The mechanism of cell differentiation in Bacillus subtilis.

Sporulation in Bacillus subtilis serves as a model for the development of two different cell types from a single cell. Although much information has been accumulated about the mechanisms that initiate the developmental programmes, important questions remain that can be answered only by quantitative analysis. Here we develop, with the help of existing and new experimental results, a mathematical model that reproduces published in vitro experiments and explains how the activation of the key transcription factor is regulated. The model identifies the difference in volume between the two cell types as the primary trigger for determining cell fate. It shows that this effect depends on the allosteric behaviour of a key protein kinase and on a low rate of dephosphorylation by the corresponding phosphatase; both predicted effects are confirmed experimentally.

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.

Physical evidence for the induced release of the Bacillus subtilis transcription factor, sigma(F), from its inhibitory complex.

The release of the transcription factor sigma(F) from its inhibitory complex with SpoIIAB is a key regulatory step in the control of sporulation in Bacillus subtilis as it initiates a pattern of differential gene expression in the mother cell and prespore compartments. The sigma(F).SpoIIAB complex is dissociated by the unphosphorylated form of the protein SpoIIAA, the alternative binding partner of SpoIIAB. Here, we employ fluorescence spectroscopy to examine the mechanism by which SpoIIAA acts on the sigma(F).SpoIIAB complex. We constructed a mutant of sigma(F), sigma(F)-W46L, which displayed a reproducible fluorescence response on binding to SpoIIAB. Using this mutant we were able to quantify the amount of sigma(F) bound to SpoIIAB in real time. The results provide physical evidence for the "induced release" mechanism of sigma(F) activation. We demonstrate that SpoIIAA interacts directly with the sigma(F).SpoIIAB complex, greatly decreasing the affinity of SpoIIAB for sigma(F) and thus causing the release of the latter. We also demonstrate that sigma(F) is released before SpoIIAA is phosphorylated and that release occurs on a similar time scale to the binding of SpoIIAA to SpoIIAB.

Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor sigmaF in Bacillus subtilis.

SigmaF, the first compartment-specific sigma factor of sporulation, is regulated by an anti-sigma factor, SpoIIAB (AB) and its antagonist SpoIIAA (AA). AB can bind to sigmaF in the presence of ATP or to AA in the presence of ADP; in addition, AB can phosphorylate AA. The ability of AB to switch between its two binding partners regulates sigmaF. Early in sporulation, AA activates sigmaF by releasing it from its complex with AB. We have previously proposed a reaction scheme for the phosphorylation of AA by AB which accounts for AA's regulatory role. A crucial feature of this scheme is a conformational change in AB that accompanies its switch in binding partner. In the present study, we have studied three AB mutants, all of which have amino-acid replacements in the nucleotide-binding region; AB-E104K (Glu104-->Lys) and AB-T49K (Thr49-->Lys) fail to activate sigmaF, and AB-R105A (Arg105-->Ala) activates it prematurely. We used techniques of enzymology, surface plasmon resonance and fluorescence spectroscopy to analyse the defects in each mutant. AB-E104K was deficient in binding to AA, AB-T49K was deficient in binding to ADP and AB-R105A bound ADP exceptionally strongly. Although the release of sigmaF from all three mutant proteins was impaired, and all three failed to undergo the wild-type conformational change when switching binding partners, the phenotypes of the mutant cells were best accounted for by the properties of the respective AB species in forming complexes with AA and ADP. The behaviour of the mutants enables us to propose convincing mechanisms for the regulation of sigmaF in wild-type bacteria.

High-molecular-weight complexes of RsbR and paralogues in the environmental signaling pathway of Bacillus subtilis.

Bacillus subtilis has developed an intricate signal transduction cascade to respond to the imposition of a variety of stresses on the cell. Reversible protein phosphorylation and the formation of alternative protein-protein complexes modulate the activity of sigma(B), the RNA polymerase sigma factor subunit responsible for the transcription of the general stress response genes. Some of the regulators of sigma(B), such as RsbR and RsbS, are known to associate in a 25S complex, called the stressosome, that can bind RsbT until RsbT phosphorylates target residues in RsbR and RsbS. To date, the RsbR-RsbS complex appears to be the most upstream component of the sigma(B) regulatory pathway. This large structure is thought to play an important role in sensing and/or integrating signals from different physical stresses. The roles of the paralogues of RsbR that are found in B. subtilis remain unclear. We describe here how the RsbR paralogues copurify with RsbR from B. subtilis cell lysates, and we demonstrate in vitro that the paralogues form large complexes either with RsbS or with a prepurified RsbR-RsbS binary complex. We conclude from these biochemical studies that stressosomes in B. subtilis cells contain minimally RsbS and all of the RsbT-phosphorylatable RsbR paralogues.

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.

Phosphorylation induces subtle structural changes in SpoIIAA, a key regulator of sporulation.

The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis. Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation. The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation. These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins. We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties.

Phosphorylation and RsbX-dependent dephosphorylation of RsbR in the RsbR-RsbS complex of Bacillus subtilis.

In the pathway that controls sigmaB activity, the RsbR-RsbS complex plays an important role by trapping RsbT, a positive regulator of sigmaB of Bacillus subtilis. We have proposed that at the onset of stress, RsbR becomes phosphorylated, resulting in an enhanced activity of RsbT towards RsbS. RsbT is then free to interact with and activate RsbU, which in turn ultimately activates sigmaB. In this study with purified proteins, we used mutant RsbR proteins to analyze the role of its phosphorylatable threonine residues. The results show that the phosphorylation of either of the two RsbT-phosphorylatable threonine residues (T171 and T205) in RsbR enhanced the kinase activity of RsbT towards RsbS. However, it appeared that RsbT preferentially phosphorylates T171. We also present in vitro evidence that identifies RsbX as a potential phosphatase for RsbR T205.

Evaluation of the kinetic properties of the sporulation protein SpoIIE of Bacillus subtilis by inclusion in a model membrane.

Starvation induces Bacillus subtilis to initiate a developmental process (sporulation) that includes asymmetric cell division to form the prespore and the mother cell. The integral membrane protein SpoIIE is essential for the prespore-specific activation of the transcription factor sigmaF, and it also has a morphogenic activity required for asymmetric division. An increase in the local concentration of SpoIIE at the polar septum of B. subtilis precedes dephosphorylation of the anti-anti-sigma factor SpoIIAA in the prespore. After closure and invagination of the asymmetric septum, phosphatase activity of SpoIIE increases severalfold, but the reason for this dramatic change in activity has not been determined. The central domain of SpoIIE has been seen to self-associate (I. Lucet et al., EMBO J. 19:1467-1475, 2000), suggesting that activation of the C-terminal PP2C-like phosphatase domain might be due to conformational changes brought about by the increased local concentration of SpoIIE in the sporulating septum. Here we report the inclusion of purified SpoIIE protein into a model membrane as a method for studying the effect of local concentration in a lipid bilayer on activity. In vitro assays indicate that the membrane-bound enzyme maintains dephosphorylation rates similar to the highly active micellar state at all molar ratios of protein to lipid. Atomic force microscopy images indicate that increased local concentration does not lead to self-association.

Protein-protein interactions that regulate the energy stress activation of sigma(B) in Bacillus subtilis.

Sigma(B) is an alternative sigma factor that controls the general stress response in Bacillus subtilis. In the absence of stress, sigma(B) is negatively regulated by anti-sigma factor RsbW. RsbW is also a protein kinase which can phosphorylate RsbV. When cells are stressed, RsbW binds to unphosphorylated RsbV, produced from the phosphorylated form of RsbV by two phosphatases (RsbU and RsbP) which are activated by stress. We now report the values of the K(m) for ATP and the K(i) for ADP of RsbW (0.9 and 0.19 mM, respectively), which reinforce the idea that the kinase activity of RsbW is directly regulated in vivo by the ratio of these nucleotides. RsbW, purified as a dimer, forms complexes with RsbV and sigma(B) with different stoichiometries, i.e., RsbW(2)-RsbV(2) and RsbW(2)-sigma(B)(1). As determined by surface plasmon resonance, the dissociation constants of the RsbW-RsbV and RsbW-sigma(B) interactions were found to be similar (63 and 92 nM, respectively). Nonetheless, an analysis of the complexes by nondenaturing polyacrylamide gel electrophoresis in competition assays suggested that the affinity of RsbW(2) for RsbV is much higher than that for sigma(B). The intracellular concentrations of RsbV, RsbW (as a monomer), and sigma(B) measured before stress were similar (1.5, 2.6, and 0.9 micro M, respectively). After ethanol stress they all increased. The increase was greatest for RsbV, whose concentration reached 13 micro M, while those of RsbW (as a monomer) and sigma(B) reached 11.8 and 4.9 micro M, respectively. We conclude that the higher affinity of RsbW for RsbV than for sigma(B), rather than a difference in the concentrations of RsbV and sigma(B), is the driving force that is responsible for the switch of RsbW to unphosphorylated RsbV.

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.

Analysis of the interaction between the transcription factor sigmaG and the anti-sigma factor SpoIIAB of Bacillus subtilis.

The activation of sigma(G), a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation. SpoIIAB, an anti-sigma factor involved in regulation of sigma(F), is also shown to form a complex with sigma(G) in vitro. SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:sigma(G) complex, releasing free sigma(G). The data suggest the existence of an as-yet-unknown mechanism to keep sigma(G) inactive prior to engulfment.

A supramolecular complex in the environmental stress signalling pathway of Bacillus subtilis.

SigmaB, an alternative sigma-factor of Bacillus subtilis, mediates the response of the cell to a variety of physical insults. Within the environmental stress signalling pathway RsbU, a protein phosphatase, is stimulated by its interaction with the protein kinase RsbT. In the absence of stress RsbT is expected to be trapped by an alternative binding partner, RsbS. Here, we have demonstrated that RsbS alone cannot act as an alternative partner for RsbT, but instead requires the presence of RsbR to create a high molecular mass RsbR:RsbS complex (approximately 1 MDa) able to capture RsbT. In this complex the phosphorylation state of RsbS, and not that of RsbR, controlled the binding to RsbT, whose kinase activity towards RsbS could be counterbalanced by the activity of RsbX, the phosphatase for RsbS-P. The RsbR:RsbS complex recruited RsbT from a mixture of RsbT and RsbU. The phosphorylated form of RsbR in the complex enhanced the kinase activity of RsbT towards RsbS. This supramolecular complex thus has the functional properties of an alternative partner for RsbT. Electron micrographs of this complex are presented, and the purification of the RsbR:RsbS complex from cellular extracts provides evidence for the existence of such a complex in vivo.

Binding of sigma(A) and sigma(B) to core RNA polymerase after environmental stress in Bacillus subtilis.

In Bacillus subtilis, the alternative sigma factor sigma(B) is activated in response to environmental stress or energy depletion. The general stress regulon under the control of sigma(B) provides the cell with multiple stress resistance. Experiments were designed to determine how activated sigma(B) replaces sigma(A) as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the sigma(A)-dependent stress gene clpE under sigma(B)-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of sigma(A) and sigma(B) for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of sigma(B) for E was 60-fold lower than that of sigma(A). Western blot analyses with antibodies against sigma(A), sigma(B), and E showed that, after exposure to ethanol stress, the concentration of sigma(B) was only twofold higher than those of sigma(A) and E. Thus, the concentration of sigma(B) after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of sigma(B) to E after stress.

Functional and structural characterization of RsbU, a stress signaling protein phosphatase 2C.

RsbU is a positive regulator of the activity of sigmaB, the general stress-response sigma factor of Gram+ microorganisms. The N-terminal domain of this protein has no significant sequence homology with proteins of known function, whereas the C-terminal domain is similar to the catalytic domains of PP2C-type phosphatases. The phosphatase activity of RsbU is stimulated greatly during the response to stress by associating with a kinase, RsbT. This association leads to the induction of sigmaB activity. Here we present data on the activation process and demonstrate in vivo that truncations in the N-terminal region of RsbU are deleterious for the activation of RsbU. This conclusion is supported by comparisons of the phosphatase activities of full-length and a truncated form of RsbU in vitro. Our determination of the crystal structure of the N-terminal domain of RsbU from Bacillus subtilis reveals structural similarities to the regulatory domains from ubiquitous protein phosphatases and a conserved domain of sigma-factors, illuminating the activation processes of phosphatases and the evolution of "partner switching." Finally, the molecular basis of kinase recruitment by the RsbU phosphatase is discussed by comparing RsbU sequences from bacteria that either possess or lack RsbT.