Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F～P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F～P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.     

Molecular analysis of Phr peptide processing in Bacillus subtilis

In Bacillus subtilis, an export-import pathway regulates production of the Phr pentapeptide inhibitors of Rap proteins. Processing of the Phr precursor proteins into the active pentapeptide form is a key event in the initiation of sporulation and competence development. The PhrA (ARNQT) and PhrE (SRNVT) peptides inhibit the RapA and RapE phosphatases, respectively, whose activity is directed toward the Spo0F approximately P intermediate response regulator of the sporulation phosphorelay. The PhrC (ERGMT) peptide inhibits the RapC protein acting on the ComA response regulator for competence with regard to DNA transformation. The structural organization of PhrA, PhrE, and PhrC suggested a role for type I signal peptidases in the processing of the Phr preinhibitor, encoded by the phr genes, into the proinhibitor form. The proinhibitor was then postulated to be cleaved to the active pentapeptide inhibitor by an additional enzyme. In this report, we provide evidence that Phr preinhibitor proteins are subject to only one processing event at the peptide bond on the amino-terminal end of the pentapeptide. This processing event is most likely independent of type I signal peptidase activity. In vivo and in vitro analyses indicate that none of the five signal peptidases of B. subtilis (SipS, SipT, SipU, SipV, and SipW) are indispensable for Phr processing. However, we show that SipV and SipT have a previously undescribed role in sporulation, competence, and cell growth.     

Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis

In the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis, the opposing activities of histidine kinases and aspartyl phosphate phosphatases determine the cell's decision whether to continue with vegetative growth or to initiate the differentiation process. Regulated dephosphorylation of the Spo0A and Spo0F response regulators allows a variety of negative signals from physiological processes that are antithetical to sporulation to impact on the activation level of the phosphorelay. Spo0F approximately P is the known target of two related phosphatases, RapA and RapB. In addition to RapA and RapB, a third member of the Rap family of phosphatases, RapE, specifically dephosphorylated the Spo0F approximately P intermediate in response to competence development. RapE phosphatase activity was found to be controlled by a pentapeptide (SRNVT) generated from within the carboxy-terminal domain of the phrE gene product. A synthetic PhrE pentapeptide could (i) complement the sporulation deficiency caused by deregulated RapE activity of a phrE mutant and (ii) inhibit RapE-dependent dephosphorylation of Spo0F approximately P in in vitro experiments. The PhrE pentapeptide did not inhibit the phosphatase activity of RapA and RapB. These results confirm previous conclusions that the specificity for recognition of the target phosphatase is contained within the amino acid sequence of the pentapeptide inhibitor.     

Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.     

Biochemical characterization of aspartyl phosphate phosphatase interaction with a phosphorylated response regulator and its inhibition by a pentapeptide

The RapA and RapB proteins are aspartyl phosphate phosphatases that specifically dephosphorylate the Spo0F approximately P intermediate response regulator of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The approximately 48-kDa His-tag derivative proteins were purified by metal affinity chromatography, and their molecular and biochemical characteristics were studied. RapA and RapB were found to be dimers in solution. Enzymatic activity was strongly dependent upon maintaining reducing conditions during purification and storage. RapA phosphatase activity on Spo0F approximately P is inhibited in vivo by a pentapeptide generated from the phrA gene. Native gel assays demonstrated that the RapA dimer forms a stable complex with two molecules of Spo0F approximately P or with its PhrA pentapeptide inhibitor. The pentapeptide was shown to displace Spo0F approximately P from a preformed complex with RapA. The structural organization of Rap phosphatases in tetratricopeptide repeats provides insights on the mechanisms of RapA interaction with its substrate and its inhibitor.     

A free terminal carboxylate group is required for PhrA pentapeptide inhibition of RapA phosphatase

In the Bacillus subtilis phosphorelay signal transduction system for sporulation initiation, signals competing with the differentiation process are interpreted by aspartyl-phosphate phosphatases that specifically dephosphorylate the Spo0F or Spo0A response regulators. The RapA phosphatase is regulated by the PhrA pentapeptide that directly and specifically inhibits its activity. PhrA specificity for RapA inhibition is dependent upon the amino acid sequence of the peptide. Here we show that the pentapeptide affinity for the phosphatase requires a free carboxylate group at the C-terminal amino acid. A free C-terminal carboxylic acid PhrA pentapeptide inhibits RapA phosphatase activity at a 1:1 ratio and it is approximately 200 fold more active than a C-terminal amide peptide. Therefore, coordination of the terminal carboxylate group appears to be critical for peptide binding to RapA.     

Structural basis of response regulator dephosphorylation by Rap phosphatases

Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic "switch" residue to an internal position when the β4-α4 loop adopts an active-site proximal conformation.     

Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.     

Negative regulation of Bacillus anthracis sporulation by the Spo0E family of phosphatases

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the Spo0F intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A approximately P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation-deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A approximately P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.     

An atypical Phr peptide regulates the developmental switch protein RapH

Under conditions of nutrient limitation and high population density, the bacterium Bacillus subtilis can initiate a variety of developmental pathways. The signaling systems regulating B. subtilis differentiation are tightly controlled by switch proteins called Raps, named after the founding members of the family, which were shown to be response regulator aspartate phosphatases. A phr gene encoding a secreted pentapeptide that regulates the activity of its associated Rap protein was previously identified downstream of 8 of the chromosomally encoded rap genes. We identify and validate here the sequence of an atypical Phr peptide, PhrH, by in vivo and in vitro analyses. Using a luciferase reporter bioassay combined with in vitro experiments, we found that PhrH is a hexapeptide (TDRNTT), in contrast to the other characterized Phr pentapeptides. We also determined that phrH expression is driven by a promoter lying within rapH. Unlike the previously identified dedicated σ(H)-driven phr promoters, it appears that phrH expression most likely requires σ(A). Furthermore, we show that PhrH can antagonize both of the known activities of RapH: the dephosphorylation of Spo0F and the sequestration of ComA, thus promoting the development of spores and the competent state. Finally, we propose that PhrH is the prototype of a newly identified class of Phr signaling molecules consisting of six amino acids. This class likely includes PhrI, which regulates RapI and the expression, excision, and transfer of the mobile genetic element ICEBs1.     

Sporulation during Growth in a Gut Isolate of Bacillus subtilis

Sporulation by Bacillus subtilis is a cell density-dependent response to nutrient deprivation. Central to the decision of entering sporulation is a phosphorelay, through which sensor kinases promote phosphorylation of Spo0A. The phosphorelay integrates both positive and negative signals, ensuring that sporulation, a time- and energy-consuming process that may bring an ecological cost, is only triggered should other adaptations fail. Here we report that a gastrointestinal isolate of B. subtilis sporulates with high efficiency during growth, bypassing the cell density, nutritional, and other signals that normally make sporulation a post-exponential-phase response. Sporulation during growth occurs because Spo0A is more active per cell and in a higher fraction of the population than in a laboratory strain. This in turn, is primarily caused by the absence from the gut strain of the genes rapE and rapK, coding for two aspartyl phosphatases that negatively modulate the flow of phosphoryl groups to Spo0A. We show, in line with recent results, that activation of Spo0A through the phosphorelay is the limiting step for sporulation initiation in the gut strain. Our results further suggest that the phosphorelay is tuned to favor sporulation during growth in gastrointestinal B. subtilis isolates, presumably as a form of survival and/or propagation in the gut environment.     

Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis

Summary Spore formation in the Gram-positive bacterium Bacillus subtilis is a last resort adaptive response to starvation. To initiate sporulation, the key regulator in this process, Spo0A, needs to be activated by the so-called phosphorelay. Within a sporulating culture of B. subtilis, some cells initiate this developmental program, while other cells do not. Therefore, initiation of sporulation appears to be a regulatory process with a bistable outcome. Using a single cell analytical approach, we show that the autostimulatory loop of spo0A is responsible for generating a bistable response resulting in phenotypic variation within the sporulating culture. It is demonstrated that the main function of RapA, a phosphorelay phosphatase, is to maintain the bistable sporulation gene expression. As rapA expression is quorum regulated, it follows that quorum sensing influences sporulation bistability. Deletion of spo0E, a phosphatase directly acting on Spo0A approximately P, resulted in abolishment of the bistable expression pattern. Artificial induction of a heterologous Rap phosphatase restored heterogeneity in a rapA or spo0E mutant. These results demonstrate that with external phosphatases, B. subtilis can use the phosphorelay as a tuner to modulate the bistable outcome of the sporulating culture. This shows that B. subtilis employs multiple pathways to maintain the bistable nature of a sporulating culture, stressing the physiological importance of this phenomenon.     

Structural Basis of Rap Phosphatase Inhibition by Phr Peptides

Two-component systems, composed of a sensor histidine kinase and an effector response regulator (RR), are the main signal transduction devices in bacteria. In Bacillus, the Rap protein family modulates complex signaling processes mediated by two-component systems, such as competence, sporulation, or biofilm formation, by inhibiting the RR components involved in these pathways. Despite the high degree of sequence homology, Rap proteins exert their activity by two completely different mechanisms of action: inducing RR dephosphorylation or blocking RR binding to its target promoter. However the regulatory mechanism involving Rap proteins is even more complex since Rap activity is antagonized by specific signaling peptides (Phr) through a mechanism that remains unknown at the molecular level. Using X-ray analyses, we determined the structure of RapF, the anti-activator of competence RR ComA, alone and in complex with its regulatory peptide PhrF. The structural and functional data presented herein reveal that peptide PhrF blocks the RapF-ComA interaction through an allosteric mechanism. PhrF accommodates in the C-terminal tetratricopeptide repeat domain of RapF by inducing its constriction, a conformational change propagated by a pronounced rotation to the N-terminal ComA-binding domain. This movement partially disrupts the ComA binding site by triggering the ComA disassociation, whose interaction with RapF is also sterically impaired in the PhrF-induced conformation of RapF. Sequence analyses of the Rap proteins, guided by the RapF-PhrF structure, unveil the molecular basis of Phr recognition and discrimination, allowing us to relax the Phr specificity of RapF by a single residue change.