SKN7 of Candida albicans: mutant construction and phenotype analysis

Abstract

The SKN7 two-component response regulator gene of Candida albicans was deleted, and the phenotype of the mutant was established. This mutant exhibited impaired growth on Spider agar and 10% serum agar compared to wild-type and gene-reconstituted strains. The skn7 mutant was sensitive to H(2)O(2) in vitro, but its virulence was only mildly attenuated. A comparison of the Skn7p and Ssk1p response regulators of C. albicans is discussed.

FIG. 1.

(A) The cassette used to disrupt the SKN7 allele. (B) The SKN7-URA3-hisG cassette used to reintroduce a wild-type allele into strain N2 (skn7/skn7) lacking the URA3. The primers used to amplify the flanking sequences are indicated (_). A 1.29-kb section of the gene, including the receiver domain, was deleted. Also, the 0.68-kb 3′ end of the gene was used as a probe for the Southern hybridizations. (C) Southern blot analysis of strains obtained during the disruption of SKN7. Genomic DNA was digested with KpnI and HindIII and probed with the 0.68-kb Msc1-HindIII PCR product (panel A). The size of each hybridizing band is indicated. The Ura3⁺ heterozygote (N1), the Ura3⁻ heterozygote (N1.5), and the Ura3⁺ null mutant (N2) are shown (see text for details). (D) PCR of four clones of the gene-reconstituted strain (R15) indicates a single PCR product of 3.05 kb. Strain R15 was confirmed by Southern hybridizations.

FIG. 2.

Phenotypes of the parent (CAF2; row 1), and heterozygote (N1; row 2), null (N2; row 3), and reconstituted (R15; row 4) strains of C. albicans. All strains are shown on 10% serum agar (top), M-199 (pH 7.5) (middle), or Spider agar (bottom). All cultures were incubated for 3 to 5 days at 37°C on each medium.

FIG. 3.

Growth of CAF2, N1, N2, and R15 at 30°C for 48 h on YPD agar containing 1 mM t-butyl hydroperoxide or 3 mM hydrogen peroxide. Plates were spotted with yeast cells of each strain at concentrations of 10⁵ (left) to 10 (right) cells in a total volume of 5 μl.