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

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.

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. (10.1093/nar/gku194) where it was proposed that all archaeal VirB4 sequences arise from a transfer from bacteria within the MPF_FATA group. b) Re-creation of MPF_FATA and MPF_FA phylogeny including VirB4 sequences from integrated elements in other archaeal genomes, and the VirB4 homologue from pT33–3. Phylogeny rooted with MPF_F 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.

Fig. 1 |. Overview of pT33–3 and evidence for CRISPR spacer targeting.

a, Plasmid map of pT33–3, with predicted ORFs indicated by grey block arrows. virB4 and virD4 homologues highlighted in blue; potential origins of transfer highlighted in orange. CRISPR spacers of Thermococcales are indicated by black arrowheads on the interior. b, Phylogenetic tree of Thermococcales species used/described in this work. Phylogeny based on RpoB with Methanococcales as an outgroup. Number of CRISPR spacers targeting pT33–3 and ability to receive pT33–3 under laboratory conditions are indicated. ND, not determined.

Fig. 2 |. Interspecies transfer of pT33–3.

a, In bacterial CPs, the plasmid-encoded T4SS forms a pore bridging the cytoplasm of donor and recipient cells through which the CP is actively transferred. Phenotypes selected in this work are indicated. b, pT33–3 readily transfers from T. kodakarensis donor to species not naturally competent for DNA uptake. Individual points indicate conjugation efficiency in a single biological replicate. c, pT33–3 transfers from T. kodakarensis to T. nautili; non-conjugative shuttle vector (pLC70) does not, indicating that transfer ability is plasmid-encoded (not host-encoded). d, Transfer of pT33–3 to non-competent species requires homologues of known T4SS proteins, p0019 (VirB4 homologue) and p0132 (VirD4 homologue). Transfer also requires cell-to-cell contact, being blocked by a 0.2 μm filter.

Fig. 3 |. pT33–3 mediated mobilization of shuttle vectors.

a, T4SSs encoded by CPs mobilize other DNAs encoding their cognate origin of transfer (oriT). Phenotypes selected in this work are indicated. b, A shuttle vector encoding pT33–3 oriT (poriT2) is mobilized to non-competent donor species in the presence of pT33–3. c, Plasmid DNA isolated from transconjugants has identical HindIII digestion patterns to the that of the donor strain (T. kodakarensis). Representative data from a single experiment are shown, but identical results were obtained in 4 replicate experiments.

Fig. 4 |. pT33–3 mediated genome modification.

a, pT33–3 oriT allows mobilization of non-replicative DNA to recipient organisms. Here, homology arms allow allelic exchange with the chromosome, facilitating genetic modification of recipients. Phenotypes selected in this work are indicated. b, Deletion of pyrF from T. litoralis via pT33–3-mediated mobilization. Top: diagnostic PCR for deletion shows loss of wild-type (WT) gene and replacement with a pop-in/pop-out MevR cassette. Bottom: resulting strains form colonies on 5-FOA, as expected for a ΔpyrF genotype. c, Same as b but with a P. abyssi recipient. Growth curves show resistance to 5-FOA. d, S. marinus, a Crenarchaeon, receives DNA designed to replace the apt gene with a MevR cassette. Top: diagnostic PCR of knockout. Bottom: phylogenetic tree based on 16S ribosomal DNA, indicating phylogenetic distance between T. kodakarensis donor (Thermococci, labelled red), and S. marinus recipient (Desulfurococcales, labelled blue). Representative data from a single experiment are shown in b–d, but identical knockouts were obtained in 3 biological replicates.