FIG 4.

BycA is involved in the Mucor hypha-yeast transition under aerobic conditions. (A) Reverse transcriptase quantitative PCR showed that in the absence of calcineurin function (cnbRΔ and WT + FK506; yeast morphology), bycA expression was 6-fold higher than the level seen with the WT (hyphal morphology) with calcineurin function, suggesting that calcineurin regulates bycA expression at the mRNA level. When the Mucor WT was grown anaerobically under high-CO₂ conditions (see Materials and Methods), it grew as yeast; however, there was no significant difference in bycA expression compared to the level seen with the WT-hypha group. One-way analysis of variance (ANOVA) data were significant (P = 0.0001). Dunnett’s post hoc test was used to compare cnbRΔ, WT + FK506, WT-yeast results with those seen with the WT-hypha group (*, P < 0.05; ns, not statistically significant; undet., undetected). As expected, no expression of the bycA gene was detected in either of the bycAΔ mutants. (B) When WT, cnbRΔ, bycAΔ, and bycAΔ cnbRΔ mutants were grown anaerobically overnight under high-CO₂ conditions, they all exhibited a yeast morphology, thereby suggesting that neither calcineurin nor BycA has a role in anaerobic morphological pathways (scale bar = 20 μm). (C) Crude protein extracts (0.5 μg) were used to measure overall PKA activity. The WT-hypha and the bycAΔ mutant showed significantly lower PKA activity than WT-yeast (grown anaerobically). In WT + FK506 and the cnbRΔ mutant, PKA activity remained higher; however, the bycAΔ cnbRΔ mutant showed lower PKA activity despite possessing no calcineurin function. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group with WT-yeast (*, P < 0.05). (D) Crude extracts from 60 mg biomass were used to measure overall cAMP activity. Yhe WT + FK506 and cnbRΔ strains showed significantly lower cAMP activity than WT-yeast, thereby suggesting that under aerobic conditions, PKA activity is elevated in Mucor yeast in a cAMP-independent manner. One-way ANOVA data were significant (P < 0.0001). Dunnett’s multiple-comparison test was used to compare each group with WT-yeast (*, P < 0.05).