with TMP but not irradiated with UV were treated in the same way. Plasmid DNA was extracted using the NucleoSpin Plasmid kit (Macherey Nagel) following the manufacturer's protocol. Then, 600 ng of plasmid per sample was digested 40 min at 37°C with FastDigest BglII restriction enzyme (ThermoFisher). Digested DNA was purified using Nucleospin Gel and PCR kit (Macherey Nagel)

following the manufacturer's protocol. Final elution was done with 30 µl NE buffer (5 mM Tris/HCl,pH 8.5). Half of each plasmid sample was heated 5 min at 95°C in a thermocycler to denature DNA and then immediately chilled on ice. Samples were analysed by electrophoresis on 1% agarose gel in 0.5X TAE buffer (20 mM Tris, 10 mM acetic acid, 0.5 mM EDTA). Gels were stained in a 3X Gelred (Biotium)

bath and imaged with a Typhoon Imager (Amersham) using Cy3 channel.

In vivo chromosomal DNA crosslinking with psoralen

The protocol for psoralen crosslinking of T. kodakarensis DNA is based on the protocol published by Kouzine and collaborators [START_REF] Drolet | DNA Topoisomerases: Methods and Protocols[END_REF] with modifications. Trimethylpsoralen (Sigma-Aldrich, ref.

T6137) was diluted up to saturation (0.9 mg/ml) in absolute ethanol. For initial screening of

DISCUSSION

The use of psoralen derivatives for genome wide mapping of supercoiling had produced major results over the last decades. Coupled to microarrays or next generation sequencing, this technique revealed the distribution of supercoiling within large topological domains down to precise genes or promoters in some bacteria and eukaryotes. We applied this approach to T. kodakarensis, to perform the first mapping of DNA supercoiling in an archaeon and in a hyperthermophile.

We found that the maximal TMP concentration producing linear increase of DNA crosslinks was 2 to 4 fold lower than those previously described for bacteria and eukaryotes. This may reflect an existence of a particular DNA topology in T. kodakarensis and/or efficient crossing of psoralen through the cellular envelope. In the same experimental conditions, the crosslink/non-crosslink ratio is similar for the four analysed strains. This suggests that the global amount of negative supercoiling is, in average, the same in all strains and that the differences, if any, may lie in the local distribution of negative supercoiling across the genome. The capacity of histones to assemble in polymers also seems not to be affected in strains that express gyrase (excess negative supercoiling) or that lack reverse gyrase (no positive supercoiling activity). The mapping of negative supercoiling is required to interpret this result since, again, the effects on chromatin distribution and structure may be local rather than global.

In conclusion, the sequencing results of TMP treated samples, coupled to the MNase-seq results, when available should give first insight of the general organization of DNA supercoiling in T. kodakarensis.

The use of TK∆RG and TKgyrAB strains in this study should give us precious information about the importance of reverse gyrase in DNA topology regulation as well as the consequences of the introduction of pervasive negative supercoiling activity to chromosomal DNA topology of T.

kodakarensis.

Conclusions and Perspectives

I. Evolutive history of gyrase in Archaea

Gyrase is ubiquitous in Bacteria but mainly restricted to Cluster II Euryarchaeota in Archaea. This distribution was thought to reflect a single horizontal gene transfer from Bacteria to Archaea leading to the emergence of mesophilic lifestyle in Archaea (Raymann et al. 2014;López-García et al. 2015). But the discovery of many new archaeal lineages over the last decade, complexified this view by revealing a wider distribution of gyrase (Adam et al. 2017). To clarify the evolution of gyrase in archaea, we reconstructed gyrase phylogeny in prokaryotes, using an up to date taxonomic sampling including the newly discovered archaeal lineages.

We conclude that the gyrase is present in three major archaeal phyla, Euryarchaea, DPANN and Asgard but never found in TACK archaea. The topology of gyrase tree recapitulates faithfully the consensus phylogeny of bacteria and archaea including the recovery of the two major bacterial clades Terrabacteria and Gracilicutes. Such topology indicates predominantly vertical evolution of gyrase. Our results are compatible with a single transfer of gyrase genes from bacteria to the ancestor of Cluster II Euryarchaea and subsequent dissemination via only few secondary gene transfers early through diversification of Archaea. From there we conclude that the establishment of gyrase in a naïve organism is not common in agreement with profound impact of this enzyme on essential cellular DNA transactions. We also propose that the gyrase phylogeny can help establish the temporal order of emergence of major bacterial and archaeal lineages, thus helping understand the most distant evolution of organisms. Finally, the short branches separating archaeal and bacterial clades are compatible with the emergence of the gyrase post LUCA in the lineage leading to bacteria.

II. Consequences of gyrase acquisition by a naïve archaeon

The development of reliable genetic tools in T. kodakarensis gave us the opportunity to describe the consequences of gyrase acquisition by a naïve organism. Besides, this organism is a good model mimicking the ancestral state of Euryarchaeota such that we could recapitulate as faithfully as possible the ancestral event of gyrase emergence in archaeal domain. We investigated the DNA topology, the growth and the transcriptome of the T. kodakarensis gyrase expressing strain.

We successfully introduced an active gyrase in a hyperthermophilic archaeon, and we thus conclude that such acquisition is possible even when the recipient is a modern organism. We also confirmed that gyrase can access the chromatinized genome of T.

kodakarensis and that its supercoiling activity deregulates massively the expression of genes (about 10% of all T. kodakarensis annotated genes), suggesting that most of these genes are supercoiling sensitive. Despite a change of DNA topology from positive (σ = +0.0077) to negative (σ = -0.0327), we did not observe marked growth defect for the gyrase expressing strain. Since the level of supercoiling is tightly regulated in Bacteria and can undergo only little changes (about 1.1 -1.4 fold) without toxicity, the 5-fold increase in negative supercoiling in T. kodakarensis is impressive. Our data also show that the endogenous topoisomerases are not used by T. kodakarensis to reestablish the native level of supercoiling thus suggesting an existence of an alternative mechanism for control of DNA supercoiling.

We propose that this mechanism could rely on high histone coverage observed in this organism with dense chromatin constraining most of the artificially introduced negative supercoiling, protecting the genome from excessive heat-induced melting and preserving DNA transactions.