Autonomously replicating linear plasmids facilitate the analysis of replication origin function in Candida albicans

Abstract

The ability to generate autonomously replicating plasmids has been elusive in Candida albicans, a prevalent human fungal commensal and pathogen. Instead, plasmids generally integrate into the genome. Here, we assessed plasmid and transformant properties, including plasmid geometry, transformant colony size, four selectable markers, and potential origins of replication for their ability to drive autonomous plasmid maintenance. Importantly, linear plasmids with terminal telomere repeats yielded many more autonomous transformants than circular plasmids with the identical sequences. Furthermore, we could distinguish by colony size, transient, autonomously replicating and chromosomally integrated transformants (tiny, medium and large, respectively). Candida albicans URA3 and a heterologous marker, ARG4, yielded many transient transformants indicative of weak origin activity; replication of plasmid carrying heterologous LEU2 marker was highly dependent upon the addition of a bona fide origin sequence. Several bona fide chromosomal origins, with an origin fragment of ~100 bp as well as a heterologous origin, panARS, from Kluyveromyces lactis drove autonomous replication, yielding moderate transformation efficiency and plasmid stability. Thus, C. albicans maintains linear plasmids that yield high transformation efficiency and are maintained autonomously in an origin-dependent manner. ImportanceCircular plasmids are important tools for molecular manipulation in model fungi such as bakers yeast, yet, in Candida albicans, an important yeast pathogen of humans, prior studies were not able to generate circular plasmids that were autonomous (duplicated without inserting themselves into the chromosome). Here, we found that linearizing circular plasmids with sequences from telomeres, the chromosome ends, allows the plasmids to duplicate and segregate in C. albicans. We used this system to identify chromosomal sequences that facilitate the initiation of plasmid replication (origins) and to show that a ~100 bp fragment of a C. albicans origin, as well as an origin sequence from a distantly related yeast, can both function as origins in C. albicans. Thus, the requirements for plasmid geometry, but not necessarily for origin sequences, differ between C. albicans and bakers yeast.