Genetic Dissection of the Sporulation Protein SpoIIE and Its Role in Asymmetric Division in Bacillus subtilis

A hallmark of the process of sporulation by the gram-positive bacterium Bacillus subtilis is the formation of an asymmetrically positioned septum that divides the developing cell into dissimilar-sized progeny called the forespore (the smaller cell) and the mother cell (33). One of the important challenges in the sporulation field is to understand the molecular mechanisms that bring about this switch from medial to polar division. Cytokinesis in bacteria is mediated by the tubulin-like protein FtsZ, which forms a ring-like structure, the cytokinetic or Z-ring, at the future site of cell division (11, 29, 30). Previous work has shown that asymmetric division is accompanied by the formation of bipolar Z-rings and that the formation of these rings involves a helical intermediate of the cytokinetic protein that may be responsible for redeploying molecules of FtsZ from the midcell position to the poles (6, 25). One of the polar Z-rings is converted into a division septum, whereas the other Z-ring is blocked from undergoing cytokinesis by the action of the sporulation genes spoIID, spoIIM, and spoIIP, which are synthesized in the mother cell as a consequence of polar division (10, 34). Redeploying FtsZ from the midcell to the poles is effected by a sporulation-specific increase in FtsZ levels (via a promoter that is recognized by the sporulation regulatory protein σH) and by the activation of the gene for the sporulation protein SpoIIE (via the sporulation regulatory protein Spo0A) (6, 15, 16, 38). Evidence that enhanced FtsZ levels and synthesis of SpoIIE are sufficient to cause polar division comes from the observation that artificial induction of spoIIE during growth in cells harboring an extra copy of ftsZ is sufficient to bring about a switch from medial to asymmetric division (6). Furthermore, a spoIIE mutation prevents polar septum formation in cells engineered to produce a constitutively active form of Spo0A during growth (22). SpoIIE is an 827-amino-acid-long protein that consists of an N-terminal domain (region I) with 10 membrane-spanning segments (3), a C-terminal domain that is homologous to the PP2C family of phosphatases domain (region III) (1, 27), and a central domain (region II) that shows little similarity to other, nonorthologous proteins in the databases (Fig. ​(Fig.1A).1A). Cytological evidence demonstrates that SpoIIE colocalizes with the Z-ring and that it depends on FtsZ for this colocalization (4, 26). Biochemical evidence additionally indicates that the interaction between SpoIIE and FtsZ is direct (28). Whereas FtsZ and other division proteins exit the developing septum during cytokinesis, SpoIIE remains associated with the polar septum after cytokinesis is complete, when it plays a critical role in the activation of the forespore-specific transcription factor σF. SpoIIE triggers the activation of σF by catalyzing (via its PP2C-like phosphatase domain) the conversion of the inactive phosphoprotein SpoIIAA-P to its active, dephosphorylated form SpoIIAA. SpoIIAA, in turn, reacts with a complex of σF and the anti-σF factor SpoIIAB to effect the release of the transcription factor from its inhibitor (19). Thus, SpoIIE is a dual-function protein. It interacts with the cytokinetic machinery to promote polar division, and it becomes incorporated into the resulting polar septum, where it participates directly in the pathway leading to the activation in the forespore of the transcription factor σF. FIG. 1. Frequency of polar septation during vegetative growth and sporulation. (A) Schematic diagram representing SpoIIE. The roman numerals represent the membrane-spanning region (I), the central region (II), and the conserved PP2C-like phosphatase region (III). ... Here we are concerned with the role of SpoIIE in polar division. This is a challenging problem to address because asymmetric division is a composite consequence of the appearance of SpoIIE and a sporulation-specific increase in FtsZ levels. Thus, a mutant lacking SpoIIE but otherwise unimpaired in sporulation-specific FtsZ synthesis is only delayed in septum formation; it eventually produces polar septa at an efficiency of about 50% that of the wild type (5, 6, 20). Further complicating efforts to study the role of SpoIIE in polar division are its indirect effects on septation via its role in the activation of σF. The absence of σF activity in a SpoIIE mutant leads to the formation of aberrant (disporic) sporangia with septa at both poles (20), and premature activation of σF from excess SpoIIE activity prevents polar septation at either pole (8, 18). To investigate the role of SpoIIE in asymmetric division specifically, we took advantage of a previously described system for producing polar septa during growth (6). This system involves the use of cells that harbor an extra copy of the gene for FtsZ and are engineered to produce SpoIIE in response to an inducer. Such cells undergo asymmetric division robustly when expression of spoIIE is induced but at only a low level when it is not. Moreover, in such cells the contribution of SpoIIE to asymmetric division is uncoupled from its role in the activation of σF. Using this system, we find that neither the phosphatase activity of SpoIIE nor the membrane-spanning segments in its N-terminal region (I) contribute measurably to polar septation but that both the central region (II) of SpoIIE and at least one residue in the C-terminal region (III) play a critical role in the switch to polar division.