Bacillus subtilis mutations that alter the pathway of phosphorylation of the anti-anti-sigmaF 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 sigmaF and thus allowing activation of sigmaF when needed. Site-directed mutagenesis of SpoIIAA has allowed us to isolate two mutants that cannot activate sigmaF 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 sigmaF.

Cytological and biochemical characterization of the FtsA cell division protein of Bacillus subtilis.

The actin-like protein FtsA is present in many eubacteria, and genetic experiments have shown that it plays an important, sometimes essential, role in cell division. Here, we show that Bacillus subtilis FtsA is targeted to division sites in both vegetative and sporulating cells. As in other organisms FtsA is probably recruited immediately after FtsZ. In sporulating cells of B. subtilis FtsZ is recruited to potential division sites at both poles of the cell, but asymmetric division occurs at only one pole. We have now found that FtsA is recruited to only one cell pole, suggesting that it may play an important role in the generation of asymmetry in this system. FtsA is present in much higher quantities in B. subtilis than in Escherichia coli, with approximately one molecule of FtsA for five of FtsZ. This means that there is sufficient FtsA to form a complete circumferential ring at the division site. Therefore, FtsA may have a direct structural role in cell division. We have purified FtsA and shown that it behaves as a dimer and that it has both ATP-binding and ATP-hydrolysis activities. This suggests that ATP hydrolysis by FtsA is required, together with GTP hydrolysis by FtsZ, for cell division in B. subtilis (and possibly in most eubacteria).

NMR studies of the interactions of SpoIIAA with its partner proteins that regulate sporulation in Bacillus subtilis.

SpoIIAA is a key component in the network of interactions that regulate the first sporulation-specific transcription factor, sigma(F), in Bacillus subtilis. In its unphosphorylated form SpoIIAA is either phosphorylated by or forms a non-covalent complex with SpoIIAB, whereas in its phosphorylated form it is dephosphorylated by SpoIIE. In this work we present NMR studies of the SpoIIAA(2).SpoIIAB complex and of mutant proteins that are deficient in their ability to interact with SpoIIAB or SpoIIE. The NMR studies of the SpoIIAA(2).SpoIIAB complex allowed us to define a contiguous patch that is perturbed upon complex formation. By examining the chemical shift perturbations in the mutant proteins we have identified more specific areas that contain residues critical for the SpoIIAB and SpoIIE interactions.

Fate of the SpoIIAB*-ADP liberated after SpoIIAB phosphorylates SpoIIAA of Bacillus subtilis.

Phosphorylation of SpoIIAA catalyzed by SpoIIAB helps to regulate the first sporulation-specific sigma factor, sigma(F), of Bacillus subtilis. The steady-state rate of phosphorylation is known to be exceptionally slow and to be limited by the return of the protein kinase, SpoIIAB, to a catalytically active state. Previous work from this laboratory has suggested that, after catalyzing the phosphorylation, SpoIIAB is in a form (SpoIIAB*) that does not readily release ADP. We now show that the rate of release of ADP from the SpoIIAB*-ADP complex was much diminished by the presence of unreacted SpoIIAA, suggesting that SpoIIAA can form a long-lived ternary complex with SpoIIAB*-ADP in which the SpoIIAB* form is stabilized. In kinetic studies of the phosphorylation of SpoIIAA, the ternary complex SpoIIAA-SpoIIAB*-ADP could be distinguished from the short-lived complex SpoIIAA-SpoIIAB-ADP, which can be readily produced in the absence of an enzymatic reaction.

Direct interaction between the cell division protein FtsZ and the cell differentiation protein SpoIIE.

SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate in Bacillus subtilis. First, SpoIIE is needed for the normal formation of the asymmetrically positioned septum that forms early in sporulation and separates the mother cell from the prespore compartment. Secondly, SpoIIE is essential for the activation of the first compartment-specific transcription factor sigma(F) in the prespore. After initiation of sporulation, SpoIIE localizes to the potential asymmetric cell division sites near one or both cell poles. Localization of SpoIIE was shown to be dependent on the essential cell division protein FtsZ. To understand how SpoIIE is targeted to the asymmetric septum we have now analysed its interaction with FtsZ in vitro. Using the yeast two-hybrid system and purified FtsZ, and full-length and truncated SpoIIE proteins, we demonstrate that the two proteins interact directly and that domain II and possibly domain I of SpoIIE are required for the interaction. Moreover, we show that SpoIIE interacts with itself and suggest that this self-interaction plays a role in assembly of SpoIIE into the division machinery.

Genotype, phenotype, and protein structure in a regulator of sporulation: effects of mutations in the spoIIAA gene of Bacillus subtilis.

SpoIIAA, a phosphorylatable protein, is essential to the regulation of sigmaF, the first sporulation-specific transcription factor of Bacillus subtilis. The solution structure of SpoIIAA has recently been published. Here we examine four mutant SpoIIAA proteins and correlate their properties with the phenotypes of the corresponding B. subtilis mutant strains. Two of the mutations severely disrupted the structure of the protein, a third greatly diminished the rate of its phosphorylation and abolished dephosphorylation, and the fourth left phosphorylation unaffected but reduced the rate of dephosphorylation about 10-fold.

Purification, kinetic properties, and intracellular concentration of SpoIIE, an integral membrane protein that regulates sporulation in Bacillus subtilis.

SpoIIE is a bifunctional protein which controls sigmaF activation and formation of the asymmetric septum in sporulating Bacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.

Replacement of vegetative sigmaA by sporulation-specific sigmaF as a component of the RNA polymerase holoenzyme in sporulating Bacillus subtilis.

Soon after asymmetric septation in sporulating Bacillus subtilis cells, sigmaF is liberated in the prespore from inhibition by SpoIIAB. To initiate transcription from its cognate promoters, sigmaF must compete with sigmaA, the housekeeping sigma factor in the predivisional cell, for binding to core RNA polymerase (E). To estimate the relative affinity of E for sigmaA and sigmaF, we made separate mixtures of E with each of the two sigma factors, allowed reconstitution of the holoenzyme, and measured the concentration of free E remaining in each mixture. The affinity of E for sigmaF was found to be about 25-fold lower than that for sigmaA. We used quantitative Western blotting to estimate the concentrations of E, sigmaA, and sigmaF in sporulating cells. The cellular concentrations of E and sigmaA were both about 7.5 microM, and neither changed significantly during the first 3 h of sporulation. The concentration of sigmaF was extremely low at the beginning of sporulation, but it rose rapidly to a peak after about 2 h. At its peak, the concentration of sigmaF was some twofold higher than that of sigmaA. This difference in concentration cannot adequately account for the replacement of sigmaA holoenzyme by sigmaF holoenzyme in the prespore, and it seems that some further mechanism-perhaps the synthesis or activation of an anti-sigmaA factor-must be responsible for this replacement.

Solution structure of SpoIIAA, a phosphorylatable component of the system that regulates transcription factor sigmaF of Bacillus subtilis.

The establishment of differential gene expression in sporulating Bacillus subtilis involves four protein components, one of which, SpoIIAA, undergoes phosphorylation and dephosphorylation. We have used NMR spectroscopy to determine the solution structure of the nonphosphorylated form of SpoIIAA. The structure shows a fold consisting of a four-stranded beta-sheet and four alpha-helices. Knowledge of the structure helps to account for the phenotype of several strains of B. subtilis that carry known spoIIAA mutations and should facilitate investigations of the conformational consequences of phosphorylation.

Properties of the phosphorylation reaction catalyzed by SpoIIAB that help to regulate sporulation of Bacillus subtilis.

Phosphorylation of SpoIIAA on Ser-58 catalyzed by SpoIIAB is important in the regulation of sporulation of Bacillus subtilis. Nucleotide binding experiments showed that the affinity of SpoIIAB for ATP was greatly increased in the presence of SpoIIAA or a mutant SpoIIAA in which Ser-58 had been changed to alanine. Study of the phosphorylation reaction showed that the Km for ATP and the Ki for ADP were both about 1 microM. The kinetics of phosphorylation of SpoIIAA by SpoIIAB were biphasic, comprising a rapid phase (leading to phosphorylation of 1 mol of SpoIIAA/mol of SpoIIAB) followed by a slower, steady-state phase. In the steady state, the rate-determining step proved to be the dissociation of a SpoIIAB-ADP complex. The rate of this dissociation was not affected significantly by changes in the concentration of ATP.

Sequencing and phylogenetic analysis of the spoIIA operon from diverse Bacillus and Paenibacillus species.

In order to clone the spoIIA operon from three different Bacillus and Paenibacillus species, we designed two sets of PCR primers based on three previously published Bacillus spoIIA sequences. One set of primers corresponded to the C-terminal region of SpoIIAB and a region near the middle of SpoIIAC. These primers were used to amplify the corresponding region of spoIIA from Bacillus stearothermophilus and Paenibacillus polymyxa (previously called Bacillus polymyxa [see Ash, C., Priest, F.G., Collins, M.D., 1993. Molecular identification of ribosomal-RNA group 3 bacilli using a PCR probe test - proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 64, 253-260]. The other set of primers, corresponding to an N-terminal and a C-terminal region of SpoIIAC, was used for B. sphaericus. The PCR products were used as probes for Southern blotting of homologous chromosomal DNA. DNA corresponding to spoIIA from the three organisms was identified by screening chromosomal DNA libraries, and cloned. Sequence analysis showed that all spoIIA sequences were conserved, but conservation was strongest in SpoIIAC and least strong in SpoIIAA. In the promoter the -35 region was conserved well but the -10 region rather poorly. Within the proteins, certain regions were particularly strongly conserved, suggesting that they are essential to the function of the protein. Phylogenetic analysis of spoIIA suggested that B. stearothermophilus is close to B. subtilis and B. licheniformis, but that P. polymyxa and B. sphaericus are remote from B. subtilis.

Contribution of partner switching and SpoIIAA cycling to regulation of sigmaF activity in sporulating Bacillus subtilis.

sigmaF, the first compartment-specific transcription factor in sporulating Bacillus subtilis, is negatively regulated by an anti-sigma factor, SpoIIAB. SpoIIAB has an alternative binding partner, SpoIIAA. To see whether (as has been proposed) SpoIIAB's binding preference for SpoIIAA or sigmaF depends on the nature of the adenine nucleotide present, we used surface plasmon resonance to measure the dissociation constants of the three complexes SpoIIAA-SpoIIAB-ADP, sigmaF-SpoIIAB-ADP, and sigmaF-SpoIIAB-ATP. The results suggested that SpoIIAB's choice of binding partner is unlikely to depend on the ATP/ADP ratio in the cell. The intracellular concentrations of sigmaF, SpoIIAB, SpoIIAA, and SpoIIAA-phosphate (SpoIIAA-P) were measured by quantitative immunoblotting between 0 and 3 h after the beginning of sporulation (t0 to t3). sigmaF and SpoIIAB were barely detectable at t0, but their concentrations increased in parallel to reach maxima at about t1.5. SpoIIAA-P increased steadily to a maximum at t3, but nonphosphorylated SpoIIAA was detectable only from t1.5, reached a maximum at t2.5, and then declined. Kinetic studies of the phosphorylation of SpoIIAA catalyzed by SpoIIAB suggested that the reaction was limited by a very slow release of one of the products (SpoIIAA-P or ADP) from SpoIIAB, with a turnover of about once per 20 min. This remarkable kinetic property provides an unexpected mechanism for the regulation of sigmaF. We propose that when SpoIIE (which dephosphorylates SpoIIAA-P) is active at the same time as SpoIIAB, SpoIIAA cycles repeatedly between the phosphorylated and nonphosphorylated forms. This cycling sequesters SpoIIAB in a long-lived complex and prevents it from inhibiting sigmaF.

Protein conformational change and nucleotide binding involved in regulation of sigmaF in Bacillus subtilis.

We have studied the ability of three mutant forms of SpoIIAA, containing amino acid substitutions at the site of phosphorylation (serine 58), to interact with SpoIIAB. Native gel analysis revealed that SpoIIAAS58A could form a complex with SpoIIAB in the presence of ADP and more strongly in the presence of ATP. SpoIIAAS58N did not form a complex with SpoIIAB in the presence of ADP but displayed some interaction with SpoIIAB in the presence of ATP. SpoIIAAS58D was unable to form a complex with SpoIIAB in the presence of either ADP or ATP. Corresponding differences were found in the behavior of the three mutant proteins when studied by gel permeation with high-performance liquid chromatography and limited proteolysis. SpoIIAAS58A behaved like the wild-type SpoIIAA, SpoIIAAS58D like SpoIIAA-P, and SpoIIAAS58N in a way that was intermediate between the behaviors of SpoIIAA and SpoIIAA-P. Limited proteolysis was also used to show that on binding of ADP or ATP SpoIIAB undergoes a shift in conformation. The affinity of SpoIIAB for ADP and ATP was determined by limited proteolysis in the presence of a wide range of nucleotide concentrations. The results indicated that SpoIIAB has approximately equal affinity for ADP and for ATP.

The SpoIIAA protein of Bacillus subtilis has GTP-binding properties.

SpoIIAA is the first protein of the spoIIA operon. Here we show that SpoIIAA can bind and hydrolyze GTP. The protein also accepts ATP, but with lower affinity. GDP competes poorly for binding of GTP. The GTPase activity of SpoIIAA is within the range found for other GTP-binding proteins.

Nucleotide sequence of the Bacillus coagulans homologue of the spoIIA operon of Bacillus subtilis.

The spoIIA locus of Bacillus coagulans (Bc) was cloned into pTZ18R and the nucleotide sequence was determined. To clone the operon, one PCR primer corresponding to the C-terminal region of SpoIIAB, and a second corresponding to a region near the middle of SpoIIAC, were designed on the basis of the three previously published Bacillus spoIIA sequences. The Bc spoIIA sequence contains three open reading frames coding for putative proteins of 116, 147 and 252 aa.

Bifunctional protein required for asymmetric cell division and cell-specific transcription in Bacillus subtilis.

During sporulation in Bacillus subtilis an asymmetric cell division gives rise to unequal progeny called the prepore and the mother cell. Gene expression in the prespore is initiated by cell-specific activation of the transcription factor sigma(F). Three proteins participate in the regulation of sigma(F) activity. The first, SpoIIAB, is an inhibitor of sigma(F), that is, an anti-sigma factor. SpoIIAB is also a protein kinase that catalyzes phosphorylation of the second regulatory protein SpoIIAA (the anti-anti-sigma factor), and thus inactivates it. A third protein, SpoIIE, was shown recently to be able to dephosphorylate SpoIIAA-P in vitro. Here we show that SpoIIE is a bifunctional protein with two critical roles in the establishment of cell fate. First, we confirm by the use of in vivo experiments that it regulates the release of sigma(F) activity by dephosphorylating SpoIIAA-P. Second, we show that SpoIIE is needed for normal formation of the asymmetric septum that separates the prespore from the mother cell. Combination of these two functions in a single polypeptide may serve to couple the release of the cell-specific transcription factors with the formation of the differentiating cells.

Control of the cell-specificity of sigma F activity in Bacillus subtilis.

Sporulation in Bacillus subtilis is a simple developmental system involving the differentiation of two cell types that are formed by an asymmetric cell division. Major changes in the pattern of transcription during sporulation are brought about by the synthesis of new sigma factors (sigma), which are subunits of RNA polymerase that determine promoter specificity. Transcription in the smaller prespore cell type is initiated by a sigma factor called sigma F, the activity of which is subject to tight spatial and temporal control. It is negatively regulated by an anti-sigma factor, SpoIIAB, which is in turn controlled by an anti-anti-sigma factor, SpoIIAA. SpoIIAA and SpoIIAB participate in two contrasting reactions in vitro. In the presence of ATP, the proteins interact transiently and SpoIIAA is inactivated by phosphorylation on a specific serine residue; SpoIIAA then remains free to inhibit sigma F. In the presence of ADP, SpoIIAA binds tightly to SpoIIAB, and sigma F is set free. Release of sigma F activity in vivo might thus be effected by a prespore-specific reduction in the ATP/ADP ratio. Genetic experiments have implicated a fourth protein, called SpoIIE, in this system. It now appears that SpoIIE has two important and independent functions in the establishment of the prespore-specific transcription by sigma F. First it regulates sigma F activity, probably acting as a phosphatase to regenerate the active, non-phosphorylated form of SpoIIAA. Second it controls the formation of the septum that generates the prespore compartment. Combination of these two functions in a single polypeptide may provide a means of coupling gene expression with morphogenesis.

Establishing differential gene expression in sporulating Bacillus subtilis: phosphorylation of SpoIIAA (anti-anti-sigmaF) alters its conformation and prevents formation of a SpoIIAA/SpoIIAB/ADP complex.

Sigma-factor F (sigmaF) is a key transcription factor that initiates prespore development in Bacillus subtilis. Its activity is controlled by an anti-sigma factor, SpoIIAB, which is also a protein kinase that phosphorylates the anti-anti-sigma factor SpoIIAA. We have examined our earlier prediction that SpoIIAA must undergo a major change in its properties when phosphorylated. Upon gel filtration in the presence of ADP, SpoIIAA-P was eluted from a Superdex column much later than SpoIIAB, whereas SpoIIAA was coeluted with SpoIIAB, indicating the formation of a protein/protein complex. The complex contained ADP, and had two monomers of SpoIIAA to each SpoIIAB dimer. Its dissociation constant was 13 mu M. Gel permeation on high-performance liquid chromatography (HPLC) suggested an apparent molecular mass for SpoIIAA-P which was much higher (23.5 kDa) than that of SpoIIAA (15.8 kDa), but Ferguson plots showed that SpoIIAA-P was not a phosphorylated dimer of SpoIIAA. Our tentative conclusion, that SpoIIAA and SpoIIAA-P differ markedly in conformation, was confirmed by the results of partial digestion with chymotrypsin.

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.

Role of interactions between SpoIIAA and SpoIIAB in regulating cell-specific transcription factor sigma F of Bacillus subtilis.

Genetic experiments have suggested that sigma F, the first compartment-specific transcription factor in sporulating B. subtilis, is regulated by an anti-sigma factor SpoIIAB and an anti-anti-sigma factor SpoIIAA. Previously, we reported biochemical results demonstrating that SpoIIAB is both a phosphokinase whose substrate is SpoIIAA and an inhibitor of sigma F-directed transcription. We now show that in the presence of SpoIIAB and ATP or ADP, SpoIIAA can undergo two alternative reactions. When ATP is present, SpoIIAA is phosphorylated rapidly and completely to SpoIIAA-phosphate, and SpoIIAB is immediately released; but in the presence of ADP, SpoIIAA forms a long-lasting complex with SpoIIAB. ADP is an inhibitor of the phosphorylation by ATP. Furthermore, we have mutated SpoIIAA at residue Ser 58, the target for phosphorylation, to aspartate or alanine. SpoIIAAS58D, which apparently resembles SpoIIAA-phosphate, is unable to make a complex with SpoIIAB and is devoid of anti-anti-sigma F activity, whereas SpoIIAAS58A, which cannot be phosphorylated, makes complexes with SpoIIAB in the presence of ADP or ATP and has constitutive anti-anti-sigma F activity both in vivo and in vitro. It seems likely that the alternative reactions of SpoIIAA and SpoIIAB, involving ADP or ATP, regulate the anti-anti-sigma capacity of SpoIIAA and hence the activity of sigma F.