Abstract.
Commonly used protocols for the transformation of the dimorphic human fungal pathogen Candida albicans rely on established methods for the yeast Saccharomyces cerevisiae. With respect to transformation efficiency, however, there is a great difference between these two organisms when using the lithium acetate procedure. Here we present a modified version of this protocol for use with C. albicans. Among the different parameters tested, two turned out to be particularly relevant and, when combined, resulted in an up to 10-fold increase in transformation efficiency (400–500 integrative transformants) compared with previous protocols: first, adjusting the heat shock applied to the cells to 44 °C for C. albicans instead of 42 °C for S. cerevisiae and, second, treating C. albicans cells with lithium acetate in an overnight incubation instead of for 30 min as used for S. cerevisiae. With these modifications, the lithium acetate procedure becomes a very efficient and reliable tool for C. albicans transformation.
Similar content being viewed by others
References
Bird D, Bradshaw R (1997) Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 255:219–225
Braun BR, Johnson AD (1997) Control of filament formation in Candida albicans by the transcriptional repressor TUP1. Science 4277:105–109
Cannon RD, Jenkinson HF, Sheperd MG (1990) Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae. Mol Gen Genet 221:210–218
De Backer MD, Maes D, Vandonick S, Logghe M, Contreras R, Luyten WH (1999) Transformation of Candida albicans by electroporation. Yeast 15:1609–1618
Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728
Gerami-Nejad M, Berman J, Gale CA (2001) Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans. Yeast 18:859–864
Gietz RD, Woods RA (2001) Genetic transformation of yeast. BioTechniques 30:816–831
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360
Herreros E, Garcia-Saez MI, Nombela C, Sanchez M (1992) A reorganized Candida albicans DNA sequence promoting homologous non-integrative genetic transformation. Mol Microbiol 6:3567–3574
Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci USA 75:1929–1933
Hull CM, Raisner RM, Johnson AD (2000) Evidence for mating of the "asexual" yeast Candida albicans in a mammalian host. Science 289:307–310
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168
Magee BB, Magee PT (2000) Induction of mating in Candida albicans by construction of MTLa and MTLalpha strains. Science 289:310–313
Pla J, Perez-Diaz RM, Navarro-Garcia F, Sanchez M, Nombela C (1995) Cloning of the Candida albicans HIS1 gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector. Gene 165:115–120
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Sanglard D, Ischer F, Monod M, Bille J (1996) Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 40:2300–2305
Thompson JR, Register E, Curotto J, Kurtz M, Kelly R (1998) An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast 14:565–571
Wendland J, Ayad-Durieux Y, Knechtle P, Rebischung C, Philippsen P (2000) PCR-based gene targeting in the filamentous fungus Ashbya gossypii. Gene 242:381–391
Wilson RB, Davis D, Mitchell AP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181:1868–1874
Wright MC, Philippsen P (1991) Replicative transformation of the filamentous fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS elements. Gene 109:99–105
Yehuda H, Droby S, Wisniewski M, Goldway M (2001) A transformation system for the biocontrol yeast, Candida oleophila, based on hygromycin B resistance. Curr Genet 40:282–287
Acknowledgement.
The authors wish to thank Erika Kothe for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by J. Heitman
Rights and permissions
About this article
Cite this article
Walther, A., Wendland, J. An improved transformation protocol for the human fungal pathogen Candida albicans . Curr Genet 42, 339–343 (2003). https://doi.org/10.1007/s00294-002-0349-0
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-002-0349-0