UC San Diego

Sporulation in Bacillus subtilis begins with asymmetric division, which generates a smaller forespore, which eventually becomes the mature spore, and a larger mother cell, which lyses after spore development. During engulfment, the mother cell membranes move up and around the forespore, fusing at the top and releasing the forespore into the mother cell cytoplasm.

This dramatic cellular reorganization requires two protein machineries. The first, SpoIID, SpoIIM, and SpoIIP, is expressed in the mother cell and is essential for engulfment. They are proposed to act as a motor, pulling the mother cell membranes around the forespore, using peptidoglycan as a track while they degrade it. The second, SpoIIQ and SpoIIIAH, acts as a ratchet that constitutes a back-up mechanism for membrane migration. SpoIIQ is produced in the forespore and SpoIIIAH in the mother cell, leaving the only possible site of interaction at the asymmetric septum, where they form foci and arcs around the forespore during membrane migration.

I investigated the role of SpoIID by identifying and characterizing spoIID mutants unable to support sporulation and using biochemical and cell biological experiments to define the role of SpoIID in engulfment. I utilized timelapse fluorescence microscopy and antibiotics to demonstrate that peptidoglycan synthesis provides a second backup mechanism that facilitates engulfment and is required in the absence of SpoIIQ. I examined interactions between SpoIIQ/SpoIIIAH and SpoIID/SpoIIM/SpoIIP using Fluorescence Recovery After Photobleaching (FRAP) analysis and found that foci characteristic of SpoIIQ localization depend on SpoIID/SpoIIM/SpoIIP, while immobilization of SpoIIQ depends on SpoIIIAH.

These results allow a more complete model for membrane migration and protein localization during engulfment. Specifically, my data suggest that SpoIID cycles in and out of the complex at the leading edge of the engulfing membrane with peptidoglycan degradation activity required for both release from the septum and movement of the complex around the forespore. It also suggests that SpoIID/SpoIIM/SpoIIP interact with SpoIIQ and localize it to arcs at the septum, where it recruits SpoIIIAH. Finally, if SpoIIQ is deleted, peptidoglycan synthesis becomes required for membrane migration. Thus, engulfment depends on redundant mechanisms for both membrane movement and protein localization.