Joanna Clarkson

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.

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, σF, which is activated only in the prespore. Regulation relies on the interactions of four proteins –σF, 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, σF is held in an inactive complex with SpoIIAB and ATP; SpoIIAA is in its phosphorylated form. To disrupt the complex so as to liberate σF 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 σF. 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 σF. We show that the system is tuned to be highly efficient in its use of components and extremely economical in conserving ATP.

Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti‐anti‐σF factor SpoIIAA lead to a Spo− phenotype

Sigma‐F, the first sporulation‐specific transcription factor of Bacillus subtilis, is regulated by an anti‐sigma factor SpoIIAB, which can also act as a protein kinase that phosphorylates the anti‐anti‐sigma factor SpoIIAA. The time course of phosphorylation reaction is biphasic, a fact that has been interpreted in terms of a mechanism for sequestering SpoIIAB away from σF and thus allowing activation of σF when needed. Site‐directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate σF and which are therefore Spo−. The two mutant SpoIIAA proteins, SpoIIAAL61A and SpoIIAAL90A, are phosphorylated with linear kinetics; in addition they are less able to form the stable non‐covalent complex that wild‐type SpoIIAA makes with SpoIIAB in the presence of ADP. The phosphorylated form of SpoIIAAL90A was hydrolysed by the specific phosphatase SpoIIE at the same rate as wild‐type SpoIIAA‐P, but the rate of hydrolysis of SpoIIAAL61A‐P was much slower. The secondary structure and the global fold of the mutant proteins were unchanged from the wild type. The results are interpreted in terms of a model for the wild type in which SpoIIAB, after phosphorylating SpoIIAA, is released in a form that is tightly bound to ADP and which then makes a ternary complex with an unreacted SpoIIAA. We propose that it is the inability to make this ternary complex that deprives the mutant cells of a means of keeping SpoIIAB from inhibiting σF.

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 σG 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.

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.

Studies of SpoIIAB mutant proteins elucidate the mechanisms that regulate the developmental transcription factor σF 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 AAs 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.