The SAT1 flipper, an optimized tool for gene disruption in Candida albicans - PubMed (original) (raw)
The SAT1 flipper, an optimized tool for gene disruption in Candida albicans
Oliver Reuss et al. Gene. 2004.
Abstract
The construction of Candida albicans mutants by targeted gene disruption usually depends on the use of nutritional markers for the selection of prototrophic transformants from auxotrophic host strains, but it is becoming increasingly evident that this strategy may cause difficulties in the interpretation of mutant phenotypes. Here, we describe a new method for inactivating both alleles of a target gene in C. albicans wild-type strains to obtain homozygous null mutants. The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants and a C. albicans-adapted FLP gene that allows the subsequent excision of the cassette, which is flanked by FLP target sequences, from the genome. Two rounds of integration/excision generate homozygous mutants that differ from the wild-type parent strain only by the absence of the target gene, and reintegration of an intact gene copy for complementation of mutant phenotypes is performed in the same way. Transformants are obtained after only 1 day of growth on a selective medium, and integration into the target locus occurs with high specificity after adding homologous flanking sequences on both sides of the cassette. FLP-mediated excision of the SAT1 flipper cassette is achieved by simply growing the transformants for a few hours in medium without selective pressure, and nourseothricin-sensitive (NouS) derivatives can easily be identified by their slower growth on indicator plates containing a low concentration of nourseothricin. We demonstrate the use of the system by deleting the OPT1 gene, which encodes an oligopeptide transporter, in the C. albicans model strain SC5314. The null mutants became resistant to the toxic peptide KLLEth, and reintroduction of an intact OPT1 copy restored susceptibility. The SAT1 flipping method provides a highly efficient method for gene disruption in C. albicans wild-type strains, which eliminates currently encountered problems in the genetic analysis of this important human fungal pathogen.
Similar articles
- Gene deletion in Candida albicans wild-type strains using the SAT1-flipping strategy.
Sasse C, Morschhäuser J. Sasse C, et al. Methods Mol Biol. 2012;845:3-17. doi: 10.1007/978-1-61779-539-8_1. Methods Mol Biol. 2012. PMID: 22328364 - A family of oligopeptide transporters is required for growth of Candida albicans on proteins.
Reuss O, Morschhäuser J. Reuss O, et al. Mol Microbiol. 2006 May;60(3):795-812. doi: 10.1111/j.1365-2958.2006.05136.x. Mol Microbiol. 2006. PMID: 16629678 - Isogenic strain construction and gene targeting in Candida dubliniensis.
Staib P, Moran GP, Sullivan DJ, Coleman DC, Morschhäuser J. Staib P, et al. J Bacteriol. 2001 May;183(9):2859-65. doi: 10.1128/JB.183.9.2859-2865.2001. J Bacteriol. 2001. PMID: 11292806 Free PMC article. - Protoplasts fusion hybrids from Candida albicans morphological mutants.
Nombela C, Pomés R, Gil C. Nombela C, et al. Crit Rev Microbiol. 1987;15(1):79-85. doi: 10.3109/10408418709104450. Crit Rev Microbiol. 1987. PMID: 3319424 Review. - Natural auxotrophic heterozygosity in Candida albicans.
Poulter RT. Poulter RT. Crit Rev Microbiol. 1987;15(1):97-101. doi: 10.3109/10408418709104452. Crit Rev Microbiol. 1987. PMID: 3319426 Review.
Cited by
- An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.
Ishii M, Yamada T, Ohata S. Ishii M, et al. AMB Express. 2024 Aug 31;14(1):96. doi: 10.1186/s13568-024-01755-8. AMB Express. 2024. PMID: 39215862 Free PMC article. - Candida tropicalis PMT2 Is a Dispensable Gene for Viability but Required for Proper Interaction with the Host.
Hernández-Chávez MJ, Martínez-Duncker I, Clavijo-Giraldo DM, López-Ramirez LA, Mora-Montes HM. Hernández-Chávez MJ, et al. J Fungi (Basel). 2024 Jul 20;10(7):502. doi: 10.3390/jof10070502. J Fungi (Basel). 2024. PMID: 39057387 Free PMC article. - Development of a Shuttle Vector That Transforms at High Frequency for the Emerging Human Fungal Pathogen: Candida auris.
Determann B 2nd, Fu J, Wickes BL. Determann B 2nd, et al. J Fungi (Basel). 2024 Jul 11;10(7):477. doi: 10.3390/jof10070477. J Fungi (Basel). 2024. PMID: 39057362 Free PMC article. - A fungal metabolic regulator underlies infectious synergism during Candida albicans-Staphylococcus aureus intra-abdominal co-infection.
Paul S, Todd OA, Eichelberger KR, Tkaczyk C, Sellman BR, Noverr MC, Cassat JE, Fidel PL Jr, Peters BM. Paul S, et al. Nat Commun. 2024 Jul 9;15(1):5746. doi: 10.1038/s41467-024-50058-w. Nat Commun. 2024. PMID: 38982056 Free PMC article. - Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.
Ramírez-Zavala B, Hoffmann A, Krüger I, Schwanfelder S, Barker KS, Rogers PD, Morschhäuser J. Ramírez-Zavala B, et al. mSphere. 2024 Jul 30;9(7):e0038824. doi: 10.1128/msphere.00388-24. Epub 2024 Jun 28. mSphere. 2024. PMID: 38940507 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials