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 .