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

A conjugative plasmid isolated from Thermococcus enables interspecies transfer across Archaea, whether naturally competent or not, and at temperatures up to 100 degrees C. 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 degrees 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.

Automated summary

A conjugative plasmid isolated from Thermococcus enables the transfer of DNA between Archaea, regardless of their natural competence or the temperature, through type IV secretion systems. This is the first self-transmissible plasmid identified in a Euryarchaeon and it is also found in other Thermococcales species. The plasmid is a bona fide conjugative plasmid that requires cell-to-cell contact and is dependent on plasmid-encoded type IV secretion system-like genes. It can transfer to various Thermococcales species and propagate at temperatures of up to 100 degrees C. It has also been used to develop a genetic toolkit for modifying Archaeal genomes and has allowed targeted genome modification in previously untransformable Thermococcales species and interphylum transfer to a Crenarchaeon.