Phenotypic plasticity: The role of a phosphatase family Rap in the genetic regulation of Bacilli

In the last two decades, an increasing number of bacterial species have been recognized that are able to generate a phenotypically diverse population that shares an identical genotype. This ability is dependent on a complex genetic regulatory network that includes cellular and environmental signals, as well as stochastic elements. Among Bacilli, a broadly distributed family of Rap (Response‐regulator aspartyl phosphate) phosphatases is known to modulate the function of the main phenotypic heterogeneity regulators by controlling their phosphorylation. Even more, their related extracellular Phr (Phosphatase regulator) peptides function as signals, creating a cell–cell communication network that regulates the phenotypic development of the entire population. In this review, we examine the role that the Rap phosphatases and their Phr peptides play in the regulation of Bacillus subtilis phenotypic differentiation, and in other members of the Bacillus genus. We also highlight the contribution of these regulatory elements to the fitness of bacterial cells and mobile genetic elements, for example, prophages and conjugative vectors.


| INTRODUC TI ON
Bacteria in natural settings are constantly exposed to changing environmental conditions, and they must adapt to those changes to survive. Developing a phenotypically heterogeneous population is a strategy that bacteria utilize to increase their environmental fitness, and as a survival mechanism (Smits et al., 2007). This is due to the benefit for the population as a whole driven by cell-level phenotypic differences. Phenotypic heterogeneity can allow specific cells to survive sudden environmental changes that kill other members of the population. It can also lead to division of labor between individuals, which can increase the population's growth rate and facilitate the development of new biological functions (Ackermann, 2015;Davis & Isberg, 2016;West & Cooper, 2016;Zhang et al., 2016). In the last decade, the study of phenotypic heterogeneity among microbial populations and communities has become a major research focus, and new techniques and models are being generated to explore this facet of microbiology (Ackermann, 2015;Claessen et al., 2014;Jo et al., 2022;Shank, 2018).
Bacillus subtilis is a Gram-positive non-pathogenic bacterium that has been studied for over a century in a wide range of topics (Kovács, 2019), and has become a model organism for the study of bacterial differentiation, including community movement on semisolid agar surfaces, swarming and sliding (Grau et al., 2015;Hölscher & Kovács, 2017;Kearns, 2010), sporulation (Errington, 2003;Khanna et al., 2020;Tan & Ramamurthi, 2014), and biofilm formation (Arnaouteli | 21 GALLEGOS-MONTERROSAandKOVÁCS et al., 2021;Kovács & Dragoš, 2019;Mhatre et al., 2014). An interesting characteristic of B. subtilis, both under planktonic and biofilm conditions, is that its cells divide into discrete subpopulations, each with a different phenotype although all still possessing the same genotype (Kovács & Kuipers, 2013;Veening et al., 2008). This phenotypic differentiation leads to division of labor or bet hedging, providing an important ecological advantage to this bacterium (de Jong et al., 2011;Dragoš et al., 2018;Jautzus et al., 2022;Van Gestel et al., 2015). In the following sections, we address the role that the family of Rap phosphatases and their Phr peptides play in the regulation of B. subtilis phenotypic differentiation among Bacilli, their mechanism of action and structural functionality, as well as the ecological and genetic reasons that may explain their wide distribution in this genus.

| A FAMILY OF REG UL ATORY PH OS PH ATA S E S
The genetic network of B. subtilis contains multiple cross-talk points between the activities of several global regulators that guarantees that the population differentiates accordingly to its environmental conditions (López & Kolter, 2010). Furthermore, B. subtilis possesses a family of cell-cell communication Rap phosphatases that finetunes this intertwined genetic network (Perego, 2013).
The Rap phosphatases are conserved proteins (>25% of sequence identity) of ca. 380 amino acids that are able to hinder the phosphorylation of global regulators and therefore influencing the expression of hundreds of genes (Perego, 2013;Pottathil & Lazazzera, 2003).
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 (Perego et al., 1994;Veening et al., 2005). Some Rap phosphatases can also bind to their target transcriptional regulators, forming a complex that can no longer adhere to DNA (Bongiorni et al., 2005;Core & Perego, 2003). Shortly after the discovery of the first Rap phosphatases (RapA and RapB), it was recognized that a small gene directly following rapA 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, phosphatase regulator A (Perego & Hoch, 1996). It has been early realized that Phr peptides have important function in affecting different developmental pathways of B. subtilis (Lazazzera et al., 1999;Solomon et al., 1995Solomon et al., , 1996. Subsequent studies have later revealed a wide variety of rap genes 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. The rap-phr gene pairs are often recognized as cassettes, and the production of their respective proteins is translationally coupled Pedreira et al., 2022;Reizer et al., 1997). Once produced, the Rap phosphatases can immediately exert their regulatory function, either by dephosphorylating or preventing the DNA-binding of their target transcriptional regulator ( Figure 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 Nterminal portion, followed by signal peptide cleavage domains and hydrophilic C-terminal domains (Stephenson et al., 2003). 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 F I G U R E 1 General regulatory mechanism of Rap-Phr pairs. (i) the Rap protein is produced, and (ii) carries its regulatory role intracellularly; meanwhile, (iii) pre-Phr proteins are produced, (iv) processed and exported out of the cell as Phr peptides; (v) upon reaching threshold concentrations, (vi) mature Phr peptides can be imported into the cell via the Opp permease, and (vii) inhibit its cognate Rap protein. Rap proteins are visualized using the RapF crystal structure (PDB doi: 10.2210/pdb4I9E/pdb), the red and green colors indicate two units of a protein dimer. The prePhr is depicted using the structure based on AlphaFold as displayed in SubtiWiki (Pedreira et al., 2022). The figure was prepared on BioRe nder.com. oligopeptide permease (Lazazzera et al., 1999;LeDeaux et al., 2006;Solomon et al., 1995). 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 (Gallego del Sol & Marina, 2013;Neiditch et al., 2017;Perego, 2013;Pottathil & Lazazzera, 2003).

| MA S TER REG UL ATOR S OF PHENOT YPI C D IFFERENTIATI ON IN B . SUBTILIS
In B. subtilis, the response regulators Spo0A, ComA, and DegU are recognized as the master switches that control the development of population heterogeneity (Kovács, 2016). The activity of these three heterogeneity modulators depends on the ratios of the respective proteins in their non-phosphorylated and phosphorylated states (phosphorylated regulators are henceforth indicated with ~P). In general, the phosphorylation state affects the regulator's affinity for the promotor regions of the genes that they regulate (Kobayashi, 2007;Kovács, 2016). Delicate modulation of these ratios allows B. subtilis to develop a heterogeneous population, where cells adapt to small environmental differences (micro-niches) within the population, especially in the spatial structure of biofilms (Arnaouteli et al., 2021;López et al., 2009;Shank & Kolter, 2011).
Spo0A is a transcriptional regulator that controls the expression of hundreds of genes and operons in B. subtilis important for biofilm matrix production and the generation of spores (Errington, 2003).
Spo0A is phosphorylated via a phosphorelay that can be initiated by any of five known independent histidine kinases (KinA, KinB, KinC, KinD, and KinE;Arnaouteli et al., 2021;Jiang et al., 2000). Once activated by their cognate signals, the Kin kinases phosphorylate the response regulator Spo0F, which in turn transfers the phosphoryl group to a secondary response regulator, Spo0B, which finally phosphorylates Spo0A. The amount of Spo0A~P in the cells determines which of its target genes are expressed, toxins are expressed at low Spo0A~P and sporulation is activated at high Spo0A~P level in the cells (Fujita et al., 2005;Kovács, 2016).
The comXQPA genes encode a quorum sensing (QS) system, including the global regulator, ComA protein (López et al., 2009). QS is a common cell communication strategy that relies on the production and detection of extracellular autoinducer signaling molecules by cells of the same species (Waters & Bassler, 2005;Whiteley et al., 2017). The ComA QS system comprises the ComX peptide as its autoinducer and the membrane-localized ComP histidine kinase as the sensor. The extracellular ComX signal activates ComP, leading to autophosphorylation and transfer of the phosphate to ComA (Grossman, 1995;Mielich-Süss & Lopez, 2015). Once phosphorylated, ComA controls the production of surfactin (an important surfactant lipopeptide), and the development of competence in B.
The third major master switch of population heterogeneity in B. subtilis, DegU is the response regulator of the DegS/U two component system. DegS is a cytoplasmic sensor histidine kinase that directly phosphorylates DegU. The DegU regulon is extensive and includes genes associated with motility (e.g., flagellum production) and biofilm formation (e.g., the hydrophobin protein BslA and exoenzymes needed for substrate degradation ;Murray et al., 2009). Non-phosphorylated DegU activates competence development. Depending on the relative amount of DegU~P, different sets of genes are transcribed that provide a smooth transition from surface spreading to settlement during biofilm development (Dahl et al., 1991;Kobayashi, 2007;Kovács, 2016).

| REG UL ATORY FUN C TION AND MECHANIS MS OF THE R AP-PHR C A SS E T TE S
The regulatory function and mechanism of action of the Rap phosphatases and Phr peptides has been intensively studied. The known Rap phosphatases have been studied in diverse strains, finding an apparent redundancy in their function: most Rap phosphatases act upon Spo0F~P, ComA~P, or both (Bischofs et al., 2009;Grossman, 1995), in addition to specific Rap phosphatases influencing DegU (Ogura et al., 2002). Furthermore, these investigations have revealed that certain Rap-Phr cassettes are encoded on plasmids, and that these regulatory modulators are a common feature of other members of the Bacillus genus (Boguslawski et al., 2015;Cardoso et al., 2019;Koetje et al., 2003;Yang et al., 2015). Table 1 presents the known function of those Rap phosphatases that have been studied or reported independently, along with their possible action mechanism.
All known Rap phosphatases share a high sequence homology; however, they regulate structurally distinct targets (Neiditch et al., 2017;Pottathil & Lazazzera, 2003). Initial structural predictions of Rap phosphatases based on amino acid sequence suggested a two-domain architecture consisting of an N-terminal 3-helix bundle domain connected to a tetratricopeptide repeat (TPR) domain. This structure is strikingly different from other known bacterial phosphatases (Neiditch et al., 2017;Parashar et al., 2011;Parashar, Jeffrey, et al., 2013). TPR domains consist of 3-16 repeats of a degenerate 34 amino acid sequence motif, these repeats create a right-handed superhelix structure with an internal ligand-binding concave surface. TPR domains are known to function as protein-protein interaction domains (Blatch & Lässle, 1999).
The regulatory mechanism of the Phr peptides has also been structurally studied. Binding of Rap proteins to their cognate Phr peptides is mediated by their C-terminal TPR domains, and causes a pronounced rotation of the N-terminal 3-helix bundle; this creates two helix-turnhelix structures that pack against the existing C-terminal TPR domain.
This rearrangement generates a compression along the whole TPR superhelical axis, which causes the loss of the ligand-binding concave surface normally present in Rap proteins. Furthermore, the Phr peptides can interact with the residues of multiple TPR repeats (up to six, in the case of RapF-PhrF complexes), leading to intramolecular interactions that stabilize the "closed" conformation of the Rap protein (Gallego del Sol & Marina, 2013;Parashar, Jeffrey, et al., 2013).
These multi-TPR motif interactions confer a high specificity to Rap-Phr binding, with some Phr residues determining protein anchoring and orientation, and others mediating the interaction with the residues of the Rap protein. Gallego del Sol and Marina (2013) demonstrated that specific residues of RapF are required to bind its PhrF inhibitor, and that these residues are independent from the ability of RapF to bind to its target regulator ComA. The conservation of similar residues among Rap proteins, and additional experimental evidence from previous studies (Diaz et al., 2012) suggest that this is a common Phr-binding mechanism for all Rap proteins.
Interestingly, a few known rap genes lack the concomitant gene for a specialized Phr peptide, but can be regulated by Phr peptides produced by other Rap-Phr cassettes (see SubtiWiki http://subti wiki.uni-goett ingen.de; Pedreira et al., 2022). This is the case of RapB, which lacks a specialized Phr but is regulated by PhrC in vitro Perego, 1997). Another example is rapJ, which is not followed by a phr gene. RapJ plays its regulatory role by dephosphorylating Spo0F~P, and it binds to PhrC, forming a complex that is no longer able to interact with Spo0F~P
Omer Bendori et al. (2015) showed that this single amino acid substitution is responsible for the observed resistance of RapP to inhibition by PhrP, and that this inhibition could be restored by repairing the  is an efficient mechanism for individual organisms to acquire genes, regardless of functionality (Ochman et al., 2000;Soucy et al., 2015).

| D IS TRIBUTI ON AND D IVER S IT Y OF THE R AP-PHR FAMILY IN THE BACILLUS G EN US
HGT of Rap-Phr cassettes is heightened due to the fact that many rap-phr genes are encoded within mobile genetic elements (Bongiorni et al., 2006;Butala & Dragoš, 2022;Meijer et al., 2021;Mori et al., 2021;Parashar, Konkol, et al., 2013;Rösch et al., 2014;Singh et al., 2013). Importantly, Rap-Phr can also control mobile elements, as RapI-PhrI sensory system activates gene expression within the ICEBs1 mobile genetic element, therefore contributing to excision and transfer (Auchtung et al., 2005). In addition, the genes related to natural competence for the uptake of DNA from the environment are widely conserved in Bacilli (Kovács et al., 2009  . Furthermore, some Rap-Phr cassettes are able to regulate the mobility of the genetic element that contains them, be them plasmids Mori et al., 2021;Singh et al., 2013), or transposons (Auchtung et al., 2005(Auchtung et al., , 2007. These features could then favor a rapid expansion of Rap-Phr cassettes through HGT among Bacilli. Similarly, experimental selection for spores of B. subtilis increases the copy number of a cryptic prophage, phi3T, that carries Rap and Phr proteins (Dragoš, Priyadarshini, et al., 2021;Martin et al., 2017).
Interestingly, certain prophages, like SPβ, that are similar to phi3T do not carry such rap gene, but encode a biosynthetic gene cluster for a bacteriocin, sublancin that presumably benefit the fitness of the host bacterium (Butala & Dragoš, 2022;Dragoš, Andersen, et al., 2021;Dragoš, Priyadarshini, et al., 2021;Floccari & Dragoš, 2023). The Rap protein coded within the phi3T prophage has been also hypothesized to contribute to phage fitness (Bernard et al., 2021). Further, genome analysis combined with targeted experimental validation revealed that diversification of the autoinducer Phr peptides might be driven by promiscuous duplication events followed by adjustment of the Phr peptide in accordance with the respective evolutionary change of its cognate Rap phosphatase (Even-Tov, Omer .
A second factor that can help explain the diversity of the Rap-Phr family is functional diversification through social selection.
Experimental analyses and modeling suggest that acquisition of additional Rap-Phr system is facilitated by a facultative social cheating mechanism in B. subtilis (Even-Tov, Omer Bendori, Valastyan, et al., 2016). At low frequency, a strain harboring an extra Rap-Phr system acts as a cheater (i.e., it exploits the public good produced by the corresponding wild-type), while at high frequency, it adheres to cooperation without fitness loss. Such social selection in combination with HGT ensures the diversification and maintenance of multiple copies of Rap-Phr systems in Bacilli. Interestingly, the advantage of having multiple quorum sensing systems, that is, ComX-dependent activation of ComP/ComA system and Phr-affected Rap phosphatases, is not only apparent in B. subtilis, but also present in other bacteria, for example, in Vibrio harveyi (Even-Tov, Omer Bendori, Valastyan, et al., 2016). The ComX and Phr-based cell-cell communication systems work in concert to adjust developmental pathways of B. subtilis (Lazazzera et al., 1999;Solomon et al., 1995). Importantly, the signaling molecules of these alternative quorum sensing systems might diffuse distinctly within the cells' environment, therefore, defining the potential range of communication (Van Gestel et al., 2021). identified to influence the expression of rap-phr genes (Table 2 and Figure 3). This leads to regulatory differences among Rap proteins with the same target. As an example, both RapA and RapB dephosphorylate Spo0F~P, however, rapA expression is promoted by QSdependent ComA, while rapB seems to be promoted only by the house-keeping sigma factor σ A (Comella & Grossman, 2005;Jarmer et al., 2001;Mueller et al., 1992). This difference means that RapB will be produced earlier and more consistently than RapA, leading to differences in the Spo0A/Spo0A~P ratio in the cell population. A second level of fine-tuning is given by the Phr peptides, which may comprise the most diverse family of cell-cell communication autoinducers known to date .  (Nordgaard et al., 2021). In biofilm settings, the process-export-import regulatory pathway of Phr peptides provides B. subtilis cells with the opportunity to detect and integrate further environmental signals into their complex gene regulatory network.

| SO CIAL REG UL ATI ON OF B . SUBTILIS PHENOT YPIC ADAP TAB ILIT Y THROUG H R AP PHOS PHATA S E S
Cells in a biofilm live in micro-niches that lead to population heterogeneity (Christensen et al., 2002;López et al., 2009;Martin et al., 2016;Otto et al., 2020), thus, the biofilm subpopulations will secrete different types and amounts of mature Phr peptides to the extracellular milieu. Furthermore, the flexibility of Phr peptides to serve as cell-cell communication signals has been demonstrated by the ability of non-producing cells to detect the Phr signals produced by other cells (Pottathil & Lazazzera, 2003;Veening et al., 2005).
Therefore, Phr peptides can form a biofilm-spanning communication network, where each biofilm subpopulation can participate in the developmental process of their neighbors.

| R AP PHOS PHATA S E S AFFEC T S TR AIN FITNE SS THROUG H TIMING OF S P ORUL ATION
B. subtilis strains have been isolated from diverse environment, including soil, animals, plants and aquatic habitats (Connor et al., 2010;Earl et al., 2008;Kovács, 2019). Interestingly, although B. subtilis strains commonly show conservation among their main population heterogeneity regulators (Spo0A, ComA, DegU; Earl et al., 2008;Serra et al., 2014), they show high variation among their Rap-Phr cassette content .
Rap phosphatases determine the phenotypic memory of B. subtilis spores, the timing of spore formation and germination speed: the earlier the spores are formed, the faster these spores germinate driven by higher level of alanine dehydrogenase (i.e., high-quality spores), while delayed establishment of spores lead to higher number of spores in the population with reduced revival ability (i.e., high-quantity spores; Mutlu et al., 2018). Overexpression of rapA gene slows sporulation, vegetative cells have more time to grow and multiply, which increases the spores yield. However, this gives rise to a lower fraction of spores germinating (Mutlu et al., 2018). The differences in the number and the diversity of the Rap-Phr family play an important role for the environmental adaptability of specific strains of B. subtilis by allowing them to fine-tune their metabolism to different ecological niches. For example, strains isolated from the digestive tract of animals can show differences in the timing of sporulation initiation influenced by Rap-Phr cassette variation (Serra et al., 2014). This variation effectively serves as an adaptation that allows them to sporulate at optimum rates according to the ecological niche in which they live (Serra et al., 2014). Ultimately, these differences in the amount of Rap phosphatases and the timing of sporulation eventually influence the quality-yield spore tradeoff in natural isolates (Mutlu et al., 2020). along with the wild-type strain was cultivated either as biofilms or as planktonic cultures for 2-or 5-days, and spores were selected for re-inoculation. The initial hypothesis was that longer cultivation time (5 days instead of 2 days) allows enrichment of rap mutants that permit longer growth phase before cells are committed to sporulation.

| CON CLUDING REMARK S
The efficiency of bacterial adaptation depends on the regulatory pathways that enable the cell to sense and respond to the external environment that encompasses both abiotic and biotic factors.
Biotic factors include the bacterial population itself and its density. Bacilli evolved to integrate population density using the Rap-Phr cell-cell communication pathways that eventually diverged to modulate distinct, but partly overlapping, regulatory systems in the bacteria. Systemic dissection of the Rap-Phr systems (i.e., single-and multiple-deletions of the casettes) in different Bacillus species under diverse conditions, including their natural environments, will reveal their impact on the ecology of this group of microorganisms.

AUTH O R CO NTR I B UTI O N S
Ramses Gallegos-Monterrosa: Writing-original draft. Ákos T.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

E TH I C S S TATEM ENT
This article does not contain any studies with human participants or animals performed.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

F I G U R E 3
Complexity of Rap-Phr network. Top row depicts the regulators that influence the expression of rap-phr genes. The activities of proteins ComA, Spo0F, and DegU are hindered by corresponding Rap proteins (bellow). For details, see also Tables 1 and 2. The figure was prepared on BioRe nder.com.