FIG 6.

Ace2 represses respiration and is expressed at high levels under embedded conditions. (A) The indicated strains were incubated at 30°C in YP plus 2% sucrose either on top of the agar or embedded within the agar plate. The respiratory activity of the colonies was determined using the triphenyl tetrazolium chloride overlay assay as described in Materials and Methods. The increased respiratory activity of the colony is indicated by the deeper red color. (B) Expression of ACE2 and EFG1 by qRT-PCR was determined for WT strains in stationary-phase planktonic culture or incubated in embedded agar. The bars indicate the mean fold change between embedded cells and planktonic stationary-phase cells for three biological replicates assayed in triplicate. Standard deviations are indicated by the error bars. (C and D) A strain containing the EFG1 promoter fused to GFP and the ACE2 promoter fused to mCherry were examined under stationary-phase (C) and embedded (D) conditions by fluorescence microscopy, and the pixel density of the signal per cell for GFP and mCherry was determined as described in Materials and Methods (n ≥ 100). Each data point represents a single cell with the GFP and mCherry signal denoted in arbitrary units. (E) The pACE2-mCherry/pEFG1-GFP strain was incubated embedded in solid agar medium; the cells were scraped into a microcentrifuge tube and examined under bright-field, GFP, and mCherry filters.