Advertisement

Chemical Transformation of Candida albicans

  • Sophie Bachellier-Bassi
  • Christophe d’Enfert
Chapter
  • 2.3k Downloads
Part of the Fungal Biology book series (FUNGBIO)

Abstract

Genetic transformation is an essential technique for studying gene function in the yeast Candida albicans. Although less efficient than spheroplasting- or electroporation-techniques, the lithium acetate-based protocol described below is easy to perform, requires basic lab equipment and reliably yields good number of the expected transformants. This method can also be adapted for high-throughput analysis purposes.

Keywords

Candida albicans Transformation Integration Lithium acetate Heat-shock 

Notes

Acknowledgement

We wish to thank Anne Neville and Mélanie Legrand for critical reading of the manuscript.

References

  1. Basso L, Bartiss A, Mao Y, Gast C, Coelho P, Snyder M, Wong B (2010) Transformation of Candida albicans with a synthetic hygromycin B resistance gene. Yeast 27:1039–1048PubMedCrossRefGoogle Scholar
  2. Brand A, MacCallum D, Brown A, Gow N, Odds F (2004) Ectopic expression of URA3 can influence the virulence phenotypes and proteome of Candida albicans but can be overcome by targeted reintegration of URA3 at the RPS10 locus. Eukaryot Cell 3:900–909PubMedCrossRefPubMedCentralGoogle Scholar
  3. Cabral V, Chauvel M, Firon A, Legrand M, Nesseir A, Bachellier-Bassi S, Chaudhari Y, Munro C, d’Enfert C (2012) Modular gene over-expression strategies for Candida albicans. Methods Mol Biol 845:227–244PubMedCrossRefGoogle Scholar
  4. Chauvel M, Nesseir A, Cabral V, Znaidi S, Goyard S, Bachellier-Bassi S, Firon A, Legrand M, Diogo D, Naulleau C, Rossignol T, d’Enfert C (2012) A versatile overexpression strategy in the pathogenic yeast Candida albicans: identification of regulators of morphogenesis and fitness. PLoS One 7:e45912PubMedCrossRefPubMedCentralGoogle Scholar
  5. De Backer MD, Maes D, Vandoninck S, Logghe M, Contreras R, Luyten WHML (1999) Transformation of Candida albicans by electroporation. Yeast 15:1609–1618PubMedCrossRefGoogle Scholar
  6. Delorme E (1989) Transformation of Saccharomyces cerevisiae by electroporation. Appl Environ Microbiol 55:2242–2246PubMedPubMedCentralGoogle Scholar
  7. Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728PubMedPubMedCentralGoogle Scholar
  8. Gerami-Nejad M, Zacchi L, McClellan M, Matter K, Berman J (2013) Shuttle vectors for facile gap repair cloning and integration into a neutral locus in Candida albicans. Microbiology 159:569–579CrossRefGoogle Scholar
  9. Gietz R, Schiestl R, Willems A, Woods R (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360PubMedCrossRefGoogle Scholar
  10. Hickman MA, Zeng G, Forche A, Hirakawa MP, Abbey D, Harrison DM, Wang Y-MM, Su C-H, Bennett RJ, Wang Y, Berman J (2013) The ‘obligate diploid’ Candida albicans forms mating-competent haploids. Nature 494:55–59PubMedCrossRefPubMedCentralGoogle Scholar
  11. Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedPubMedCentralGoogle Scholar
  12. Kawai S, Pham T, Nguyen H, Nankai H, Utsumi T, Fukuda Y, Murata K (2004) Molecular insights on DNA delivery into Saccharomyces cerevisiae. Biochem Biophys Res Commun 317:100–107PubMedCrossRefGoogle Scholar
  13. Köhler GA, White TC, Agabian N (1997) Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid. J Bacteriol 179:2331–2338PubMedPubMedCentralGoogle Scholar
  14. Kurtz MB, Cortelyou MW, Miller SM, Lai M, Kirsch DR (1987) Development of autonomously replicating plasmids for Candida albicans. Mol Cell Biol 7:209–217PubMedPubMedCentralGoogle Scholar
  15. Mitrikeski PT (2013) Yeast competence for exogenous DNA uptake: towards understanding its genetic component. Antonie Van Leeuwenhoek 103:1181–1207PubMedCrossRefGoogle Scholar
  16. Murad AMA, Lee PR, Broadbent ID, Barelle CJ, Brown AJP (2000) CIp10, an efficient and convenient integrating vector for Candida albicans. Yeast 16:325–327PubMedCrossRefGoogle Scholar
  17. Noble S, Johnson A (2005) Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans. Eukaryot Cell 4:298–309PubMedCrossRefPubMedCentralGoogle Scholar
  18. Noble S, Johnson A (2007) Genetics of Candida albicans, a diploid human fungal pathogen. Annu Rev Genet 41:193–211PubMedCrossRefGoogle Scholar
  19. Pham T, Kawai S, Kono E, Murata K (2011) The role of cell wall revealed by the visualization of Saccharomyces cerevisiae transformation. Curr Microbiol 62:956–961PubMedCrossRefGoogle Scholar
  20. Pla J, Pérez-Díaz R, Navarro-García F, Sánchez 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 165:115–120PubMedCrossRefGoogle Scholar
  21. Prelich G (2012) Gene overexpression: uses, mechanisms, and interpretation. Genetics 190:841–854PubMedCrossRefPubMedCentralGoogle Scholar
  22. Reuß O, Vik A, Kolter R, Morschhäuser J (2004) The SAT1 flipper, an optimized tool for gene disruption in Candida albicans. Gene 341:119–127PubMedCrossRefGoogle Scholar
  23. Schiestl R, Gietz R (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346PubMedCrossRefGoogle Scholar
  24. Selmecki A, Bergmann S, Berman J (2005) Comparative genome hybridization reveals widespread aneuploidy in Candida albicans laboratory strains. Mol Microbiol 55:1553–1565PubMedCrossRefGoogle Scholar
  25. Shen J, Guo W, Köhler J (2005) CaNAT1, a heterologous dominant selectable marker for transformation of Candida albicans and other pathogenic Candida species. Infect Immun 73:1239–1242PubMedCrossRefPubMedCentralGoogle Scholar
  26. Staab JF, Sundstrom P (2003) URA3 as a selectable marker for disruption and virulence assessment of Candida albicans genes. Trends Microbiol 11:69–73PubMedCrossRefGoogle Scholar
  27. 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 14:565–571PubMedCrossRefGoogle Scholar
  28. Tsuchiya E, Shakuto S, Miyakawa T, Fukui S (1988) Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei. J Bacteriol 170:547–551PubMedPubMedCentralGoogle Scholar
  29. Walther A, Wendland J (2003) An improved transformation protocol for the human fungal pathogen Candida albicans. Curr Genet 42:339–343PubMedCrossRefGoogle Scholar
  30. Walther A, Wendland J (2008) PCR-based gene targeting in Candida albicans. Nat Protoc 3:1414–1421PubMedCrossRefGoogle Scholar
  31. Wilson R, Davis D, Mitchell A (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181:1868–1874PubMedPubMedCentralGoogle Scholar
  32. Wirsching S, Michel S, Morschhäuser J (2000) Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates. Mol Microbiol 36:856–865PubMedCrossRefGoogle Scholar
  33. Zheng H-Z, Liu H-H, Chen S-X, Lu Z-X, Zhang Z-L, Pang D-W, Xie Z-X, Shen P (2005) Yeast transformation process studied by fluorescence labeling technique. Bioconjug Chem 16:250–254PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Sophie Bachellier-Bassi
    • 1
  • Christophe d’Enfert
    • 1
  1. 1.Fungal Biology and Pathogenicity UnitInstitut PasteurParisFrance

Personalised recommendations