Characterization of sporulation histidine kinases of Bacillus anthracis

The initiation of sporulation in Bacillus species is regulated by the phosphorelay signal transduction pathway, which is activated by several histidine sensor kinases in response to cellular and metabolic signals. Comparison of the protein components of the phosphorelay between Bacillus subtilis and Bacillus anthracis revealed high homology in the phosphorelay orthologs of Spo0F, Spo0B, and Spo0A. The sensor domains of sensor histidine kinases are poorly conserved between species, making ortholog recognition tenuous. Putative sporulation sensor histidine kinases of B. anthracis were identified by homology to the HisKA domain of B. subtilis sporulation sensor histidine kinases, which interacts with Spo0F. Nine possible kinases were uncovered, and their genes were assayed for complementation of kinase mutants of B. subtilis, for ability to drive lacZ expression in B. subtilis and B. anthracis, and for the effect of deletion of each on the sporulation of B. anthracis. Five of the nine sensor histidine kinases were inferred to be capable of inducing sporulation in B. anthracis. Four of the sensor kinases could not be shown to induce sporulation; however, the genes for two of these were frameshifted in all B. anthracis strains and one of these was also frameshifted in the pathogenic pXO1-bearing Bacillus cereus strain G9241. It is proposed that acquisition of plasmid pXO1 and pathogenicity may require a dampening of sporulation regulation by mutational selection of sporulation sensor histidine kinase defects. The sporulation of B. anthracis ex vivo appears to result from any one or a combination of the sporulation sensor histidine kinases remaining.     

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.     

Spo0B of Bacillus anthracis - a protein with pleiotropic functions

Spo0B is an important component of the phosphorelay signal transduction pathway, the pathway involved in the initiation of sporulation in Bacillus subtilis. Bioinformatic, phylogenetic and biochemical studies showed that Spo0B of Bacillus anthracis has evolved from citrate/malate kinases. During the course of evolution, Spo0B has retained the characteristic histidine kinase boxes H, N, F, G(1) and G(2), and has acquired nucleotide-binding domains, Walker A and Walker B, of ATPases. Owing to the presence of these domains, autophosphorylation and ATPase activity was observed in Spo0B of B. anthracis. Mutational studies showed that among the six histidine residues, His13 of the H-box is involved in the autophosphorylation activity of Spo0B, whereas Lys33 of the Walker A domain is associated with the ATPase activity of the protein. Thermodynamic and binding studies of the binding of Mg-ATP to Spo0B using isothermal titration calorimetry (ITC) suggested that the binding is driven by favorable entropy changes and that the reaction is exothermic, with an apparent dissociation constant (K(d)) equal to 0.02 mm. The value of the dissociation constant (K(d) = 0.05 mm) determined by the intrinsic fluorescence of trytophan of Spo0B was similar to that obtained by ITC studies. The purified Spo0B of B. anthracis also showed nucleoside diphosphate kinase-like activity of phosphate transfer from nucleoside triphosphate to nucleoside diphosphate. This is the first evidence for Spo0B of B. anthracis as an enzyme with histidine kinase and ATPase activities, which may have important roles to play in sporulation and pathogenesis.     

A transient interaction between two phosphorelay proteins trapped in a crystal lattice reveals the mechanism of molecular recognition and phosphotransfer in signal transduction

BACKGROUND: Spo0F and Spo0B specifically exchange a phosphoryl group in a central step of the phosphorelay signal transduction system that controls sporulation in Bacilli. Spo0F belongs to the superfamily of response regulator proteins and is one of 34 such proteins in Bacillus subtilis. Spo0B is structurally similar to the phosphohistidine domain of histidine kinases, such as EnvZ, and exchanges a phosphoryl group between His30 and Asp54 on Spo0F. Information at the molecular level on the interaction between response regulators and phosphohistidine domains is necessary to develop a rationale for how phospho-signaling fidelity is maintained in two-component systems. RESULTS: Structural analysis of a co-crystal of the Spo0F response regulator interacting with the Spo0B phosphotransferase of the phosphorelay signal transduction system of B. subtilis was carried out using X-ray crystallographic techniques. The association of the two molecules brings the catalytic residues from both proteins into precise alignment for phosphoryltransfer. Upon complex formation, the Spo0B conformation remains unchanged. Spo0F also retains the overall conformation; however, two loops around the active site show significant deviations. CONCLUSIONS: The Spo0F-Spo0B interaction appears to be a prototype for response regulator-histidine kinase interactions. The primary contact surface between these two proteins is formed by hydrophobic regions in both proteins. The Spo0F residues making up the hydrophobic patch are very similar in all response regulators suggesting that the binding is initiated through the same residues in all interacting response regulator-kinase pairs. The bulk of the interactions outside this patch are through nonconserved residues. Recognition specificity is proposed to arise from interactions of the nonconserved residues, especially the hypervariable residues of the beta4-alpha4 loop.     

Pentapeptide regulation of aspartyl-phosphate phosphatases

Aspartyl-phosphate phosphatases are integral components of the phosphorelay signal transduction system for sporulation initiation in Bacillus subtilis. The Rap and Spo0E families of protein phosphatases specifically dephosphorylate the sporulation response regulators Spo0F and Spo0A, respectively. The phosphatases interpret regulatory signals antithetical to sporulation and the Rap phosphatases are subject to inactivation by specific pentapeptides generated from an inactive peptide precursor. Additional regulatory signals are brought about by the complex activation circuit that generates the Phr pentapeptide inhibitors of Rap phosphatases. Phr peptide's recognition of the Rap phosphatase targets is remarkably specific. Specificity is dictated by the amino acid sequence of the pentapeptide. The identification of tetratricopeptide repeats in the Rap proteins may explain the mechanism by which Phr peptides bind to and inhibit the activity of Rap phosphatases.     

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.     

Heterologous expression of Bacillus intermedius gene of glutamyl endopeptidase in Bacillus subtilis strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

A unique GTP-dependent sporulation sensor histidine kinase in Bacillus anthracis

The Bacillus anthracis BA2291 gene codes for a sensor histidine kinase involved in the induction of sporulation. Genes for orthologs of the sensor domain of the BA2291 kinase exist in virulence plasmids in this organism, and these proteins, when expressed, inhibit sporulation by converting BA2291 to an apparent phosphatase of the sporulation phosphorelay. Evidence suggests that the sensor domains inhibit BA2291 by titrating its activating signal ligand. Studies with purified BA2291 revealed that this kinase is uniquely specific for GTP in the forward reaction and GDP in the reverse reaction. The G1 motif of BA2291 is highly modified from ATP-specific histidine kinases, and modeling this motif in the structure of the kinase catalytic domain suggested how guanine binds to the region. A mutation in the putative coiled-coil linker between the sensor domain and the catalytic domains was found to decrease the rate of the forward autophosphorylation reaction and not affect the reverse reaction from phosphorylated Spo0F. The results suggest that the activating ligand for BA2291 is a critical signal for sporulation and in a limited concentration in the cell. Decreasing the response to it either by slowing the forward reaction through mutation or by titration of the ligand by expressing the plasmid-encoded sensor domains switches BA2291 from an inducer to an inhibitor of the phosphorelay and sporulation.     

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.     

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.