SummaryAt the onset of sporulation in Bacillus subtilis, two potential division sites are assembled at each pole, one of which will be used to synthesize the asymmetrically positioned sporulation septum. Using the vital stain FM 4-64 to label the plasma membrane of living cells, we examined the fate of these potential division sites in wild-type cells and found that, immediately after the formation of the sporulation septum, a partial septum was frequently synthesized within the mother cell at the second potential division site. Using time-lapse deconvolution microscopy, we were able to watch these partial septa first appear and then disappear during sporulation. Septal dissolution was dependent on E activity and was partially inhibited in mutants lacking the E -controlled proteins SpoIID, SpoIIM and SpoIIP, which may play a role in mediating the degradation of septal peptidoglycan. Our results support a model in which E inhibits division at the second potential division site by two distinct mechanisms: inhibition of septal biogenesis and the degradation of partial septa formed before E activation.

A hallmark of bacterial endospore formation is engulfment, during which the membrane of one cell (the mother cell) migrates around the future spore, enclosing it in the mother cell cytoplasm. Bacteria lack proteins required for eukaryotic phagocytosis, and previously proteins required for membrane migration remained unidentified. Here we provide cell biological and genetic evidence that three membrane proteins synthesized in the mother cell are required for membrane migration as well as for earlier steps in engulfment. Biochemical studies demonstrate that one of these proteins, SpoIID, is a cell wall hydrolase, suggesting that membrane migration in bacteria can be driven by membrane-anchored cell wall hydrolases. We propose that the bacterial cell wall plays a role analogous to that of the actin and tubulin network of eukaryotic cells, providing a scaffold along which proteins can move.[Keywords: Bacillus subtilis; sporulation; membrane movement; peptidoglycan hydrolysis; protein localization] Supplemental material is available at http://www.genesdev.org.

A key step in the Bacillus subtilis spore formation pathway is the engulfment of the forespore by the mother cell, a phagocytosis-like process normally accompanied by the loss of peptidoglycan within the sporulation septum. We have reinvestigated the role of SpoIIB in engulfment by using the fluorescent membrane stain FM 4-64 and deconvolution microscopy. We have found that spoIIB mutant sporangia display a transient engulfment defect in which the forespore pushes through the septum and bulges into the mother cell, similar to the situation in spoIID, spoIIM, and spoIIP mutants. However, unlike the sporangia of those three mutants, spoIIB mutant sporangia are able to complete engulfment; indeed, by time-lapse microscopy, sporangia with prominent bulges were found to complete engulfment. Electron micrographs showed that in spoIIB mutant sporangia the dissolution of septal peptidoglycan is delayed and spatially unregulated and that the engulfing membranes migrate around the remaining septal peptidoglycan. These results demonstrate that mother cell membranes will move around septal peptidoglycan that has not been completely degraded and suggest that SpoIIB facilitates the rapid and spatially regulated dissolution of septal peptidoglycan. In keeping with this proposal, a SpoIIB-myc fusion protein localized to the sporulation septum during its biogenesis, discriminating between the site of active septal biogenesis and the unused potential division site within the same cell.Bacillus subtilis is a gram-positive bacterium which, under conditions of nutrient deprivation, undergoes a developmental process known as sporulation (for review, see references 10 and 46). During sporulation, a septum is positioned near the pole instead of the midcell site used for vegetative division, resulting in the production of two daughter cells of different sizes and fates, a smaller forespore and a larger mother cell. Shortly after the onset of differential gene expression in these two cells, the septum between them begins to migrate around the forespore until the leading edges of the membrane meet on the distal side of the forespore and fuse, releasing the forespore into the mother cell cytoplasm (Fig. 1A). After the completion of this phagocytosis-like process (known as engulfment), the forespore is enclosed in the mother cell and bounded by two membranes, its original cytoplasmic membrane and a membrane derived from the engulfing mother cell membrane. It is between these two membranes that the specialized spore cell wall (the cortex) is synthesized, while the multilayered spore coat is assembled around the forespore within the mother cell cytoplasm.Although engulfment is an essential part of the spore formation pathway of B. subtilis and its endospore-forming relatives, the mechanism by which the membranes move around the forespore remains poorly understood. However, it appears that thinning or removal of peptidoglycan between the septal membranes is necessary to allow movement of the mother cell membrane around the forespore (16, 31). ...

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.

We have isolated a UV-induced temperature-sensitive mutant ofMycobacterium smegmatis that fails to grow at 42°C and exhibits a filamentous phenotype following incubation at the nonpermissive temperature, reminiscent of a defect in cell division. Complementation of this mutant with an M. smegmatis genomic library and subsequent subcloning reveal that the defect lies within the M. smegmatis dnaG gene encoding DNA primase. Sequence analysis of the mutant dnaG allele reveals a substitution of proline for alanine at position 496. Thus, dnaG is an essential gene in M. smegmatis, and DNA replication and cell division are coupled processes in this species. Characterization of the sequences flanking the M. smegmatis dnaG gene shows that it is not part of the highly conserved macromolecular synthesis operon present in other eubacterial species but is part of an operon with a dgt gene encoding dGTPase. The organization of this operon is conserved in Mycobacterium tuberculosis andMycobacterium leprae, suggesting that regulation of DNA replication, transcription, and translation may be coordinated differently in the mycobacteria than in other bacteria.