FIGURE 7: Progress in our understanding of SpoIIIE-mediated chromosome translocation. (A) Fluorescence microscopy images of a wild-type (WT, left) and of a spoIIIE mutant (spoIIIE, right) strain of B. subtilis. The upper panels show the DNA stained with DAPI (blue), and the lower panels the overlay of DAPI-stained DNA and the membranes stained with FM 4-64 (red). In the upper panel, forespores are indicated by single arrowheads, and mother cells by double arrowheads. Wild-type forespores contain a complete chromosome. However, forespores of spoIIIE mutant strains contain only ~30% of a chromosome, and the rest remains trapped in the mother cell. (B) Fluorescence microscopy image of a sporulating cell producing a SpoIIIE-GFP fusion during chromosome translocation. The upper panel shows the DNA stained with DAPI (blue) and the GFP signal, and the lower panel shows in addition the membranes stained with FM 4-64 (red). SpoIIIE forms a focus (green) at septal midpoint, where the chromosome is trapped. (C) SpoIIIE visualized by super-resolution microscopy (PALM) in living cells with thicker polar septa. SpoIIIE forms two foci (dual foci), which are separated by a distance equivalent to the septal thickness, indicating that one cluster is present at one side of the septum and the other at the opposite side. Reproduced from [101]. (D) Model for the organization and function of the SpoIIIE translocation complex at the septal midpoint. SpoIIIE forms two side-by-side channels spanning both septal membranes (red lines), thereby allowing the simultaneous transport of both arms of the chromosome from the mother cell (MC) to the forespore (FS). Translocation is powered by SpoIIIE motor domains at the mother-cell side of the septum (green circles), which are activated to export the chromosome to the forespore. Forespore motor domains (red circles) remain inactive. (E) Cryo-electron microscopy of a wild-type sporulating cell (top), and of a spoIIIE mutant (bottom). Membranes are annotated as follows: forespore membrane, pink; mother-cell membrane, purple. Chromosome translocation is required to maintain the shape of the forespore. In the absence of chromosome translocation, the forespore appears deflated. Reproduced with permission from [100].

100. Lopez-Garrido J, Ojkic N, Khanna K, Wagner FR, Villa E, Endres RG, and Pogliano K (2018). Chromosome translocation inflates Bacillus forespores and impacts cellular morphology. Cell 172(4). 10.1016/j.cell.2018.01.027

101. Shin JY, Lopez-Garrido J, Lee S-HH, Diaz-Celis C, Fleming T, Bustamante C, and Pogliano K (2015). Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum. Elife 4: e06474. 10.7554/eLife.06474