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A self-transmissible plasmid from a hyperthermophile that facilitates genetic modification of diverse Archaea

Nature Microbiology volume 8, pages 1339–1347 (2023)Cite this article

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Abstract

Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated conjugation has been well-studied in bacteria, information is sparse in Archaea and known representatives exist only in the Sulfolobales order of Crenarchaeota. Here we present the first self-transmissible plasmid identified in a Euryarchaeon, Thermococcus sp. 33-3. The 103 kbp plasmid, pT33-3, is seen in CRISPR spacers throughout the Thermococcales order. We demonstrate that pT33-3 is a bona fide conjugative plasmid that requires cell-to-cell contact and is dependent on canonical, plasmid-encoded T4SS-like genes. Under laboratory conditions, pT33-3 transfers to various Thermococcales and transconjugants propagate at 100 °C. Using pT33-3, we developed a genetic toolkit that allows modification of phylogenetically diverse Archaeal genomes. We demonstrate pT33-3-mediated plasmid mobilization and subsequent targeted genome modification in previously untransformable Thermococcales species, and extend this process to interphylum transfer to a Crenarchaeon.

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Fig. 1: Overview of pT33-3 and evidence for CRISPR spacer targeting.
Fig. 2: Interspecies transfer of pT33-3.
Fig. 3: pT33-3 mediated mobilization of shuttle vectors.
Fig. 4: pT33-3 mediated genome modification.

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Data availability

The genome sequence for Thermococcus sp. 33-3 has been deposited with ENA (number GCA_946300405). Sequences of all plasmids, genomes and primers used in this study are available in Dryad at https://doi.org/10.5061/dryad.2jm63xst9.

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Acknowledgements

We thank N. Soler for advice while writing this manuscript. This project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no.772725, project HiPhore to P.F.), under the European Union’s Seventh Framework Program (grant agreement no. 340440, project EVOMOBIL to P.F.) and by the National Institutes of Health (R35GM118160 to M.T.).

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Authors and Affiliations

  1. Unité de Biologie Moléculaire du Gène chez les Extrêmophiles (BMGE), Département de Microbiologie, Institut Pasteur, Paris, France

    Ryan J. Catchpole, Patrick Forterre & Violette Da Cunha

  2. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris-Sud, Univ. Paris-Saclay, Gif-sur-Yvette, Cedex, France

    Ryan J. Catchpole, Evelyne Marguet, Jacques Oberto, Patrick Forterre & Violette Da Cunha

  3. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA

    Ryan J. Catchpole & Michael Terns

  4. Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, Evry, France

    Valérie Barbe, Ghislaine Magdelenat & Violette Da Cunha

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Contributions

R.J.C. and E.M. performed the experiments, V.B. and G.M. performed the sequencing, R.J.C., J.O. and V.D.C. performed bioinformatic analyses. M.T., P.F., J.O. and V.D.C. supervised aspects of the project and provided essential expert analysis. R.J.C. wrote the manuscript, with input from M.T., P.F., J.O. and V.D.C.

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Correspondence to Ryan J. Catchpole or Violette Da Cunha.

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Extended data

Extended Data Fig. 1 Similarity of T. sp. 33-3 to other species.

Genome alignment between Thermococcus. sp. 33-3 and closely related strains or species, T. nautili (a); T. henrietii EXT12c (b); and T. sp. 26-2 (c). Dots (which form into lines) indicate sequence identity between corresponding regions of the chromosome for the two indicated species. Discontinuities indicate indels; direction changes for example change from pointing north-east to south-east, indicate large-scale inversions.

Extended Data Fig. 2 Bioinformatic identification of pT33-3 origin of replication and transfer.

Plot of cumulative GC skew [(G-C)/(G + C)] and keto excess [(G + T)-(C + A)] for pT33-3. Sharp inflection points are often indicative of origins of replication, origins of transfer, or replication termination sites. These sites are indicated by vertical dashed lines.

Extended Data Fig. 3 Interference assays with CRISPR WT and null strains of T. kodakarensis.

Diamonds indicate data from a single biological replicate (for WT with target plasmid, overlapping points obscure data, n = 3). Wild-type (TS559) T. kodakarensis is unable to be transformed by plasmids encoding a sequence complementary to spacers in its CRISPR array (target plasmid). In contrast, non-target plasmids transform at ~100cfu/μg DNA. Deletion of genes encoding all CRISPR-associated (Cas) proteins abolishes this targeting activity, restoring transformation with target-encoding plasmids.

Extended Data Fig. 4 Deletion of genes homologous to bacterial T4SS.

PCR of knockouts for predicted transfer genes, p0019 and p0132. PCR was carried out using primers binding to pT33-3 outside the homology arms used in pop-in/pop-out recombination.

Extended Data Fig. 5 Transfer rates of genetic markers between T. kodakarensis strains does not require conjugation.

a) T. kodakarensis incubated with purified plasmid DNA (presented as transformants per fg DNA for comparable scale) - T. kodakarensis is naturally competent for DNA uptake. b) Transfer of a non-conjugative plasmid from a T. kodakarensis donor to a plasmid-free recipient – plasmids transfer between T. kodakarensis strains occurs by simple co-culturing. c) Transfer of a chromosomal prototrophic marker from a T. kodakarensis donor to an auxotrophic recipient - chromosomal markers transfer between T. kodakarensis strains. d) Transfer of a chromosomal prototrophic marker from a T. gammatolerans donor to an auxotrophic T. kodakarensis recipient - chromosomal markers are unable to transfer between T. gammatolerans and T. kodakarensis, suggesting allelic exchange is mediated by homologous recombination. e) Transfer of a chromosomal prototrophic marker from a T. kodakarensis donor to an auxotrophic recipient where the marker is encoded at a locus encoding a second prototrophic marker – transfer chromosomal markers requires a suitable receptive genomic site (non-essential). Individual biological replicates (-, n = 3) are presented with average and standard deviation indicated by error bars. g-i) In contrast to T. kodakarensis, T. nautili is unable to receive a shuttle vector by co-culturing, whereas pT33-3 readily transfers (see main text and Fig. 2c).

Extended Data Fig. 6 Identification of oriT-encoding region of pT33-3.

1.5 kb regions surrounding GC-skew/keto excess minima/maxima were cloned into a shuttle vector and transfer observed in the presence of pT33-3 from T. kodakarensis donors. a) oriT1 candidate region. While plasmids encoding this region readily transferred between T. kodakarensis strains, minimal transfer was observed to T. nautili and T. gammatolerans recipients. b) oriT2 candidate region. Plasmids encoding this region readily transferred from T. kodakarensis to T. kodakarensis, T. nautili, and T. gammatolerans, indicating this region encodes the oriT of pT33.3. c) oriT2 region was split into four overlapping fragments. Plasmids encoding both oriT2.1 and oriT2.2 regions transferred to the non-competent recipients, whereas oriT2.3 and oriT2.4 did not, indicating the oriT is encoded by the overlap region between oriT2.1 and oriT2.2. d) The 300 bp overlap between oriT2.1 and oriT2.2 (named oriT300) also confers mobilization ability to plasmids, indicating that this region encodes the oriT of pT33.3.

Extended Data Fig. 7 Phylogeny of VirB4.

a) Figure adapted from Figure 6 of Guglielmini et al.33 (10.1093/nar/gku194) where it was proposed that all archaeal VirB4 sequences arise from a transfer from bacteria within the MPFFATA group. b) Re-creation of MPFFATA and MPFFA phylogeny including VirB4 sequences from integrated elements in other archaeal genomes, and the VirB4 homologue from pT33-3. Phylogeny rooted with MPFF outgroup. pT33-3 VirB4 groups within a clade of Crenarchaeal sequences, suggesting pT33-3 arose from an inter-phylum transfer. Supported branches, computed by aLRT >80 or UFBoot >95, are indicated by dots at nodes (the full tree is provided as an extended data file). The scale bar indicates the average number of substitutions per site.

Extended Data Fig. 8 Strategy employed for allelic exchange in non-competent recipient cells.

a) Schematic diagram of mobilizable plasmid (pMob) encoding pT33-3 oriT sequences and homologous recombination + selectable marker cassette for allelic exchange. b) Transfer of pMob initiating at oriT1 and terminating at oriT2 results in transfer of a non-replicative DNA encoding recombination/marker cassette. This can be a substrate for homologous recombination and allelic exchange with the recipient chromosome. c) Transfer of pMob initiating at oriT2 and terminating at oriT1 results in transfer of a replicative DNA without any selectable marker. Transformant selection on drug-containing media renders these cells non-viable.

Extended Data Fig. 9 PCR screen of S. marinus colonies following conjugation mediated mobilisation of a recombination substrate.

Eight colonies were screened using primers binding to the S. marinus chromosome, outside the homology arms used in allelic exchange. Clean allelic exchange (Δapt::SimR) was observed in 2/8 colonies. Data from a representative replicate is shown, but similar data obtained on 3 occasions.

Supplementary information

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Supplementary Table

Bioinformatic prediction of pT33-3 ORF function. A combination of BLAST and HHblits was used to predict functions for each ORF in pT33-3. BLAST was performed against the non-redundant protein database. Hits with E-value < 0.01 are highlighted in yellow. HHblits was performed against the UniProt database; however, the majority of hits returned ‘hypothetical protein’ or ‘uncharacterized protein’. Thus, hits were further limited to those with functional annotations.

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Catchpole, R.J., Barbe, V., Magdelenat, G. et al. A self-transmissible plasmid from a hyperthermophile that facilitates genetic modification of diverse Archaea. Nat Microbiol 8, 1339–1347 (2023). https://doi.org/10.1038/s41564-023-01387-x

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