SpoIIE Phosphatase Research Articles

Cell division machinery drives cell-specific gene activation during differentiation in Bacillus subtilis

When faced with starvation, the bacterium Bacillus subtilis transforms itself into a dormant cell type called a "spore". Sporulation initiates with an asymmetric division event, which requires the relocation of the core divisome components FtsA and FtsZ, after which the sigma factor σF is exclusively activated in the smaller daughter cell. Compartment-specific activation of σF requires the SpoIIE phosphatase, which displays a biased localization on one side of the asymmetric division septum and associates with the structural protein DivIVA, but the mechanism by which this preferential localization is achieved is unclear. Here, we isolated a variant of DivIVA that indiscriminately activates σF in both daughter cells due to promiscuous localization of SpoIIE, which was corrected by overproduction of FtsA and FtsZ. We propose that the core components of the redeployed cell division machinery drive the asymmetric localization of DivIVA and SpoIIE to trigger the initiation of the sporulation program.

Metal-dependent SpoIIE oligomerization stabilizes FtsZ during asymmetric division in Bacillus subtilis.

SpoIIE is a bifunctional protein involved in asymmetric septum formation and in activation of the forespore compartment-specific transcription factor σF through dephosphorylation of SpoIIAA-P. The phosphatase activity of SpoIIE requires Mn2+ as a metal cofactor. Here, we show that the presence of a metal cofactor also influences SpoIIE oligomerization and asymmetric septum formation. Absence of Mn2+ from sporulation medium results in a delay of the formation of polar FtsZ-rings, similar to a spoIIE null mutant. We purified the entire cytoplasmic part of the SpoIIE protein, and show that the protein copurifies with bound metals. Metal binding both stimulates SpoIIE oligomerization, and results in the formation of larger oligomeric structures. The presence of SpoIIE oligomers reduces FtsZ GTP hydrolysis activity and stabilizes FtsZ polymers in a light scattering assay. Combined, these results indicate that metal binding is not just required for SpoIIE phosphatase activity but also is important for SpoIIE's role in asymmetric septum formation.

A Membrane-Embedded Amino Acid Couples the SpoIIQ Channel Protein to Anti-Sigma Factor Transcriptional Repression during Bacillus subtilis Sporulation.

SpoIIQ is an essential component of a channel connecting the developing forespore to the adjacent mother cell during Bacillus subtilis sporulation. This channel is generally required for late gene expression in the forespore, including that directed by the late-acting sigma factor σ(G) Here, we present evidence that SpoIIQ also participates in a previously unknown gene regulatory circuit that specifically represses expression of the gene encoding the anti-sigma factor CsfB, a potent inhibitor of σ(G) The csfB gene is ordinarily transcribed in the forespore only by the early-acting sigma factor σ(F) However, in a mutant lacking the highly conserved SpoIIQ transmembrane amino acid Tyr-28, csfB was also aberrantly transcribed later by σ(G), the very target of CsfB inhibition. This regulation of csfB by SpoIIQ Tyr-28 is specific, given that the expression of other σ(F)-dependent genes was unaffected. Moreover, we identified a conserved element within the csfB promoter region that is both necessary and sufficient for SpoIIQ Tyr-28-mediated inhibition. These results indicate that SpoIIQ is a bifunctional protein that not only generally promotes σ(G)activity in the forespore as a channel component but also specifically maximizes σ(G)activity as part of a gene regulatory circuit that represses σ(G)-dependent expression of its own inhibitor, CsfB. Finally, we demonstrate that SpoIIQ Tyr-28 is required for the proper localization and stability of the SpoIIE phosphatase, raising the possibility that these two multifunctional proteins cooperate to fine-tune developmental gene expression in the forespore at late times. Cellular development is orchestrated by gene regulatory networks that activate or repress developmental genes at the right time and place. Late gene expression in the developing Bacillus subtilis spore is directed by the alternative sigma factor σ(G) The activity of σ(G)requires a channel apparatus through which the adjacent mother cell provides substrates that generally support gene expression. Here we report that the channel protein SpoIIQ also specifically maximizes σ(G)activity as part of a previously unknown regulatory circuit that prevents σ(G)from activating transcription of the gene encoding its own inhibitor, the anti-sigma factor CsfB. The discovery of this regulatory circuit significantly expands our understanding of the gene regulatory network controlling late gene expression in the developing B. subtilis spore.

Complexity of bacterial phosphorylation interaction network.

Complexity of bacterial phosphorylation interaction network.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

Characterization of a novel inhibitory feedback of the anti-anti-sigma SpoIIAA on Spo0A activation during development in Bacillus subtilis.

Compartmentalized gene expression during sporulation is initiated after asymmetric division by cell-specific activation of the transcription factors sigmaF and sigmaE. Synthesis of these sigma factors, and their regulatory proteins, requires the activation (phosphorylation) of Spo0A by the phosphorelay signalling system. We report here a novel regulatory function of the anti-anti-sigmaF SpoIIAA as inhibitor of Spo0A activation. This effect did not require sigmaF activity, and it was abolished by expression of the phosphorelay-independent form Spo0A-Sad67 indicating that SpoIIAA directly interfered with Spo0A approximately P generation. IPTG-directed synthesis of the SpoIIE phosphatase in a strain carrying a multicopy plasmid coding for SpoIIAA and its specific inhibitory kinase SpoIIAB blocked Spo0A activation suggesting that the active form of the inhibitor was SpoIIAA and not SpoIIAA-P. Furthermore, expression of the non-phosphorylatable mutant SpoIIAAS58A (SpoIIAA-like), but not SpoIIAAS58D (SpoIIAA-P-like), completely blocked Spo0A-dependent gene expression. Importantly, SpoIIAA expressed from the chromosome under the control of its normal spoIIA promoter showed the same negative effect regulated not only by SpoIIAB and SpoIIE but also by septum morphogenesis. These findings are discussed in relation to the potential contribution of this novel inhibitory feedback with the proper activation of sigmaF and sigmaE during development.

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.

Septation, dephosphorylation, and the activation of sigmaF during sporulation in Bacillus subtilis.

Cell-specific activation of transcription factor sigmaF during sporulation in Bacillus subtilis requires the formation of the polar septum and the activity of a serine phosphatase (SpoIIE) located in the septum. The SpoIIE phosphatase indirectly activates sigmaF by dephosphorylating a protein (SpoIIAA-P) in the pathway that controls the activity of the transcription factor. By use of a SpoIIE-GFP fusion protein in time-course and time-lapse experiments and by direct visualization of septa in living cells, we show that SpoIIE is present in the predivisional sporangium, where it often localizes near both cell poles in structures known as E-rings. We also present evidence consistent with the view that SpoIIE is present in both progeny cells after polar division. These findings are incompatible with a model for the control of sigmaF activity in which the phosphatase is simply sequestered to one cell. Instead, we conclude that the function of SpoIIE is subject to regulation, and we present evidence that this occurs in two stages. The first stage, which involves the phosphatase function of SpoIIE, depends on the cell division protein FtsZ and could correspond to the FtsZ-dependent assembly of SpoIIE into E-rings. The second stage occurs after the dephosphorylation of SpoIIAA-P and is dependent on the later-acting, cell-division protein DivIC. Evidence based on the use of modified and mutant forms of the phosphatase protein indicates that SpoIIE blocks the capacity of unphosphorylated SpoIIAA to activate sigmaF until formation of the polar septum is completed.

The Bacillus subtilis regulator protein SpoIIE shares functional and structural similarities with eukaryotic protein phosphatases 2C

Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn 2+ or Mg 2+. Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins. D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity. The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2Cα can also be applied to the bacterial protein phosphatase 2C-like protein.

The SpoIIE phosphatase, the sporulation septum and the establishment of forespore-specific transcription in Bacillus subtilis: a reassessment.

Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the sigma F factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of sigma F is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant sigma F activity, indicating that the septum requirement for activating sigma F can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.

Transient gene asymmetry during sporulation and establishment of cell specificity in Bacillus subtilis.

Sporulation in Bacillus subtilis is initiated by an asymmetric division generating two cells of different size and fate. During a short interval, the smaller forespore harbors only 30% of the chromosome until the remaining part is translocated across the septum. We demonstrate that moving the gene for sigmaF, the forespore-specific transcription factor, in the trapped region of the chromosome is sufficient to produce spores in the absence of the essential activators SpoIIAA and SpoIIE. We propose that transient genetic asymmetry is the device that releases SpoIIE phosphatase activity in the forespore and establishes cell specificity.

Establishment of prespore-specific gene expression in Bacillus subtilis: localization of SpoIIE phosphatase and initiation of compartment-specific proteolysis.

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor sigma E and the other dependent on the prespore-specific sigma factor sigma F.

Prespore-specific gene expression in Bacillus subtilis is driven by sequestration of SpoIIE phosphatase to the prespore side of the asymmetric septum.

The spoIIE gene is essential for the compartment-specific activation of transcription factor sigmaF 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 sigmaF 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 sigmaF activation.

Disappearance of the sigma E transcription factor from the forespore and the SpoIIE phosphatase from the mother cell contributes to establishment of cell-specific gene expression during sporulation in Bacillus subtilis.

We used immunofluorescence microscopy to investigate mechanisms governing the establishment of cell-specific gene transcription during sporulation in the bacterium Bacillus subtilis. The transcription factors sigma E and sigma F are synthesized shortly after the start of sporulation but do not become active in directing gene transcription until after polar division, when the activity of sigma E is confined to the mother cell and the activity of sigma F is restricted to the forespore. We show that shortly after septation, sigma E and its proprotein precursor pro-sigma E appear to be absent from the forespore and that a null mutation in spoIIIE, a gene known to be required for the translocation of a chromosome into the forespore, allows sigma E and/or pro-sigma E to persist and sigma E to become active in the forespore. These findings suggest that the loss of sigma E/pro-sigma E from the forespore contributes to the compartmentalization of sigma E-directed gene transcription. We also investigated the distribution of SpoIIE, a regulatory phosphatase required for the activation of sigma F which exhibits a bipolar pattern of localization shortly after the start of sporulation. Normally, SpoIIE rapidly disappears from the sporangium, first from the mother-cell pole and then from the forespore pole. Here we show that a null mutation in spoIIIE causes the SpoIIE phosphatase to persist at both poles. The persistence of the SpoIIE phosphatase at the mother-cell pole could explain the lack of compartmentalization of sigma F activity observed in a spoIIIE null mutant. We conclude that the establishment of cell-specific gene transcription involves the loss of sigma E/pro-sigma E from the forespore and the loss of the SpoIIE phosphatase from the mother-cell pole and that both processes are dependent upon the SpoIIIE protein.

Compartmentalized distribution of the proteins controlling the prespore-specific transcription factor sigmaF of Bacillus subtilis.

Differential gene expression during sporulation in the prespore and mother cell of Bacillus subtilis is dependent on the correct timing and localization of the activity of specific transcription (sigma) factors. The first sigma factor activated is sigmaF, which directs gene expression specifically in the prespore compartment. Release of sigmaF activity is tightly controlled through a series of complex interactions involving an anti-sigma factor, SpoIIAB, an anti-anti-sigma factor SpoIIAA and a phosphoprotein phosphatase SpoIIE. In vitro studies have shown that SpoIIAB binds to sigmaF, preventing transcription of the sigmaF regulon, and that it can also phosphorylate SpoIIAA, thereby inactivating it. However, non-phosphorylated SpoIIAA can displace sigmaF from SpoIIAB. The SpoIIE phosphatase provides a means of reactivating SpoIIAA-P. We have directly determined the cellular distributions of sigmaF, SpoIIAB, SpoIIAA-P and SpoIIAA during sporulation, using recently developed immunofluorescence methods. While sigmaF activity is restricted to the prespore, the protein is present in both compartments. As development proceeds the sigmaF signal disappears. The anti-sigma factor SpoIIAB is also distributed throughout both cells and rapidly disappears from both cellular compartments soon after sigmaF becomes active. Disappearance of SpoIIAB seems to be closely associated with the activation of the second prespore-specific sigma factor sigmaF. The distribution of phosphorylated SpoIIAA closely mimics that of SpoIIAB, being non-compartmentalized and disappearing soon after sigmaF activation occurs. Significantly, the active, non-phosphorylated form of the anti-anti-sigma factor, SpoIIAA, accumulates in the prespore just before sigmaF becomes active. These results support the hypothesis that the accumulation of SpoIIAA within the prespore is the single most important requirement for activation of sigmaF.