Research Article | Open Access

Volume 2022 |Article ID 9802168 | https://doi.org/10.34133/2022/9802168

Superior Conjugative Plasmids Delivered by Bacteria to Diverse Fungi

Ryan R. Cochrane,¹ Arina Shrestha,¹ Mariana M. Severo de Almeida,¹ Michelle Agyare-Tabbi,² Stephanie L. Brumwell,¹ Samir Hamadache,¹ Jordyn S. Meaney,¹ Daniel P. Nucifora,¹ Henry Heng Say,¹ Jehoshua Sharma,² Maximillian P. M. Soltysiak,³ Cheryl Tong,¹ Katherine Van Belois,³ Emma J. L. Walker,¹ Marc-André Lachance,³ Gregory B. Gloor,¹ David R. Edgell,¹ Rebecca S. Shapiro,² and Bogumil J. Karas iD ¹

¹Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada N6A 5C1
²Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
³Department of Biology, The University of Western Ontario, London, Ontario, Canada N6A 5B7

Abstract

Fungi are nature’s recyclers, allowing for ecological nutrient cycling and, in turn, the continuation of life on Earth. Some fungi inhabit the human microbiome where they can provide health benefits, while others are opportunistic pathogens that can cause disease. Yeasts, members of the fungal kingdom, have been domesticated by humans for the production of beer, bread, and, recently, medicine and chemicals. Still, the great untapped potential exists within the diverse fungal kingdom. However, many yeasts are intractable, preventing their use in biotechnology or in the development of novel treatments for pathogenic fungi. Therefore, as a first step for the domestication of new fungi, an efficient DNA delivery method needs to be developed. Here, we report the creation of superior conjugative plasmids and demonstrate their transfer via conjugation from bacteria to 7 diverse yeast species including the emerging pathogen Candida auris. To create our superior plasmids, derivatives of the 57 kb conjugative plasmid pTA-Mob 2.0 were built using designed gene deletions and insertions, as well as some unintentional mutations. Specifically, a cluster mutation in the promoter of the conjugative gene traJ had the most significant effect on improving conjugation to yeasts. In addition, we created Golden Gate assembly-compatible plasmid derivatives that allow for the generation of custom plasmids to enable the rapid insertion of designer genetic cassettes. Finally, we demonstrated that designer conjugative plasmids harboring engineered restriction endonucleases can be used as a novel antifungal agent, with important applications for the development of next-generation antifungal therapeutics.