A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.

SummaryEngulfment in Bacillus subtilis is mediated by two complementary systems, SpoIID, SpoIIM and SpoIIP (DMP), which are essential for engulfment, and the SpoIIQ-SpoIIIAGH (Q-AH) zipper, which provides a secondary engulfment mechanism and recruits other proteins to the septum. We here identify two mechanisms by which DMP localizes to the septum. The first depends on SpoIIB, which is recruited to the septum during division and provides a septal landmark for efficient DMP localization. However, sporangia lacking SpoIIB ultimately localize DMP and complete engulfment, suggesting a second mechanism for DMP localization. This secondary targeting pathway depends on SpoIVFA and SpoIVFB, which are recruited to the septum by the Q-AH zipper. The absence of a detectable localization phenotype in mutants lacking only SpoIVFAB (or Q-AH) suggests that SpoIIB provides the primary DMP localization pathway while SpoIVFAB provides a secondary pathway. In keeping with this hypothesis, the spoIIB spoIVFAB mutant strain has a synergistic engulfment defect at septal thinning (which requires DMP) and is completely defective in DMP localization. Thus, the Q-AH zipper both provides a compensatory mechanism for engulfment when DMP activity is reduced, and indirectly provides a compensatory mechanism for septal localization of DMP when its primary targeting pathway is disrupted.

Summary Bacillus subtilis sporulation depends on the forespore membrane protein SpoIIQ, which interacts with the mother cell protein SpoIIIAH at the septum to localize other sporulation proteins. It has remained unclear how SpoIIQ localizes. We demonstrate that localization of SpoIIQ is achieved by two pathways: SpoIIIAH and the SpoIID, SpoIIM, SpoIIP engulfment proteins. SpoIIQ shows diffuse localization only in a mutant lacking both pathways. Super-resolution imaging shows that in the absence of SpoIIIAH, SpoIIQ forms fewer, slightly larger foci than in wild type. Surprisingly, photobleaching experiments demonstrate that, although SpoIIQ localizes without SpoIIIAH, it is no longer immobilized, and is therefore able to exchange subunits within a localized pool. SpoIIQ mobility is further increased by the additional absence of the engulfment proteins. However an enzymatically inactive SpoIID protein immobilizes SpoIIQ even in the absence of SpoIIIAH, indicating that complete septal thinning is not required for SpoIIQ localization. This suggests that SpoIIQ interacts with both SpoIIIAH and the engulfment proteins or their peptidoglycan cleavage products. They further demonstrate that apparently normal localization of a protein without a binding partner can mask dramatic alterations in protein mobility. We speculate that SpoIIQ assembles foci along the path defined by engulfment proteins degrading peptidoglycan.

No abstract

No abstract