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\begin{document}

\title{Phenotypic plasticity: the role of a phosphatase family in the genetic
regulation of Bacilli}



\author[1]{Ákos T. Kovács}%
\author[2]{Ramses Gallegos-Monterrosa}%
\affil[1]{Danmarks Tekniske Universitet}%
\affil[2]{Universidad Nacional Autonoma de Mexico Instituto de Biotecnologia}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
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
\emph{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 quorum sensing 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 \emph{B. subtilis} phenotypic differentiation, and in
other members of the \emph{Bacillus} genus. We also highlight the
contribution of these regulatory elements to the fitness of bacterial
cells and mobile genetic elements, e.g. prophages and conjugative
vectors.%
\end{abstract}%



\sloppy


Phenotypic plasticity: the role of a phosphatase family in the genetic
regulation of \emph{Bacilli}

Ramses Gallegos-Monterrosa\textsuperscript{1} and \selectlanguage{ngerman}Ákos T.
Kovács\textsuperscript{2,*}

\textsuperscript{1} Departamento de Microbiología Molecular, Instituto
de Biotecnología, Universidad Nacional Autónoma de México, C.P. 62210,
Cuernavaca, Morelos, México.

\textsuperscript{2} Bacterial Interactions and Evolution Group, DTU
Bioengineering, Technical University of Denmark, 2800 Kgs. Lyngby,
Denmark.

*Correspondence: Ákos T. Kovács, Technical University of Denmark, 2800
Kgs. Lyngby, Denmark. Email: atkovacs@dtu.dk

Running title: Rap phosphatase regulatory network of \emph{Bacilli}

\textbf{Abstract}

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\emph{Bacilli} , a broadly distributed family of Rap (\emph{R}
esponse-regulator \emph{a} spartyl \emph{p} hosphate) phosphatases is
known to modulate the function of the main phenotypic heterogeneity
regulators by controlling their phosphorylation. Even more, their
related extracellular Phr (\emph{Ph} osphatase\emph{r} egulator)
peptides function as quorum sensing 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 \emph{B.
subtilis} phenotypic differentiation, and in other members of the
\emph{Bacillus} genus. We also highlight the contribution of these
regulatory elements to the fitness of bacterial cells and mobile genetic
elements, e.g. prophages and conjugative vectors.

\textbf{Keywords}

Rap phosphatase, differentiation, phenotypic heterogeneity, Bacillus,
biofilm



\textbf{Introduction}

Bacteria in natural settings are constantly exposed to changing
environmental conditions, and they must adapt to those changes in order
to survive. Developing a phenotypically heterogeneous population is a
strategy that bacteria utilize to increase their environmental fitness,
and as a survival mechanism . 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 labour between individuals, which can increase
the population's growth rate and facilitate the development of new
biological functions . 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 .

\emph{Bacillus subtilis} is a Gram-positive non-pathogenic bacterium
that has been studied for over a century in a wide range of topics , and
has become a model organism for the study of bacterial differentiation,
including community movement on semi-solid agar surfaces, swarming and
sliding , sporulation , and biofilm formation . An interesting
characteristic of \emph{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 . This phenotypic differentiation leads to division of labor or
bet hedging, providing an important ecological advantage to this
bacterium . In the following sections we address the role that the
family of Rap phosphatases and their Phr peptides play in the regulation
of \emph{B. subtilis} phenotypic differentiation among \emph{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.

\textbf{Master regulators of phenotypic differentiation in \emph{B.
subtilis}}

In \emph{B. subtilis} , the response regulators DegU, Spo0A, and ComA
are recognized as the master switches that control the development of
population heterogeneity . 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 \selectlanguage{english}~P). In general, the phosphorylation state
affects the regulator's affinity for the promotor regions of the genes
that they regulate . Delicate modulation of these ratios allows \emph{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 .

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 to
motility (e.g. flagellum production) and biofilm formation (e.g.
hydrophobin protein and exoenzymes needed for substrate degradation) .
Non-phosphorylated DegU activates competence development, while
depending on the relative amount of DegU\selectlanguage{english}~P different sets of genes of
its regulon are transcribed that provide a smooth transition from
surface spreading to settlement during biofilm development .

Spo0A is a transcriptional regulator that controls the expression of
hundreds of genes and operons in \emph{B. subtilis} important among
others for biofilm matrix production and the generation of spores .
Spo0A is phosphorylated via a phosphorelay that can be initiated by any
of five known independent histidine kinases (KinA, KinB, KinC, KinD, and
KinE) . 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. Like DegU, the amount of Spo0A\selectlanguage{english}~P in the cells
determines which of its target genes are expressed .

The third major master switch of population heterogeneity in \emph{B.
subtilis} , ComA, is directly modulated by a quorum sensing (QS) system
. 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 . ComA-related QS system uses the
ComX peptide as its autoinducer and the membrane localized ComP
histidine kinase as sensor. The extracellular ComX signal activates
ComP, leading to autophosphorylation and transfer of phosphate to ComA .
Once phosphorylated, ComA controls the production of surfactin (an
important surfactant lipopeptide), and the development of competence in
\emph{B. subtilis} cells .

\textbf{A family of regulatory phosphatases}

The genetic network of \emph{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, \emph{B. subtilis} possesses a
family of QS-regulated Rap phosphatases that fine-tunes this
intertwinned genetic network .

The Rap phosphatases are conserved proteins (\textgreater{}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\selectlanguage{english}~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
following\emph{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, \emph{ph}
osphatase\emph{r} egulator A .

Subsequent studies have later revealed a wide variety of \emph{rap}genes
in the genome of \emph{B. subtilis} , most of which are followed (and
slightly overlapped) by \emph{phr} genes that code for small proteins of
ca. 40 amino acids known as Phr pro-peptides. The\emph{rap-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
\emph{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 .

\textbf{Regulatory function and mechanisms of the Rap-Phr cassettes}

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\selectlanguage{english}~P,
ComA\selectlanguage{english}~P, or both . 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\emph{Bacillus} genus . 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 . 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 . TPR domains consist
of 3 to 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 . Rap proteins appear to possess 6
canonical TPR motifs distributed along most of their length, with a
further non-canonical TPR motif separating TPR5 and TPR6 .
Parashar\emph{et al.} found that Rap proteins undergo a major
conformational change in their N-terminal domain when complexed with
their target proteins: the N-terminal 3-helix bundle is flipped and
merged with the existing C-terminal TPR domains . Further comparison of
the crystal structures of RapI, RapH and RapF (these last two in complex
with their target proteins) revealed that these conformational changes
can generate different interacting surfaces that block their target's
active site (in the case of Spo0F), or DNA-binding domain (in the case
of ComA) .

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-turn-helix structures that pack against the existing C-teminal 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 . 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
, suggest that this is a common Phr-binding mechanism for all Rap
proteins.

Interestingly, a few known \emph{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://subtiwiki.uni-goettingen.de) . This is the case of RapB, which
lacks a specialized Phr but is regulated by PhrC \emph{in vitro} .
Another example is \emph{rapJ} , which is not followed by a
\emph{phr}gene. RapJ plays its regulatory role by dephosphorylating
Spo0F\selectlanguage{english}~P, and it binds to PhrC, forming a complex that is no longer able
to interact with Spo0F\selectlanguage{english}~P . Moreover, at least one Rap protein is known
to be insensitive to regulation by its cognate Phr peptide.
The\emph{rapP-phrP} cassette is encoded in the pBS32 plasmid present in
the undomesticated strain of \emph{B. subtilis} NCIB 3610. RapP
regulates biofilm formation, sporulation, and competence development by
directly dephosphorylating Spo0F\selectlanguage{english}~P , and by a ComA-dependent mechanism .
However, RapP is not inhibited by PhrP, either when PhrP is
overexpressed\emph{in vivo,} or tested \emph{in vitro} with exogenously
added peptides derived from the C-terminal sequence of \emph{phrP} .
Conspicuously, RapP of \emph{B. subtilis} NCIB 3610 shows an
asparagine-to-threonine mutation at position 236 that is not present in
the corresponding \emph{rapP} alleles of other \emph{Bacillus} strains.
Omer Bendori \emph{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 N236T mutation. Similarly, the plasmid encoded Rap63-Phr63
and Rap8-Phr8 modules synergistically moderate sporulation and biofilm
formation of \emph{B. thuringiensis} .

The structural insights from these studies suggest that there is a
delicate balance between peptide-recognition specificity and regulatory
plasticity in the Rap-Phr family. Studies that use various synthetic Phr
peptides to investigate Rap-Phr interactions have found that, although
usually one peptide shows strong affinity for a given Rap protein
(normally the one coded in the same \emph{rap-phr} cassette), other Phr
peptides also show a partial ability to regulate the same Rap
protein\emph{in vitro} and \emph{in vivo} . These alternative Phr are
usually synthetized comprising the last 5 or 6 residues of the
C-terminal end of Phr pro-peptides . However, it is important to
consider that these results have been observed using artificial
laboratory conditions and, in particular, \emph{in vitro} experiments
include the testing of a very limited number of peptides at a time. In
natural settings, a complex network of Phr peptide cross-talk and
co-regulation might exist among the populations of \emph{Bacilli} to
modulate the function of Rap phosphatases.

\textbf{Distribution and diversity of the Rap-Phr family in
the\emph{Bacillus} genus}

The Rap-Phr family of regulatory proteins is highly diversified and
widespread in the \emph{Bacillus} genus. Rap homologs have been found in
the genomes of \emph{B. subtilis} , \emph{Bacillus
amyloliquefaciens,Bacillus pumilus, Bacillus anthracis, Bacillus cereus,
Bacillus halodurans} , \emph{Bacillus stearothermophilus,} and
\emph{Bacillus clausii} . Also, one of these Rap-Phr cassettes has been
studied heterologously in another \emph{Bacillus} species (RapQ-PhrQ),
and was found to be both functional and comparable to the native Rap
phosphatases of the host . These reports support the idea that the Rap
proteins and their Phr peptides play a common regulatory role in the
entire \emph{Bacillus} genus.

Interestingly, \emph{Bacillus} species that have at least one Rap-Phr
cassette normally possess multiple of these regulatory elements encoded
in their genomes. The first study to identify RapA as a Spo0F
phosphatase already established RapA and RapB as members of a
protein-aspartate phosphatase family with multiple members within the
same organism: \emph{B. subtilis} strain JH642 . Subsequent studies have
identified further members of this phosphatase family in diverse\emph{B.
subtilis} strains (see Table 1). In 2016, Even-Tov \emph{et al} .
compared over 400 \emph{Bacillus} genomes, searching for Rap homologs
based on their conserved N-terminal 3-helix bundle and C-terminal TPR
domain structure. They found over 2500 \emph{rap}homologs among the
studied genomes, and that \emph{B. subtilis} strains have, on average,
11 \emph{rap} genes per strain, while \emph{B. cereus}strains usually
possess around 6 of these phosphatases .

How has the Rap-Phr family achieved such a widespread presence and
diversity among \emph{Bacilli} ? There are two main factors that can be
considered when answering this question. First, bacteria commonly pass
on genes among sibling cells or cells from closely related species. This
ability, known as horizontal gene transfer (HGT), is an efficient
mechanism for individual organisms to acquire genes, regardless of
functionality . HGT of Rap-Phr cassettes is heightened due to the fact
that many \emph{rap-phr} genes are encoded within mobile genetic
elements . In addition, the genes related to natural competence for the
uptake of DNA from the environment are widely conserved in\emph{Bacilli}
. Even-Tov \emph{et al} . recently estimated that up to 75\% of Rap-Phr
cassettes may be mobile, based on a GC-content comparison with their
host strain . Furthermore, some Rap-Phr cassettes are able to regulate
the mobility of the genetic element that contains them, be them plasmids
, or transposons . These features could then favor a rapid expansion of
Rap-Phr cassettes through HGT among\emph{Bacilli} . Similarly,
experimental selection for spores of \emph{B. subtilis} increases the
copy number of a cryptic prophage, phi3T, that carries Rap and Phr
proteins . Interestingly, certain prophages, like SP\selectlanguage{greek}β, \selectlanguage{english}that are similar
to phi3T do not carry such \emph{rap} gene, but encode a biosynthetic
gene cluster for a bacteriocin that presumably benefit the fitness of
the host bacterium . The Rap protein coded within the phi3T prophage has
been also hypothesized to contribute to phage fitness . 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 .

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 \emph{B. subtilis} . At low frequency, a strain
harbouring an extra Rap-Phr system acts a cheater (i.e. exploiting the
public good produced by the wild type), while at high frequency it
returns to cooperation without fitness loss. Such social selection
processes in combination with HGT ensure the diversification and
maintainance of multiple copies of Rap-Phr systems in \emph{Bacilli} .

\textbf{Social regulation of \emph{B. subtilis} phenotypic adaptability
through Rap phosphatases}

Rap phosphatases fine-tune the sociomicrobiology of \emph{B. subtilis}by
modulating the activity of the master regulators Spo0A, ComA and DegU
and therefore their regulons (Fig. 2). This fine-tuning occurs at
different levels: first, the Rap phosphatases themselves that may appear
at first sight to have directly redundant regulatory roles, are
expressed under different conditions. This leads to regulatory
differences among Rap proteins with the same target. As an example, both
RapA and RapB dephosphorylate Spo0F~P, however, \emph{rapA} expression
is promoted by QS-dependent ComA, while \emph{rapB} seems to be promoted
only by the house-keeping sigma factor \selectlanguage{greek}σ\selectlanguage{english}\textsuperscript{A} . 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 QS
autoinducers known to date . QS systems are used to coordinate social
behavior that is most effective if a large number of cells participate,
such as expression of virulence, or production of a biofilm matrix. The
different Rap-Phr pairs have distinct influence on biofilm development
of \emph{B. subtilis} and colonization of the roots of the model
plant\emph{Arabidopsis thaliana} . In biofilm settings, the
process-export-import regulatory pathway of Phr peptides
provides\emph{B. subtilis} cells with the opportunity to detect and
integrate further environmental signals into their complex gene
regulatory network. Cells in a biofilm live in micro-niches that lead to
population heterogeneity , 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 cell .
Therefore, Phr peptides can form a biofilm-spanning communication
network, where each biofilm subpopulation can participate in the
developmental process of their neighbors.

\emph{B. subtilis} is an environmentally ubiquitous bacterium, with
numerous strains obtained from soil, animals, plants and aquatic
habitats . Interestingly, although \emph{B. subtilis} strains commonly
show conservation among their main population heterogeneity regulators
(Spo0A, ComA, DegU) , they show high variation among their Rap-Phr
cassette content . Rap phosphatases determine the phenotypic memory
of\emph{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) . Overexpression of \emph{kinA} gene
decelerates sporulation and therefore increase the spores yield,
however, this gives rise to a lower fraction of spores growing out . 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 \emph{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. This
variation effectively serves as an adaptation that allows them to
sporulate at optimum rates according to the ecological niche in which
they live . Ultimately, these differences in the amount of Rap
phosphatases and the timing of sporulation eventually influence the
quality-yield spore tradeoff in natural isolates .

Motivated by these observations related to the divergence in\emph{rap}
-\emph{phr} modules and their influence on timing of sporulation,
Gallegos-Monterrosa and colleagues constructed all possible combinations
of single and double \emph{rap} -\emph{phr} deletions in\emph{B.
subtilis} NCIB 3610 and tested the fitness of these strains in a
selection experiment . A mixture of the single and double mutants along
with the wild-type strain was cultivated either as biofilms or as
plaktonic cultures for 2- or 5-days, and spores were selected for
re-inoculation again. After 9 cycles of spore selection, the abundance
of strains in each culture condition was examined, which revealed that a
shorter incubation time selects for higher diversity of strains, while
longer cultivation selects for specific combination of
\emph{rap-phr}deletions . Additionally, fitness assays using the mutants
that were selected under these conditions, e.g. derivative carrying a
\emph{rapB}or \emph{rapH} deletion in addition to other \emph{rap}
-\emph{phr}mutation, demonstrated the competitiveness of these strains
compared to the wild-type strain. However, as these evolution
experiments included spore selection, the cryptic phi3T prophage and its
encoded Rap protein could have potentially influenced the selection
dynamics . Nevertheless, this experimental approach highlighted the
complexity of how the different Rap-Phr systems contribute to fitness of
\emph{B. subtilis} .

\textbf{Concluding remarks}

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.\emph{Bacilli} evolved to integrate population density using the
Rap-Phr quorum sensing 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 \emph{Bacillus} species
under diverse conditions, including their natural environments, will
reveal their impact on the ecology of this group of microorganisms.

\textbf{AUTHOR CONTRIBUTIONS}

Ramses Gallegos-Monterrosa: Writing -- original draft. \selectlanguage{ngerman}Ákos T. Kovács:
Writing -- original draft; Funding acquisition.

\textbf{ACKNOWLEDGEMENTS}

Work In the laboratory of ÁTK is supported by Danish National Research
Foundation (DNRF137) for the Center for Microbial Secondary Metabolites,
and Novo Nordisk Foundation within the INTERACT project of the
Collaborative Crop Resiliency Program (NNF19SA0059360).

\textbf{ORCID}

Ramses Gallegos-Monterrosa 0000-0002-2366-0912

Ákos T. Kovács 0000-0002-4465-1636

\textbf{Tables and Figures}

\textbf{TABLE 1} Known Rap-Phr proteins and their regulatory role
in\emph{B. subtilis} .\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Rap} \textbf{protein}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Phr} \textbf{peptide}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Gene cluster localization}\strut
\end{minipage} & \begin{minipage}[b]{0.24\columnwidth}\raggedright\strut
\textbf{Target regulator and action mechanism}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapA\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrA\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapB\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Blocks binding of ComA to DNA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapD\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Unknown\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Inhibition of ComA activity\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapE\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrE\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapF\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrF\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Blocks binding of ComA to DNA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapG\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrG\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Blocks binding of DegU to DNA. Possible inhibition of ComA
activity\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapH\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrH\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P. Blocks binding of ComA to DNA.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapI\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrI\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P. Involved in the regulation of mobile
genetic elements.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapJ\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrC\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Possible dephosphorylation of Spo0F~P\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapK\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrK\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Chromosome\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Inhibition of ComA activity\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RapLS20\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Unknown\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Possible dephosphorylation of Spo0F~P. Regulatory effect on sporulation.
Involved in the regulation of mobile genetic elements.\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
RapP\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
PhrP (RapP is resistant to PhrP)\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P. Inhibition of ComA activity\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
RapQ\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
PhrQ\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Inhibition of ComA activity. Possible dephosphorylation of Spo0F~P.
Originally identified in \emph{Bacillus amyloliquefaciens.}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rap40\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phr40\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Unknown\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Rap50\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Phr50\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.22\columnwidth}\raggedright\strut
Unknown\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Rap60\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Phr60\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Plasmid\strut
\end{minipage} & \begin{minipage}[t]{0.24\columnwidth}\raggedright\strut
Dephosphorylation of Spo0F~P. Inhibition of ComA activity\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{References}

\textbf{Figure legends}

\textbf{FIGURE 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), while prePhr is depicted using
the structure based on AlphaFold as displayed in SubtiWiki . The figure
was prepared on BioRender.com.

\textbf{FIGURE 2} Known cellular functions regulated by Rap phosphatases
in \emph{B. subtilis} . The centeral protein structure shows the
conserved architecture of Rap phosphatases (RapF crystal structure, PDB
doi: 10.2210/pdb4I9E/pdb). The biofilm colony images of wild type (WT)
and \selectlanguage{greek}Δ\selectlanguage{english}\emph{rapP} mutant are taken from Nordgaard \emph{et al.} (2021),
scale bar represents 5 mm. The figure was prepared on BioRender.com.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-1/Fig-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig-2-Rap-review/Fig-2-Rap-review}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

