Conjugative plasmids (CP) are self-transmissible mobile genetic elements which 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 upon 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 which 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.

Illumina sequencing of DNA isolated from cultures, or Sanger sequencing of plasmid DNA where appropriate.

Sequences are provided in fasta format for use with any text editor or DNA viewing software.