FIG 4.

Altered ergosterol content renders strains sensitive to alkaline pH. (A) The reduced growth rate of the sre1Δ mutant strain in liquid growth medium at pH 8 can be rescued through the addition of exogenous ergosterol in a dose-dependent manner. Growth rate of indicated strains was assessed by changes in OD₅₉₅ in biological triplicate every 10 min for 42 h at 30°C. Ergosterol was added as indicated. One-way ANOVA and Dunnett’s multiple-comparison test were run on the last time point under each condition compared to the pH 8-alone condition to determine statistical significance. **, P value < 0.003; ***, P value < 0.0005; ns, not significant. (B) Other sterol-related mutants exhibit alkaline pH sensitivity. Two deletion mutants related to ergosterol biosynthesis in C. neoformans (erg4Δ and cnag_00490Δ) display a pH sensitivity when grown on pH 8 growth medium. Indicated strains were serially diluted onto YPD medium and YPD-150 mM HEPES pH 8. Growth was compared to WT and assessed after 3 days. (C) erg6Δ C. neoformans mutant also exhibits alkaline pH sensitivity when grown on pH 8 medium. Indicated strains were serially diluted onto YPD medium and YPD-150 mM HEPES pH 8. Growth was compared to WT and reconstituted strains and assessed after 3 days. (D) Candida species ergosterol mutants reveal similar pH-sensitive phenotypes. C. albicans and C. lusitaniae wild-type strains and strains with mutations in various components of ergosterol biosynthesis were serially diluted onto YPD medium and YPD-150 mM HEPES pH 8 and 8.5 as well as YES medium with 7 mM CoCl₂. Growth was compared to WT and assessed after 2 days.