We here present genetic and cell biological evidence that SpoIIB is incorporated into the septum during division and serves directly or indirectly as a landmark for localization of SpoIIM and then SpoIIP and SpoIID to the septum. Thus, SpoIIB appears to play a role similar to Caulobacter crescentus TipN, which localizes to the septum and remains at the cell pole to serve as a landmark for polar localization (Huitema et al., 2006; Lam et al., 2006). We also show that a localization hierarchy exists within the engulfment machinery, with SpoIIM being required to recruit and retain SpoIIP at the septum, which is required together with SpoIID to move the machinery around the forespore. Our results are similar to those recently published by Chastanet and Losick (2007), although our studies have allowed the identification of a second, SpoIIB-independent, pathway for DMP localization. Specifically, when we eliminated the synergistic engulfment defect observed in spoIIB strains expressing GFP–SpoIIP (by also expressing untagged SpoIIP), we noted that localization was partially rescued, with engulfing sporangia showing almost wild-type localization of GFP–SpoIIP at the leading edge of the engulfing membrane. This localization depended on SpoIVFAB, which in turn depends on the Q-AH zipper for localization (Blaylock et al., 2004; Doan et al., 2005; Jiang et al., 2005). The DMP engulfment proteins therefore appear to have two distinct mechanisms by which they can reach the sporulation septum, SpoIIB, which assembles a septal landmark for DMP during cytokinesis, and the Q-AH zipper (via SpoIVFAB), which assembles after cytokinesis (Fig. 7). Interestingly, while the Q-AH zipper proteins and SpoIVFAB are present in the genomes of all endospore-forming bacteria, SpoIIB is present only in the Bacilli, not in the Clostridia (Stragier, 2002). This suggests either that DMP localization in the Clostridia depends entirely on the Q-AH zipper via SpoIVFAB or that the Clostridia have another primary pathway for DMP localization that can substitute for SpoIIB. We recently reported that the Q-AH zipper mediates engulfment in cells whose cell walls have been enzymatically removed (Broder and Pogliano, 2006). This protoplast engulfment does not require SpoIVFAB, SpoIIB or DMP, indicating that the requirement for Q-AH for protoplast engulfment is not mediated by their ability to localize DMP or SpoIVFAB to the septum. However, we have here shown that in intact cells, the Q-AH zipper also makes a second apparently distinct contribution to engulfment, by localizing SpoIVFAB, which can mediate DMP localization in the absence of SpoIIB. Three observations suggest that SpoIVFAB might also interact with DMP in cells containing SpoIIB. First, the absence of SpoIVFAB (or SpoIIQ or SpoIIIAGH) results in the release of a small amount of SpoIIP from the septum into the mother cell cytoplasmic membrane (Fig. 4), suggesting that SpoIVFAB is necessary for the efficient retention of SpoIIP at the septum. Second, the absence of SpoIVFAB (or SpoIIQ or SpoIIIAGH) results in the loss of the foci that SpoIIP normally assembles around the forespore, although localization to the leading edge of the engulfing membrane is maintained (likely by SpoIIB). Finally, Doan et al. (2005) reported that SpoIVFA is slightly delocalized in the absence of DMP, suggesting an interaction between SpoIVFA and DMP. Thus, although the interaction between SpoIVFAB and the DMP proteins has not yet been detected biochemically, evidence is building that this interaction occurs in wild-type cells. If this is indeed the case, then this interaction would bring proteins involved in engulfment together with proteins involved in activating engulfment-dependent gene expression. The localization studies presented here also suggest that there are two distinct populations of SpoIIP (and likely also SpoIID), one recruited to the leading edge of the engulfing membrane by SpoIIB and a second that interacts with the protein complex containing the Q-AH zipper and the σK-processing machinery (SpoIVFA, SpoIVFB and BofA). We emphasize that it remains unclear if the SpoIIB-dependent or SpoIVFAB-dependent localization pathways for DMP are mediated by their direct protein–protein interaction with the DMP proteins (or a subset). While the physical interaction between SpoIID and SpoIIP has been demonstrated by both affinity chromatography (Chastanet and Losick, 2007) and co-immunoprecipitation (Fig. 2), we have thus far failed to detect any robust and repeatable interaction between SpoIIB or SpoIVFAB with SpoIID, SpoIIM or SpoIIP (although we have not tested all possible combinations, as described in the Experimental procedures). Indeed, it seems possible that certain steps in the localization hierarchy might be mediated indirectly, for example, by other unknown proteins that interact with the Q-AH zipper or SpoIIB or by one protein modifying the bacterial cell wall in a manner that allows a second protein to bind. Consistent with this latter proposal, the engulfment proteins include two cell wall hydrolases likely capable of binding the wall (SpoIID and SpoIIP), although the specificity or affinity of this interaction has not yet been determined. In addition, SpoIIB and SpoIIP show limited sequence similarity with the CwlC amidase (Errington et al., 2003; Soding et al., 2005; Chastanet and Losick, 2007) and both SpoIVFA and SpoIIQ show some sequence similarity to the M23 family of endopeptidases (Rudner and Losick, 2002), some of which cleave peptide cross-bridges in peptidoglycan. It is therefore possible that these proteins modify the peptidoglycan in a manner that allows subsequent proteins to bind in a modification-specific manner. Peptidoglycan is the perfect molecule to serve as a landmark in bacterial cells, as unlike membrane proteins, it is immobile for long periods of time (until it is degraded or recycled) so that modifications (amidation, distinct peptide cross-linking or trimming reactions) could remain in the same cellular location. It is also tempting to speculate that such modifications are necessary to allow SpoIID and SpoIIP to cleave peptidoglycan, as these enzymes must be subject to strict spatial and temporal control, as their unregulated activity could lead to cell lysis.