A family of regulatory phosphatases
The genetic network of B. subtilis contains multiple cross-talk points between the activities of the three main regulators described above that guarantees that population differentiates accordingly to its environmental conditions . Furthermore, B. subtilis possesses a family of QS-regulated Rap phosphatases that fine-tunes this intertwinned genetic network .
The Rap phosphatases are conserved proteins (>25% of sequence identity) of ca. 380 amino acids that are able to hinder the phosphorylation of Spo0A, DegU, or ComA, thus preventing or delaying the expression of genes that depends of high levels of the phosphorylated versions of these regulators . Early studies showed that the primary function of these proteins is to directly dephosphorylate their target regulators. The exception is the regulation of Spo0A, where the cognate Rap phosphatases act on upstream members of the phosphorelay, such as Spo0F∼P . Some Rap phosphatases can also bind to their target transcriptional regulators, forming a complex that can no longer adhere to DNA . Shortly after the discovery of the first Rap phosphatases (RapA and RapB), it was recognized that a small gene directly followingrapA was involved in RapA regulation: its expression results in the production of a five-amino acid peptide that binds and inhibits the activity of RapA, and thus was called PhrA, ph osphataser egulator A .
Subsequent studies have later revealed a wide variety of rapgenes in the genome of B. subtilis , most of which are followed (and slightly overlapped) by phr genes that code for small proteins of ca. 40 amino acids known as Phr pro-peptides. Therap-phr gene pairs are often recognized as cassettes, and the production of their respective proteins is translationally coupled . Once produced, the Rap phosphatases can immediately exert their regulatory function, either by dephosphorylating or preventing the DNA-binding of their target transcriptional regulator (Fig. 1). The pro-peptides encoded by the phr genes follow a more complicated path to become active. Phr pro-peptides contain export signal sequences in their N-terminal portion, followed by cleavage peptidase signal domains and hydrophilic C-terminal domains. The Phr pro-peptides are mobilized to the cell membrane, where they are processed by peptidases that produce 5-6 amino acid Phr peptides in the extracellular space. The mature Phr peptides, upon reaching threshold concentrations, are imported back into the cell by the Opp oligopeptide permease. Once inside the cell, Phr peptides can finally directly bind to their respective cognate Rap phosphatase and induce a conformational change that blocks Rap activity .