A Threshold Mechanism Governing Activation of the Developmental Regulatory Protein σF in Bacillus subtilis

Abstract

The RNA polymerase sigma factor F is a developmental regulatory protein that is activated in a cell-specific manner following the formation of the polar septum during the process of spore formation in the bacterium Bacillus subtilis. Activation of F depends on the membrane-bound phosphatase SpoIIE, which localizes to the septum, and on the formation of the polar septum itself. SpoIIE is responsible for dephosphorylating and thereby activating the phosphoprotein SpoIIAA, which, in turn, triggers the release of F from the anti-F factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause F activation as SpoIIAA reaches substantial levels in mutants blocked in polar septation. We now describe mutants of SpoIIE, SpoIIAA, and SpoIIAB that break the dependence of F activation on polar division. Analysis of these mutants indicates that unphosphorylated SpoIIAA must reach a threshold concentration in order to trigger the release of F from SpoIIAB. Evidence is presented that this threshold is created by the action of SpoIIAB, which can form an alternative, long lived complex with SpoIIAA. We propose that formation of the SpoIIAASpoIIAB complex serves as a sink that traps SpoIIAA in an inactive state and that only when unphosphorylated SpoIIAA is in excess to the sink does activation of F take place.

Compartmentalization of Gene Expression duringBacillus subtilisSpore Formation

“…Consistent with the checkpoint model, a number of studies have found missense mutations in SpoIIE that uncouple asymmetric division from F activation (32,73,111). Indeed, one study tested the in vitro phosphatase activity of the mutant proteins and found that it was similar to that of the wild-type protein (73), further supporting the concept that SpoIIE is subject to a phosphatase-independent regulatory step that couples asymmetric division to F activation.…”

“…However, how the cell prevented the dephosphorylated SpoIIAA from activating F in the predivisional cell remained mysterious. A recent study has provided evidence that SpoIIAB, it its ADP-bound state, can serve as a "sink" absorbing SpoIIAA in the cell until a threshold level is reached (32). Precisely why asymmetric division allows the threshold level of dephosphorylated SpoIIAA to be crossed in the prespore is unknown; some speculate that the phosphatase activity of SpoIIE is stimulated by asymmetric division (73), whereas others think that equivalent inheritance of SpoIIE in the two compartments creates a favorable SpoIIE/SpoIIAA-PO 4 ratio in the prespore (32).…”

FisB mediates membrane fission during sporulation in Bacillus subtilis

“…Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto Immobilon-P membranes (Millipore), and blocked in 5% nonfat milk in phosphate-buffered saline (PBS) and 0.5% Tween-20. The blocked membranes were probed with affinity-purified anti-His (1:1000; Santa Cruz Biotechnology), anti-FisB (1:10,000), and anti-s F (15,000) (Carniol et al 2004) diluted into 3% BSA in 13 PBS-0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat anti-rabbit G (Bio-Rad) and the Western Lightning reagent kit as described by the manufacturer (PerkinElmer).…”

First-passage time approach to controlling noise in the timing of intracellular events

“…T iming of events in many cellular processes, such as cell-cycle control (1-3), cell differentiation (4,5), sporulation (6,7), apoptosis (8,9), development (10,11), temporal order of gene expression (12)(13)(14), and so on, depend on regulatory proteins reaching critical threshold levels. Triggering of these events in single cells is influenced by fluctuations in protein levels that arise naturally due to noise in gene expression (15)(16)(17)(18).…”