Cytosolic linear DNA plasmids inSaccharomycopsisspecies

Abstract

Some budding yeast species contain cytosolic linear DNA plasmids (also called virus-like elements, VLEs) that code for killer toxins that can kill other yeasts. The toxins are anticodon nucleases that cleave a specific tRNA in the cells being attacked, stopping translation. The best known plasmids of this type are the pGKL1/pGKL2 system of Kluyveromyces lactis. pGKL1 is a killer plasmid encoding the toxin zymocin (gamma-toxin) which cleaves tRNA-Glu, and pGKL2 is a helper plasmid required for replication and transcription of pGKL1. Here, we investigated similar plasmids in the genus Saccharomycopsis that were originally described in the 1980s. Saccharomycopsis has undergone an evolutionary change of its genetic code, from CUG-Leu to CUG-Ser translation, which we hypothesized could have been driven by a tRNA-cleaving toxin encoded by a cytosolic plasmid. We sequenced a three-plasmid system in S. crataegensis, consisting of apparent killer, immunity, and helper plasmids. The killer plasmid contains genes coding for putative alpha/beta (chitin-binding) and gamma (ribonuclease) toxin subunits, but the gamma-toxin gene is damaged in all the isolates we examined. We inferred the sequence of the intact S. crataegensis gamma-toxin and expressed it in Saccharomyces cerevisiae and Kluyveromyces marxianus, but it did not cause a growth defect. We also identified free plasmids, or plasmids integrated into the nuclear genome, in nine other Saccharomycopsis species, including a case of recent interspecies transfer of a plasmid. Our results show that many yeasts in the CUG-Ser2 clade contain, or have in the past contained, plasmids related to those that carry anticodon nucleases.