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. 2022 Apr 26;11:e74275. doi: 10.7554/eLife.74275

Figure 3. Effects of alanine substitutions in GFPΔ27BofA during Bacillus subtilis sporulation.

(A) Effects of GFPΔ27BofA variants (N48A, N61A, and T64A) on Pro-σK cleavage. Wild-type strain PY79, a spoIVB165 null mutant, a spoIVB165 bofA::erm double mutant, and the double mutant with PbofA-gfpΔ27bofA integrated at amyE to express GFPΔ27BofA with no substitution (none) or the indicated Ala substitution, were starved to induce sporulation. Samples collected at 4 and 5 hr poststarvation (PS) were subjected to immunoblot analysis with antibodies against SpoIVFA, GFP, SpoIVFB, and Pro-σK. The graph shows quantification of the cleavage ratio [σK/(Pro-σK+σK)] for three biological replicates. Error bars, 1 standard deviation. Student’s two-tailed t-tests were performed to compare certain cleavage ratios (p values). (B) Localization of GFPΔ27BofA and the three variants. Samples collected at 3 hr PS were treated with FM 4–64 to stain membranes. Confocal microscopy images of fluorescence from GFPΔ27BofA, membranes, and merged images are shown for representative sporangia with discrete (no substitution in GFPΔ27BofA, designated ‘none’), partial (N48A and T64A), or no forespore (FS) localization (N61A). Scale bar, 1 μm. The percentage of sporangia (44–93 counted; nonsporulating cells were not counted) with each localization pattern is shown.

Figure 3—source data 1. Immunoblot images (annotated) and quantification of cleavage assays (Figure 3A), and confocal microscopy images (see the readme file) and a table of the numbers of sporangia in which FS localization of GFPΔ27BofA and the three variants were counted (Figure 3B).
Figure 3—source data 2. Immunoblot images (raw) (Figure 3A).

Figure 3.

Figure 3—figure supplement 1. GFPΔ27BofA variants are intact during Bacillus subtilis sporulation.

Figure 3—figure supplement 1.

A spoIVB165 bofA::erm double mutant with PbofA-gfpΔ27bofA or the indicated mutant version integrated ectopically at amyE, were starved to induce sporulation. Samples collected at 3 hr poststarvation were subjected to immunoblot analysis with antibodies against GFP. The star (*) indicates very small amounts of potential breakdown species of GFPΔ27BofA and the variants detectable in the long exposure.
Figure 3—figure supplement 1—source data 1. Immunoblot images (raw and annotated).