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Journal of Bacteriology, June 2001, p. 3761-3769, Vol. 183, No. 12
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.
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
*
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.
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