During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor cr F. Three proteins participate in the regulation of orF activity. The first, SpoIIAB, is an inhibitor of OrF, that is, an anti-or factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-or factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of orF activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.

SummaryThe actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

The spoIIE gene is essential for the compartment-specific activation of transcription factor F during sporulation in Bacillus subtilis. SpoIIE is a membrane protein that is targeted to the potential sites of asymmetric septation near each pole of the sporulating cell. The cytoplasmic carboxy-terminal domain of SpoIIE contains a serine phosphatase that triggers the release of F in the prespore compartment after septation. To understand how septum-located SpoIIE is activated selectively in the prespore, we examined the distribution of a SpoIIE-GFP fusion protein. We show that the polar bands of SpoIIE protein actually form sequentially and that the most prominent band develops at the pole where the prespore forms. We also show that the protein is sequestered to the prespore side of the asymmetric septum. Sequestration of SpoIIE into the prespore compartment provides a mechanism that could explain the cell specificity of F activation.

reviewed by Errington, 1996;Losick and Dworkin, 1999 and SpoIIAB (Gholamhoseinian and Piggot, 1989). At I.Lucet and A.Feucht contributed equally to this work first, σ F is held in an inactive complex by the anti-σ factor SpoIIAB (Duncan and Losick, 1993;Min et al., 1993; SpoIIE is a bifunctional protein with two critical roles Alper et al., 1994). SpoIIAB is also a protein kinase, in the establishment of cell fate in Bacillus subtilis.which phosphorylates the anti-anti-σ factor SpoIIAA on First, SpoIIE is needed for the normal formation of a specific serine residue, thereby maintaining it in an the asymmetrically positioned septum that forms early inactive state (Min et al., 1993;Diederich et al., 1994; in sporulation and separates the mother cell from the Najafi et al., 1995;Duncan et al., 1996; Magnin et al., prespore compartment. Secondly, SpoIIE is essential 1996). After asymmetric septation, σ F remains inactive in for the activation of the first compartment-specific the mother cell, but in the prespore it is released from transcription factor σ F in the prespore. After initiation SpoIIAB (Margolis et al., 1991). This release is triggered of sporulation, SpoIIE localizes to the potential asymby SpoIIE, a membrane-bound phosphatase, which metric cell division sites near one or both cell poles.dephosphorylates SpoIIAA-P and allows it to bind Localization of SpoIIE was shown to be dependent on SpoIIAB, thereby releasing σ F (Duncan et al., 1995; the essential cell division protein FtsZ. To understand Arigoni et al., 1996; Feucht et al., 1996). σ F is then how SpoIIE is targeted to the asymmetric septum we free to associate with core RNA polymerase and direct have now analysed its interaction with FtsZ in vitro.transcription. Thus, SpoIIE plays a key role in σ F activation Using the yeast two-hybrid system and purified FtsZ, by regulating the phosphorylation state of SpoIIAA. and full-length and truncated SpoIIE proteins, we SpoIIE is an integral membrane protein and has a multidemonstrate that the two proteins interact directly and domain structure (Figure 1) ; Arigoni that domain II and possibly domain I of SpoIIE are et al., 1999). It contains 10 membrane-spanning segments required for the interaction. Moreover, we show that in its N-terminal domain (domain I), which targets the SpoIIE interacts with itself and suggest that this selfprotein to the membrane. The large central domain interaction plays a role in assembly of SpoIIE into the (domain II) is poorly conserved and its function is division machinery.unknown. The C-terminal domain (domain III) contains Keywords: Bacillus subtilis/cell division/oligomerization/ serine phosphatase activity and is structurally related to protein-protein interaction/sporulation eukaryotic PP2C protein phosphatases (Adler et al., 1997). PP2C phosphatases are involved in regulating stress response pathways in both prokaryotes and eukaryotes (Das et al., 1996). SpoIIE is synthesized before asymmetric

Differentiation in the spore-forming bacterium Bacillus subtilis is governed by the sequential activation of five sporulation-specific transcription factors. The early mother-cell-specific transcription factor, s E , directs the transcription of many genes that contribute to the formation of mature, dormant spores. In this study, DNA microarrays were used to identify genes belonging to the s E regulon. In total, 171 genes were found to be under the control of s E . Of these, 101 genes had not previously been described as being s E dependent. Disruption of some of the previously unknown genes ( ydcC, yhaL, yhbH, yjaV and yqfD) resulted in a defect in sporulation.