Structure of the Bacillus cell fate determinant SpoIIAA in phosphorylated and unphosphorylated forms

BACKGROUND: The asymmetric cell division during sporulation in Bacillus subtilis gives rise to two compartments: the mother cell and the forespore. Each follow different programs of gene expression coordinated by a succession of alternate RNA polymerase sigma factors. The activity of the first of these sigma factors, sigmaF, is restricted to the forespore although sigmaF is present in the predivisional cell and partitions into both compartments following the asymmetric septation. For sigmaF to become active, it must escape from a complex with its cognate anti-sigma factor, SpoIIAB. This relief from SpoIIAB inhibition requires the dephosphorylation of the anti-sigma factor antagonist, SpoIIAA. The phosphorylation state of SpoIIAA is thus a key determinant of sigmaF activity and cell fate. RESULTS: We have solved the crystal structures of SpoIIAA from Bacillus sphaericus in its phosphorylated and unphosphorylated forms. The overall structure consists of a central beta-pleated sheet, one face of which is buried by a pair of alpha helices, while the other is largely exposed to solvent. The site of phosphorylation, Ser57, is located at the N terminus of helix alpha2. The phosphoserine is exceptionally well defined in the 1.2 A electron density maps, revealing that the structural changes accompanying phosphorylation are slight. CONCLUSIONS: Comparison of unphosphorylated and phosphorylated SpoIIAA shows that covalent modification has no significant effect on the global structure of the protein. The phosphoryl group has a passive role as a negatively charged flag rather than the active role it plays as a nucleus of structural reorganization in many eukaryotic signaling systems.     

Efficient regulation of sigmaF, the first sporulation-specific sigma factor in B.subtilis

Differential gene expression is established in the prespore and mother-cell compartments of Bacillus subtilis through the successive activation of a series of cell-type-specific sigma factors. Crucial to the success of this process is the control of the first prespore-specific sigma factor, sigmaF. sigmaF is regulated by the proteins SpoIIAB, SpoIIAA and SpoIIE. SpoIIAB forms an inhibitory complex with sigmaF, which can be dissociated by interaction with SpoIIAA. During this interaction SpoIIAA is phosphorylated. SpoIIE is a membrane-bound phosphatase that dephosphorylates SpoIIAA, thereby re-activating it. It is not understood how sigmaF is activated specifically in the prespore but not in the mother cell. Here, we use a recently developed fluorescence spectroscopy technique to follow in real time the formation of sigmaF.SpoIIAB complexes and their dissociation by SpoIIAA. We show that complete activation of sigmaF is induced by a tenfold increase in SpoIIE activity. This result demonstrates that relatively small changes in SpoIIE activity, which could arise from asymmetric septation, can achieve the all-or-nothing response in sigmaF activity required by the cell. For long-term sigmaF activation, we find that sustained SpoIIE activity is required to counteract the activity of SpoIIAB. Even though the continual phosphorylation and dephosphorylation of SpoIIAA by these two enzymes will expend some ATP, the formation of SpoIIAA.SpoIIAB.ADP complexes greatly diminishes the rate of the phosphorylation reaction, and thus minimizes the wastage of energy. These features provide a very efficient system for regulating sigmaF.     

Characterization of a morphological checkpoint coupling cell-specific transcription to septation in Bacillus subtilis

Early in the process of spore formation in Bacillus subtilis, asymmetric cell division produces a large mother cell and a much smaller prespore. Differentiation of the prespore is initiated by activation of an RNA polymerase sigma factor, sigmaF, specifically in that cell. sigmaF is controlled by a regulatory cascade involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor, SpoIIAA, and a membrane-bound phosphatase, SpoIIE, which converts the inactive, phosphorylated form of SpoIIAA back to the active form. SpoIIE is required for proper asymmetric division and much of the protein is sequestered into the prespore during septation. Importantly, activation of sigmaF is dependent on formation of the asymmetric septum. We have now characterized this morphological checkpoint in detail, using strains affected in cell division and/or spoIIE function. Surprisingly, we found that significant dephosphorylation of SpoIIAA occurred even in the absence of septation. This shows that the SpoIIE phosphatase is at least partially active independent of the morphological event and also that cells can tolerate significant levels of unphosphorylated SpoIIAA without activating sigmaF. We also describe a spoIIE mutant in which the checkpoint is bypassed, probably by an increase in the dephosphorylation of SpoIIAA. Taken together, the results support the idea that sequestration of SpoIIE protein into the prespore plays an important role in the control of sigmaF activation and in coupling this activation to septation.     

Fluorescence and kinetic analysis of the SpoIIAB phosphorylation reaction, a key regulator of sporulation in Bacillus subtilis

Sporulation in Bacillus subtilis provides a valuable model system for studying differential gene expression. The anti-sigma factor SpoIIAB is a bifunctional protein, responsible for regulating the activity of the first sporulation-specific sigma factor, sigma(F). SpoIIAB can either bind to (and thus inhibit) sigma(F) or phosphorylate the anti-anti-sigma factor SpoIIAA. The phosphorylation reaction follows an unusual time course in which a pre-steady-state phase is succeeded by a slower steady-state phase. Previous experiments have shown that in the steady-state phase SpoIIAB is unable to inhibit sigma(F). A fluorescent derivative of SpoIIAB (AB-F97W) was made that was indistinguishable from the wild type in its interactions with SpoIIAA and sigma(F). AB-F97W exhibited distinctive changes in its fluorescence intensity when bound to ATP, ADP, or SpoIIAA. By following changes in the fluorescence properties of AB-F97W during the phosphorylation reaction, we confirmed a previous hypothesis that during the steady-state phase the predominant species are SpoIIAA.SpoIIAB.ADP complexes. The formation of these complexes is responsible for the slowing of the reaction, an important feature during sporulation since it reduces the loss of ATP in the nutrient-deprived cell. We also show that, to form a complex with SpoIIAA and ADP during the reaction, SpoIIAB must undergo a change in state which increases its affinity for ADP, and that this change in state is stimulated by its interaction with SpoIIAA. We derive a model of the reaction using previously determined kinetic and binding constants, and relate these findings to the known structure of SpoIIAB.     

Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis.

Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [gamma-32P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58.     

Novel Mycobacterium tuberculosis anti-sigma factor antagonists control sigmaF activity by distinct mechanisms

The aetiological agent of tuberculosis, Mycobacterium tuberculosis, encodes 13 sigma factors, as well as several putative anti-, and anti-anti- sigma factors. Here we show that a sigma factor that has been previously shown to be involved in virulence and persistence processes, sigmaF, can be specifically inhibited by the anti-sigma factor UsfX. Importantly, the inhibitory activity of UsfX, in turn, can be negatively regulated by two novel anti-anti-sigma factors. The first anti-anti-sigma factor seems to be regulated by redox potential, and the second may be regulated by phosphorylation as it is rendered non-functional by the introduction of a mutation that is believed to mimic phosphorylation of the anti-anti-sigma factor. These results suggest that sigmaF activity might be post-translationally modulated by at least two distinct pathways in response to different possible physiological cues, the outcome being consistent with the bacteria's ability to adapt to diverse host environments during disease progression, latency and reactivation.     

A threshold mechanism governing activation of the developmental regulatory protein sigma F in Bacillus subtilis

The RNA polymerase sigma factor sigma(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 sigma(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 sigma(F) from the anti-sigma(F) factor SpoIIAB. Paradoxically, however, the presence of unphosphorylated SpoIIAA is insufficient to cause sigma(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 sigma(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 sigma(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 SpoIIAA-SpoIIAB 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 sigma(F) take place.     

A possible extended family of regulators of sigma factor activity in Streptomyces coelicolor

SCO4677 is one of a large number of similar genes in Streptomyces coelicolor that encode proteins with an HATPase_c domain resembling that of anti-sigma factors such as SpoIIAB of Bacillus subtilis. However, SCO4677 is not located close to genes likely to encode a cognate sigma or anti-anti-sigma factor. SCO4677 was found to regulate antibiotic production and morphological differentiation, both of which were significantly enhanced by the deletion of SCO4677. Through protein-protein interaction screening of candidate sigma factor partners using the yeast two-hybrid system, SCO4677 protein was found to interact with the developmentally specific σ^F, suggesting that it is an antagonistic regulator of σ^F. Two other proteins, encoded by SCO0781 and SCO0869, were found to interact with the SCO4677 anti-σ^F during a subsequent global yeast two-hybrid screen, and the SCO0869-SCO4677 protein-protein interaction was confirmed by coimmunoprecipitation. The SCO0781 and SCO0869 proteins resemble well-known anti-anti-sigma factors such as SpoIIAA of B. subtilis. It appears that streptomycetes may possess an extraordinary abundance of anti-sigma factors, some of which may influence diverse processes through interactions with multiple partners: a novel feature for such regulatory proteins.     

Differential gene expression in genetically identical sister cells: the initiation of sporulation in Bacillus subtilis

Early in sporulation, the cell divides asymmetrically to give two sister compartments, a smaller prespore and a larger mother cell. Differential gene expression in these compartments depends on the regulation of the first sporulation-specific sigma factor, sigma(F), which is activated only in the prespore. Regulation relies on the interactions of four proteins -sigma(F), its antisigma SpoIIAB (which also has protein kinase activity), the anti-antisigma SpoIIAA and the protein phosphatase SpoIIE. Before asymmetric division, and in the mother cell after division, sigma(F) is held in an inactive complex with SpoIIAB and ATP; SpoIIAA is in its phosphorylated form. To disrupt the complex so as to liberate sigma(F) in the prespore, dephosphorylated SpoIIAA is needed, and this is made available by SpoIIE. Thereafter, SpoIIAB and SpoIIE are active simultaneously in the prespore, cycling SpoIIAA through phosphorylated and non-phosphorylated forms. This cycle detains SpoIIAB in a state in which it cannot inhibit sigma(F). Results from biophysical techniques, mathematical simulations and enzyme kinetics have now helped to elucidate the dynamics of the protein-protein interactions involved. An understanding of these dynamics largely accounts for the regulation of sigma(F). We show that the system is tuned to be highly efficient in its use of components and extremely economical in conserving ATP.