Flipping chromosomes in deep-sea archaea

Abstract

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.

Fig 1. Genomic dotplots and synteny analysis.

Genomic dotplots (A) between T. kodakarensis and T. nautili and (B) between T. onnurineus and T. sp. 4557. All genomes are centered on their putative predicted origin of replication [33]. C. The two synteny breaks in the genomic alignment between T. onnurineus and T. sp. 4557 (Panel B) were further analyzed. Gene order conservation and recombination endpoints of the two major inversions were identified using composite images generated by the SyntTax web tool. Inversion “1” occurred between tRNA^Leu (GQS_t10759) and tRNA^Thr (GQS_t10745) genes; T. sp. 4557 GQS_t10759 gene is orthologous to the T. nautili tRNA^Leu gene (BD01_0018) which corresponds to the chromosomal attachment site of plasmid pTN3. Inversion “2” (Panel B) occurred between tRNA^Leu (GQS_t10807) and tRNA^Gly (GQS_t10803) genes.

Fig 2. Dimer formation.

Supercoiled (SC) plasmids pUC18 and pJO322 carrying the Leu2-88 fragment (S5A Fig) were incubated with Int^pTN3 in a standard reaction (see Materials and methods) and compared with linearized pJO322 by agarose gel electrophoresis. The integrase has no effect on pUC18 with the exception of the production of a faint linear species (indicated by an arrow). The integrase increases considerably the formation of plasmid pJO322 dimers and to a lower extent that of multimers. No increase in the formation of open circular (OC) form was observed.

Fig 3. Int^ptn3 excision and integration.

Plasmid pMC479 carries two copies of tRNA^Leu cloned in direct orientation and separated by a 762bp spacer fragment (see Material and methods). The direct repeats consist of the minimal tRNA^Leu 2–44 and the longer tRNA^Leu 2–88, both proficient in dimerization reactions. Plasmid pMC479 was incubated with Int^pTN3 in a standard reaction (see Materials and methods). The NdeI restriction enzyme generates two fragments of 3207 and 1366bp respectively in pMC479. Upon incubation with Int^pTN3, NdeI digestion generates additional fragments of 2358bp corresponding to recombined pMC479* and 849bp corresponding to the circularized spacer and recombined att site. Both constitute the products of the excision reaction. A larger 4056bp fragment is generated as well and corresponds to the recombination product generated by integration of the 3207 and 849bp species. The relative intensity of the bands is compatible with an expected equilibrium reaction.

Fig 4. TKV4 excision in vitro and in vivo.

A PCR amplification assay was designed to assert artificial Int^pTN3-mediated TKV4 circularization (Panel A). The assay was first performed in vitro on four samples of purified T. kodakarensis genomic DNA incubated with wild type Int^pTN3 or inactive Int^pTN3 Y428A mutated enzyme in a standard reaction analyzed by agarose gel electrophoresis (see Materials and methods). Only reactions using wild-type enzyme generated a 1710bp band of the expected excision size (Panel B). The same TKV4 excision reaction was tested in vivo by transforming T. kodakarensis KUW1 with shuttle plasmids pRC524 (expressing wild type integrase) and pRC526 (expressing mutated Int^pTN3Y428A) or with the vector alone (Panel C). Total DNA was extracted from the transformants and amplified as described above. In this in vivo experiment, both enzymes were TKV4 excision-proficient (Panel D).

Fig 5. Int^ptn3 inversion.

Plasmid pMC477 carries two copies of tRNA^Leu cloned in inverted orientation and separated by a 892bp spacer fragment (see Material and methods). The inverted repeats consist of the minimal tRNA^Leu 2–44 and the longer tRNA^Leu 2–88, both proficient in dimerization reactions. Plasmid pMC473 carries tRNA^Leu 2–44 and tRNA^Thr GQS_t10745, in inverted orientation as well. Both plasmids were incubated with Int^pTN3 in a standard reaction (see Materials and methods). The NdeI restriction enzyme generates in each case two fragments of 2796 and 1777bp. Upon incubation with Int^pTN3, NdeI digestion of pMC477 generates additional fragments of 2358 and 2215bp corresponding to the recombinant pMC477*. As for the integration/excision reactions, the relative intensity of the bands is compatible with an expected equilibrium reaction. We could not detect any inversion between tRNA^Leu and tRNA^Thr in plasmid pMC473.

Fig 6. Laboratory inversions events.

A. Dotplot analysis of the original isolate of T. nautili (GenBank accession NZ_CP007264) and the same organism after 66 generations (S2 Dataset). B. Dotplot analysis of the original isolate of T. 5–4 (GenBank accession CP021848) and the same organism after 66 generations (S4 Dataset). C. One of the possible sequential inversion scenarios leading to T. nautili 66G (Panel A), drawn to scale. The arrows direction reflects the chromosomal segment orientation in the original T. nautili strain. Genomic coordinates are indicated and the identifiers of the genes bordering each inversion are boxed.

Fig 7. Int^pTN3-promoted low sequence specificity reactions on archaeal sequences.

Int^pTN3 catalyzes inversion on linear DNA substrates between archaeal gene pairs separated by a Kanamycin resistance determinant. White arrowheads refer to original fragments and black arrowheads indicate inversions products. A. Inversion between two identical copies of tRNA^leu gene GQS_t10759 from T. sp. 4557. B. Inversion between tRNA^Gly genes BD01_1557 and BD01_1976 from T. nautili. C. Inversion between chemotaxis genes BD01_1166 and BD01_1584 from T. nautili. Int^pTN3 concentration multipliers refer to the standard assay described in Materials and Methods. The detailed DNA sequences involved in these reactions are illustrated in S8 Fig.

Fig 8. Int^pTN3-promoted low sequence specificity reactions on exogenous sequences.

A. Low sequence specificity reactions mimicking homologous DNA integration are visualized by the accumulation of multimers of increasing size only when the reaction occurs in the presence of wild-type Int^pTN3. A linear pBR322 species generated by Int^pTN3-generated double-strand cleavage is visible and migrates close to a control plasmid digested by the EcoRI endocnuclease. OC and SC refer to the open circle and supercoiled DNA forms, respectively. B. Int^pTN3 catalyzes inversion on linear DNA substrates between two inverted E. coli lacZ gene segments of varying sizes separated by a Kanamycin resistance determinant. The sequence identity between the inverted segment amounts to 250, 175 and 100bp respectively in plasmids pCB574, pCB572 and pCB538 (see Materials and methods). White arrowheads refer to original fragments and black arrowheads indicate inversions products. Int^pTN3 concentration multipliers refer to the standard assay described in Materials and Methods.

Fig 9. pTN3-like integrated elements in Thermococcales.

The presence of pTN3-like integrated elements was investigated in all completely sequenced Thermococcales genomes by synteny analysis using the SyntTax web server [42]. In addition to T. nautili, the genomes of T. guaymasensis DSM11113, T. eurythermalis A501, T. kodakarensis KOD1, T. barophilus CH5, and T. cleftensis CL1 carry an extensive genomic region corresponding to plasmid pTN3 shown on top. Each arrow corresponds to an individual gene numbered according to GenBank annotations. The consistent gene color code illustrates orthology across organisms while white color indicates its absence. As indicated by a blue dotted line, conservation of synteny is clearly visible on the right border and limited by the gene encoding pTN3 C-ter integrase and its remnants. Truncated N-terminal-encoding integrase genes constitute pseudogenes lacking a stop codon and are therefore not annotated. Genetic divergence appears stronger on the left border.