Fig. 9.
The role of Ena1 and Nha1 in capsule and melanin production. (A) For capsule production measurement, each strain (WT H99 strain and hog1, ena1, ena1 nha1, and nha1 mutants) was spotted and cultured on DME agar medium at 37°C for 2 days. Capsules were visualized by India ink staining (upper panel) and the relative capsule volume was measured by calculating the ratio of the length of packed cell volume phase per length of total volume phase (lower panel). Three independent experiments with technical triplicates were performed. Statistical analysis was performed by using Bonferroni multiple comparison test. *, P < 0.01 and NS, not significant (P > 0.05). (B) Each C. neoformans strain was spotted, cultured on Niger seed medium containing concentration of glucose 0.1% at 30°C or 37°C for 1 day, and photographed. (C) The role of Ena1 and Nha1 in controlling LAC1 expression. WT H99 strain and ena1 nha1 mutants grown to a logarithmic phase (OD600nm = 1.0) in YPD liquid medium (zero time control) were shifted to YNB liquid medium (without glucose), and further incubated at 30°C. Northern blot analysis was performed with total RNA isolated from each cell grown at the indicated time points. Each membrane was hybridized with the LAC1 specific probe, washed, and developed. Subsequently the same membrane was deprobed, re-hybridized with the ACT1 specific probe, washed, and developed. The expression levels of LAC1 relative to ACT1 (LAC1/ACT1) were measured via phosphoimager analysis.