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Journal of Bacteriology, June 2001, p. 3761-3769, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3761-3769.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Analysis of Phase-Specific Gene Expression at the Single-Cell Level in the White-Opaque Switching System of Candida albicans

Anja Strauß, Sonja Michel, and Joachim Morschhäuser*

Zentrum für Infektionsforschung, Universität Würzburg, D-97070 Würzburg, Germany

Received 22 January 2001/Accepted 26 March 2001

The opportunistic fungal pathogen Candida albicans can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and evasion of host defense mechanisms. Phenotypic switching has been studied intensively for the white-opaque switching system of strain WO-1. To facilitate the molecular analysis of phenotypic switching, we have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles were sequentially inactivated using the MPAR-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid (MPA) resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. To investigate a possible cell type-independent switching in the expression of individual phase-specific genes, two different reporter genes that allowed the analysis of gene expression at the single-cell level were integrated into the genome, using URA3 as a selection marker. Fluorescence microscopic analysis of cells in which a GFP reporter gene was placed under the control of phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells and that the white-phase-specific WH11 gene was detectably expressed only in white cells. When MPAR was used as a reporter gene, it conferred an MPA-resistant phenotype on opaque but not white cells in strains expressing it from the SAP1 promoter, which was monitored at the level of single cells by a significantly enlarged size of the corresponding colonies on MPA-containing indicator plates. Similarly, white but not opaque cells became MPA resistant when MPAR was placed under the control of the WH11 promoter. The analysis of these reporter strains showed that cell type-independent phase variation in the expression of the SAP1 and WH11 genes did not occur at a detectable frequency. The expression of these phase-specific genes of C. albicans in vitro, therefore, is tightly linked to the cell type.


* Corresponding author. Mailing address: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-31 21 52. Fax: 49-931-31 25 78. E-mail: joachim.morschhaeuser{at}mail.uni-wuerzburg.de.


Journal of Bacteriology, June 2001, p. 3761-3769, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3761-3769.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.



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