| This thesis addresses the connection between a determinant of cell fate and the formation of a septum in the bacterium Bacillus subtilis. The cell fate determinant is SpoIIE, a membrane bound phosphatase that localizes to the septum and that couples the activation of the developmental transcription factor sigmaF to septum formation. Work reported here and elsewhere reveals that the restriction of SpoIIE to the septum develops in stages. In the predivisional sporangium, SpoIIE assembles into rings (E-rings) coincident with rings of the cell division protein FtsZ (Z-rings) near both cell poles. As one polar Z-ring constricts to form the sporulation septum, SpoIIE invades the septum at that pole and subsequently disappears from the distal pole.;A key feature of sigmaF regulation is that after the formation of the asymmetrically-positioned sporulation septum, sigma F directs gene expression only in the smaller of the two daughter cells. One model for the compartmentalization of sigmaF activity requires the sequestration of SpoIIE to the smaller cell after septation. Instead, work presented here indicates that the total level of SpoIIE in each daughter cell is comparable and that the concentration of SpoIIE in the smaller cell exceeds that in the larger cell. This observation suggests that the cell-specificity of sigmaF activation stems in part either from the high concentration of SpoIIE in the smaller cell or the presence of an inhibitor in the larger cell.;SpoIIE contains a large N-terminal membrane spanning domain and a soluble C-terminal phosphatase domain. I have shown that both are needed for SpoIIE localization and I have identified a patch of residues (Y622 D686 L687) in the soluble domain that contributes to SpoIIE localization and phosphatase function. The localization of some mutants was improved in the presence of wild type SpoIIE, suggesting that molecules of SpoIIE interact.;The SpoIIE phosphatase activates sigmaF indirectly by converting SpoIIAA-P to SpoIIAA, which then induces the release of sigma F from an inhibitory protein. By examining SpoIIE-dependent dephosphorylation of SpoIIAA-P and sigmaF activity in mutants blocked at septation, I have discovered that sigmaF activity is coupled to septation in two steps. First, in the predivisional cell and partially dependent upon the assembly of Z-rings, SpoIIE dephosphorylates SpoIIAA-P. Evidence for the second step of sigmaF regulation comes from the observation that a mutant that is blocked at a late stage of septation accumulates high levels of unphosphorylated SpoIIAA, even though sigmaF fails to become active. From this and from experiments using mutant and modified forms of SpoIIE, I conclude that sigmaF is regulated by SpoIIE at a step subsequent to the dephosphorylation of SpoIIAA-P. |
