Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>

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, transfor...

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Autores principales:	Swati Bijlani, Mathuravani A. Thevandavakkam, Hung-Ji Tsai, Judith Berman
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CaURA3 linear plasmids replication replication origins telomere repeats Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/be10dce569c14a3bb901c608f45e0ae6
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spelling	oai:doaj.org-article:be10dce569c14a3bb901c608f45e0ae62021-11-15T15:22:22ZAutonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>10.1128/mSphere.00103-192379-5042https://doaj.org/article/be10dce569c14a3bb901c608f45e0ae62019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00103-19https://doaj.org/toc/2379-5042ABSTRACT 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; the replication of the plasmid carrying the 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. IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s 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 an ∼100-bp fragment of a C. albicans origin and 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 baker’s yeast.Swati BijlaniMathuravani A. ThevandavakkamHung-Ji TsaiJudith BermanAmerican Society for MicrobiologyarticleCaURA3linear plasmidsreplicationreplication originstelomere repeatsMicrobiologyQR1-502ENmSphere, Vol 4, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CaURA3 linear plasmids replication replication origins telomere repeats Microbiology QR1-502
spellingShingle	CaURA3 linear plasmids replication replication origins telomere repeats Microbiology QR1-502 Swati Bijlani Mathuravani A. Thevandavakkam Hung-Ji Tsai Judith Berman Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
description	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; the replication of the plasmid carrying the 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. IMPORTANCE Circular plasmids are important tools for molecular manipulation in model fungi such as baker’s 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 an ∼100-bp fragment of a C. albicans origin and 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 baker’s yeast.
format	article
author	Swati Bijlani Mathuravani A. Thevandavakkam Hung-Ji Tsai Judith Berman
author_facet	Swati Bijlani Mathuravani A. Thevandavakkam Hung-Ji Tsai Judith Berman
author_sort	Swati Bijlani
title	Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
title_short	Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
title_full	Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
title_fullStr	Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
title_full_unstemmed	Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>
title_sort	autonomously replicating linear plasmids that facilitate the analysis of replication origin function in <italic toggle="yes">candida albicans</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/be10dce569c14a3bb901c608f45e0ae6
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Autonomously Replicating Linear Plasmids That Facilitate the Analysis of Replication Origin Function in <italic toggle="yes">Candida albicans</italic>

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