Figure 7. σ^G is active when synthesized prior to the completion of engulfment.

(A) σ^G activity was assessed by microscopy using a fluorescence reporter (P_sspE-gfp; false-colored green in the lower panel) in a ΔsigG mutant (ΔsigG, BTD3004), a wild-type background (wt, BTD3002), a ΔsigG mutant containing one copy of P_spoIIQ-sigG (P_IIQ-sigG, BTD3007), or three copies of P_spoIIQ-sigG (3× P_IIQ-sigG, BCM791), and a ΔsigG, ΔspoIIIAE double mutant that contains three copies of P_spoIIQ-sigG (ΔE, BCM816). Sporulating cells were monitored at hour 2 of sporulation. The membranes from the same field were visualized using the dye TMA-DPH (false-colored red) and merged with the GFP signal. The membrane dye inefficiently traverses the lipid bilayer and therefore reports on the engulfment status of the forespore [54]. Forespores that stain weakly with TMA-DPH (white carets) have been completely engulfed by the mother cell. Forespores that have not yet completed engulfment have strong signal due to the two membranes surrounding the spore. Forespore that have σ^G activity but have not completed engulfment are indicated (yellow carets). Scale bar, 1 µm. Similar results were obtained with a P_sspB-gfp reporter (not shown). (B) Immunoblot analysis of whole cell lysates from sporulating cells. The levels of σ^G were analyzed in sporulating cells from the same strains described in A. σ^F levels were monitored to control for efficiency of sporulation. σ^A levels were monitored to control for loading.