Siyuan Niu
email: siyuan.niu@lirmm.fr

Adrien Suau

Gabriel Staffelbach

Aida Todri-Sanial

A Hardware-aware Heuristic for the Mapping Problem in the NISQ Era

Keywords: connections., Different physical qubits: various calibration data., Qubit mapping problem: Adapting a quantum program to given hardware connectivity

• 𝑞 1 , 𝑞 2 and 𝑞 1 , 𝑞 3

•NISQ devices.

•Connectivity constraint: Nearest-neighbor

Motivation

• Choose 𝑞 1 , 𝑞 2 because of the lower error rate.

• Final mapping • {𝑞 0 → 𝑄 0 , 𝑞 1 → 𝑄 2 , 𝑞 2 → 𝑄 1 , 𝑞 3 → 𝑄 3 , 𝑞 4 → 𝑄 4 }

Methods

• Hardware-Aware (HA) mapping transition algorithm. • Distance matrix 𝐷 = α 1 × 𝑆 + 𝛼 2 × 𝜀 + 𝛼 3 × 𝑇 • S: SWAP matrix, ε: SWAP error matrix, T: SWAP execution time matrix

•Hardware-Aware (HA) mapping transition algorithm.

•Cost function

𝐻 =

1 𝐹 e matrix 𝐷 = α 1 × 𝑆 + 𝛼 2 × 𝜀 + 𝛼 3 × 𝑇

• S: SWAP matrix, ε: SWAP error matrix, T: SWAP execution time matrix

• Selection between SWAP and Bridge gate.

• Hardware-aware Simulated Annealing (HSA) initial mapping.

• Hardware-aware get_neighbor method.

=

SWAP gate Bridge gate

=

Results

• Comparison of number of additional gates and fidelity on IBM Q 20 Almaden.

Number of additional gates improved by 32%.

Fidelity improved by 23%.

Conclusion

•Map the most used qubit of the mapped circuit to the most connected physical qubit.

•Apply CNOT gates on qubits that are directly connected and with reliable interconnects. •If a CNOT cannot be applied on two neighbor qubits, apply on two qubits whose distance is two.

  Introduction (a) Original circuit. (b) IBM Q 5 Valencia. (c) Updated circuit.

  This work was supported in part by the Region of Occitanie, Direction de la Recherche, du Transfert Technologique et de l'Enseignment Superieur, France, under the Grant "Quantum CAD", in part by a Research Collaboration Grant between TOTAL, LIRMM, and CERFACS, and in part by the QuantUM Initiative of the Region Occitanie, University of Montpellier, and IBM Montpellier.


Sofia Medvedeva

David Brandt

Virginija Cvirkaite-Krupovic

Ying Liu

Konstantin Severinov

Sonoko Ishino

Yoshizumi Ishino

David Prangishvili

Jörn Kalinowski

Mart Krupovic
email: mart.krupovic@pasteur.fr

New insights into the diversity and evolution of the archaeal mobilome from three complete genomes of Saccharolobus shibatae

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Terrestrial acidic hydrothermal environments are dominated by Archaea and their viruses [START_REF] Wang | Archaeal extrachromosomal genetic elements[END_REF][START_REF] Prangishvili | The enigmatic archaeal virosphere[END_REF]Munson-McGee et al., 2018a;[START_REF] Lewis | The biology of thermoacidophilic archaea from the order Sulfolobales[END_REF]. Members of the order Sulfolobales are particularly abundant in such habitats and thrive in sulfuric thermal springs and solfataras around the globe [START_REF] Kvist | Archaeal diversity in Icelandic hot springs[END_REF][START_REF] Kozubal | Microbial iron cycling in acidic geothermal springs of yellowstone national park: integrating molecular surveys, geochemical processes, and isolation of novel fe-active microorganisms[END_REF]Munson-McGee et al., 2018b;[START_REF] Liu | New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures[END_REF]. The order Sulfolobales includes 7 genera (Acidianus, Metallosphaera, Saccharolobus, Sulfolobus, Sulfurisphaera, Stygiolobus, Sulfodiicoccus and Sulfuracidifex) with more than 30 species in total [START_REF] Counts | Life in hot acid: a genome-based reassessment of the archaeal order Sulfolobales[END_REF]. Besides variation in optimal growth temperature (65-88°C) and pH (0.7 -4.5), Sulfolobales members are characterized by different morphological and genetic features, and metabolic capacities, such as oxidation/reduction of sulfur and iron, oxygen tolerance, sugar catabolism, etc.

A notable characteristic of the Sulfolobales is the remarkable diversity of their mobile genetic elements (MGE), including unique groups of viruses, plasmids and transposons, which collectively play a profound role in the hosts' genome evolution by gene capture and horizontal gene transfer [START_REF] Zillig | Genetic elements in the extremely thermophilic archaeon Sulfolobus[END_REF][START_REF] Brügger | Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements[END_REF][START_REF] Lipps | Plasmids and viruses of the thermoacidophilic crenarchaeote Sulfolobus[END_REF][START_REF] Wang | Archaeal extrachromosomal genetic elements[END_REF][START_REF] Krupovic | Viruses of archaea: Structural, functional, environmental and evolutionary genomics[END_REF]. The MGEs provide hotspots for genomic rearrangements and promote species diversification through evolutionary arms race [START_REF] Redder | Mutations and rearrangements in the genome of Sulfolobus solfataricus P2[END_REF]. To cope with constant onslaught by MGEs, especially viruses, the Sulfolobales customary carry CRISPR-Cas and toxin-antitoxin systems which are concentrated in genomic regions called defense islands [START_REF] Makarova | Comparative genomics of defense systems in archaea and bacteria[END_REF]. The adaptive CRISPR-Cas immunity system consists of CRISPR arrays, which contain spacers, small DNA fragments some of which are derived from MGEs, and CRISPR-associated (cas) genes. The cas genes form two major, functionally distinct modules: the adaptation module captures new MGE fragments and incorporates them into CRISPR arrays, whereas the interference module recognizes and cleaves the DNA or RNA containing sequences matching acquired fragments. The Sulfolobales species typically encode CRISPR-Cas systems of types I and III, with 1-2 adaptation modules, 1-5 interference modules and 2-6 CRISPR arrays. The toxin-antitoxin systems function in a different fashion. Although in archaea, the role of toxin-antitoxin systems has not been studied in detail, it has been shown in bacteria that upon virus infection, toxins induce cell dormancy or death (i.e., altruistic suicide), thereby blocking the virus spread in the population [START_REF] Lopatina | Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy[END_REF].

Insertion sequences (IS) are transposable elements capable of relocation from one genomic position to another with the help of transposases encoded within the IS boundaries (for autonomous transposons) or somewhere else in the genome (for non-autonomous transposons). IS elements are particularly abundant in the genomes of members of the genus Saccharolobus [START_REF] Redder | Non-autonomous mobile elements in the crenarchaeon Sulfolobus solfataricus[END_REF][START_REF] Redder | Mutations and rearrangements in the genome of Sulfolobus solfataricus P2[END_REF] and are often upregulated in response to various forms of stress, including virus infection [START_REF] Quax | Massive activation of archaeal defense genes during viral infection[END_REF]. The plasmids of Sulfolobales are generally divided into larger conjugative and small nonconjugative ones, depending on the presence of conserved genes involved in conjugation, including ATPases VirB4/TrbE and VirD4/TraG [START_REF] Prangishvili | Conjugation in archaea: frequent occurrence of conjugative plasmids in Sulfolobus[END_REF][START_REF] Stedman | pING family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus: insights into recombination and conjugation in Crenarchaeota[END_REF][START_REF] Erauso | Two novel conjugative plasmids from a single strain of Sulfolobus[END_REF][START_REF] Lipps | Plasmids and viruses of the thermoacidophilic crenarchaeote Sulfolobus[END_REF]. Other conserved genes carried by conjugative plasmids encode replication-and transcription regulation-related proteins. There is no consistent classification system for non-conjugative plasmids and they are typically assigned to types based on the major replication protein they encode. Plasmids of the most common type, pRN-like (represented by pRN1 plasmid of Saccharolobus [formerly Sulfolobus] islandicus), encode a large replication protein consisting of the N-terminal primasepolymerase domain and the C-terminal superfamily 3 helicase domain [START_REF] Lipps | Plasmids and viruses of the thermoacidophilic crenarchaeote Sulfolobus[END_REF][START_REF] Lipps | Structure and function of the primase domain of the replication protein from the archaeal plasmid pRN1[END_REF].

Viruses infecting members of the Sulfolobales are classified into eight different families, which exhibit a broad range of morphologies, gene contents and infection strategies [START_REF] Prangishvili | The enigmatic archaeal virosphere[END_REF][START_REF] Krupovic | Viruses of archaea: Structural, functional, environmental and evolutionary genomics[END_REF]Munson-McGee et al., 2018a). The first Sulfolobales virus, Sulfolobus spindle-shaped virus 1 (SSV1), the prototype member of the family Fuselloviridae, was discovered in 1982 together with its host Saccharolobus (formerly known as Sulfolobus) shibatae B12 [START_REF] Yeats | A plasmid in the archaebacterium Sulfolobus acidocaldarius[END_REF][START_REF] Grogan | Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov[END_REF]. SSV1 is integrated in the tRNA-Arg gene of Sa. shibatae B12 [START_REF] Reiter | Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements[END_REF][START_REF] Schleper | The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA[END_REF] and its reactivation is induced by UV-irradiation [START_REF] Schleper | The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA[END_REF][START_REF] Fusco | A standardized protocol for the UV induction of Sulfolobus spindle-shaped virus 1[END_REF], which leads to budding of spindle-shaped virus particles covered with a lipid-containing envelope [START_REF] Quemin | Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein, and host-derived lipids[END_REF][START_REF] Quemin | Eukaryoticlike virus budding in Archaea[END_REF]. Following the discovery of SSV1, numerous related viruses, infecting different species of the Saccharolobus and Acidianus, have been isolated around the globe, testifying to the ubiquity and importance of the Fuselloviridae in extreme geothermal habitats [START_REF] Wiedenheft | Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses[END_REF][START_REF] Redder | Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism[END_REF][START_REF] Krupovic | Unification of the globally distributed spindle-shaped viruses of the Archaea[END_REF][START_REF] Goodman | Comparative genetic and genomic analysis of the novel fusellovirus Sulfolobus spindle-shaped virus 10[END_REF][START_REF] Pauly | Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus[END_REF]. Fuselloviruses have been extensively studied from structural, biochemical and genomic standpoints [START_REF] Lawrence | Structural and functional studies of archaeal viruses[END_REF][START_REF] Contursi | Molecular biology of fuselloviruses and their satellites[END_REF][START_REF] Quemin | Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein, and host-derived lipids[END_REF][START_REF] Stedman | Structural insights into the architecture of the hyperthermophilic Fusellovirus SSV1[END_REF][START_REF] Pauly | Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus[END_REF]Baquero et al., 2020a), with SSV1 being one of the best characterized archaeal viruses overall. Recently, two new viruses infecting Sa. shibatae strains, S38A and BEU9, have been isolated along with their viruses, namely, Sulfolobus polyhedral virus 1 (SPV1) [START_REF] Liu | A novel type of polyhedral viruses infecting hyperthermophilic archaea[END_REF], and Sulfolobus filamentous virus 1 (SFV1) [START_REF] Liu | Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile[END_REF]. Both virus-host pairs were isolated from the Beppu hot spring complex from which the B12 strain has been isolated more than 30 years prior [START_REF] Liu | New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures[END_REF]. SPV1 and SFV1 have been structurally characterized and classified into families Portogloboviridae and Lipothrixviridae, respectively [START_REF] Liu | Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile[END_REF]Wang et al., 2019a). Interestingly, despite unrelated virion architectures (i.e., icosahedral versus enveloped helical capsids), the dsDNA genomes of both viruses adopt the A-form in the virions, suggesting that this unusual DNA conformation is one of the adaptations to high temperature environments (Wang et al., 2020b). Furthermore, it has been shown that SPV1 genome carries mini-CRISPR arrays and apparently deploys them to eliminate other coinfecting MGEs, including closely related viruses [START_REF] Medvedeva | Virus-borne mini-CRISPR arrays are involved in interviral conflicts[END_REF].

The Sa. shibatae strains and their viruses, and in particular, B12-SSV1, became important models for studying archaeal transcription [START_REF] Martin | SAV 1, a temperate u.v.-inducible DNA virus-like particle from the archaebacterium Sulfolobus acidocaldarius isolate B12[END_REF][START_REF] Reiter | Gene expression in archaebacteria: physical mapping of constitutive and UV-inducible transcripts from the Sulfolobus virus-like particle SSV1[END_REF][START_REF] Palm | Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae[END_REF][START_REF] Qureshi | Factor requirements for transcription in the Archaeon Sulfolobus shibatae[END_REF][START_REF] Qureshi | Sequence-specific DNA binding by the S. shibatae TFIIB homolog, TFB, and its effect on promoter strength[END_REF][START_REF] Wojtas | Structural and functional analyses of the interaction of archaeal RNA polymerase with DNA[END_REF], the mechanisms of viral genome integration [START_REF] Clore | The SSV1 viral integrase is not essential[END_REF], UV-inducible gene expression and more. The SSV1 genome has been harnessed for developing vectors for protein expression, genetic manipulation and gene silencing in Saccharolobus species [START_REF] Stedman | Genetic requirements for the function of the archaeal virus SSV1 in Sulfolobus solfataricus: construction and testing of viral shuttle vectors[END_REF][START_REF] Jonuscheit | A reporter gene system for the hyperthermophilic archaeon Sulfolobus solfataricus based on a selectable and integrative shuttle vector[END_REF][START_REF] Zebec | CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus[END_REF]. Sa. shibatae itself also served as a valuable model for investigating different aspects of adaptation to high temperature environments, such as DNA relaxation by topoisomerases [START_REF] Bergerat | Purification of a DNA topoisomerase II from the hyperthermophilic archaeon Sulfolobus shibatae. A thermostable enzyme with both bacterial and eucaryal features[END_REF][START_REF] Jaxel | Reverse gyrase gene from Sulfolobus shibatae B12: gene structure, transcription unit and comparative sequence analysis of the two domains[END_REF], RNA stability [START_REF] Zhang | Exploring the influence of hyperthermophilic protein Ssh10b on the stability and conformation of RNA by molecular dynamics simulation[END_REF] and served as a source for discovery of thermostable proteins with potential industrial applications [START_REF] Van | Cloning and characterization of glycogendebranching enzyme from hyperthermophilic archaeon Sulfolobus shibatae[END_REF][START_REF] Boyce | Expression and characterisation of a thermophilic endo-1,4-betaglucanase from Sulfolobus shibatae of potential industrial application[END_REF]. However, the application of these models has been strongly limited by the lack of the complete genome sequence for any of the Sa. shibatae strains. Consequently, Sa. shibatae was overshadowed by its close relatives with available full genome sequences, namely, Saccharolobus solfataricus and Sa. islandicus (She et al., 2001b;[START_REF] Guo | Genome analyses of Icelandic strains of Sulfolobus islandicus, model organisms for genetic and virus-host interaction studies[END_REF]Zhang et al., 2018), which are widely used to study the diversity of archaeal MGEs (viruses, plasmids, insertion sequences), virus-host interactions, defense and anti-defense systems.

To expand the number of available models for the studies of archaeal cell biology, metabolism and virushost interactions, here we present the complete genome sequences of all three isolated Sa. shibatae strains, B12, S38A and BEU9. Analysis and comparison of the three genomes coupled with mapping of a large set of CRISPR spacers sequenced directly from the hot spring from which these strains have been isolated revealed a diverse mobilome associated with Sa. shibatae, including novel conjugative and cryptic plasmids, virus-like elements and transposons. Comparative genomics of these elements provided nontrivial insights into the evolution of archaeal MGE, including a remarkable case of virus evolution into a plasmid. Our data suggest a frequent horizontal transfers of MGE over large phylogenetic distances, crossing the genus boundary, which we attribute to the pressure exerted by CRISPR targeting.

RESULTS AND DISCUSSION

Core genome and strain-specific genes To better understand the relationship between Sa. shibatae and other members of the Sulfolobales as well as to gain further insights into virus-host interactions in archaea, we have determined the genome sequences of three Sa. shibatae strains, namely, B12 [START_REF] Yeats | A plasmid in the archaebacterium Sulfolobus acidocaldarius[END_REF], S38A [START_REF] Liu | A novel type of polyhedral viruses infecting hyperthermophilic archaea[END_REF] and BEU9 [START_REF] Liu | Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile[END_REF]. A combination of short-read and long-read sequencing yielded complete circular chromosomes for all three strains. The genomes were similar in size -2,879,035 bp for B12, 2,791,785 bp for S38A and 2,740,857 bp for BEU9with the variation stemming from differences in the content of mobile genetic elements integrated into various chromosomal loci (see below). The three genomes have a GC% content of 35.4-35.7 and share genome-wide average nucleotide identity (ANI) of 98.8-99.2% (≥ 99.7% identity in their 16S rRNA genes). Phylogenetic analysis based on concatenated alignments of ribosomal proteins and the B subunit of RNA polymerase generally confirmed the established relationships between different genera in the order Sulfolobales and showed that Sa. shibatae belongs to a clade containing Sa. solfataricus and Sa. islandicus, as reported previously [START_REF] Grogan | Isolate B12, which harbours a virus-like element, represents a new species of the archaebacterial genus Sulfolobus, Sulfolobus shibatae, sp. nov[END_REF][START_REF] Dai | Genome Sequencing of Sulfolobus sp. A20 from Costa Rica and Comparative Analyses of the Putative Pathways of Carbon, Nitrogen, and Sulfur Metabolism in Various Sulfolobus Strains[END_REF]. However, unlike in the previous 16S rRNA-based phylogenies, where Sa. shibatae typically occupies a basal position relative to Sa. solfataricus and Sa. islandicus species [START_REF] Dai | Genome Sequencing of Sulfolobus sp. A20 from Costa Rica and Comparative Analyses of the Putative Pathways of Carbon, Nitrogen, and Sulfur Metabolism in Various Sulfolobus Strains[END_REF], in our present analysis, Sa. shibatae formed a sister group to Sa. islandicus (Fig. 1A), suggesting a closer evolutionary relationship.

Each Sa. shibatae genome encodes ~3200 genes, which can be assigned to ~2000 functional groups using arCOG classification [START_REF] Makarova | Archaeal Clusters of Orthologous Genes (arCOGs): An Update and Application for Analysis of Shared Features between Thermococcales, Methanococcales, and Methanobacteriales[END_REF]. The three Sa. shibatae strains share more than 93% of arCOG clusters (Fig. 1B), which represent the core genome. The majority of strain-specific arCOGs are concentrated within the hypervariable regions of the corresponding genomes (Fig. 1C). Similar hypervariable regions have been previously observed in Sa. islandicus [START_REF] Jaubert | Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon[END_REF], where they occupy the same genomic position as in Sa. shibatae (Fig. 1C). The core genome of Sa. shibatae is also similar to that of Sa. islandicus, which has been extensively analyzed elsewhere [START_REF] Reno | Biogeography of the Sulfolobus islandicus pan-genome[END_REF][START_REF] Jaubert | Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon[END_REF]Zhang et al., 2018) and will not be considered here. Instead, we will focus on the hypervariable regions and mobile genetic elements. Similar to Sa. islandicus, the hypervariable regions of Sa. shibatae contain multiple insertion sequences (IS elements), toxin-antitoxin systems, CRISPR-Cas systems and distinct clusters of metabolic genes, which appear to contribute different adaptive capabilities to the three Sa. shibatae strains. Strain B12 contains the largest number of strain-specific genes and encompasses a ~100 kbp region encoding multiple glycosyltransferases and membrane proteins, which could be components of a secretion system. A similar region encompassing numerous glycosyltransferases has been previously described in several S. acidocaldarius strains and in Sa. islandicus HVE10/4 [START_REF] Counts | Life in hot acid: a genome-based reassessment of the archaeal order Sulfolobales[END_REF]. By contrast, BEU9 carries a putative operon (livKFGH) constituting the leucine, isoleucine, and valine (LIV) branched-chain amino acid transport system [START_REF] Ribardo | Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport[END_REF], not found in the two other strains.

One of the notable features in the hypervariable region of the S38A genome is the presence of several [NiFe]-hydrogenase clusters, which are not found in other Sa. shibatae strains. The [NiFe]-hydrogenase clusters typically consist of large and small hydrogenase subunits, membrane subunits and several maturation factors [START_REF] Søndergaard | HydDB: A web tool for hydrogenase classification and analysis[END_REF]. Based on the sequence similarities of the large subunits, hydrogenases are classified into ~30 types [START_REF] Søndergaard | HydDB: A web tool for hydrogenase classification and analysis[END_REF]. In Sa. shibatae S38A, we identified four different [NiFe]-hydrogenase types, namely, 1g, 1h, 2d and 2e (Fig. 2). Type 1g includes membranebound hydrogenases specific of Crenarchaeota, which were shown to participate in anaerobic reduction of elemental sulfur [START_REF] Laska | Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens[END_REF], whereas type 1h hydrogenases are oxygen-tolerant enzymes widespread in soil bacteria which oxidize atmospheric hydrogen [START_REF] Greening | Atmospheric hydrogen scavenging: from enzymes to ecosystems[END_REF]. Functions of the type 2e and 2d hydrogenases are not known yet. Despite playing an important role in respiration and sulfur metabolism, [NiFe]-hydrogenases display patchy distribution in Sulfolobales genomes (present in 8 out of 21 Sa. islandicus genomes, 0/4 S. acidocaldarius, 2/2 Sa. solfataricus, 4/7 Metallosphaera, 8/8 Acidianus and 1/2 Sulfurisphaera). Notably, the 1h, 2d and 2e cluster of S38A display closest sequence similarity to the corresponding genes of Sa. islandicus HVE10/4, whereas the 1g cluster has been apparently lost in the latter strain (Fig. 2). Based on the genome analysis it appears that, unlike B12 and BEU9, S38A is capable of anaerobic growth by reduction of elemental sulfur, emphasizing that closely related strains can display considerable variation in metabolic capacities.

Defense systems CRISPR-Cas systems

The hypervariable regions of the three Sa. shibatae strains encompass overlapping but distinct sets of CRISPR-Cas systems. Each strain carries four CRISPR arrays (39-134 spacers in length), several adaptation modules (I-A and I-D types) as well as complete and incomplete interference modules which can be assigned to I-A, I-D, III-B or III-D types based on the presence of signature genes (Fig. 3A). A type I-A system with adaptation and interference modules, and two cognate divergently oriented CRISPR arrays is present in all three strains as well as in Sa. islandicus strains (Fig. 3A). By contrast, all other CRISPR-Cas systems display more patchy distribution. Namely, two III-B type interference modules are shared by BEU9 and S38A, whereas I-A and III-D interference modules are shared by S38A and B12. The latter two strains also share two additional I-A adaptation modules with two additional CRISPR arrays. Notably, although spacer contents in these CRISPR arrays differ between the two strains, S38A and B12 share two adjacent, leader-distal spacers (Fig. 3A), indicative of the common ancestry of the corresponding arrays. In addition, the BEU9 strain carries I-D adaptation and interference modules along with two CRISPR arrays with CRISPR repeat sequences not found in B12 or S38A, and a III-B interference module (Fig. 3A). The provenance of these additional I-D and III-B CRISPR-Cas systems remains unclear. All complete III-B modules belong to the Cmr-β subtype, most closely similar to the corresponding module of Sa. solfataricus P2 [START_REF] Yu | CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms[END_REF]. The fact that type III interference modules are not accompanied by cognate adaptation modules suggests that they function using the CRISPR spacers of type I systems in a PAM-independent manner, as demonstrated for other Saccharolobus species [START_REF] Deng | A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus[END_REF][START_REF] Yu | CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms[END_REF].

CRISPR spacers represent an archive of past virus-host interactions. The CRISPR arrays of the three Sa. shibatae strains collectively include 898 spacers, of which 97 (10.8%) could be matched (>85% identity) to known viruses and other MGEs of Sulfolobales (Fig. 3B). The majority of these spacers (n=67) target rod-shaped virus SBRV1 (family Rudiviridae), which has been sequenced (but not isolated) from the same enrichment culture from which Sa. shibatae BEU9 and S38A were isolated [START_REF] Liu | New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures[END_REF]. Notably, some of the SBRV1-matching spacers are adjacent to each other in the CRISPR arrays (Figure 3B), indicating that they were acquired consecutively, possibly in the course of primed adaptation. The extensive level of CRISPR targeting suggests that SBRV1 is able to escape CRISPR-Cas immunity by high mutation or replication rate or an anti-CRISPR mechanism. Rudivirus SIRV2 (and many other rudiviruses) is known to encode at least three different anti-CRISPR (Acr) proteins, neutralizing type I-D and type III systems [START_REF] He | Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity[END_REF][START_REF] Bhoobalan-Chitty | Inhibition of Type III CRISPR-Cas Immunity by an Archaeal Virus-Encoded Anti-CRISPR Protein[END_REF][START_REF] Athukoralage | An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity[END_REF]. None of these Acrs have homologs in SBRV1, suggesting that the virus may encode novel proteins to block the CRISPR-Cas immunity. Notably, orthologs of the proteins constituting the adhesive type IV pili, which are recognized as host receptors by all known rudiviruses (Wang et al., 2019b;[START_REF] Rowland | Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus[END_REF]Wang et al., 2020a), are conserved in all three Sa. shibatae strains.

Other viruses targeted by Sa. shibatae CRISPR spacers were previously identified as infecting one of three sequenced strains: fusellovirus SSV1 (5 spacers in S38A and 1 spacer in BEU9, albeit the latter with 84% identity), lipothrixvirus SFV1 (1 spacer in B12 and 1 spacer in S38A) and portoglobovirus SPV1 (2 spacers in B12 and 2 spacers in BEU9). Notably, S38A, the host of SPV1, contains one spacer which is 95% identical to the protospacer in the SPV1 genome. In addition, rudivirus SIRV10, fusellovirus SSV9 as well as bicaudaviruses SMV1 and SMV3 were targeted by one CRISPR spacer each (Fig. 3B). Overall, CRISPR targeting pattern is consistent with the adaptation to local viral communities, with viruses originating from the same hot spring being targeted more extensively than those isolated from geographically remote hot springs (Table S1), as has been observed previously for rudiviruses isolated from other geographic locations [START_REF] Bautista | Differentiation and structure in Sulfolobus islandicus rod-shaped virus populations[END_REF]. Except for the above mentioned pair of spacers shared by S38A and B12, the three Sa. shibatae strains do not contain shared spacers. Notably, we identified a spacer in S38A which targets a gene encoding an ABC-transporter in B12 and BEU9 (100% identity); the ortholog of this gene is not present in S38A, perhaps because it has been lost due to selftargeting by CRISPR.

Toxin-antitoxin systems

Toxin-antitoxin (TA) systems can function in antiviral defense by inducing cell dormancy or death, thereby preventing virus spread in the population [START_REF] Lopatina | Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy[END_REF]. Indeed, it has been shown that both CRISPR-Cas and TA systems are strongly upregulated during infection of Sa. islandicus [START_REF] Quax | Massive activation of archaeal defense genes during viral infection[END_REF]. The complement of TA systems is similar in all three Sa. shibatae strains (Table 1; Table S2). The identified toxins belong to RelE, PIN (PilT N-terminal) and HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) families and all are predicted to function as ribonucleases. These toxins are associated with structurally and functionally distinct antitoxins (Table 1), emphasizing the modular evolution of TA systems [START_REF] Makarova | Comparative genomics of defense systems in archaea and bacteria[END_REF]. Toxins of the PIN and HEPN are conserved in all Sa. islandicus strains [START_REF] Jaubert | Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon[END_REF], whereas RelE family toxins are present in Sa. tokodaii and certain Sa. islandicus strains [START_REF] Makarova | Archaeal Clusters of Orthologous Genes (arCOGs): An Update and Application for Analysis of Shared Features between Thermococcales, Methanococcales, and Methanobacteriales[END_REF].

Sa. shibatae strains are associated with a diverse mobilome Transposable elements

As in the case of Sa. solfataricus and Sa. islandicus, Sa. shibatae genomes are heavily infested by IS elements. The IS elements of Saccharolobus have been implicated in insertional mutagenesis and genome rearrangements, which are reported to occur at relatively high frequencies [START_REF] Martusewitsch | High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements[END_REF][START_REF] Blount | New insertion sequences of Sulfolobus: functional properties and implications for genome evolution in hyperthermophilic archaea[END_REF][START_REF] Redder | Mutations and rearrangements in the genome of Sulfolobus solfataricus P2[END_REF]. Using the ISsaga tool [START_REF] Varani | ISsaga is an ensemble of web-based methods for high throughput identification and semi-automatic annotation of insertion sequences in prokaryotic genomes[END_REF], we identified 10 families of autonomous and 5 families of non-autonomous transposons (Fig. 4; Table S3). The identified autonomous transposons of the IS1, IS5, IS6, IS256, IS630, ISH3 families encode DDEtype transposases, whereas IS110 encode evolutionarily related transposase of the DEDD family. By contrast, IS200/IS605 and IS607 encode distinct transposases belonging to the tyrosine and serine recombinase superfamilies, respectively [START_REF] Siguier | Bacterial insertion sequences: their genomic impact and diversity[END_REF]. The non-autonomous transposons, namely, TnpB and miniature inverted repeat transposable elements (MITE), lack the transposase genes and are mobilized by the transposases of their autonomous siblings. The overall number and diversity of IS elements is comparable with that reported for Sa. islandicus and Sa. solfataricus [START_REF] Brügger | Shuffling of Sulfolobus genomes by autonomous and non-autonomous mobile elements[END_REF][START_REF] Redder | Mutations and rearrangements in the genome of Sulfolobus solfataricus P2[END_REF][START_REF] Guo | Genome analyses of Icelandic strains of Sulfolobus islandicus, model organisms for genetic and virus-host interaction studies[END_REF][START_REF] Jaubert | Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon[END_REF], although there is notable variation between the three Sa. shibatae strains. In particular, IS110 and IS256, and to a lesser extent, IS1 and IS5 are considerably more abundant in S38A and BEU9 than in B12, suggesting active proliferation of these IS families following the diversification of the common ancestor of S38A and BEU9 from that of B12. Furthermore, IS200/IS605 and IS607 family elements were not present in B12. Interestingly, a B12 CRISPR array of type I-A carries a spacer matching the IS200/IS605 found in S38A and BEU9. This observation suggests that CRISPR system might participate in controlling the intragenomic transposon proliferation.

Integrated mobile genetic elements

We have previously shown that MGE integrated in archaeal genomes can be efficiently identified by matching the CRISPR spacers across the corresponding cellular genomes [START_REF] Medvedeva | Virus-borne mini-CRISPR arrays are involved in interviral conflicts[END_REF]. Thus, we mapped a large collection of CRISPR spacers sequenced from the uncultivated Sulfolobales community of the Beppu thermal field (~40,000 unique spacer sequences; [START_REF] Medvedeva | Virus-borne mini-CRISPR arrays are involved in interviral conflicts[END_REF]) and spacers extracted from completely sequenced Sulfolobales genomes (~10,000 spacers) onto the Sa. shibatae genomes. As expected, CRISPR spacer hits (protospacers) were unevenly distributed across the Sa. shibatae genomes, with certain genomic islands being matched by multiple spacers (Fig. 5). The regions displaying elevated density of protospacers were further analyzed for the presence of features characteristic of integrated (i)MGEs, namely, genes encoding transposases, integrases and virus-or plasmid-specific proteins. To define the precise boundaries of the iMGE, the prospective regions were searched for the presence of direct repeats corresponding to the attachment (att) sites. In total, we predicted 14 iMGE (Fig. 5, Table 2, Table S4), among which only fusellovirus SSV1 has been described previously [START_REF] Palm | Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae[END_REF][START_REF] Schleper | The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA[END_REF]. Somewhat surprisingly, considering the high insertional mutagenesis rate in Sulfolobus [START_REF] Martusewitsch | High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements[END_REF], the genome sequence of SSV1 determined in this study was identical to that obtained 30 years ago [START_REF] Palm | Complete nucleotide sequence of the virus SSV1 of the archaebacterium Sulfolobus shibatae[END_REF]. In addition to SSV1, B12 contains five other iMGE (E1-E5), whereas S38A and BEU9 carry four iMGEs each. The iMGEs vary in size from 6.7 kbp (for BEU9-E2) to 37.8 kbp (for B12-E5). In SSV1-infected cells, the virus genome is present in both extrachromosomal and integrated forms and the former is considered to be essential for the excision of the latter (She et al., 2001a). Mapping of the paired-end reads revealed that not only SSV1 is present in both integrated and extrachromosomal forms, but the same is true for B12-E5 and BEU9-E1 (Fig. S1). No significant increase in sequencing coverage was observed for these iMGEs, suggesting that they are maintained in the cells at low copy number.

Sa. shibatae iMGE target tRNA genes for integration

Two types of recombinases, both belonging to the tyrosine recombinase superfamily, were reported for iMGE of Sulfolobales [START_REF] She | Archaeal integrases and mechanisms of gene capture[END_REF]. The pNOB8-like integrase gene remains intact upon iMGE integration, because the att site is located adjacent to the integrase gene. By contrast, in the case of the SSV-like integrases, the att site is located within the integrase gene and upon integration, the gene is split into two separate gene fragments flanking the integrated element. The identified Sa. shibatae iMGEs encode both types of integrases, but all have integrated into the genome by recombining with the 3'-distal regions of diverse tRNA genes (Table 2). Although protein-coding genes and intergenic regions are also known to serve as integration sites for archaeal viruses and plasmids (Krupovic et al., 2010a;Krupovic et al., 2010b;[START_REF] Kazlauskas | Novel families of archaeo-eukaryotic primases associated with mobile genetic elements of Bacteria and Archaea[END_REF][START_REF] Krupovic | Integrated mobile genetic elements in Thaumarchaeota[END_REF], the tRNA genes are by far the most common att sites [START_REF] Badel | Archaeal tyrosine recombinases[END_REF]. Notably, we found that the SSV1 genome occupied not only the previously reported tRNA-Arg (CCG) gene [START_REF] Reiter | Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements[END_REF][START_REF] Schleper | The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA[END_REF], but was also integrated into a secondary target site, tRNA-Arg (TCG). Interestingly, in the latter case, SSV1 shared the target site with B12-E4 and the two elements were integrated in tandem. In vitro studies have shown that for efficient recombination, SSV1 integrase does not require nucleotide sequences flanking the att homology region itself [START_REF] Muskhelishvili | SSV1-encoded site-specific recombination system in Sulfolobus shibatae[END_REF]. Thus, the same tRNA gene, in principle, can accommodate multiple consecutive integrations by the same or different iMGEs as is indeed observed. Notably, despite recognizing the same tRNA gene on the host chromosome, SSV1 and B12-E4 encode SSV-like and pNOB8-like integrases, respectively, suggesting convergent evolution of the integration specificity in the two elements. Similarly, B12-E1 and B12-E2 also recognize overlapping att sites and are tandemly integrated into the same tRNA-Thr (GGT) gene (Fig. 5A, Table 2). Tandem integrations have been previously described for euryarchaeal and thaumarchaeal iMGEs, including viruses, plasmids and casposons [START_REF] Krupovic | Archaeal proviruses TKV4 and MVV extend the PRD1-adenovirus lineage to the phylum Euryarchaeota[END_REF][START_REF] Krupovic | Integrated mobile genetic elements in Thaumarchaeota[END_REF], and it has been suggested that recombination between such tandemly integrated elements promotes their modular genome evolution [START_REF] Badel | Archaeal tyrosine recombinases[END_REF].

Diversity and evolution of Sa. shibatae iMGE

Based on the presence of signature genes, Sa. shibatae iMGEs can be grouped into three major classes: (i) viruses and virus-derived iMGEs; (ii) conjugative plasmids; (iii) cryptic non-conjugative iMGEs. Below we will describe the three iMGE classes in more detail.

Viruses and virus-derived elements

As mentioned above, the three Sa. shibatae strains serve as hosts for three distinct viruses (SSV1, SFV1 and SPV1) representing different families. Among these, only SSV1 was identified as a provirus, even though icosahedral portoglobovirus SPV1 also establishes a chronic infection in S38A and does not appear to lyse the host cell upon virion egress. This is consistent with the lack of the integrase gene in either SPV1 or filamentous lipothrixvirus SFV1 [START_REF] Liu | A novel type of polyhedral viruses infecting hyperthermophilic archaea[END_REF][START_REF] Liu | Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile[END_REF].

Strains S38A and BEU9 carry CRISPR spacers targeting SSV1 (5 in S38A and 1 in BEU9; Fig. 3B, Table S1) and are immune to this virus (Fig. S2). However, both strains carry nearly identical iMGEs, S38A-E3 and BEU9-E4, respectively, which exclusively encode genes found in fuselloviruses, including SSV1 (Fig. 6A). Comparative genomic analysis has shown that S38A-E3/BEU9-E4 have evolved from a fusellovirus ancestor through the loss of a block of nine genes, including those encoding the major and minor capsid proteins. Notably, the deletion has resulted in the fusion of the 5'-terminal region of a gene encoding for the putative host recognition protein VP4 [START_REF] Quemin | Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein, and host-derived lipids[END_REF] and the 3'-terminal fragment of gene b277, producing a novel chimeric gene (Fig. 6A). Such recombination-based mechanism could play an important role in the emergence of new archaeal virus genes lacking homologs in sequence databases, which typically constitute ~75% of the archaeal virus genomes [START_REF] Krupovic | Viruses of archaea: Structural, functional, environmental and evolutionary genomics[END_REF]. Indeed, it has been previously noted that fusellovirus genomes are prone to recombination [START_REF] Redder | Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism[END_REF][START_REF] Pauly | Diversified local CRISPR-Cas immunity to viruses of Sulfolobus islandicus[END_REF][START_REF] Zhang | Novel Sulfolobus fuselloviruses with extensive genomic variations[END_REF].

Interestingly, S38A and BEU9 CRISPR spacers target SSV1 in the structural module, suggesting that the deletion of the nine-gene block in S38A-E3/BEU9-E4 has been instigated by CRISPR targeting. We have previously shown that CRISPR targeting drives the evolution of archaeal virus genomes (Medvedeva et al., 2019) and our present results further support this conclusion. The integrase and helicase genes of S38A-E3/BEU9-E4 are intact, which should allow this element to excise from the host genome, replicate and reintegrate into the tRNA-Arg (GCG) gene, but not to form virions for spread between the cells. Thus, S38A-E3/BEU9-E4 exists like an integrative plasmid, rather than a virus. We note that S38A-E3/BEU9-E4 presents one of the most explicit examples of an evolutionary transition between two types of MGEs, that is, between a virus and a plasmid, which appears to be a recurrent phenomenon underlying the diversification of the viral world [START_REF] Kazlauskas | Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids[END_REF].

Importantly, S38A-E3/BEU9-E4 emergence might not be an isolated case of virus-to-plasmid transition involving fuselloviruses. Notably, the replication protein (WP_012954641) of pSSVi [START_REF] Guo | A superfamily 3 DNA helicase encoded by plasmid pSSVi from the hyperthermophilic archaeon Sulfolobus solfataricus unwinds DNA as a higher-order oligomer and interacts with host primase[END_REF], a satellite plasmid of Sa. solfataricus P2 which, in the presence of fuselloviruses SSV1 or SSV2, is packaged into spindle-shaped virus particles and spreads in the population [START_REF] Wang | A novel Sulfolobus non-conjugative extrachromosomal genetic element capable of integration into the host genome and spreading in the presence of a fusellovirus[END_REF], is closely related to the helicase of S38A-E3/BEU9-E4 (66.73% identity over 526 aa alignment). Thus, pSSVi might have also evolved from a fusellovirus by losing the structural module and hence its unexplained ability to hijack the fusellovirus particles might be an ancestral property of this MGE. We predict that upon coinfection with a fusellovirus, S38A-E3/BEU9-E4 might behave as a satellite, akin to pSSVi, and be packed into the viral particles.

Conjugative plasmids

Sa. shibatae B12 and BEU9 carry two novel conjugative plasmids, B12-E5 (pB12E5) and BEU9-E1 (pBEU9E1), both detected in extrachromosomal and integrated forms (Fig. S1). The two plasmids are only distantly related to each other or to the previously reported plasmids (Fig. 6B). Nevertheless, their gene contents are similar to those of other conjugative plasmids of Sulfolobales [START_REF] Prangishvili | Conjugation in archaea: frequent occurrence of conjugative plasmids in Sulfolobus[END_REF][START_REF] Stedman | pING family of conjugative plasmids from the extremely thermophilic archaeon Sulfolobus islandicus: insights into recombination and conjugation in Crenarchaeota[END_REF][START_REF] Erauso | Two novel conjugative plasmids from a single strain of Sulfolobus[END_REF]. Both plasmids encode a number of diverse transcription regulators, pNOB8-like integrases, segregation proteins (ParA and/or ParB) as well as the components of conjugation machinery, namely, ATPases VirB4/TrbE and VirD4/TraG, and the transmembrane poreforming subunit VirB6/TrbL (Fig. 6B). The plasmids also encode several putative toxins, including an mRNA interferase and a GCN5-related N-acetyl-transferase, which might be components of toxin-antitoxin systems and promote plasmid stabilization through the post-segregational killing mechanism. Notably, both plasmids carry a gene encoding a homolog of the fusellovirus SSV1 protein D63, which was proposed to function as an adaptor in macromolecular assembly [START_REF] Kraft | Structure of D-63 from sulfolobus spindle-shaped virus 1: surface properties of the dimeric fourhelix bundle suggest an adaptor protein function[END_REF], suggesting a horizontal gene transfer between Sa. shibatae viruses and plasmids.

Cryptic non-conjugative plasmids

Most of the identified iMGEs belong to this category and likely represent medium-sized (7-14 kb), nonconjugative plasmids (Table 2, Table S4, Fig. 7A). Based on the encoded DNA replication proteins, these plasmids can be divided into those replicating by the rolling-circle mechanism (B12-E2, S38A-E2 and BEU9-E2) and pRN-like plasmids (B12-E1, B12-E3, B12-E4, S38A-E4), which encode characteristic primases-polymerases of the archaeo-eukaryotic primase superfamily [START_REF] Lipps | Molecular biology of the pRN1 plasmid from Sulfolobus islandicus[END_REF][START_REF] Kazlauskas | Novel families of archaeo-eukaryotic primases associated with mobile genetic elements of Bacteria and Archaea[END_REF], whereas S38A-E1/BEU9-E3 do not encode recognizable replication proteins, except for a superfamily (SF) 1 helicase. The latter element lacks a gene for an identifiable integrase and hence might be inactive.

The plasmids and viruses which use rolling-circle mechanism of DNA replication are widespread in bacteria and archaea [START_REF] Kazlauskas | Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids[END_REF], but thus far have not been described in Crenarchaeota, including Sulfolobales. The replication mechanism used by these MGEs is initiated by the MGE-encoded rolling-circle replication endonuclease (RCRE), which nicks the double-stranded replicative intermediate and attaches covalently to the 5' end of the nicked strand through the catalytic tyrosine residue, whereas the free 3' end serves as a primer for the synthesis of the new strand; once the full new circle is synthesized, RCRE catalyzes the ligation (and liberation) of the ssDNA circle. Based on the presence of two or one tyrosine residues in the catalytic motif 3, RCRE are broadly classified into superfamilies 1 and 2 [START_REF] Koonin | Computer-assisted dissection of rolling circle DNA replication[END_REF]. The RCRE encoded by B12-E2/S38A-E2 (the two elements are 95% identical) and BEU9-E2 contain 1 and 2 tyrosine residues in motif 3 (Fig. 7B). However, closer examination of the B12-E2/S38A-E2 protein showed that the lack of the second tyrosine is due to truncation of the protein, which likely renders it inactive. Consistently, the integrase encoded by this MGE is inactivated by frameshift mutations (Fig. 7A). By contrast, the RCRE encoded by BEU9-E2 appears to be intact, with all signature motifs being conserved (Fig. 7B). BLASTP searches showed that BEU9-E2 RCRE is most closely related to the previously uncharacterized homologs from iMGE present in the genomes of several crenarchaeal species as well as in Sulfolobus monocaudavirus 2, although the latter appears to be inactivated. More distant homologs, retrieved during the second PSI-BLAST iteration, are encoded by plasmids and viruses of Euryarchaeota, including haloarchaeal pleolipoviruses HRPV-1 and HRPV-2 [START_REF] Senčilo | Related haloarchaeal pleomorphic viruses contain different genome types[END_REF], provirus of Methanococcus voltae A3 [START_REF] Krupovic | Archaeal proviruses TKV4 and MVV extend the PRD1-adenovirus lineage to the phylum Euryarchaeota[END_REF], Thermococcus prieurii plasmid pTP2 [START_REF] Gorlas | Living side by side with a virus: characterization of two novel plasmids from Thermococcus prieurii, a host for the spindle-shaped virus TPV1[END_REF] and diverse integrated elements. Collectively, these results extend the presence of the rolling-circle plasmids to Crenarchaeota.

The pRN-like plasmids, typified by the Sa. islandicus plasmid pRN1, encode large fusion proteins consisting of the N-terminal primase-polymerase of the archaeo-eukaryotic primase superfamily (PrimPol) and the C-terminal SF3 helicase domains [START_REF] Lipps | Structure and function of the primase domain of the replication protein from the archaeal plasmid pRN1[END_REF]. Similar PrimPol-domain proteins are widespread in bacterial and archaeal MGEs, and comprise many different families [START_REF] Kazlauskas | Novel families of archaeo-eukaryotic primases associated with mobile genetic elements of Bacteria and Archaea[END_REF]. The PrimPol-helicase proteins encoded by the integrative Sa. shibatae plasmids display considerable sequence diversity and do not form a monophyletic clade in maximum likelihood phylogenetic analysis (Fig. 7C). Instead, the four plasmids cluster with different groups of pRN-like plasmids from different species of the order Sulfolobales. The B12-E1 plasmid forms a clade with the Sa. islandicus elements, including plasmid pXZ1 which, similar to B12-E1, encodes an integrase and has been experimentally demonstrated to integrate into a tRNA gene [START_REF] Peng | Evidence for the horizontal transfer of an integrase gene from a fusellovirus to a pRN-like plasmid within a single strain of Sulfolobus and the implications for plasmid survival[END_REF]. By contrast, the protein encoded by B12-E4 is most closely related to the homologs of integrative elements from Sulfurisphaera tokodaii and Sulfurisphaera ohwakuensis TA-1, whereas B12-E3 encodes a divergent protein which forms a separate branch in the phylogenetic tree. Finally, S38A-E4 is at the base of the clade including S. neozealandicus plasmid pORA1 (NC_006906) and Sa. solfataricus plasmid pIT3 (NC_005907) as well as integrated elements of Sa. islandicus. Overall, this analysis shows that pRN-like elements of Sa. shibatae are very diverse and their evolution does not follow that of the host species.

Patterns of MGE distribution in Sulfolobales

To understand the patterns of iMGE distribution in Sulfolobales, we systematically assessed the presence/absence of iMGEs related to those of Sa. shibatae in other Sulfolobales members and overlaid this information with the CRISPR targeting data (Fig. 8). This analysis revealed a complex network of MGE distributions in different strains, which did not correspond to the phylogenetic or geographical distance between the strains harboring the corresponding MGE. For instance, BEU9-E4/S38A-E3 was not found in B12, but was instead present in Sa. islandicus strains Y.G.57.14 and L.S.2.15 isolated in the USA [START_REF] Reno | Biogeography of the Sulfolobus islandicus pan-genome[END_REF]; BEU9-E2 was not found in either of the two other Sa. shibatae strains, but was identified in Sa. islandicus L.S.2.15 and A. brierleyi strains; B12-E4 was restricted to B12, but its relatives were detected in two species of the genus Sulfurisphaera (S. tokodai and S. ohwakuensis). The mechanism underlying the transfer of non-conjugative and non-viral MGEs between different species remains unknown. One possibility is that the MGEs are exchanged through extracellular membrane vesicles. Indeed, it has been recently demonstrated that membrane vesicles produced by Saccharolobus species mediate intercellular transfer of plasmid DNA in the laboratory and similar vesicles were observed in environmental samples [START_REF] Liu | Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments[END_REF].

The patchy distribution of the iMGEs in Sa. shibatae strains could be to a considerable extent attributed to CRISPR targeting, that is, strains devoid of a particular element harbor CRISPR spacers matching this MGE. Furthermore, CRISPR targeting occurred across the genus boundary, with MGE from Sa. shibatae being matched by spacers from S. tokodai and Sa. islandicus, and vice versa (Fig. 8). Thus, CRISPR targeting might not be sufficient for host assignment of the Sulfolobales MGEs, at least, not when a precision above the family rank is sought. The same conclusion has been reached based on the analysis of the lytic rod-shaped viruses infecting Saccharolobus, Metallosphaera and Acidianus species (Baquero et al., 2020b). Nevertheless, analysis of the CRISPR spacers points to local adaptations of the hosts to their MGEs, with species from the same geographical location having more matching spacers against local viruses than species from remote locations (Table S1). Collectively, these data indicate that host switching by MGEs is relatively frequent in Sulfolobales and, to a large part, is driven by the CRISPR targeting.

EXPERIMENTAL PROCEDURES

Strains and their cultivation Sa. shibatae strains B12 [START_REF] Yeats | A plasmid in the archaebacterium Sulfolobus acidocaldarius[END_REF], S38A [START_REF] Liu | A novel type of polyhedral viruses infecting hyperthermophilic archaea[END_REF] and BEU9 [START_REF] Liu | Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile[END_REF] were grown aerobically (120 rpm) at 76°C in the Sulfolobus growth medium as described previously (Zillig et al., 1993). Plaque assays with SSV1 on the lawns of Sa. solfataricus P2 (known host and positive control) and Sa. shibatae strains S38A and BEU9 were performed as described previously [START_REF] Quemin | Sulfolobus spindle-shaped virus 1 contains glycosylated capsid proteins, a cellular chromatin protein, and host-derived lipids[END_REF].

Genome purification and sequencing

The DNA was extracted from exponentially growing cultures of Sa. shibatae strains B12, S38A and BEU9 as described previously [START_REF] Zaparty | Hot standards" for the thermoacidophilic archaeon Sulfolobus solfataricus[END_REF]. The DNA was sequenced in the framework of the Virus-X project [START_REF] Aevarsson | Going to extremes -a metagenomic journey into the dark matter of life[END_REF], using the Oxford Nanopore GridION Mk1 sequencer with FLO-MIN106D flow cells and SQK-RBK004 libraries, as well as the Illumina MiSeq and Nextera libraries. Both were carried out according to the manufacturer's instructions. Hybrid genome assemblies were done using Unicycler v0.4, which includes short-read assembly, contig-bridging with long reads and a final polishing step [START_REF] Wick | Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads[END_REF].

Identification of insertion sequences IS elements were predicted and classified into families using the ISsaga platform [START_REF] Varani | ISsaga is an ensemble of web-based methods for high throughput identification and semi-automatic annotation of insertion sequences in prokaryotic genomes[END_REF]. The 'probable false-positive' predicted by ISsaga were excluded from the final results. Exact coordinates for all identified IS elements are provided in Supporting Information Table S3.

Mapping of the integration sites

The precise boundaries of integration were defined based on the presence of direct repeats corresponding to attachment sites or target site duplications. The direct and inverted repeats were searched for using Unipro UGENE [START_REF] Okonechnikov | Unipro UGENE: a unified bioinformatics toolkit[END_REF]. Whenever possible, additional validation of the MGE integration sites was obtained by comparing sequences of genomes containing the putative iMGEs with those of closely related genomes that do not contain such insertions using blastn algorithm and Mauve full-genome alignments. iMGE related to those of Sa. shibatae and integrated in the genomes of other members of the Sulfolobales were searched for using tblastx. The elements were considered related if the length of the alignment generated by tblastx was > 70% of the length of the corresponding Sa. shibatae iMGE.

Annotation of the MGE genes

For each analyzed gene, the functional annotations were assigned using the PSI-BLAST program with position specific scoring matrixes derived from arCOG alignments [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF][START_REF] Makarova | Archaeal Clusters of Orthologous Genes (arCOGs): An Update and Application for Analysis of Shared Features between Thermococcales, Methanococcales, and Methanobacteriales[END_REF]. To detect remote homology, additional searches were performed using PSI-BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] against the non-redundant protein database at NCBI and HHpred against the PDB, CDD, SCOPe and Pfam databases available through the MPI Bioinformatics Toolkit [START_REF] Gabler | Protein Sequence Analysis Using the MPI Bioinformatics Toolkit[END_REF] (Table S4).

Phylogenetic analysis

For phylogenetic analysis, MCP and ATPase sequences from each (pro)virus were concatenated and aligned using MAFFT [START_REF] Katoh | MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization[END_REF]. Poorly aligned (low information content) positions were removed using trimal [START_REF] Capella-Gutierrez | trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses[END_REF] with the gap threshold of 0.2. The final alignment contained 1034 positions. The maximum likelihood phylogenetic tree was constructed using the IQ-tree program with the automatic selection of the best-fit substitution model for a given alignment [START_REF] Minh | IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era[END_REF]. The best model identified by IQ-tree was VT +F +I +G4. The tree was mid-point rooted for convenient visualization. The branch support was assessed using SH-aLRT implemented in IQ-tree.

Genome comparisons

The genomes of MGEs were compared and visualized using EasyFig v2.1 with tblastx algorithm [START_REF] Sullivan | Easyfig: a comparison visualizer[END_REF]. The complete genomes of the Sa. shibatae strains and Sa. islandicus were compared using progressive-Mauve with default parameters [START_REF] Darling | progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement[END_REF]. Average nucleotide identity (ANI) and GC% content were calculated using OrthoANIu at https://www.ezbiocloud.net/tools/ani.

CRISPR-Cas prediction and protospacer search

Genome sequences of Sa. shibatae strains were analyzed by CRISPRCasFinder with default parameters [START_REF] Couvin | CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins[END_REF]. Findings with evidence level 4 were considered as CRISPR arrays and corresponding spacer sequences were extracted. Spacers sequences were re-oriented with respect to proposed transcription direction. Protospacer search against the database of all isolated viruses and plasmids of Sulfolobales was performed by blastn algorithm with the following parameters: word_size 7, minimal e-value 0.001. The dataset of CRISPR spacers from hot springs has been described previously [START_REF] Medvedeva | Virus-borne mini-CRISPR arrays are involved in interviral conflicts[END_REF].

arCOG assignment

For the arCOG assignment, the arCOG database (ar18) was downloaded from https://ftp.ncbi.nih.gov/pub/wolf/COGs/arCOG/tmp.ar18/. Proteins from Sa. shibatae were matched to the proteins from the ar18 database with BLASTP (E-value 1e-05, max_target_seqs 1) and the best hit for each Sa. shibatae protein sequence was recovered. All arCOGs annotated in the best hit were assigned to the corresponding Sa. shibatae proteins.

Accession numbers

The sequence data have been submitted to the GenBank database under the project number PRJNA716249 with the following accession numbers: Sa. shibatae B12 chromosome (CP077717), Sa. shibatae B12 plasmid pB12E5 (CP077716), Sa. shibatae S38A chromosome (CP077713), Sa. shibatae BEU9 chromosome (CP077715), Sa. shibatae BEU9 plasmid pBEU9E1 (CP077714).

Figure 1 .

 1 FIGURES AND LEGENDS

Figure 2 .

 2 Figure 2. A locus of Sa. shibatae S38A genome containing the [NiFe]-hydrogenase clusters. The S38A locus is aligned with the corresponding loci in Sa. islandicus HVE10/4 and Sulfolobus sp. S-194. Genes constituting the [NiFe]-hydrogenase clusters are color coded with the key provided on the right.

Figure 3 .

 3 Figure 3. The diversity of CRISPR-Cas systems in Sa. shibatae strains. A. Schematic representation of the composition and arrangement of the CRISPR-Cas systems in the three Sa. shibatae strains. Sa. islandicus M.16.4 is shown for comparison. Numbers above the colored boxes indicate the number of spacers in the corresponding CRISPR arrays, whereas black triangles denote the leader-proximal end of the array. The sequences of the CRISPR repeats characteristic of the three types of arrays are colored similarly and are shown at the bottom of the panel. B. CRISPR arrays. Triangles correspond to individual spacers, with those colored matching diverse viruses and integrated elements (listed on the right).

Figure 4 .

 4 Figure 4. Diversity and distribution of insertion sequences in Sa. shibatae strains. MITE, miniature inverted repeat transposable elements.

Figure 5 .

 5 Figure 5. Integrated mobile genetic elements of Sa. shibatae. The circular genomes of the three Sa. shibatae strains are depicted as yellow rings with the distribution of CRISPR spacers depicted as vertical bars colored according to their density in the genome (scale is provided in the center of each genome map). The location of integrated mobile genetic elements is indicated with red bars and the zoom-in on the distribution of CRISPR spacers targeting the corresponding elements is shown in the outer circle, with the spacers originating from the environmental dataset[START_REF] Medvedeva | Virus-borne mini-CRISPR arrays are involved in interviral conflicts[END_REF] and those extracted from genome sequences shown in blue and red, respectively. The secondary integration site of SSV1 is shown in grey.

Figure 6 .

 6 Figure 6. Viral and conjugative elements integrated in Sa. shibatae genomes. A. Genome maps of fuselloviruses SSV1 and SSV8, and a derived integrative element S38A-E3/BEU9-E4. Genes shared between SSV1 and S38A-E3/BEU9-E4 are shown in green, whereas those shared with SSV8 are in yellow. Genes encoding structural proteins of fuselloviruses (VP1-VP4) are shown in blue. FuselloGrey shading connects genes displaying sequence similarity at the protein level, with the percent of sequence identity depicted with different shades of grey (see scale on the right). Int-N and Int-C, N-and C-terminal fragments of the integrase. B. Integrative and conjugative plasmids of Sa. shibatae. Genes encoding proteins conserved in archaeal conjugative elements are shown in red. Grey shading connects genes displaying sequence similarity at the protein level, with the percent of sequence identity depicted with different shades of grey (see scale on the right).

Figure 7 .

 7 Figure 7. Cryptic plasmids of Sa. shibatae. A. Genome maps of cryptic plasmids. Gene encoding genome replication proteins and integrases are shown in red and green, respectively. Abbreviations: RHH, ribbon-helix-helix; PrimPol, primase-polymerase; SF1 and SF3, superfamily 1 and 3, respectively; (w)HTH, (winged) helix-turn-helix; REase, restriction endonuclease; MTase, methyltransferase; PAPS, phosphoadenosine phosphosulfate; DDE, DDE superfamily transposase. B. Alignment of the three conserved motifs (I-III) of superfamily I rolling circle replication initiation proteins encoded by euryarchaeal plasmids and viruses with the corresponding motifs from the putative replication protein of crenarchaeal integrated elements, including those from Sa. shibatae. Note the mutations in the proteins encoded by the virus SMV2 and Sa. shibatae B12 element E2. C. Maximum likelihood phylogenetic tree of PrimPol-SF3 helicase proteins encoded by diverse plasmids and integrated mobile genetic elements in the order Sulfolobales. Elements of Sa. shibatae are shown on grey background. The tree was constructed using the automatic optimal model selection (VT +F +I +G4) and is rooted at the midpoint. Numbers at the nodes represent represent SH-aLRT support values.

Figure 8 .

 8 Figure 8. Relationships between integrated (green circles) and extrachromosomal (yellow circles) mobile genetic elements of Sa. shibatae and other members of the Sulfolobales. Identical and similar elements are connected with solid and broken green lines, respectively. Red arrows indicate targeting of the corresponding elements by CRISPR spacers carried by particular strains. SFV1 and SPV1 do not integrate into the cellular genome and are thus depicted adjacent to their respective hosts.

Table 1 .

 1 Toxin-antitoxin systems of Sa. shibatae.

	Toxin	Antitoxin	B12 S38A BEU9
	RelE (2 arCOGs)	?	4	5	5
	PIN domain (7 arCOGs) ?	6	8	9
	PIN domain (8 arCOGs) AbrB (8 arCOGs)	8	8	9
	PIN domain (5 arCOGs) RHH/CopG (7 arCOGs) 7	8	8
	HEPN (6 arCOGs)	MNT (7 arCOGs)	16	17	13

Table 2 .

 2 Integrated mobile genetic elements of Sa. shibatae.

	Element	Nucleotide	Length	Integration	att	MGE type	Replication
	name	coordinates	(bp)	site	length		protein
	B12-E1	635422..649821	14400	tRNA-Thr	48 bp	Cryptic	PrimPol-SF3
				(GGT)		plasmid	helicase
	B12-E2	649777..660711	10935	tRNA-Thr	43 bp	Cryptic	RCRE
				(GGT)		plasmid
	B12-E3	2221911..2229307	7397	tRNA-Ala	46 bp	Cryptic	PrimPol-SF3
				(CGC)		plasmid	helicase
	B12-E4	2405111..2413149	8039	tRNA-Arg	43 bp	Cryptic	PrimPol-SF3
				(TCG)		plasmid	helicase
	B12-E5*	2612035..2649874	37840	tRNA-Glu	67 bp	Conjugative	Replication
	(pB12E5)			(TTC)		plasmid	protein A
	SSV1*	2839680..2855188	15508	tRNA-Arg	44 bp	Fusellovirus	DnaA-like
				(CCG/TCG)			ATPase
	BEU9-E1*	45765..83514	37749	tRNA-His	49 bp	Conjugative	Replication
	(pBEU9E1)			(GTG)		plasmid	protein A
	BEU9-E2	141911..148661	6750	tRNA-Val	43 bp	pNOB8-like	RCRE
				(CAC)
	BEU9-E3	186480..200539	14059	tRNA-Trp	30 bp	incomplete	SF1 helicase?
				(CCA)
	BEU9-E4	2499412..2506668	7256	tRNA-Arg	45 bp	virus-derived	helicase
				(GCG)
	S38A-E1	142104..154888	12784	tRNA-Trp	30 bp	incomplete	SF1 helicase?
				(CCA)
	S38A-E2	648710..659573	10863	tRNA-Thr	51 bp	incomplete	RCRE
				(GGT)
	S38A-E3	2539711..2546966	7255	tRNA-Arg	45 bp	virus-derived	helicase
				(GCG)
	S38A-E4	2775115..2785077	9962	tRNA-Val	52 bp	pNOB8-like	PrimPol-SF3
				(GAC)			helicase
	* -detected as extrachromosomal elements. RCRE, rolling circle replication endonuclease; SF, superfamily

ACKNOWLEDGMENTS

This work was supported by l'Agence Nationale de la Recherche (Grant ENVIRA, ANR-17-CE15-0005-01) and the Emergence(s) project MEMREMA from Ville de Paris (to M.K.); the European Union's Horizon 2020 research and innovation program under grant agreement 685778, project VIRUS X (to D.P. and J.K.). Y.L. was a recipient of the Pasteur-Roux-Cantarini Fellowship from Institut Pasteur. S.M. was partly supported by the Metchnikov fellowship from Campus France.

Supplementary tables

Table S2. Toxin-antitoxin systems identified in the three Sa. shibatae strains.

Table S3. Insertion sequences identified in the three Sa. shibatae strains. Nucleotide coordinates for each IS element are provided.

Table S4. Functional annotation of the genes carried by the identified mobile genetic elements integrated in Sa. shibatae genomes.