Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in Candida...

Area of Science:

• Microbiology
• Molecular Biology
• Fungal Genetics

Background:

• Autonomous plasmid replication is crucial for genetic manipulation in fungi but has been challenging in *Candida albicans*.
• Previous attempts to create self-replicating plasmids in *C. albicans* primarily resulted in genomic integration, limiting their utility.
• Understanding plasmid maintenance mechanisms is essential for advancing genetic research and therapeutic strategies involving *C. albicans*.

Purpose of the Study:

• To investigate factors influencing autonomous plasmid maintenance in *Candida albicans*.
• To identify plasmid geometries and DNA sequences that promote stable, self-replicating plasmids.
• To characterize the role of replication origins in driving plasmid replication and stability.

Main Methods:

• Assessed properties of circular and linear plasmids, including their geometry and the presence of telomere repeats.
• Evaluated the efficacy of four selectable markers and various potential replication origins.
• Differentiated transformants based on colony size to distinguish between transient, autonomous, and integrated plasmids.

Main Results:

• Linear plasmids with telomere repeats significantly outperformed circular plasmids in generating autonomous transformants.
• Transformant colony size reliably indicated plasmid status: tiny for transient, medium for autonomous, and large for integrated.
• Both a ~100 bp fragment of a *C. albicans* chromosomal origin and a heterologous origin (*panARS*) from *Kluyveromyces lactis* supported autonomous replication.

Conclusions:

• Linear plasmid geometry, particularly with terminal telomere repeats, is key to achieving high transformation efficiency and autonomous maintenance in *C. albicans*.
• Autonomous replication is origin-dependent, with specific chromosomal and heterologous origins effectively driving plasmid maintenance.
• This study establishes a robust system for generating and maintaining linear, autonomously replicating plasmids in *C. albicans*, facilitating future genetic studies and applications.