Figure 4
Dependence of spore differentiation on PhyA. (A) Spore differentiation of normal Ax3 (phyA+) and phyA– cells in submerged conditions was quantitated as a function of time and [O2], by counting Calcofluor-positive spores in a hemacytometer. Data are from a single representative experiment. Note that data points for Ax3/21% O2 and the phyA– strains overlap at the bottom. (B) Complementation of phyA– cells by PhyA overexpressed under control of either the prestalk (ecmA) or prespore (cotB) cell specific promoter, and partial rescue of sporulation by expression of the catalytic domain of protein kinase A (PKAcat) under control of its own promoter. Spores were counted after incubation in 100% O2 for 72 h. Data represent the average and standard error of the mean (SEM) of 2–3 independent trials. (C) Effect of PhyA overexpression in normal (phyA+) cells as a function of O2. Spores were counted after 72 h. Data represent the average and SEM of 3–5 independent trials. (D) Typical cell cyst-like structures from samples in panel A were imaged using transmitted light. An increasing fraction of cells enter aggregates at higher O2, as inferred from fewer single cells in the background, and darker cores correlate with increased spore formation. (E) Cells developed for the indicated time were analyzed for the prespore cell differentiation markers SP85, and SP96 and SP75, based on Western blotting with mAb 5F5 and mAb 83.5, respectively.