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Eukaryot Cell. 2005 Aug;4(8):1328-42.

Tetracycline-inducible gene expression and gene deletion in Candida albicans.

Author information

1
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.

Abstract

The genetic analysis of Candida albicans, the major fungal pathogen of humans, is hampered by its diploid genome, the absence of a normal sexual cycle, and a nonstandard codon usage. Although effective methods to study gene function have been developed in the past years, systems to control gene expression in C. albicans are limited. We have established a system that allows induction of gene expression in C. albicans by the addition of tetracycline (Tet). By fusing genetically modified versions of the reverse Tet repressor from Escherichia coli and the transcription activation domain of the Gal4 protein from Saccharomyces cerevisiae, a C. albicans-adapted reverse Tet-dependent transactivator (rtTA) was created that was expressed from the constitutive ADH1 or the opaque-specific OP4 promoter. To monitor Tet-inducible gene expression, the caGFP reporter gene was placed under the control of a Tet-dependent promoter, obtained by fusing a minimal promoter from C. albicans to seven copies of the Tet operator sequence. Fluorescence of the cells demonstrated that gene expression could be efficiently induced by the addition of doxycycline in yeast, hyphal, and opaque cells of C. albicans. The Tet-inducible gene expression system was then used to manipulate the behavior of the various growth forms of C. albicans. Tet-induced expression of a dominant-negative CDC42 allele resulted in growth arrest as large, multinucleate cells. Filamentous growth was efficiently inhibited under all tested hyphal-growth-promoting conditions by Tet-inducible expression of the NRG1 repressor. Tet-induced expression of the MTLa1 gene in opaque cells of an MTLalpha strain forced the cells to switch to the white phase, whereas Tet-induced expression of the MTLa2 transcription factor induced shmooing. When the ecaFLP gene, encoding the site-specific recombinase FLP, was placed under the control of the Tet-dependent promoter, Tet-inducible deletion of genes which were flanked by the FLP target sequences was achieved with high efficiency to generate conditional null mutants. In combination with the dominant selection marker caSAT1, the Tet-inducible gene expression system was also applied in C. albicans wild-type strains, including drug-resistant clinical isolates that overexpressed the MDR1, CDR1, and CDR2 multidrug efflux pumps. This system, therefore, allows a growth medium-independent, Tet-inducible expression and deletion of genes in C. albicans and provides a convenient, versatile new tool to study gene function and manipulate cellular behavior in this model pathogenic fungus.

PMID:
16087738
PMCID:
PMC1214539
DOI:
10.1128/EC.4.8.1328-1342.2005
[Indexed for MEDLINE]
Free PMC Article

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