pSB3, a member of the 2-micron family of plasmids, replicates and is efficiently partitioned in multiple budding yeast species.

The plasmids pSB3 of Zygosaccharomyces rouxii and the 2-micron circle of Saccharomyces cerevisiae belong to a small family of multi-copy yeast DNA plasmids. Members are similarly organized, with each encoding a conserved version of Flp, the recombinase required for copy number amplification. In contrast, proteins and loci required for partitioning seemed to differ. For the 2-micron circle, partitioning involves association of plasmid proteins Rep1 and Rep2 with each other and with the plasmid STB locus. Here, updated sequencing revealed all members encode a Rep1 protein more conserved than previously reported with pSB3 Rep1 the largest due to a long internal non-conserved region. pSB3 partitioning protein C, despite not resembling Rep2, was found to associate with pSB3 Rep1  in vivo, with amino-terminal domains of each sufficient for this interaction. Plasmid inheritance assays identified a region upstream of the REP1 gene as the pSB3 partitioning locus (PAR) and showed pSB3 could replicate and partition in several budding yeast species including Lachancea waltii, S. cerevisiae, Torulaspora delbrueckii, and Zygosaccharomyces bailii. In one-hybrid protein-DNA interaction assays, pSB3 Rep1 and C were found to require each other for association with pSB3 PAR and the plasmid gene promoters and did not recognize 2-micron STB. Taken together, these results support the mechanism of partitioning and regulation of plasmid gene expression by partitioning proteins being conserved between pSB3 and 2-micron. Furthermore, molecular tools developed in this study provide the basis for developing new plasmid vectors based on pSB3 that can be effectively inherited in multiple budding yeast species.