FIG. 6.
SpoIIIAE processing is required for σG activation. (A) Expression patterns of a PsspE-cfp fusion in DSM cultures of strains expressing wild-type spoIIIAE (AH9337) or the spoIIIAEA24K allele (AH9338) from the spoIIIA promoter. Samples were collected at the indicated times (in hours) after the onset of sporulation, stained with the membrane dye FM4-64, and observed by fluorescence microscopy. Lowercase letters indicate fluorescence intensity patterns (low [a] or high [b]). (B) Quantitative analysis of the PsspE-cfp expression patterns observed at hours 4 and 6 of sporulation for strains AH9337 (black) and AH9338 (dark gray) and in mutants with deletion of the spoIIIAE (AH9336; white) or spoIIIG (AH6566; light gray) allele. Note that the last two strains are not shown in panel A, for simplicity, and that no fluorescence was detected for the ΔsigF mutant AH9335 (not shown). At least 200 cells were scored for the fluorescence patterns designated by low intensity (class a; pixel intensity of <300; see Material and Methods) or high intensity (class b; pixel intensity of >300). (C) An anti-SpoIIIAE immunoblot of a membrane fraction prepared from cells of MB24 (wild type; lane 1), AH2468 (ΔspoIIIAE; lane 2), AH9257 (PspoIIIA-spoIIIAE; lane 3), AH9272 (PspoIIIA-spoIIIAEA24K; lane 4), and AH9262 (PspoIIIA-SPsleB-ΔSPspoIIIAE; lane 5), at hour 3 of sporulation in DSM. The black and white arrowheads show the positions of the precursor and mature forms of SpoIIIAE, respectively. Bar, 1 μm.