# Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group

Priscilla de F. Cardoso, Stephane Perchat, Laurival A. Vilas-Boas, Didier Lereclus, Gislayne T. Vilas-Bôas

► To cite this version:

Priscilla de F. Cardoso, Stephane Perchat, Laurival A. Vilas-Boas, Didier Lereclus, Gislayne T. Vilas-Bôas. Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group. Current Genetics, 2019, 10.1007/s00294-019-00993-9. http://02625540

HAL Id: hal-02625540

https://hal.inrae.fr/hal-02625540

Submitted on 17 Nov 2023

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Diversity of the Rap-Phr quorum-sensing systems in the *Bacillus cereus* group

Priscilla de F. Cardoso<fnref n="1" />,<fnref n="2" /> · Stéphane Perchat<fnref n="2" /> · Laurival A. Vilas-Boas<fnref n="1" /> · Didier Lereclus<fnref n="2" /> · Gislayne T. Vilas-Bôas<fnref n="1" />

Received: 7 December 2018 / Revised: 10 May 2019 / Accepted: 13 May 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

## Abstract

Bacteria of the *Bacillus cereus* group colonize several ecological niches and infect different hosts. *Bacillus cereus*, a ubiquitous species causing food poisoning, *Bacillus thuringiensis*, an entomopathogen, and *Bacillus anthracis*, which is highly pathogenic to mammals, are the most important species of this group. These species are closely related genetically, and their specific toxins are encoded by plasmids. The infectious cycle of *B. thuringiensis* in its insect host is regulated by quorum-sensing systems from the RNPP family. Among them, the Rap-Phr systems, which are well-described in *Bacillus subtilis*, regulate essential processes, such as sporulation. Given the importance of these systems, we performed a global in silico analysis to investigate their prevalence, distribution, diversity and their role in sporulation in *B. cereus* group species. The *rap-phr* genes were identified in all selected strains with 30% located on plasmids, predominantly in *B. thuringiensis*. Despite a high variability in their sequences, there is a remarkable association between closely related strains and their Rap-Phr profile. Based on the key residues involved in RapH phosphatase activity, we predicted that 32% of the Rap proteins could regulate sporulation by preventing the phosphorylation of Spo0F. These Rap are preferentially located on plasmids and mostly related to *B. thuringiensis*. The predictions were partially validated by in vivo sporulation experiments suggesting that the residues linked to the phosphatase function are necessary but not sufficient to predict this activity. The wide distribution and diversity of Rap-Phr systems could strictly control the commitment to sporulation and then improve the adaptation capacities of the bacteria to environmental changes.

**Keywords** Rap-Phr · *Bacillus cereus* group · RNPP · Sporulation · Phosphatase

## Introduction

Several bacterial processes are regulated by quorum sensing, a cell-cell communication that enables bacteria to regulate their fate with regard to the population density. The Rap proteins and their cognate Phr peptide inhibitors are quorum-sensing systems present in the *Bacillus cereus* group but not

extensively studied in these bacteria. The *B. cereus* group comprises at least seven species (*Bacillus cereus* sensu stricto, *Bacillus thuringiensis*, *Bacillus anthracis*, *Bacillus weihenstephanensis*, *Bacillus mycoides*, *Bacillus pseudo-mycoides* and *Bacillus cytotoxicus*) of rod-shaped, spore-forming, Gram-positive bacteria that are found in diverse ecological niches and able to colonize different hosts (Liu et al. 2015). Due to the complex phylogeny of the group, as phylogenetic clades are polyphyletics and species are paraphyletics, its taxonomy continues to be debated (Bazinet 2017; Guinebretière et al. 2008; Helgason et al. 2000; Liu et al. 2015; Raymond, 2017; Tourasse et al. 2011).

The three main species of the *B. cereus* group have a significant impact on human activity. *B. cereus* is a ubiquitous and opportunistic bacterium and includes strains that cause food poisoning with vomiting or diarrhea and severe local infections, such as endophthalmitis or periodontitis (Callegan et al. 2003; Ehling-schulz et al. 2006; Stenfors Arnesen et al. 2008). *B. thuringiensis* is the world's most

Communicated by M. Kupiec.

**Electronic supplementary material** The online version of this article (https://doi.org/10.1007/s00294-019-00993-9) contains supplementary material, which is available to authorized users.

<figure><img src="image_2.png" /></figure>

Gislayne T. Vilas-Bôas
gtvboas@gmail.com

<footnote n="1">¹ Depto. Biologia Geral, Universidade Estadual de Londrina, Londrina, Brazil</footnote>

<footnote n="2">² Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France</footnote>
used biopesticide due to its production of insecticidal tox-
ins (designated as Cry proteins) specifically pathogenic to
a wide range of insects (Schnepf et al. 1998). *B. anthracis*
is a mammal pathogen, including humans, and is the caus-
ative agent of anthrax (Liu et al. 2014). Although pheno-
typically different, these species are closely related geneti-
cally (Rasko et al. 2005), and the species determinants are
encoded by plasmid genes (Vilas-Boas et al. 2007). For
*B. cereus*, the enzymatic complex involved in cereulide
(emetic toxin) synthesis is encoded by pCER270 (Ehling-
Schulz et al. 2006). Strains are identified as *B. thuringien-
sis* if they produce a crystal inclusion during sporulation
due to the presence of plasmids carrying genes encoding
Cry toxins, generally active against insects or nematodes
(Deng et al. 2014; Schnepf et al. 1998). The high toxic-
ity of *B. anthracis* is due to toxins and its capsule, which
are encoded by genes located on the plasmids pXO1 and
pXO2, respectively (Kolstø et al. 2009).

Several microorganisms behaviors, such as biofilm for-
mation, sporulation, motility, genetic exchange (compe-
tence and conjugation), and virulence factor production,
are regulated by quorum sensing (QS), a cell–cell com-
munication process that allows bacteria and eukaryotic
microorganisms to coordinate their biological processes
based on the population density (Polke and Jacobsen 2017;
Rutherford and Bassler 2012). In Gram-positive bacteria,
this communication is done by signaling oligopeptides
that are recognized by cognate regulators, such as the
QS systems of the RNPP family (from Rap, NprR, PlcR,
and PrgX) (Declerck et al. 2007). These regulators are
formed by tetratricopeptide repeat (TPR) domains that are
structural motifs of degenerated residues that mediate pro-
tein–protein and protein–peptide interactions (D’Andrea
and Regan 2003). The activity of these cytoplasmic regu-
lators is activated (NprR and PlcR) or inhibited (Rap) by
secreted, matured, and re-imported peptides that func-
tion as signaling molecules (Perchat et al. 2011; Perego
and Hoch 1996; Pottathil and Lazazzera 2003; Slamti
and Lereclus 2002). The genes encoding these signaling
peptides are located directly downstream from the coding
sequence of their cognate RNPP regulator, and the two
genes are transcribed in the same orientation (Declerck
et al. 2007). Except for the Rap proteins, RNPP regula-
tors have an HTH (helix-turn-helix) DNA-binding domain,
allowing them to function as transcriptional regulators
(Declerck et al. 2007). During the infectious cycle of *B.
thuringiensis* in insect larvae, three QS systems are suc-
cessively activated (Slamti et al. 2014): (i) PlcR–PapR
regulates the virulence stage by controlling the expression
of virulence genes; (ii) NprR–NprX regulates the necro-
trophic stage, allowing bacteria to survive and to sporulate
in the insect cadaver; and (iii) Rap–Phr regulates the initia-
tion of the sporulation process.

Sporulation is essential for survival and dispersion of a
wide variety of organisms (Huang and Hull 2017). In *Bacil-
lus subtilis*, this differentiation process is regulated by a
complex pathway (Sonenshein 2000), in which Spo0A is
the major regulator of sporulation that must be phosphoryl-
ated to be active. External signals, such as starvation, are
detected by different sporulation kinases (KinA to KinE),
which phosphorylate the Spo0F response regulator (Bur-
bulys et al. 1991). The phosphoryl group is then transferred
through the phosphorelay from Spo0F to the phosphotrans-
ferase Spo0B, and then to Spo0A (Jiang et al. 2000a). Cer-
tain Rap proteins indirectly inhibit the phosphorylation of
Spo0A by dephosphorylating Spo0F and thus impair the
initiation of sporulation (Perego and Hoch 1996).

Eleven *rap* genes (from *rapA* to *rapK*) were identified
on the chromosome of the *B. subtilis* 168 strain. Functional
studies have shown that RapA, RapB, RapE, RapH, RapI,
and RapJ can dephosphorylate Spo0F (Jiang et al. 2000b;
2011, Parashar et al. 2011, 2013a; Perego et al. 1996; Smits
et al. 2007). RapC, RapD, RapF, RapH, and RapK regulate
competence by inhibiting ComA (Auchtung et al. 2006;
Bongiorni et al. 2005; Core and Perego 2003; Ogura and
Fujita 2007; Smits et al. 2007), RapG regulates extracel-
lular protease production by inhibiting DegU (Ogura et al.
2003), and RapI also regulates the mobility of the ICEBs1
genetic element (Auchtung et al. 2005). *rap*-*phr* genes have
also been identified in *B. subtilis* plasmids. These plasmid
systems are involved in the regulation of proteolytic enzyme
production (Koetje et al. 2003), sporulation, competence,
biofilm formation (Parashar et al. 2013b), and plasmid con-
jugation (Singh et al. 2013). The activities of Rap proteins
are inhibited by their cognate Phr peptides. Phr-encoding
genes are located downstream from the *rap* genes and are
generally co-transcribed, although many *phr* genes have a
secondary promoter (Perego and Brannigan 2001; McQuade
et al. 2001). The pro-Phr are secreted and processed in the
extracellular environment. The mature Phr are then internal-
ized within the bacterial cells by oligopeptide permeases and
bind to Rap proteins to inhibit their activity (Perego 1997).

The Rap–Phr systems are also present in bacteria of the *B. cereus* group. Bongiorni et al. (2006) have identified five *rap* genes in the *B. anthracis* A2012 strain, among which only two were shown to inhibit sporulation. More recently, Fazion et al. (2018) characterized the Rap–Phr system from a small plasmid (pHT8_1) of the *B. thuringiensis* HD73 strain and demonstrated its involvement in the regulation of sporulation in insect larvae.

In this study, we performed an overview of the Rap–Phr
systems in the *B. cereus* group, including their identifica-
tion, distribution, and prediction of their sporulation activity.
We show that the Rap–Phr systems are widespread in all
strains, in both chromosomes and plasmids, and with great
sequence variability. A comparison between the *B. cereus*

<figure><img src="image_3.png" /></figure>
and *B. thuringiensis* strains showed that plasmid Rap-Phr systems are more frequently present in *B. thuringiensis* than in *B. cereus*. One-third of the Rap proteins were predicted to have a sporulation function and these Rap proteins are preferentially located on plasmids and, therefore, are mainly present in *B. thuringiensis*.

# Materials and methods

## Bacterial genomes

The *B. cereus* group strains with a complete genome sequence available in the NCBI Genome database (http://www.ncbi.nlm.nih.gov/genome/) on April 2015 were selected. The species classification was considered as found in the database in the moment of data collection. The main available features (chromosome size and GC content, MLST sequence type, size and number of plasmids, and the proportion of the total genome they represent) of the genomes from the 49 selected strains are presented in Table 1 and Online Resource Table 1.

## Construction of the rap-phr database

Each selected genome sequence was screened for the presence of *rap* genes. Three strategies were used: (i) using ‘rap’ and ‘response regulator aspartate phosphatase’ as keywords; (ii) using each identified sequence for a sequence similarity search by BLASTn against all selected genomes and; (iii) using all chromosomal Rap protein sequences from the *B. subtilis* 168 strain as a query for a tBLASTn alignment with *B. cereus* group genomes. All protein sequences were analyzed using InterProScan (http://www.ebi.ac.uk/interpro/sequence-search) and SMART (http://smart.embl-heidelberg.de) servers for the identification of domains and motifs (Jones et al. 2014; Letunic et al. 2015). The detection of TPR domains and the absence of HTH DNA-binding domains were used as the main criteria to validate putative sequences as Rap proteins. Sequences shorter than 333 amino acids or showing uncharacteristic domains for Rap proteins were excluded.

Identification of *phr* genes was performed considering the gene organization: (i) short open reading frames (encoding 35–120 aa) and location, (ii) overlapping the *rap* gene (up to 10 bp) at the 3' terminal end or located immediately downstream (up to 100 bp) from the *rap* gene, and (iii) transcription from the same DNA strand as the *rap* gene. When a *phr* gene was not identified by this strategy, the downstream region of *rap* was scanned for small open reading frames (ORFs) checking for putative unannotated *phr* using the VectorNTI software (Invitrogen). The identified systems were numbered according to their location (first the

chromosomal genes, then the plasmid ones). The chromosomal genes were numbered according to their order from the replication origin.

A 20 kb region around the *rap* genes (10 kb upstream and 10 Kb downstream) was analyzed using the ISfinder database (Siguier et al. 2005) to verify the presence of mobile elements within these genomic regions.

## Rap protein clustering

Phylogenetic trees were constructed with Rap protein sequences according to the location of their genes in the genome: (i) total (all sequences), (ii) chromosome, and (iii) plasmids. The DAMBE program (Xia 2013) was used to gather sequences with 100% identity. Unique Rap sequences were aligned using the MUSCLE algorithm, and MEGA 6 (Tamura et al. 2013) was used to build phylogenetic trees by the Neighbor-Joining Method with the best model corrections for each alignment. RapH from *B. subtilis* 168 was used as outgroup in the Total tree, which was visualized on iTOL—Interactive Tree Of Life (Letunic and Bork 2019). For Rap clustering delimitation, a value of 0.8 was used as the cutoff using the average distance of the number of amino acid substitutions for chromosomal and plasmid trees. These trees were visualized on MEGA 6. For the Phr peptide, identical sequences recognition was done as described for the Rap proteins.

## Multilocus sequencing type (MLST) tree

According to the scheme of Tourasse et al. (2006), sequences of MLST housekeeping genes of all selected genomes were obtained from the ‘University of Oslo’s *Bacillus cereus* group MultiLocus and MultiData Typing website (http://mlstoslo.uio.no)’. Sequences of *adk*, *glpT*, *glpF*, *panC*, *pycA*, *ccpA*, and *pta* genes were downloaded already concatenated. Alignment and phylogenetic tree development were performed as described for Rap proteins.

## Plasmids construction and growth conditions

To assess the effect of Rap proteins on sporulation, seven plasmid *rap* genes (*rap6*-BtHD1, *rap8*-BtHD1, *rap10*-BtHD1, *rap6*-Bt407, *rap7*-Bt407, *rap8*-Bt407 and *rap7*-BtHD73) and three chromosomal *rap* genes (*rap1*-BcATCC14579, *rap2*-BcATCC14579 and *rap5*-BtHD73) were cloned in the plasmid pHT315-*P*<sub>xylA</sub>, a multi-copy vector with xylose-inducible promoter (Grandvalet et al. 2001). All genes were amplified by PCR using primers listed in Online Resource Table 2 and ligated to the plasmid pHT315-*P*<sub>xylA</sub> using the appropriate restriction sites. For cloning steps, these plasmids were transformed in *Escherichia coli* K-12 strain TG1 and then in the
<table><caption>**Table 1** Main features of the 49 selected genomes from the *B. cereus* group strains</caption><thead><tr><th rowspan="2">Strain (named as in NCBI database)</th><th rowspan="2">Release date</th><th rowspan="2">Assembly</th><th colspan="2">Chromosome</th><th colspan="3">Plasmids</th><th rowspan="2">STs (MLST)</th></tr><tr><th>Size (Mb)</th><th>%GC</th><th>Nb</th><th>Size (Mb)</th><th>%↑ genome</th></tr></thead><tbody><tr><td colspan="9"><strong>B. cereus</strong></td></tr><tr><td>ATCC 10987</td><td>2002</td><td>GCA_000008005.1</td><td>5.22</td><td>35.6</td><td>1</td><td>0.21</td><td>4%</td><td>2</td></tr><tr><td>ATCC 14579</td><td>2003</td><td>GCA_000007825.1</td><td>5.41</td><td>35.3</td><td>1</td><td>0.01</td><td>0%</td><td>33</td></tr><tr><td>E33L</td><td>2004</td><td>GCA_000011625.1</td><td>5.30</td><td>35.4</td><td>5</td><td>0.55</td><td>10%</td><td>57</td></tr><tr><td>AH187</td><td>2008</td><td>GCA_000021225.1</td><td>5.27</td><td>35.6</td><td>4</td><td>0.33</td><td>6%</td><td>3</td></tr><tr><td>B4264</td><td>2008</td><td>GCA_000021205.1</td><td>5.42</td><td>35.3</td><td>0</td><td>-</td><td>-</td><td>-2</td></tr><tr><td>G9842</td><td>2008</td><td>GCA_000021305.1</td><td>5.39</td><td>35.3</td><td>2</td><td>0.35</td><td>6%</td><td>120</td></tr><tr><td>AH820</td><td>2008</td><td>GCA_000021785.1</td><td>5.3</td><td>35.4</td><td>3</td><td>0.28</td><td>5%</td><td>39</td></tr><tr><td>Q1</td><td>2009</td><td>GCA_000013065.1</td><td>5.21</td><td>35.6</td><td>2</td><td>0.29</td><td>6%</td><td>40</td></tr><tr><td>03BB102</td><td>2009</td><td>GCA_000022505.1</td><td>5.27</td><td>35.4</td><td>1</td><td>0.18</td><td>3%</td><td>122</td></tr><tr><td>anthracis CI</td><td>2010</td><td>GCA_000143605.1</td><td>5.2</td><td>35.4</td><td>3</td><td>0.28</td><td>5%</td><td>153</td></tr><tr><td>NC7401</td><td>2011</td><td>GCA_000283675.1</td><td>5.2</td><td>35.6</td><td>5</td><td>0.33</td><td>6%</td><td>3</td></tr><tr><td>F837/76</td><td>2011</td><td>GCA_000239195.1</td><td>5.22</td><td>35.4</td><td>2</td><td>0.07</td><td>1%</td><td>182</td></tr><tr><td>FRI-35</td><td>2012</td><td>GCA_000292415.1</td><td>5.08</td><td>35.6</td><td>4</td><td>0.3</td><td>6%</td><td>188</td></tr><tr><td>FT9</td><td>2014</td><td>GCA_000724585.1</td><td>5.22</td><td>35.5</td><td>0</td><td>-</td><td>-</td><td>191</td></tr><tr><td>03BB87</td><td>2014</td><td>GCA_000789315.1</td><td>5.46</td><td>35.3</td><td>2</td><td>0.26</td><td>5%</td><td>58</td></tr><tr><td>D17</td><td>2015</td><td>GCA_000832385.1</td><td>5.38</td><td>35.4</td><td>1</td><td>0.21</td><td>4%</td><td>179</td></tr><tr><td>FM1</td><td>2015</td><td>GCA_000832525.1</td><td>5.3</td><td>35.5</td><td>1</td><td>0.40</td><td>8%</td><td>186</td></tr><tr><td>3a</td><td>2015</td><td>GCA_000832765.1</td><td>5.27</td><td>35.4</td><td>3</td><td>0.37</td><td>7%</td><td>124</td></tr><tr><td>G9241</td><td>2015</td><td>GCA_000832805.1</td><td>5.27</td><td>35.4</td><td>3</td><td>0.45</td><td>9%</td><td>58</td></tr><tr><td>ATCC 4342</td><td>2015</td><td>GCA_000832845.1</td><td>5.27</td><td>35.4</td><td>1</td><td>0.04</td><td>1%</td><td>4</td></tr><tr><td>03BB108</td><td>2015</td><td>GCA_000832865.1</td><td>5.34</td><td>35.3</td><td>7</td><td>0.73</td><td>14%</td><td>119</td></tr><tr><td>Al Hakam<sup>a</sup></td><td>2015</td><td>GCA_000832885.1</td><td>5.23</td><td>35.8</td><td>6</td><td>0.45</td><td>9%</td><td>173</td></tr><tr><td>S2-8</td><td>2015</td><td>GCA_000835185.1</td><td>5.27</td><td>35.4</td><td>2</td><td>0.37</td><td>7%</td><td>124</td></tr><tr><td>FORC_005</td><td>2015</td><td>GCA_000978375.1</td><td>5.35</td><td>35.3</td><td>0</td><td>-</td><td>-</td><td>187</td></tr><tr><td><strong>B. cereus</strong> average</td><td></td><td></td><td>5.29</td><td>35.4</td><td>2.5</td><td>0.31</td><td>6%</td><td></td></tr><tr><td colspan="9"><strong>B. thuringiensis</strong></td></tr><tr><td>konkukian 97-27</td><td>2004</td><td>GCA_000008505.1</td><td>5.24</td><td>35.4</td><td>1</td><td>0.07</td><td>1%</td><td>59</td></tr><tr><td>Al Hakam</td><td>2006</td><td>GCA_000015065.1</td><td>5.26</td><td>35.4</td><td>1</td><td>0.06</td><td>1%</td><td>89</td></tr><tr><td>BMB171</td><td>2010</td><td>GCA_000092165.1</td><td>5.33</td><td>35.3</td><td>1</td><td>0.31</td><td>6%</td><td>152</td></tr><tr><td>finitimus YBT-020</td><td>2011</td><td>GCA_000190515.1</td><td>5.36</td><td>35.5</td><td>2</td><td>0.33</td><td>6%</td><td>155</td></tr><tr><td>chinensis CT-43</td><td>2011</td><td>GCA_000193355.1</td><td>5.49</td><td>35.4</td><td>10</td><td>0.66</td><td>12%</td><td>44</td></tr><tr><td>HD-771</td><td>2012</td><td>GCA_000292455.1</td><td>5.89</td><td>35.2</td><td>8</td><td>0.56</td><td>10%</td><td>75</td></tr><tr><td>HD-789</td><td>2012</td><td>GCA_000292705.1</td><td>5.5</td><td>35.3</td><td>6</td><td>0.84</td><td>15%</td><td>136</td></tr><tr><td>MC28</td><td>2012</td><td>GCA_000300475.1</td><td>5.41</td><td>35.4</td><td>7</td><td>1.28</td><td>24%</td><td>231</td></tr><tr><td>Bt407</td><td>2012</td><td>GCA_000306745.1</td><td>5.5</td><td>35.4</td><td>9</td><td>0.65</td><td>12%</td><td>44</td></tr><tr><td>kurstaki HD73</td><td>2013</td><td>GCA_000338755.1</td><td>5.65</td><td>35.3</td><td>7</td><td>0.27</td><td>5%</td><td>115</td></tr><tr><td>thuringiensis IS5056</td><td>2013</td><td>GCA_000341665.1</td><td>5.49</td><td>35.4</td><td>14</td><td>1.3</td><td>24%</td><td>44</td></tr><tr><td>YBT-1518</td><td>2013</td><td>GCA_000497525.2</td><td>6</td><td>35.4</td><td>6</td><td>0.68</td><td>11%</td><td>261</td></tr><tr><td>kurstaki YBT-1520</td><td>2014</td><td>GCA_000688795.1</td><td>5.6</td><td>35.3</td><td>11</td><td>0.98</td><td>18%</td><td>115</td></tr><tr><td>kurstaki HD-1</td><td>2014</td><td>GCA_000717535.1</td><td>5.63</td><td>35.3</td><td>13</td><td>1.13</td><td>20%</td><td>115</td></tr><tr><td>galleriae HD-29</td><td>2014</td><td>GCA_000803665.1</td><td>5.7</td><td>35.3</td><td>10</td><td>1.04</td><td>18%</td><td>211</td></tr><tr><td>HD1011</td><td>2015</td><td>GCA_000832485.1</td><td>5.23</td><td>35.5</td><td>4</td><td>0.86</td><td>16%</td><td>71</td></tr><tr><td>HD571</td><td>2015</td><td>GCA_000832825.1</td><td>5.26</td><td>35.4</td><td>1</td><td>0.06</td><td>1%</td><td>89</td></tr><tr><td>HD682</td><td>2015</td><td>GCA_000832925.1</td><td>5.21</td><td>35.5</td><td>3</td><td>0.08</td><td>2%</td><td>212</td></tr><tr><td>HD1002</td><td>2015</td><td>GCA_000835025.1</td><td>5.49</td><td>35.3</td><td>7</td><td>1.08</td><td>20%</td><td>136</td></tr><tr><td>morrisoni BGSC 4AA1</td><td>2015</td><td>GCA_000940785.1</td><td>5.65</td><td>35.3</td><td>6</td><td>0.53</td><td>9%</td><td>112</td></tr><tr><td><strong>B. thuringiensis</strong> average</td><td></td><td></td><td>5.49</td><td>35.4</td><td>6.4</td><td>0.64</td><td>12%</td><td></td></tr></tbody></table>
<table><caption>**Table 1** (continued)</caption><thead><tr><th rowspan="2">Strain (named as in NCBI database)</th><th rowspan="2">Release date</th><th rowspan="2">Assembly</th><th colspan="2">Chromosome</th><th colspan="3">Plasmids</th><th rowspan="2">STs (MLST)</th></tr><tr><th>Size (Mb)</th><th>%GC</th><th>Nb</th><th>Size (Mb)</th><th>%↑ genome</th></tr></thead><tbody><tr><td><i>B. anthracis</i> 'Ames Ancestor' A2084</td><td>2004</td><td>GCA_000008445.1</td><td>5.23</td><td>35.4</td><td>2</td><td>0.27</td><td>5%</td><td>1</td></tr><tr><td><i>B. weihenstephanensis</i> KBAB4<sup>b</sup></td><td>2007</td><td>GCA_000018825.1</td><td>5.26</td><td>35.6</td><td>4</td><td>0.61</td><td>12%</td><td>118</td></tr><tr><td><i>B. mycoides</i> ATCC 6462</td><td>2015</td><td>GCA_000832605.1</td><td>5.26</td><td>35.5</td><td>3</td><td>0.38</td><td>7%</td><td>133</td></tr><tr><td><i>B. pseudomycoides</i> DSM 12442</td><td>2012</td><td>GCA_000161455.1</td><td>5.78</td><td>35.4</td><td>-</td><td>-</td><td>-</td><td>132</td></tr><tr><td><i>B. cytotoxicus</i> NVH 391-98</td><td>2007</td><td>GCA_000017425.1</td><td>4.09</td><td>35.9</td><td>1</td><td>0.01</td><td>0%</td><td>117</td></tr><tr><td><i>B. cereus</i> group Average</td><td></td><td></td><td>5.35</td><td>35.4</td><td>4.1</td><td>0.46</td><td>9%</td><td></td></tr></tbody></table>

STs (Sequence Types) data was obtained from the University of Oslo's *Bacillus cereus* group MultiLocus and MultiData Typing website (http://mlstoslo.uio.no). Plasmid 'Nb' is the sum of different plasmids sequenced for each strain and '%↑ genome' is how much all these plasmids increase the genome size of that strain (in relation to chromosome size alone)

Dam⁻ Dcm⁻ *E. coli* strain ET12567 (Stratagene, La Jolla, CA, USA) by thermal shock. Finally, each constructed plasmid was transformed by electroporation (Lereclus et al. 1989) in the acrystalliferous (Cry⁻) *B. thuringiensis* var. kurstaki HD73 strain (Wilcks et al. 1998). Luria-Bertani (LB) medium was used to cultivate *E. coli* and *B. thuringiensis* at 37 °C for DNA preparation. The medium HCT was used to optimize the sporulation of *B. thuringiensis* (Lereclus et al. 1982). Antibiotics were used at the following concentration: ampicillin 100 µg/mL for *E. coli* and erythromycin 10 µg/mL for *B. thuringiensis*.

## DNA manipulation

Genomic DNA from the three *B. thuringiensis* strains (HD-1, Bt407, and HD73) and the *B. cereus* strain (ATCC14579) was extracted using the Puregene Yeast/Bact. Kit B (Qiagen, France). PCRs were performed in an Applied Biosystems 2720 Thermal cycler (Applied Biosystem, USA) with Phusion High-Fidelity or Taq DNA Polymerase (New England Biolabs, USA) and oligonucleotides (Online Resource Table 2) were synthesized by Eurofins Genomics (Germany). The QIAquick PCR Purification Kit (Qiagen, France) was used to purify the amplified DNA fragments that were subsequently treated with appropriated restriction enzymes (New England Biolabs). Digested DNA fragments were separated on 1% agarose gels and purified from gels using the QIAquick gel extraction kit (Qiagen, France). T4 DNA ligase and restriction enzymes were used following the manufacturer's recommendations (New England Biolabs). *E. coli* plasmid DNA extractions were performed using the QIAprep Spin Mini-prep Kit (Qiagen, France). DNA sequencing was carried out by GATC Biotech (Konstanz, Germany).

## Sporulation assay

The sporulation efficiency of *B. thuringiensis* HD73 strain expressing *rap* genes was determined in the sporulation-specific medium HCT supplemented with 20 mM of xylose at the beginning of stationary growth phase. After 48 h of growth at 30 °C, serial dilutions were plated before and after heat treatment for 12 min at 80 °C. The sporulation percentage was calculated as 100×the ratio between heat-resistant spores per milliliter and total viable cells per milliliter. All experiments were repeated at least three times, and the mean values (± standard error of the mean) were calculated.

## Statistical analyses

The appropriate statistical test for each data was performed in GraphPad InStat Software version 3.05. Comparisons between *B. cereus* and *B. thuringiensis* means were analyzed with *t* test while when contingency table were used to confront both species, the Fisher's exact test was used. The data obtained with sporulation assay were analyzed using one-way Analysis of Variance (ANOVA) followed by Tukey-Kramer Multiple Comparisons Test (*P* < 0.01).

## Results

### Genomic overview of the *B. cereus* group strains

To study the occurrence, the prevalence and the distribution of Rap-Phr systems in the *B. cereus* group, those genome sequences with complete assembly level available in NCBI Genome section until April 2015 were selected. Most of these genomes belong to *B. cereus*, *B. thuringiensis* or *B. anthracis*, considering the relevance

<footnote><sup>a</sup>This strain was first annotated as *B. cereus* (Johnson et al. 2015), and is now classified as *B. pseudomycoides* (strain BTZ)</footnote>

<footnote><sup>b</sup>Nowadays, *B. weihenstephanensis* strains are considered as heterotypic synonym of *B. mycoides* (Liu et al. 2018)</footnote>
of these species. All *B. cereus* and *B. thuringiensis* avail-
able strains were selected. However, as *B. anthracis* is a
clonal species (Helgason et al. 2000; Rasko et al. 2005),
only one representative genome sequence (Ames Ancestor
strain) was selected, even if around 30 genomes of this
species were available. Moreover, our preliminary results
revealed identical Rap-Phr profiles in all the *B. anthra-
cis* isolates, thus confirming the clonal aspect of the *B.
anthracis* strains (data not shown). For the other species
(*B. mycoides*, *B. pseudomycoides*, *B. weihenstephanensis*,
and *B. cytotoxicus*), only one genome for each species was
chosen. When the same strain was sequenced twice, only
one was selected. Following these criteria, 49 genomes
were used in this study (Table 1).

The GC content of these genomes was around 35%, with minor differences among the strains. In the *B. cereus* strains, the chromosomal size ranged from 5.08 to 5.46 Mb, while in *B. thuringiensis* the variation was from 5.21 to 6 Mb. All the other species showed chromosomes larger than 5 Mb, except *B. cytotoxicus* NVH 391-98, which was 4.09 Mb (Table 1). The analysis of the plasmid content of the 49 selected strains revealed a total of 197 plasmids with a size ranging from 2.1 to 502 Kb (Online Resource Table 3). These plasmids can provide a significant increase in genome size (Table 1). In *B. cereus*, the average increase of the genome size is 5% with a maximum of 12% in the strain *B. cereus* 03BB108. In *B. thuringiensis*, the average increase of the genome size was 12%, with a maximum of 24% in the *B. thuringiensis* MC28 and *B. thuringiensis* IS5056 strains.

rap-phr genes distribution

In the 49 genomes sequences, 302 *rap* genes were identi-
fied (Online Resource Data 1 and Online Resource Table 4)
whose 144 (47.7%) were correctly annotated as ‘response
regulator aspartate phosphatase’ or ‘rap’. A *phr* gene was
identified downstream from all *rap* genes, but 31 of them
were not annotated as ORFs (Online Resource Table 4). The
*rap* and *phr* genes were always located in the same DNA
strand with a slight overlapping (usually 1 or 4 nucleotides).
The average size was 1099 bp for the *rap* genes (from 1032
to 1185 bp) and 166 bp for the *phr* genes (from 120 to
330 bp).

From 2 to 16 *rap-phr* genes were identified in all strains (Fig. 1a, Online Resource Table 4). Two to eight chromosomal *rap-phr* systems were found by strain, representing 70% of all the identified *rap-phr* genes (Fig. 1b). Plasmid *rap-phr* genes were found in 27 of the 49 strains and on 65 of the 197 plasmids. Some strains harbor a large number of plasmid *rap-phr* genes, up to 12 plasmid systems for the *B. thuringiensis* strain IS 5056 (Online Resource Table 3). The size of plasmids harboring *rap-phr* genes varied from 6.88 to 502 kb. However, the occurrence of these genes is most common (70%) in plasmids larger than 70 kb (Online Resource Table 3). Also, large plasmids (> 200 kb) might contain several *rap-phr* genes, up to five, such as the pBMB422 plasmid (422.7 kb) of the *B. thuringiensis* YBT-1520 strain (Online Resources Table 3 and 4).

The average number of chromosomal *rap-phr* genes is similar between *B. cereus* and *B. thuringiensis* (4.04 and 4.45, respectively). However, the average number of plasmid *rap-phr* genes is sixfold higher in *B. thuringiensis* than in *B.*

<figure><img src="image_4.png" /><figcaption>**Fig. 1** Distribution of the identified *rap*-**phr** genes in the *B. cereus* group. **a** The number of *rap*-**phr** genes by strain with the chromosomal systems in blue and the plasmid systems in red. **b** Percentage of the *rap*-**phr** genes by replicon with chromosomes in blue and plasmids in red</figcaption></figure>

<figure><img src="image_5.png" /></figure>
cereus (3.6 and 0.6, respectively; *P* = 0.001). The percentage of plasmids harboring *rap-phr* genes is higher in *B. thuringiensis* than in *B. cereus* (38.6% versus 22.0%, respectively, *P* = 0.03). Moreover, *B. thuringiensis* strains show a similar amount of chromosomal and plasmid *rap-phr* genes, while in the *B. cereus* strains the number of chromosomal *rap-phr* genes is almost seven times higher (*P* < 0.001) than plasmid genes (Table 2).

As the genome of *B. cereus* group strains are rich in mobile and repeated elements (Kolstø et al. 2009), the presence of these elements in the vicinity of *rap-phr* genes was analyzed. A mobile element was found in the vicinity of 48% of all the *rap* genes, corresponding to 39% of the chromosomal genes and 68% of the plasmid ones (Online Resource Data 1). The prevalence of mobile elements in the 20-kb region around *rap-phr* chromosomal genes is similar between *B. cereus* and *B. thuringiensis* (Table 2). However, these elements were found in the vicinity of 36% of plasmid *rap* genes from *B. cereus* against 72% of *B. thuringiensis* ones (*P* < 0.01).

## Rap protein clustering

Among the 302 Rap and Phr proteins initially identified, we distinguished 192 different Rap protein sequences and 152 different Phr pro-peptide sequences, corresponding to 63.5% and 50.3% of all sequences, respectively. Rap protein sequences with 100% of identity were found in several strains from different species (Fig. 2). The identical sequences are always identified on different strains and on the same type of replicon (chromosome or plasmid), except for the chromosomal Rap1 from the *B. cereus* 03BB87 strain, which is identical to the plasmid Rap3 of the pBCX01 plasmid from the *B. cereus* G9241 strain. To investigate this unique case, a 10-kb region around the *rap1* gene from the *B. cereus* 03BB87 strain was analyzed with BLASTn. By this approach, we detected 99–100% identity to the pXO1 and pXO1-like plasmids. A BLAST alignment between the pXO1 plasmid and the entire *B. cereus* 03BB87 chromosome

highlighted a region corresponding to 3% of the chromosome with 98% of coverage and 99% of identity with the plasmid. Moreover, the *B. cereus* 03BB87 strain also carries a pXO1-like plasmid, the pBCX01, sharing 97% identity with pXO1 with less than 10% of coverage (data not shown). This might be due to a mistake in the genome assembly or to the integration of DNA regions from the pBCX01 plasmid in the chromosome.

Three phylogenetic trees were constructed using Rap protein sequences, according to the location of their encoding genes in the genome (all sequences, chromosome or plasmid sequences) (Fig. 2 and Online Resource Fig. 1). These trees were built by the Neighbor-Joining method with corrections based on the Jones–Taylor–Thornton matrix model that enables the assessment of the overall divergence among Rap proteins. The phylogenetic tree with all sequences showed a high number of branches with a dispersion of plasmid sequences in several branches. Closely related Rap sequences are encoded by similar kind of plasmids distributed in eight major sets (Fig. 2, sets A–H). Set A comprises *rap* genes harbored by an identical 8.5 kb plasmid present in different strains and set B by plasmids higher than 70 kb. Set C includes pXO1-like plasmids; set D plasmids larger than 200 kb, while plasmids from set E are smaller than 20 kb. Set F includes plasmids around 70 kb and set G plasmids larger than 200 kb that can also harbor Rap sequences from set B. The set H is the most versatile since it comprises two distinct subsets: one with plasmids larger than 400 kb and another with plasmids ranging from 75 to 235 kb.

The chromosomal tree is divided into 12 Rap groups (Online Resource Fig. 1a). Some groups are composed of several sequences, such as Group C6 which includes 38 Rap sequences. However, some groups have few Rap sequences, such as Group C11 with Rap4 from *B. anthracis* Ames Ancestor and Rap5 from *B. cytotoxicus* NHV391-98. Also, Group C10 is formed by three identical sequences from two *B. cereus* emetic strains (*B. cereus* AH187 and *B. cereus* NC7401) and from BcQ1. The plasmid tree is separated into nine Rap groups (Online Resource Fig. 1b). Likewise in the

<table><caption>**Table 2** Comparison between *B. cereus* and *B. thuringiensis* concerning the Rap–Phr systems</caption><thead><tr><th></th><th><i>B. cereus</i></th><th><i>B. thuringiensis</i></th><th>Significance</th></tr></thead><tbody><tr><td>Number of strains</td><td>24</td><td>20</td><td></td></tr><tr><td>Number of plasmids</td><td>59</td><td>127</td><td></td></tr><tr><td>Number of chromosome <i>rap-phr</i> genes<sup>a</sup></td><td>97 (4.0/strain)</td><td>89 (4.5/strain)</td><td>ns</td></tr><tr><td>Number of plasmid <i>rap-phr</i> genes<sup>b</sup></td><td>14 (0.6/strain)</td><td>72 (3.6/strain)</td><td>***</td></tr><tr><td>Chromosomal/plasmid <i>rap-phr</i> genes ratio<sup>c</sup></td><td>6.9</td><td>1.2</td><td>***</td></tr><tr><td>Plasmids with <i>rap-phr</i> genes<sup>c</sup></td><td>13 (22%)</td><td>49 (38%)</td><td>*</td></tr><tr><td>Chromosomal <i>rap</i> genes with nearby mobile elements<sup>a</sup></td><td>36 (37%)</td><td>35 (39%)</td><td>ns</td></tr><tr><td>Plasmid <i>rap</i> genes with nearby mobile elements<sup>d</sup></td><td>5 (36%)</td><td>52 (72%)</td><td>**</td></tr></tbody></table>

Statistical analyses: <sup>a</sup>unpaired *t* test; <sup>b</sup>Mann–Whitney test; <sup>c</sup>Fisher’s exact test; <sup>d</sup>unpaired *t* test with Welch correction. *ns* not significant; \* *P* < 0.05; \*\* *P* < 0.01; \*\*\* *P* ≤ 0.001
<figure><img src="image_6.png" /><figcaption>**Fig. 2** Phylogenetic tree of all Rap protein sequences (plasmid and chromosome sequences) inferred by Neighbor-Joining method (conducted in MEGA6) and visualized by iTOL (Letunic and Bork 2019). RapH from *B. subtilis* 168 was used as outgroup. The evolutionary distances were computed using the JTT matrix-based method and are</figcaption></figure>

in the units of the number of amino acid substitutions per site. Chromosome-encoded proteins are in blue and plasmid-encoded proteins are in red. Sets A–H represent related Rap proteins associated to the same kind of plasmid. Chromosome and plasmid groups are indicated in the two outer circles

chromosomal tree, many groups are composed of several sequences, such as Group P2 with 26 Rap proteins. However, Group P4 and Group P5 are composed of unique sequences and Group P6 by two identical sequences.

## Phr peptides

In silico determination of the mature Phr sequences is complex due to the high variability of these sequences within the *B. cereus* group. Indeed, the mature Phr already described from the *B. cereus* group revealed some differences in size and location within the pro-peptide. The BXA0205Phr from

the pXO1 plasmid (Phr5-*B. anthracis*) is a pentapeptide while the Phr8 from the pHT8_1 plasmid (Phr8-*B. thuringiensis* HD73) is an heptapeptide but both are located in the C-terminal end. Moreover, the active form of the BA3791Phr from *B. anthracis* (Phr3-*B. anthracis*) is located within the C-terminal region of its precursor, but its exact sequence was not determined (Bongiorni et al. 2006; Fazion et al. 2018). However, all these mature peptides present a positively charged residue, the typical feature of Phr active form (Pottathil and Lazazzera 2003). The Phr sequences from the *B. cereus* group present a great variability in their amino acid sequences and in their sizes (Online Resources Data 1).

<figure><img src="image_7.png" /></figure>
The mature Phr peptides from the *B. cereus* group described above were sought among the Phr sequences in our database. While the mature Phr8-*B. thuringiensis* HD73 (YAHGKDI) was identified only on identical Phr sequences, the Phr5-*B. anthracis* (GHTGG) was found in several sequences. A great number of longer Phr possess the GDTGG/GDGGG/ GETGG repetition sequences duplication described by Even-Tov et al. (2016). These sequences were defined as the putative autoinducer sequences although they are not gener- ally associated with a positively charged residue. However, the Phr peptides containing these repetitions also bear an ARPDY sequence, which could be the active form.

## The relationship between MLST phylogenetic tree and Rap distribution

The 49 selected strains are distributed into six of the seven phylogenetic clusters determined by Guinebretière et al. (2008). These clusters, established from the *Bacillus cereus* group MultiLocus and MultiData Typing website (Tourasse et al. 2011), are supported by recent results of a pangenomic study of this clade (Bazinet 2017). Moreover, the MLST data are still effective in discriminating variation of biology, ecol- ogy and host association among this group strains (Raymond and Federici 2017). The cluster III, including *B. anthracis* strains, emetic strains, and other pathogenic strains (mainly composed by *B. cereus* strains), and the cluster IV with *B. cereus* and *B. thuringiensis* strains from diverse environmen- tal sources (mainly formed by *B. thuringiensis* strains) are the more extensively represented (Fig. 3). The Rap profile of each strain was analyzed in relation to the MLST tree of the *B. cereus* group. We observed that any Rap group was not present in all strains and that a Rap group was not exclusively related to an MLST cluster. None of the strains has more than five different Rap chromosomal groups, and a same Rap group can be present more than once in the same strain. As expected, the Cluster IV (mainly composed of *B. thuringiensis* strains) has a higher number of plasmid Rap. Additionally, phylogenetically related strains show a similar Rap profile both for chromosomal and plasmid groups. For example, the closely related strains *B. thuringiensis* Bt407 Cry⁻, *B. thuringiensis* CT-43, and *B. thuringiensis* IS5056 show an identical chromosomal profile and similar plasmid profile. These three strains might derive from a same paren- tal strain, for example the *B. thuringiensis* strain 407 Cry⁺ from the serotype 1 (Lereclus et al. 1989).

## Sporulation activity prediction

Considering the importance of sporulation for the survival and dispersion of *Bacillus* and the role of some Rap-Phr systems in this process, we aimed to predict the activity of Rap proteins from the *B. cereus* group on sporulation. The

RapH residues E45, D46, Q47, L50, F58, L96, D134, E137, and Y175 have been described to be involved in the binding and the dephosphorylation of Spo0F in *B. subtilis* (Parashar et al. 2011). First, we used the sequences of Rap proteins interacting with Spo0F to define a consensual sequence of residues potentially involved in the sporulation process (Online Resource Table 5). Next, the Rap protein sequences of each chromosomal and plasmid groups were separately aligned with the RapH sequence, and the presence of the nine key residues was examined (Online Resource Table 5). Depending on the presence of these residues, the Rap proteins from the *B. cereus* group were classified as Spo⁺ (predicted phosphatase activity on Spo0F) or Spo⁻ (no predicted phosphatase activity) (Online Resource Table 4).

This analysis showed that 97 of the 302 Rap proteins display a Spo⁺ profile (32%) and the predicted Rap Spo⁺ are more frequently found in plasmids (65% of plasmid Rap) than in chromosomes (18% of chromosomal Rap) (Fig. 4a). However, there is no correlation between Rap groups and predicted sporulation function because there are groups with only Spo⁻ or Spo⁺ Rap proteins, as well as mixed groups. Most of the chromosomal groups were exclusively Spo⁻ and the plasmid groups are mainly mixed (Online Resource Table 5). Interestingly, the amount of Rap Spo⁺ is significantly higher (*P* < 0.01) in *B. thuringiensis* than in *B. cereus* (40% and 26%, respectively) (Fig. 4a). Ten *B. cereus* strains (42%) do not harbor chromosomal Rap Spo⁺, including four strains that do not have any Rap Spo⁺ at all (Online Resource Table 4). The *B. thuringiensis* HD-1 strain, widely used as a biopesticide against lepidopteran insects, has nine Rap Spo⁺. In sharp contrast, five *B. thuringiensis* strains do not have chromosomal Rap Spo⁺, and the nematicidal *B. thuringiensis* YBT1518 strain is the only one *B. thuringiensis* to have no predicted Rap Spo⁺ at all.

To validate the in silico prediction, ten Rap proteins representative of various plasmid or chromosomal groups with a predicted Spo⁺ (seven Rap) or Spo⁻ (three Rap) activity were selected to study their effect on sporulation (Online Resource Table 5). The corresponding *rap* genes were expressed under the xylose-inducible promoter *P*<sub>xylA</sub> in the *B. thuringiensis* HD73 strain, and the sporulation efficiency was measured after 48 h at 30 °C in a sporulation-specific medium (HCT). The three Rap Spo⁻ (*rap*6-BtHD1, *rap*6-Bt407, and *rap*7-BtHD73) did not inhibit the sporulation efficiency compared to the control strain, confirming their prediction. Among the Rap Spo⁺ analyzed, the expression of *rap*8-Bt407, *rap*10-BtHD1, *rap*7-Bt407, and *rap*5-HD73 strongly prevent the sporulation (*P* < 0.001) while the expression of *rap*1-BcATCC14579, and *rap*2-BcATCC14579 slightly inhibited the sporulation efficiency compared to the control strain (*P* < 0.01) (Fig. 4b, Online Resource Table 6). However, the *rap*8-BtHD1, predicted Rap Spo⁺ does not display a role in sporulation in our *B*.
<figure><img src="image_8.png" /><figcaption>**Fig. 3** Distribution of the chromosomal and plasmid Rap-Phr systems groups in relation to the MLST phylogenetic tree (*Bacillus cereus* group MultiLocus and MultiData Typing website—http://mlstoslo.uio.no). MLST clusters proposed by Guinebretière et al.</figcaption></figure>

(2008) are showed on the left. Chromosomal systems are in blue, and plasmid systems are in red. Numbers inside the boxes specify how many times (if > 1) that group is found in each strain

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 4** Activity of Rap from *B. cereus* group in sporulation. a Distribution of the Rap protein predicted as Spo+ (orange) or Spo− (green), difference between species are statistically significant (*P*<0.01—Fisher's Exact test); b efficiency of sporulation, calculated as 100×the ratio between heat-resistant spores per milliliter and</figcaption></figure>

<figure><img src="image_11.png" /></figure>

total viable cells per milliliter. The data were analyzed using one-way
analysis of variance (ANOVA) followed by Tukey–Kramer Multiple
Comparisons Test (P<0.01). * (P<0.01) and ** (P<0.001): % of
spores is statistically different from control (315xyl)

*thuringiensis* HD73 model strain in this growth condition.
Hence, the sporulation results allow us to confirm the pre-
dicted phenotype for nine of the ten tested Rap.

**Discussion**

Despite the importance of the Rap–Phr systems in the
regulation of various essential pathways, they have been
poorly studied in the bacteria of the *B. cereus* group (Bon-
giorni et al. 2006; Fazion et al. 2018; Slamti et al. 2014).
Here, we provide a complete and detailed overview of
these systems in this group, concerning their prevalence,
sequence diversity, relevant association to plasmids and
their role in sporulation. We show that the *rap* genes are
widespread in all the studied strains of the *B. cereus* group
and that a putative *phr* gene is always present immediately
downstream from all *rap* genes. The *rap–phr* genes are
always encoded on the same DNA strand but in different
transcription frames, a characteristic of the RNPP family
(Declerck et al. 2007). Genes coding for the Phr peptides
were diverse in size and the occurrence of *phr* genes two
times longer than the average could be explained by the
duplication of the region coding for the mature signaling
peptide, important for the evolutionary diversification of
Rap–Phr specificity (Even-Tov et al. 2016). The *rap–phr*
genes are located in all chromosomes and numerous plas-
mids. The total number of chromosome *rap–phr* genes is
similar between *B. cereus* and *B. thuringiensis* species.
However, chromosome size seems unrelated to the amount
of chromosomal *rap–phr* genes as the *B. cytotoxicus* NVH

391-98 strain (distantly related to other strains of the *B. cereus* group) presents the smallest chromosome and the largest number of chromosomal *rap-phr* genes.

A global analysis of *rap* genes in the genus *Bacillus*
showed that species from the *B. subtilis* group contain 11
(±2) *rap* genes in opposition to six (±3) for the *B. cereus*
group species (Even-Tov et al. 2016). In agreement with
this study, we found a similar number of *rap* genes in the
*B. cereus* group (6.2 ± 3.2). Another difference between
the two *Bacillus* groups is related to the *phr* gene occur-
rence. While 27% of the *rap* genes from *B. subtilis* strain
168 do not have an associated *phr* gene (Perego 2013), a
common pattern for the *B. subtilis* group (Even-Tov et al.
2016), a putative *phr* gene is always located downstream
from all identified *rap* genes in the *B. cereus* group. Inter-
estingly, Rap proteins from the *B. subtilis* and *B. cereus*
species constitute two independent clusters, suggesting
that the diversification of the Rap sequences occurred after
the evolutionary separation of the two bacterial groups
(Even-Tov et al. 2016). After this separation, Rap–Phr
systems from the *B. cereus* group might have been sub-
jected to genetic variations that also evolved this quorum-
sensing system to the other RNPP family systems, like
PlcR–PapR and NprR–NprX. The role of these QS systems
in the production of extracellular proteases or sporula-
tion corresponds to functions performed by some Rap–Phr
systems of *B. subtilis* (Auchtung et al. 2006; Ogura et al.
2003). Interestingly, NprR, which presents a Rap-like
structure combined with an HTH DNA-binding domain,
was suggested to be the evolutionary intermediate between
Rap proteins and the other regulators of the RNPP family
COFECUB (Comité Français d'Evaluation de la Coopération Universitaire et Scientifique avec le Brésil). Cardoso, P.F. was supported by fellowships from CAPES—finance code 001 and CAPES/COFECUB and Vilas-Boas, L. A. by CAPES/COFECUB. We would like to thank the reviewers who helped to improve this manuscript.

## References

Auchtung JM, Lee CA, Monson RE, Lehman AP, Grossman AD (2005) Regulation of a *Bacillus subtilis* mobile genetic element by intercellular signaling and the global DNA damage response. Proc Natl Acad Sci 102(35):12554–12559. https://doi.org/10.1073/pnas.0505835102

Auchtung JM, Lee CA, Grossman AD (2006) Modulation of the ComA-dependent quorum response in *Bacillus subtilis* by multiple rap proteins and Phr peptides. J Bacteriol 188(14):5273–5285. https://doi.org/10.1128/JB.00300-06

Bazinet AL (2017) Pan-genome and phylogeny of *Bacillus cereus* sensu *lato*. BMC Evol Biol 17:176. https://doi.org/10.1186/s12862-017-1020-1

Bongiorni C, Ishikawa S, Stephenson S, Ogasawara N, Perego M (2005) Synergistic regulation of competence development in *Bacillus subtilis* by two Rap-Phr systems. J Bacteriol 187(13):4353–4361. https://doi.org/10.1128/JB.187.13.4353-4361.2005

Bongiorni C, Stoessel R, Shoemaker D, Perego M (2006) Rap phosphatase of virulence plasmid pXO1 inhibits *Bacillus anthracis* sporulation. J Bacteriol 188(2):487–498. https://doi.org/10.1128/JB.188.2.487-498.2006

Burbulys D, Trach KA, Hoch JA (1991) Initiation of sporulation in *B. subtilis* is controlled by a multicomponent phosphorelay. Cell 64:545–552. https://doi.org/10.1016/0092-8674(91)90238-T

Callegan MC, Kane ST, Cochran DC, Gilmore MS, Gominet M, Lereclus D (2003) Relationship of plcR-Regulated Factors to *Bacillus* endophthalmitis virulence. Infect Immun 71(6):3116–3124. https://doi.org/10.1128/IAI.71.6.3116-3124.2003

Chao L, Qiyu B, Fuping S, Ming S, Dafang H, Guiming L, Ziniu Y (2007) Complete nucleotide sequence of pBMB67, a 67-kb plasmid from *Bacillus thuringiensis* strain YBT-1520. Plasmid 57:44–54. https://doi.org/10.1016/j.plasmid.2006.06.002

Core L, Perego M (2003) TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in *Bacillus subtilis*. Mol Microbiol 49:1509–1522. https://doi.org/10.1046/j.1365-2958.2003.03659.x

D'Andrea LD, Regan L (2003) TPR proteins: the versatile helix. Trends Biochem Sci 28(12):655–662. https://doi.org/10.1016/j.tibs.2003.10.007

Declerck N, Bouillaut L, Chaix D, Rugani N, Slamti L, Hoh F, Lereclus D, Arold ST (2007) Structure of PlcR: insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria. Proc Natl Acad Sci 104(47):18490–18495. https://doi.org/10.1073/pnas.0704501104

Deng C, Peng Q, Song F, Lereclus D (2014) Regulation of cry Gene Expression in *Bacillus thuringiensis*. Toxins 6(7):2194–2209. https://doi.org/10.3390/toxins6072194

Ehling-Schulz M, Fricker M, Grallert H, Rieck P, Wagner M, Scherer S (2006) Cereulide synthetase gene cluster from emetic *Bacillus cereus*: structure and location on a mega virulence plasmid related to *Bacillus anthracis* toxin plasmid pXO1. BMC Microbiol 6:20. https://doi.org/10.1186/1471-2180-6-20

Even-Tov E, Omer Bendori S, Pollak S, Eldar A (2016) Transient duplication-dependent divergence and horizontal transfer underlie the evolutionary dynamics of bacterial cell-cell signaling. PLoS Biol 14(12):e2000330. https://doi.org/10.1371/journal.pbio.2000330

Fazion F, Perchat S, Buisson C, Vilas-Bôas G, Lereclus D (2018) A plasmid-borne Rap-Phr system regulates sporulation of *Bacillus thuringiensis* in insect larvae. Environ Microbiol 20:145–155. https://doi.org/10.1111/1462-2920.13946

Grandvalet C, Gominet M, Lereclus D (2001) Identification of genes involved in the activation of the *Bacillus thuringiensis inhA* metalloprotease gene at the onset of sporulation. Microbiology 147(7):1805–1813. https://doi.org/10.1099/00221287-147-7-1805

Guinebretière M, Thompson FL, Sorokin A, Normand P, Dawyndt P, Ehling-Schulz M, Svensson B, Sanchis V, Nguyen-The C, Heyn-drickx M, De Vos P (2008) Ecological diversification in the *Bacil-lus cereus* Group. Environ Microbiol 10:851–865. https://doi.org/10.1111/j.1462-2920.2007.01495.x

Helgason E, Økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø A-B (2000) *Bacillus anthracis*, *Bacillus cereus*, and *Bacillus thuringiensis*—one species on the basis of genetic evidence. Appl Environ Microbiol 66(6):2627–2630

Huang M, Hull CM (2017) Sporulation: how to survive on planet Earth (and beyond). Curr Genet 63(5):831–838. https://doi.org/10.1007/s00294-017-0694-7

Jiang M, Shao W, Perego M, Hoch JA (2000a) Multiple histidine kinases regulate entry into stationary phase and sporulation in *Bacillus subtilis*. Mol Microbiol 38:535–542. https://doi.org/10.1046/j.1365-2958.2000.02148.x

Jiang M, Grau R, Perego M (2000b) Differential processing of propeptide inhibitors of rap phosphatases in *Bacillus subtilis*. J Bacteriol 182(2):303–310

Johnson SL, Daligault HE, Davenport KW, Jaissle J, Frey KG, Lader JT et al (2015) Complete Genome sequences for 35 biothreat assay-relevant *Bacillus* species. Genome Announc 3(2):e00151. https://doi.org/10.1128/genomeA.00151-15

Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C et al (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30(9):1236–1240. https://doi.org/10.1093/bioinformatics/btu031

Ko KS, Kim J-W, Kim J-M, Kim W, Chung S, Kim IJ, Kook Y-H (2004) Population structure of the *Bacillus cereus* group as determined by sequence analysis of six housekeeping genes and the *plcR* gene. Infect Immun 72(9):5253–5261. https://doi.org/10.1128/IAI.72.9.5253-5261.2004

Koetje E, Hajdo-Milasinovic A, Kiewiet R, Bron S, Tjalsma H (2003) A plasmid-borne Rap-Phr system of *Bacillus subtilis* can mediate cell-density controlled production of extracellular proteases. Microbiology 149(1):19–28. https://doi.org/10.1099/mic.0.25737-0

Kolstø AB, Tourasse NJ, Økstad OA (2009) What sets *Bacillus anthracis* apart from other *Bacillus* species? Annu Rev Microbiol 63:451–476. https://doi.org/10.1146/annurev.micro.091208.073255

Lereclus D, Lecadet MM, Ribier J, Dedonder R (1982) Molecular relationships among plasmid of *Bacillus thuringiensis*: conserved sequences through 11 crystalliferous Strains. Mol Gen Genet 186:391–398

Lereclus D, Arantes O, Chaufaux J, Lecadet M-M (1989) Transformation and expression of a cloned δ -endotoxin gene in *Bacillus thuringiensis*. FEMS Microbiol Lett 60:211–217. https://doi.org/10.1111/j.1574-6968.1989.tb03448.x

Letunic I, Bork P (2019) Interactive tree of life (iTOL) v4: recent updates and new developments. Nucl Acids Res. https://doi.org/10.1093/nar/gkz239

Letunic I, Doerks T, Bork P (2015) SMART: recent updates, new developments and status in 2015. Nucl Acids Res 43:D257–D260. https://doi.org/10.1093/nar/gku949

Liu S, Moayeri M, Leppla SH (2014) Anthrax lethal and edema toxins in anthrax pathogenesis. Trends Microbiol 22(6):317–325. https://doi.org/10.1016/j.tim.2014.02.012
Liu Y, Lai Q, Göker M, Meier-Kolthoff JP, Wang M, Sun Y et al (2015) Genomic insights into the taxonomic status of the *Bacillus cereus* group. *Sci Rep* 5:14082. https://doi.org/10.1038/srep14082

Liu Y, Lai Q, Shao Z (2018) Genome analysis-based reclassification of *Bacillus weihenstephanensis* as a later heterotypic synonym of *Bacillus mycoides*. *Int J Syst Evol Microbiol* 68:106–112. https://doi.org/10.1099/ijsem.0.002466

McQuade RS, Comella N, Grossman AD (2001) Control of a family of phosphatase regulatory genes (*phr*) by the alternate sigma factor sigma-H of *Bacillus subtilis*. *J Bacteriol* 183(16):4905–4909. https://doi.org/10.1128/JB.183.16.4905-4909.2001

Méric G, Mageiros L, Pascoe B, Woodcock DJ, Mourkas E, Lamble S et al (2018) Lineage-specific plasmid acquisition and the evolution of specialized pathogens in *Bacillus thuringiensis* and the *Bacillus cereus* group. *Mol Ecol* 27(7):1524–1540. https://doi.org/10.1111/mec.14546

Ogura M, Fujita Y (2007) *Bacillus subtilis* rapD, a direct target of transcription repression by RghR, negatively regulates srfA expression. *FEMS Microbiol Lett* 268:73–80. https://doi.org/10.1111/j.1574-6968.2006.00559.x

Ogura M, Shimane K, Asai K, Ogasawara N, Tanaka T (2003) Binding of response regulator DegU to the *aprE* promoter is inhibited by RapG, which is counteracted by extracellular PhrG in *Bacillus subtilis*. *Mol Microbiol* 49:1685–1697. https://doi.org/10.1046/j.1365-2958.2003.03665.x

Parashar V, Mirouze N, Dubnau DA, Neiditch MB (2011) Structural basis of response regulator dephosphorylation by rap phosphatases. *PLoS Biol* 9(2):e1000589. https://doi.org/10.1371/journal.pbio.1000589

Parashar V, Jeffrey PD, Neiditch MB (2013a) Conformational change-induced repeat domain expansion regulates Rap phosphatase quorum-sensing signal receptors. *PLoS Biol* 11(3):e1001512. https://doi.org/10.1371/journal.pbio.1001512

Parashar V, Konkol MA, Kearns DB, Neiditch MB (2013b) A plasmid-encoded phosphatase regulates *Bacillus subtilis* biofilm architecture, sporulation, and genetic competence. *J Bacteriol* 195(10):2437–2448. https://doi.org/10.1128/JB.02030-12

Perchat S, Dubois T, Zouhir S, Gominet M, Poncet S, Lemy C, Aumont-Nicaise M, Deutscher J, Gohar M, Nessler S, Lereclus D (2011) A cell-cell communication system regulates protease production during sporulation in bacteria of the *Bacillus cereus* group. *Mol Microbiol* 82:619–633. https://doi.org/10.1111/j.1365-2958.2011.07839.x

Perchat S, Talagas A, Zouhir S, Poncet S, Bouillaut L, Nessler S, Lereclus D (2016a) NprR, a moonlighting quorum sensor shifting from a phosphatase activity to a transcriptional activator. *Microbial Cell* 3(11):573–575. https://doi.org/10.15698/mic2016.11.542

Perchat S, Talagas A, Poncet S, Lazar N, Li de la Sierra-Gallay I, Gohar M, Lereclus D, Nessler S (2016b) How quorum sensing connects sporulation to necrotrophism in *Bacillus thuringiensis*. *PLoS Pathog* 12(8):e1005779. https://doi.org/10.1371/journal.ppat.1005779

Perego M (1997) A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay. *Proc Natl Acad Sci* 94(16):8612–8617

Perego M (2013) Forty years in the making: understanding the molecular mechanism of peptide regulation in bacterial development. *PLoS Biol* 11(3):e1001516. https://doi.org/10.1371/journal.pbio.1001516

Perego M, Brannigan JA (2001) Pentapeptide regulation of aspartyl-phosphate phosphatases. Peptides 22(10):1541–1547. https://doi.org/10.1016/S0196-9781(01)00490-9

Perego M, Hoch JA (1996) Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in *Bacillus subtilis*. *Proc Natl Acad Sci* 93(4):1549–1553

Perego M, Glaser P, Hoch JA (1996) Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in *Bacillus subtilis*. *Mol Microbiol* 19:1151–1157. https://doi.org/10.1111/j.1365-2958.1996.tb02460.x

Polke M, Jacobsen ID (2017) Quorum sensing by farnesol revisited. *Curr Genet* 63:791. https://doi.org/10.1007/s00294-017-0683-x

Pottathil M, Lazazzera BA (2003) The extracellular Phr peptide-Rap phosphatase signaling circuit of *Bacillus subtilis*. *Front Biosci* 8:d32–d45

Rasko DA, Altherr MR, Han CS, Ravel J (2005) Genomics of the *Bacillus cereus* group of organisms. *FEMS Microbiol Rev* 29:303–329. https://doi.org/10.1016/j.fmrre.2004.12.005

Raymond B (2017) The biology, ecology and taxonomy of *Bacillus thuringiensis* and related bacteria. In *Fiuza LM, Polanczyk RA, Crickmore N (Eds.), *Bacillus thuringiensis* and *Lysinibacillus sphaericus*: Characterization and use in the Field of Biocontrol*. pp 19–39. https://doi.org/10.1007/978-3-319-56678-8

Raymond B, Federici BA (2017) In defense of *Bacillus thuringiensis*, the safest and most successful microbial insecticide available to humanity—a response to EFSA. *FEMS Microbiol Ecol* 93(7):84. https://doi.org/10.1093/femsec/fix084

Rutherford ST, Bassler BL (2012) Bacterial quorum sensing: its role in virulence and possibilities for its control. *Cold Spring Harbor Perspect Med* 2(11):a012427. https://doi.org/10.1101/cshperspect.ta012427

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J et al (1998) *Bacillus thuringiensis* and its pesticidal crystal proteins. *Microbiol Mol Biol Rev* 62(3):775–806

Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M (2005) ISfinder: the reference centre for bacterial insertion sequences. *Nucl Acids Res* 34:D32–D36. https://doi.org/10.1093/nar/gkj014

Singh PK, Ramachandran G, Ramos-Ruiz R, Peiró-Pastor R, Abia D, Wu LJ, Meijer WJ (2013) Mobility of the native *Bacillus subtilis* conjugative plasmid pLS20 is regulated by intercellular signaling. *PLoS Genet* 9(10):e1003892. https://doi.org/10.1371/journal.pgen.1003892

Slamti L, Lereclus D (2002) A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the *Bacillus cereus* group. *EMBO J* 21(17):4550–4559. https://doi.org/10.1093/emboj/cdf450

Slamti L, Lereclus D (2005) Specificity and polymorphism of the PlcR-PapR quorum-sensing system in the *Bacillus cereus* group. *J Bacteriol* 187(3):1182–1187. https://doi.org/10.1128/JB.187.3.1182-1187.2005

Slamti L, Perchat S, Huillet E, Lereclus D (2014) Quorum sensing in *Bacillus thuringiensis* is required for completion of a full infectious cycle in the insect. *Toxins* 6(8):2239–2255. https://doi.org/10.3390/toxins6082239

Smits WK, Bongiorni C, Veening J, Hamoen LW, Kuipers OP, Perego M (2007) Temporal separation of distinct differentiation pathways by a dual specificity Rap-Phr system in *Bacillus subtilis*. *Mol Microbiol* 65:103–120. https://doi.org/10.1111/j.1365-2958.2007.05776.x

Sonenshein AL (2000) Control of sporulation initiation in *Bacillus subtilis*. *Curr Opin Microbiol* 3:561–566. https://doi.org/10.1016/S1369-5274(00)00141-7

Stenfors Arnesen LP, Fagerlund A, Granum PE (2008) From soil to gut: *bacillus cereus* and its food poisoning toxins. *FEMS Microbiol Rev* 32:579–606. https://doi.org/10.1111/j.1574-6976.2008.00112.x

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197

Tourasse N, Helgason E, Økstad O, Hegna I, Kolstø A (2006) The *Bacillus cereus* group: novel aspects of population structure and

<figure><img src="image_12.png" /></figure>
genome dynamics. J Appl Microbiol 101:579-593. https://doi.org/10.1111/j.1365-2672.2006.03087.x

Tourasse NJ, Helgason E, Klevan A, Sylvestre P, Moya M, Haustant M et al (2011) Extended and global phylogenetic view of the *Bacillus cereus* group population by combination of MLST, AFLP, and MLEE genotyping data. Food Microbiol 28:236-244. https://doi.org/10.1016/j.fm.2010.06.014

Van der Auwera GA, Andrup L, Mahillon J (2005) Conjugative plasmid pAW63 brings new insights into the genesis of the *Bacillus anthracis* virulence plasmid pXO2 and of the *Bacillus thuringiensis* plasmid pBT9727. BMC Genom 6:103. https://doi.org/10.1186/1471-2164-6-103

Vilas-Boas GT, Peruca APS, Arantes OMN (2007) Biology and taxonomy of *Bacillus cereus*, *Bacillus anthracis*, and *Bacillus thuringiensis*. Can J Microbiol 53:673-687. https://doi.org/10.1139/W07-029

Wilcks A, Jayaswal N, Lereclus D, Andrup L (1998) Characterization of plasmid pAW63, a second self-transmissible plasmid

in *Bacillus thuringiensis* subsp. *kurstaki* HD73. Microbiology 144(5):1263-1270. https://doi.org/10.1099/00221287-144-5-1263

Xia X (2013) DAMBE5: a comprehensive software package for data analysis in molecular biology and evolution. Mol Biol Evol 30(7):1720-1728. https://doi.org/10.1093/molbev/mst064

Yang Y, Wu HJ, Lin L, Zhu QQ, Borriss R, Gao XW (2015) A plasmid-born Rap-Phr system regulates surfactin production, sporulation and genetic competence in the heterologous host, *Bacillus subtilis* OKB105. Appl Microbiol Biotechnol 99(17):7241-7252. https://doi.org/10.1007/s00253-015-6604-3

**Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.