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

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.

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