Flipping chromosomes in deep-sea archaea
[['Large-scale genomic rearrangements allow organisms to evolve much more rapidly than through random mutation alone .', 'Rearrangements can result in the movement of genes within genomes , changes in coding strand use , loss of nonessential functions and the incorporation of foreign DNA .', 'As a result , the organization , content and processing of genetic information can be deeply altered .', 'In all three domains of life , chromosomal reorganization is mainly promoted by recombination between homologous sequences , for example between redundant ribosomal operons [1 , 2] or integrated copies of mobile elements ( ME ) such as prophages [3 , 4] , transposons [5 , 6] and insertion sequences ( IS ) [7] .', 'Such recombination can result in the DNA inversions readily observed in closely related genomes [8 , 9] .', 'In addition to homologous recombination , chromosomes can undergo rearrangement through retrotransposon-associated non-homologous recombination [10] .', 'Other elements like integrons confer rapid adaptation to bacteria in changing environments by shuffling cassette arrays encoding a variety of functions , a process involving a site-specific recombinase and two types of attachment sites [11] .', 'Further genomic rearrangement/reorganization can occur through the acquisition of new genetic material , predominantly by lateral gene transfer .', 'Such gene transfer occurs in all organisms through infection by mobile elements such as viruses or plasmids , or through the uptake of free or encapsulated DNA from the environment [12 , 13] .', 'Genomes can acquire novel genes in a fashion ranging from transient to permanent depending on the type of element and the physiological conditions of the host .', 'When ME succeed in stably inserting their genome , the inserted DNA is then replicated as part of the host chromosome .', 'The transactions between ME DNA and host genome are catalyzed by recombinases typically encoded by the elements themselves .', 'These recombinases rank in different classes based on their enzymatic activity and the specificity of their DNA targets .', 'The smallest ME are insertion sequences ( IS ) composed of a short DNA segment encoding only the enzymes involved in their transposition which can occur at many different genomic locations [14] .', 'The related transposons are larger DNA segments which can be transposed by two flanking IS and frequently carry additional genes such as antibiotic resistance determinants [15] .', 'The most frequent IS recombinases are DDE transposases which do not form covalent transposase-DNA intermediates during transposition [16] .', 'Other and typically larger ME such as plasmids and viruses encode recombinases promoting DNA transactions with a stronger DNA sequence specificity .', 'Such site-specific recombination is not only used for mobile element integration and excision in bacteria but also in the spread of antibiotic resistance by transposable elements , the control of plasmid copy number , regulation of gene expression and the resolution of concatenated chromosomes [17] .', 'Site-specific recombinases can be categorized into the serine recombinases and tyrosine recombinases ( Y-recombinases ) ; which , in contrast to DDE transposases , form covalent enzyme-DNA intermediates during recombination , albeit with markedly different mechanisms of action .', 'Before religation of the two recombining DNA strands , serine recombinases generate breaks in all strands while Y-recombinases produce two sequential single-strand breaks [17] .', 'As a rule , site-specific integration/excision reactions promoted by Y-recombinases occur via a synaptic complex composed of two DNA duplexes carrying the specific sites bound by four recombinase protomers [17] .', 'The two-recombinase pairs are activated sequentially , allowing one strand from each duplex to be exchanged at a time via two consecutive and symmetrical Holliday junctions .', 'A notable exception is Vibrio cholerae phage CTX .', 'Not only does this phage integrate into its host genome in single stranded form where two sites fold into a hairpin structure , mimicking a recombination target for the cellular XerCD chromosome resolvase; but also only requires XerC for integration [18] .', 'One of the best-studied Y-recombinases is the integrase of phage λ .', 'The primary function of this enzyme is the integration of phage DNA into the chromosome of its bacterial host ( and its excision ) .', 'This function is achieved by promoting site-specific recombination between the phage attachment site attP and its chromosomal counterpart attB [19] .', 'Under particular circumstances , the integrase of the lambdoid phage HK022 is capable of generating inversions between attP and a secondary attachment site in the HK022 left operon [3] .', 'Similarly , the primary function of the yeast FLP protein is the control of the 2μ plasmid copy number [20] by DNA inversion between two divergent 34bp FRT sites located on the plasmid [21] .', 'FLP recombinase activity has also been successfully used for integration and excision of synthetic DNA in mammalian genomes [22] .', 'The recombination activities of both λ integrase and FLP recombinase are summarized as shown in S1 Fig . Historically , this reciprocal and conservative recombination between two stringently defined double-stranded DNA sequences in each chromosome was denominated the Campbell model [23] .', 'The sequences of a considerable number of Y-recombinases have been compared to reveal the position of conserved residues and infer the location of the catalytic active site [24] .', 'They share in their C-terminal moiety a rather well conserved region of ~120 amino acids containing up to six nearly invariant amino acids R .', '. K .', '. HxxR .', '. [W/H] .', '. Y forming the active site [25 , 26] .', 'A small number of Y-recombinases have been characterized biochemically in Archaea , for example the XerA recombinase of the hyperthermophilic euryarchaeon Pyrococcus abyssi which exhibits a perfect active site consensus [27] .', 'Sequence alignments have revealed that other archaeal active sites diverge slightly from the bacterial consensus R .', '. HxxR .', '. Y [28] .', 'The integrases of viruses SSV1 isolated from the hyperthermophilic crenarchaeon Sulfolobus shibatae [29] and SSV2 from Sulfolobus islandicus [30] share the consensus R .', '. KxxR .', '. Y while the plasmidic integrase of Sulfolobus sp .', 'NOB8H2 displays R .', '. YxxR .', '. Y [28] .', 'Mobile elements therefore contribute to genome evolution through both site-specific and homologous recombination , which usually operate by distinct mechanisms and enzymatic activities .', 'Homologous recombination is also known to occur frequently between multiple IS copies resulting in large scale archaeal genomic rearrangements , as observed in both Crenarchaeota e . g . Sulfolobus islandicus [31] and Euryarchaota e . g . Pyrococcus abyssi [32] .', 'The distribution of archaeal ISs is patchy not only at the phylum level but also at genus level [9] .', 'Interestingly , genome shuffling occurs in Thermococcus [33] even if ISs are seldom found in this genus suggesting that alternative recombination mechanisms are capable of producing large-scale genomic rearrangements .', 'If site-specific recombination only requires specific nucleotide sequences targeted by a dedicated recombinase , homologous recombination on the other hand is a much more complex process .', 'In all organisms , homologous recombination constitutes one of several pathways to repair double-strand breaks .', 'In addition to DNA synthesis , it requires dedicated recombinases and their accessory factors which act on stretches of near-sequence-identical DNA .', 'In eukaryotic and bacterial cells , the enzymes and pathways involved in homologous recombination have been extensively studied ( see [34 , 35] for reviews ) , whereas archaeal homologous recombination is still an active field of investigation .', 'It is known that the initial resectioning step after double-strand break involves the Rad50–Mre11–HerA–NurA complex to generate 3’ single-strand substrates [36 , 37] .', 'The RecA paralog RadA and its accessory functions associate with this ssDNA to constitute the presynaptic filament , which will scan and pair with homologous sequences [38] .', 'In the archaeon Thermococcus kodakarensis , homologous recombination has been detected experimentally between stretches of identical DNA sequences equal to or greater than 500bp [39] .', 'To our knowledge , a direct overlap between site-specific and homologous recombination processes has not been described so far .', 'In the present work , we report the discovery and characterization of a new integrase from the hyperthermophilic archaeon Thermococcus nautili [40 , 41] capable of catalyzing both site-specific recombination and low sequence specificity recombination reactions mimicking homologous recombination .', 'The wide distribution of this particular Y-recombinase among the Thermococcus genus provides a valid rationale for the observed genomic rearrangements in these Archaea .'], ['We compared the chromosomes of the 13 completely sequenced Thermococcus species available to date by dotplot analysis and observed high levels of genome scrambling as shown in Fig 1A .', 'Strikingly , comparison of T . onnurineus and T . sp .', '4557 chromosomes by this approach revealed only two large inversions of 139/143Kb and 102/74Kb respectively ( Fig 1B & 1C ) .', 'This relatively small number of inversions facilitated the investigation of the synteny breakpoints bordering both inversions .', 'Using the SyntTax web tool [42] , a composite representation was obtained as shown in Fig 1C .', 'Gene order is conserved immediately upstream and downstream of each inversion border and was used to identify the synteny breakpoints .', 'For each inversion , the breakpoints are located within tRNA gene pairs , transcribed in opposite orientations .', 'Interestingly , T . nautili plasmid pTN3 integrates in the tRNALeu gene BD01_0018 [41 , 43] ( S2 Fig ) and this gene displays over 97% sequence identity with tRNALeu ( GQS_t10759 ) , which borders a large chromosomal inversion between T . onnurineus and T . sp .', '4557 ( Fig 1B ) .', 'The concordance between the chromosomal attachment site of the pTN3 integrase ( IntpTN3 ) and the recombination targets bordering each inversion ( in opposite orientations ) led us to define a working model to explain the formation of genomic inversions observed in the Thermococcus genus .', 'We hypothesize that the frequent genomic inversions observed in the evolution of the Thermococcales order are a result of enzymatic activity of the integrase encoded by horizontally mobile elements , such as pTN3 .', 'The integrase of pTN3 shares significant sequence similarity with canonical Y-recombinases and its predicted active site can be defined as R .', '. K .', '. AxxR .', '. Y which only slightly diverges from the consensus ( S3A Fig ) .', 'In addition , IntpTN3 displays a high degree of conservation with two biochemically characterized hyperthermophilic Y-recombinases , the archaeal IntSSV1 [44] and IntSSV2 [30] ( S3B Fig ) .', 'Thus , it seemed worthwhile to compare the enzymatic activities of IntpTN3 to those of other enzymes of the same family such as phage λ integrase and Saccharomyces cerevisiae 2μ plasmid FLP protein and to validate them against the canonical Y-recombinase model .', 'In order to characterize the activities of IntpTN3 , it was necessary to over-produce and purify the enzyme ( S4 Fig ) and to construct DNA substrates carrying appropriate attachment sites ( as determined by sequential deletions ( S5 Fig ) .', 'An integrase variant ( IntpTN3Y428A ) in which the catalytic tyrosine is substituted with an alanine was constructed , purified and tested ( S6 Fig ) .', 'We used these proteins and DNA components in a series of in vitro and in vivo experiments , detailed below , to ascertain the properties of IntpTN3 .', 'The large-scale genomic inversions observed between T . sp .', '4557 and T . onnurineus display minor gene order rearrangements near the recombination endpoints indicating that these events are not recent and might have undergone remodeling ( Fig 1C ) .', 'In order to identify more recent rearrangements , we investigated whether large-scale genomic inversions could occur spontaneously under laboratory conditions .', 'T . nautili carrying its natural plasmids was sub-cultured in two independent experiments for 60 and 66 generations ( therefore termed T . nautili 60G and 66G ) in rich liquid medium with intermittent storage at 4°C and the metagenomes of the resulting populations were completely re-sequenced .', 'We observed in both T . nautili 60G and 66G sub-cultures a high proportion of a novel rearranged genome exhibiting four new large-scale chromosomal inversions when compared to the original published T . nautili genome ( GenBank accession NZ_CP007264 ) [41] ( Fig 6A ) .', 'By mapping the frequency of the Illumina reads around the four inversion sites , we measured the incidence of the rearranged genome in the T . nautili 66G population , which was found in most cases to exceed that of the original genome ( S3 Table ) .', 'Both T . nautili 60G and 66G rearranged chromosomes were remarkably similar when compared by dotplot analysis ( S7 Fig ) .', 'Additionally , plasmid pTN3 was largely underrepresented in the T . nautili 66G sub-culture ( S3 Table ) , whereas the smaller pTN1 and pTN2 were conserved .', 'The chromosomally-integrated pTN3 copy carrying the disrupted integrase gene was also retained .', 'The chain of nested inversion events leading to these new recombined genomes could be reconstructed ( Fig 6C ) and allowed us to analyze and precisely map the recombination endpoints .', 'Each of the four genomic inversions occurred between paralogous gene pairs: between tRNAGly genes BD01_1557 and BD01_1976 , between methyl accepting chemotaxis genes BD01_1166 and BD01_1584 , between transposase genes BD01_1317 and BD01_1763 and finally between UDP-glucose-6 dehydrogenase genes BD01_1333 and BD01_1481 .', 'For each pair of paralogous genes , the inversion events always occurred between two inverted segments of DNA sharing extensive sequence identity ( S8 Fig ) .', 'However , we could not detect significant similarity between inverted DNA segments corresponding to different pairs of paralogous genes using BLAST ( e-value ≥ 0 . 075 ) .', 'Furthermore , none of these sequences could be aligned with the original pTN3 attachment site , tRNALeu ( e-value ≥ 10 ) .', 'In a control experiment , in contrast to T . nautili , the genome of a closely related organism , the plasmid-less Thermococcus sp .', '5–4 ( GenBank accession CP021848 ) remained stable when sub-cultured for 36 or 66 generations in two separate experiments ( Fig 6B and S7 Fig ) .', 'The remarkable differences in the outcome of T . nautili and T . sp 5–4 sub-culturing experiments and the observation that tRNAGly genes could recombine in these conditions suggested a causal link between IntpTN3 and genome shuffling .', 'To ascertain if the new recombinations in T . nautili 60G and 66G could have been indeed generated by IntpTN3 , we decided to test whether this integrase was able to catalyze in vitro inversions using the sequences detected at the borders of these recombination events .', 'New inversion templates pCB548 and pCB552 were thus constructed respectively carrying sequences encompassing tRNAGly genes BD01_1557 and BD01_1976 or sequence fragments from chemotaxis genes BD01_1166 and BD01_1584 ( S8 Fig ) .', 'To limit the number and size of generated fragments , an in vitro inversion assay was conducted on linear fragments originating from these plasmids and compared to a linear fragment carrying inverted attP sites derived from pCB524 .', 'Inversions could be detected with all three templates albeit with significantly longer incubation times or higher IntpTN3 concentrations for pCB548 and pCB552-derived templates as compared to pCB524 ( Fig 7 ) .', 'To confirm this recombination event , one of the products of the pCB548 template inversion reaction was further characterized by DNA sequencing and corresponded to a bona fide cross-over between BD01_1557 and BD01_1976 ( S9 Fig ) .', 'We conclude that IntpTN3 is able to catalyze low sequence specificity recombination reactions between sites that differ in sequence from its cognate att site , with the same outcome as homologous recombination events .', 'It is to be noted that IntpTN3 catalyzes these two types of reactions with a different efficiency .', 'Site-specific recombination reactions reach the equilibrium within 30 minutes whereas several hours and higher enzyme concentrations are required to detect all low sequence specificity recombinations .', 'The absence of inter-pair DNA similarity observed in T . nautili 60G and 66G chromosomal inversions prompted us to test whether IntpTN3 could catalyze recombination between homologous non-archaeal sequences .', 'The simplest experiment consisted of the incubation of cloning vector pBR322 DNA with the integrase in the same conditions as described above .', 'This recombination reaction promoted by IntpTN3 yielded a ladder of plasmid multimers produced by sequential integration , which could be readily observed by eletrophoretic migration whereas no homologous integration reaction was detected with the mutated IntpTN3Y428A ( Fig 8A ) .', 'Surprisingly , IntpTN3 generated also a double-strand cut at the pBR322 ColE1 origin of replication for which we have no explanation at this stage ( S10 Fig ) .', 'This cleavage does not constitute an intermediate step in the recombination reaction since none of IntpTN3 linear substrates shown in Fig 7 carries the ColE1 origin .', 'In addition to the homologous integration reaction , we investigated the capacity of IntpTN3 to promote inversions between homologous sequences of bacterial origin .', 'Short DNA segments of decreasing length ( 250 , 175 and 100bp , see S11 Fig ) originating from the E . coli lacZ gene were cloned in opposite orientations respective to the lacZα gene of pUC18 to generate plasmids pCB574 , pCB571 and pCB558 , respectively .', 'These templates were linearized , incubated with IntpTN3 and tested by subsequent restriction analysis .', 'In each case , IntpTN3 generated additional bands consistent with homologous inversion reactions displaying efficiencies proportional to the extent of DNA identity ( Fig 8B ) .'], ['The major mechanism producing chromosomal rearrangements is recombinational exchange between homologous sequences [46] .', 'These rearrangements often consist of DNA inversions between IS elements [9 , 46 , 47] .', 'The observation that , in the Thermococcus genus , large chromosomal inversions occur even in the absence of IS elements prompted us to investigate the molecular mechanism behind these rearrangements .', 'The presence of tRNA genes at recombination endpoints in genomes as diverse as plant chloroplasts [48 , 49] and Thermococcales [9] , combined with the fact that integrases often target tRNA genes [50] , lead us to propose a precise molecular model involving IntpTN3 to explain large-scale genomic rearrangements .', 'Using a combination of comparative genomics , in vitro analyses , and serial culturing experiments , we uncovered a mechanism and enzymatic activity responsible for the shuffling-driven chromosomal evolution in Thermococcales .', 'By means of deep comparative genomic analyses , we were able to correlate genome scrambling with the presence of a mobile element .', 'This mobile element has been identified as plasmid pTN3 , naturally present in T . nautili both as an episome and integrated in the genome [41 , 43] .', 'Plasmid pTN3 encodes the IntpTN3 integrase of the Y-recombinase superfamily capable of promoting its site-specific plasmid integration at a tRNALeu gene of its host .', 'Due to perfect DNA conservation between attB and attP attachment sites ( S2B Fig ) , an intact and presumably expressed tRNALeu is reconstituted upon pTN3 chromosomal integration .', 'We successfully reproduced , with high efficiency in a purified in vitro system , the canonical DNA reactions of integration and excision expected from a bona fide integrase .', 'Site-specific mutation of the active site tyrosine to alanine abolished these activities .', 'A positive excision reaction was also obtained in vivo by expressing wild-type IntpTN3 and the catalytic tyrosine mutant IntpTN3Y428A in T . kodakarensis KOD1 cells .', 'The genome of this strain carries the integrated episome TKV4 [45] which is remarkably similar to pTN3 ( Fig 9 ) .', 'Surprisingly , both wild-type and mutant forms of the integrase excised TKV4 in circular form .', 'This suggests that a truncated C-terminal IntTKV4 , presumably impaired in DNA-binding but carrying the catalytic tyrosine , can complement IntpTN3Y428A .', 'A plausible explanation invokes the participation of integrase dimers in the recombination reaction .', 'In this case , only the heterodimeric form would possess an active catalytic site where Tyr428 is provided by the first monomer while the second monomer contributes the remaining conserved residues .', 'This cleavage in trans was initially reported for the FLP recombinase [51 , 52] .', 'Similarly , the complementation of activity between a DNA-binding impaired mutant and a catalytic tyrosine residue mutant has been described for another archaeal integrase , IntSSV1 [44] .', 'The peculiar location of tRNALeu GQS_t10759 at the exact border of a large DNA inversion observed between the genomes of T . onnurineus and T . sp .', '4557 suggested that this inversion could have occurred by the recombinase activity of IntpTN3 .', 'In our purified system , we could obtain highly efficient DNA inversions between two inverted copies of GQS_t10759 .', 'Paradoxically , we were unable to promote inversion between tRNALeu GQS_t10759 and tRNAThr GQS_t10745 contrary to what the genomic comparisons between T . onnurineus and T . sp .', '4557 suggested .', 'An experiment of prolonged T . nautili cultivation was instrumental in elucidating the large-scale inversion mechanism in Thermococcus .', 'The strain carrying its natural plasmids was cultivated during 60 or 66 generations; total DNA was extracted from this population and sequenced in a manner similar to a metagenome .', 'We observed the high incidence in the resulting populations of a particular recombined genome with four large chromosomal inversions and a very low copy number of plasmid pTN3 encoding active IntpTN3 ( < 2/chromosome ) ( S3 Table ) .', 'This plasmid loss could have contributed to the higher fitness and spread of a particular clone in the population .', 'The four large-scale inversions occurred between four pairs of naturally occurring paralogous genes sharing at least 104bp of sequence identity in inverted orientation ( S8 Fig ) .', 'No significant sequence conservation could be detected between the four pairs .', 'We did not observe chromosomal rearrangements after prolonged incubation of Thermococcus sp .', '5–4 , which does not carry plasmids .', 'The potential causal link between pTN3 and a number of unrelated sequence pairs involved in large scale genomic shuffling in T . nautili was difficult to conciliate with the classical site-specific recombination properties we described for IntpTN3 .', 'Remarkably , by in vitro assays with this integrase , we succeeded in producing inversions between several pairs of inverted paralogous genes detected in our T . nautili sub-culturing experiments .', 'These results suggested that the recombination properties of IntpTN3 could be extended to virtually any homologous pair of DNA sequences .', 'Using exogenous pBR322 plasmid DNA or genes segments from bacterial origin , we demonstrated in vitro that IntpTN3 actively promotes low sequence specificity reactions mimicking homologous integration and inversion of any sequence pair as short as 100bp .', 'The catalytic site mutation in variant IntpTN3Y428A abolishes this particular recombination reaction as well .', 'Interestingly , cellular homologous recombination in Archaea operates according to a different pathway with dedicated enzymes [36 , 37] and in Thermococcus kodakarensis has only been reported between DNA segments of 500bp or more [39] .', 'These reactions unveiled a specific IntpTN3-generated double-strand cut at the ColE1 origin of replication carried by pBR322 and its derivatives ( S10 Fig ) .', 'At this moment , we do not have a precise rationale to explain this observation other than a potential distant secondary structure similarity between the small RNAI and RNAII encoded by the ColE1 origin and the tRNALeu encoded by IntpTN3 attB substrate .', 'Biological interactions between tRNAs and ColE1 RNAs have been reported [53] .', 'Clearly , this double-strand cleavage does not participate in any recombination reaction since we demonstrated all in vitro IntpTN3 inversions on linear DNA segments devoid of ColE1 origin .', 'The positive in vitro IntpTN3-promoted low sequence specificity recombination results explain the failure of this enzyme to promote inversion between tRNALeu GQS_t10759 and tRNAThr GQS_t10745 .', 'These sites were initially thought to constitute inversion endpoints between the genomes of T . onnurineus and T . sp .', '4557 but do not share sufficient sequence similarity to be efficiently recombined in vitro .', 'The particular positioning of these sequences in opposite orientations could have occurred through previous overlapping inversions between a different set of paralogs or by less frequent native homologous recombination .', 'We observed a similar situation in the sequence of the T . nautili 60G and 66G populations .', 'In several cases , homologous segments were in direct orientation in the original genome but became opposed due to a previous overlapping inversion therefore indicating that T . nautili 60G and 66G inversions occurred sequentially .', 'In order to investigate whether pTN3 could account for large-scale rearrangements in the Thermococcus genus , we examined by synteny analysis the distribution of pTN3-like integrated element among completely sequenced Thermococcales .', 'Out of 17 sequenced Thermococcus , and in addition to the previously reported T . kodakarensis TKV4 element [45] , five isolates were found to harbor a pTN3-related element ( Fig 9 ) .', 'The natural competence for DNA uptake of some Thermococcales such as T . kodakarensis [39] and the capacity of pTN3 to be transferred between cells using membrane vesicles [43] could explain the ubiquitous presence of this mobile element .', 'Protein sequence and structural comparisons between IntpTN3 and other hyperthermophilic archaeal integrases such as that of crenarchaeal virus SSV1 indicate that these proteins are clearly related .', 'However , IntpTN3 possesses several additional interspersed domains relative to SSV1 ( S2 and S12 Figs ) .', 'We surmise that these additional domains contribute to the low sequence specificity recombination reactions akin to homologous recombination events that we have observed .', 'By summing up all direct and indirect evidence reported here , it is very likely that the integrase encoded by pTN3-like plasmids can account for the genomic shuffling observed in the Thermococcus genus .', 'Plasmids of the pTN3 class are genetically closely related to viruses as they encode a capsid protein and a DNA packaging ATPase [43] but pTN3 virions have not be observed to date .', 'It is not clear at this stage whether plasmids or viruses equipped with an IntpTN3-like integrase have a better fitness either due to provirus maintenance or by virion spreading .', 'An integrase mimicking homologous recombination could promote viral integration into the host genome only if both viral and cellular chromosomes share significant DNA similarity .', 'This enzyme however , could facilitate integration of a virus into the genome of a closely related provirus .', 'The question arises whether an enzyme promoting genome shuffling using very short repeated segments as substrates , would be beneficial for a cellular organism .', 'On one hand , ‘wrongly’ recombined genomes would result in suboptimal gene expression programs and cells carrying scrambled genomes would display a reduced fitness and clearly be counter-selected in the population .', 'Interestingly , the presence of a pTN3-specific spacer in a T . nautili CRISPR locus strongly suggests that the presence of this plasmid is deleterious [41] .', 'On the other hand , it is also possible to envision situations where high-level genome shuffling by inversion could be advantageous .', 'Alternate gene expression patterns could increase , for instance , adaptation to rapid environmental changes .', 'In addition , for organisms such as Thermococcales where highly-expressed essential housekeeping genes maintain invariable positions [33] , genome scrambling could be beneficial by relocating “less desirable” integrated elements to chromosomal areas of reduced gene expression , therefore minimizing their impact on cellular physiology .'], ['Escherichia coli strain XL1-Blue was used for cloning , plasmid amplification and site-directed mutagenesis .', 'Overexpression of recombinant wild-type or mutant IntpTN3 was carried out in strain BL21 ( DE3 ) ( Novagen ) .', 'All E . coli strains were grown in Luria-Bertani medium supplemented with 100μg/mL ampicillin or/and 50μg/mL kanamycin when necessary .', 'T . kodakarensis KUW1 ( ΔpyrF ΔtrpE ) was grown anaerobically in ASW-YT medium [54] at 85°C .', 'Long term Thermococcus sub-culturing experiments were carried out in the same conditions by sequential 50x dilutions of stationary phase cultures into fresh media .', 'The number of generations was assessed statistically at each dilution step using a Thoma cell counting chamber under 400x magnification .', 'The plasmids used or constructed in this work are listed in S1 Table .', 'Transformation with pRC524 and pRC526 plasmids ( see below ) was performed following standard protocols [55] .', 'Plasmid-containing KUW1 strains were grown in ASW-CH medium [54] supplemented with uracil ( 10 μg/mL ) .', 'T . nautili sp .', '30–1 ( CP007264 ) was grown anaerobically at 85°C in Zillig’s broth [56] .', 'Genomic sequences were compared and aligned by dotplot analysis using Gepard [57] .', 'Conservation of gene order was assessed by synteny analysis using Absynte [58] and SyntTax [42] .', 'The original genome of Thermococcus 5–4 JCM31817 ( GenBank accession CP021848 ) and the genomes of sub-cultured T . nautili 60G and 66G and T . sp .', '5–4 36G and 66G were sequenced by Genoscope ( Centre National de Séquençage , France ) , using Illumina MiSeq .', 'Reads were assembled with Newbler ( release 2 . 9 ) and gap closure was performed by PCR , Sanger sequencing and Oxford Nanopore MinION .', 'The primary genomic sequences of rearranged T . nautili 60G , 66G and T . 5–4 36G , 66G are available in S1 , S2 , S3 and S4 Datasets , respectively .', 'These genomic sequences are compared by dotplot analysis in S7 Fig . Genomic regions corresponding to ~2000bp upstream and downstream of inversion break-points were extracted from both the ancestral T . nautili sequence , and the sub-cultured T . nautili 66G sequence .', 'Illumina sequencing reads were mapped to the ancestral sequence , and the pool of unmapped reads were mapped to the 66G sequence ( Geneious 6 . 1 . 8 ) .', 'Two positions close to the break-point which differ in base composition between ancestral and 66G sequences were chosen to classify reads as resulting from original or inverted genome sequences .', 'Bases were enumerated at these positions , and the percentage of reads corresponding to original sequences or inversions were calculated .', 'The prevalence of pTN3 in the population was determined by comparing read depth across the entire T . nautili 66G genome ( excluding the integrated pTN3 region ) to that of pTN3 ( S3 Table ) .', 'The gene encoding the integrase of the plasmid pTN3 of T . nautili 30–1 , ( gene ID: 17125032 ) was codon-optimized for expression in E . coli and synthesized by GenScript .', 'The synthetic gene contained a Strep-Tag at the 5’ end and was cloned into pET26b+ expression vector ( Novagen ) to yield pJO344 .', 'Plasmid pJO496 carrying the mutated IntpTN3Y428A was obtained by site directed mutagenesis of pJO344 with primers Int_A and Int_B ( S2 Table ) using the Agilent QuikChange Lightning Site-Directed Mutagenesis Kit .', 'Wild-type IntpTN3 and mutated In
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['One of the major mechanisms driving the evolution of all organisms is genomic rearrangement .', 'In hyperthermophilic Archaea of the order Thermococcales , large chromosomal inversions occur so frequently that even closely related genomes are difficult to align .', 'Clearly not resulting from the native homologous recombination machinery , the causative agent of these inversions has remained elusive .', 'We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements .', 'We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions , it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo , in contrast to other known tyrosine recombinases .', 'Through serial culturing , we showed that the integrase-mediated divergence of T . nautili strains occurs at an astonishing rate , with at least four large-scale genomic inversions appearing within 60 generations .', 'Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element .']
['Mobile elements ( MEs ) such as viruses , plasmids and transposons infect most living organisms and often encode recombinases promoting their insertion into cellular genomes .', 'These insertions alter the genome of their host according to two main mechanisms .', 'First , MEs provide new functions to the cell by integrating their own genetic information into the DNA of the host , at one or more locations .', 'Secondly , cellular homologous recombination will act upon multiple integrated copies and produce a variety of large-scale chromosomal rearrangements .', 'If such modifications are advantageous , they will spread into the population by natural selection .', 'Typically , enzymes involved in cellular homologous recombination and the integration of MEs are distinct .', 'We describe here a novel plasmid-encoded archaeal integrase which in addition to site-specific recombination can catalyze low sequence specificity recombination reactions akin to homologous recombination .']
['recombination reactions', 'enzymes', 'enzymology', 'plasmid construction', 'genome analysis', 'genetic elements', 'dna construction', 'dna', 'molecular biology techniques', 'homologous recombination', 'research and analysis methods', 'sequence analysis', 'genomics', 'sequence alignment', 'chromosome biology', 'proteins', 'recombinases', 'bioinformatics', 'chemistry', 'molecular biology', 'biochemistry', 'cell biology', 'nucleic acids', 'database and informatics methods', 'genetics', 'biology and life sciences', 'chemical reactions', 'physical sciences', 'dna recombination', 'mobile genetic elements', 'computational biology', 'chromosomes']