| Bacillus subtilis, a Gram-positive bacterium, sporulates when it encounters nutrient starvation. The first morphological sign of sporulation is asymmetric septum formation, dividing the cell into a smaller forespore, which eventually becomes the spore, and a larger mother cell, which eventually lyses, releasing the mature spore into the environment. A key sporulation step is engulfment, a phagocytotic-like process in which the mother cell membrane surrounds the forespore, enclosing it within the mother cell cytoplasm. The necessary engulfment factors were determined to be located in the mother cell, however, the only identified mother cell proteins, SpoIID, SpoIIM, and SpoIIP, were known to be involved in peptidoglycan thinning of the asymmetric septum. With the knowledge that previously studied septal thinning mutants were null mutations and would exhibit pheonotypic blocks at the first step each protein is required, therefore masking any possible phenotypes associated with blocks later in engulfment, I conducted further studies on SpoIID, SpoIIM, and SpoIIP. By using genetic, cell biological, and biochemical methods, I determined that they were also involved during mother cell membrane migration.; Although no new engulfment proteins were found in my genetic screens to identify new engulfment proteins, I identified novel spoIID and spoIIP mutations that affected both septal thinning and membrane migration. In addition to septal localization, functional green fluorescent protein fusions to the cytoplasmic amino-terminals of SpoIID, SpoIIM, and SpoIIP localized as foci at the leading edge of the engulfing mother cell membrane, suggesting that they functioned as a ring at the leading edge. SpoIID was identified as a peptidoglycan hydrolase, allowing us to propose that a ring-like complex of SpoIID, SpoIIM, and SpoIIP pulls the tethered mother cell membrane toward the forespore pole, driven by a processive peptidoglycan hydrolase. My latest localization studies provide evidence for interaction between SpoIIM and SpoIIP, and suggest that septal localization depends on SpoIIB, while relocalization of SpoIIM and SpoIIP to the septal disk periphery requires SpoIID. Moreover, I found that engulfment is restricted to the correct septum by MinCD, which also spatially regulate the site of cell division and the polarity of DNA translocase assembly. |
