Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide

Abstract

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 (Spo0FϳP), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNAbinding domain of ComA, rather than Spo0FϳP, 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 aminoterminal 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. T he initiation of sporulation in the Gram-positive organismBacillus subtilis is regulated by the complex phosphorelay signal transduction system. In this system, sporulation-activating signals are sensed by multiple sensor histidine kinases whose activation results in autophosphorylation and phosphoryl transfer to an intermediate component, the Spo0F response regulator. From phosphorylated Spo0F (Spo0FϳP), the phosphoryl group is then transferred to the Spo0B phosphotransferase, which then relays it to the Spo0A response regulator and transcription factor. The phosphorylation level of Spo0A in the cell is the determining factor of whether sporulation will initiate or not in response to the activating signals sensed by the kinases (7,15,22,23).In order to counteract the kinase activities and prevent untimely initiation of sporulation, a number of phosphatases exist to respond to physiological states antithetical to sporulation, such as growth and competence for DNA transformation. There are two families of phosphatases, classified by structure and substrate specificity: the Spo0E-like family and the Rap family (30,32).The members of the Spo0E family of phosphatases (Spo0E, YisI, and YnzD) are small proteins (56 to 85 amino acids) identified by a conserved sequence motif, SQ/RE/DLD, in which the aspartate in the fifth position is the essential catalytic residue.

A Plasmid-Encoded Phosphatase Regulates Bacillus subtilis Biofilm Architecture, Sporulation, and Genetic Competence

“…6C and D). Substitution mutations at positions corresponding to RapP Asp203 were previously shown to render Rap proteins resistant to the inhibitory effects of their cognate Phr peptides (8,20,27,46). Moreover, we recently showed that B. subtilis Rap proteins have aspartate or, less commonly, glutamate at the position corresponding to RapP Asp203, and these conserved acidic residues form salt bridges with conserved basic residues in their cognate Phr peptides (47).…”

Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR

“…The TPR domains, however, are conserved in comparison with PlcR and PrgX, and residues potentially involved in binding of the Phr peptides have already been identified. The role of a conserved asparagine has also been highlighted (26). Binding of Phr peptides thus most probably induces a conformational change that propagates from the TPR domain to the N-terminal domain interacting with the targeted protein.…”

CodY Regulates Expression of the Bacillus subtilis Extracellular Proteases Vpr and Mpr

“…RapA is a Spo0F phosphatase involved in the regulation of sporulation; PhrA is processed extracellularly to produce a pentapeptide that inhibits RapA activity (75). Therefore, CodY regulates the activity of RapA both at the level of rapA-phrA expression and at the level of Vpr-dependent proteolytic processing of the full-length PhrA protein.…”