Priscilla Cardoso

De Freitas

Diversidade

Gislayne T Vilas Boas

Keywords: Bacillus thuringiensis. Esporulação. Quorum sensing. Família RNPP. Plasmídeo Bacillus thuringiensis. Sporulation. Quorum sensing. RNPP family. Plasmid RNPP, Bacillus thuringiensis, Bacillus anthracis, sporulation, phosphatase

O grupo Bacillus cereus é formado por oito espécies de bactérias Gram positivas esporulantes que podem colonizar diversos nichos ecológicos. As espécies mais importantes do grupo são B. cereus, bactéria ubíqua do solo e patógeno oportunista; B. thuringiensis, entomopatógeno amplamente utilizado como biopesticida; e B. anthracis, agente etiológico do antraz. Embora apresentem fenótipos diferentes, essas espécies são próximas geneticamente e seus principais fatores de virulência são codificados por plasmídeos. O ciclo infeccioso de B. thuringiensis na larva de inseto é regulado pela ativação consecutiva de sistemas de quorum sensing da família RNPP. Dentre eles, o sistema Rap-Phr foi amplamente estudado em B. subtilis, porém apenas pontualmente explorado nas espécies do grupo B. cereus. Os sistemas Rap-Phr regulam vários processos fisiológicos bacterianos, inclusive a esporulação. O objetivo deste estudo foi analisar os sistemas Rap-Phr no grupo B. cereus, com intuito de conhecer sua distribuição, localização e diversidade a fim de obter um panorama desses sistemas neste grupo. Além disso, o possível envolvimento desses sistemas no controle do processo de esporulação foi predito com base nos dados estruturais descritos para RapH de B. subtilis. Genes rap, sempre associados a um gene phr, estão presentes em todas as 49 linhagens estudadas com uma média de seis alelos rap-phr por linhagem e 30% dos sistemas estão localizados em plasmídeos. As linhagens de B. thuringiensis possuem seis vezes mais sistemas Rap-Phr plasmidiais do que as linhagens de B. cereus. Ademais, linhagens filogeneticamente próximas apresentam um perfil similar de genes rap-phr. Um terço das proteínas Rap foram preditas como inibidoras da esporulação e estas proteínas estão preferencialmente localizadas em plasmídeos e, portanto, em linhagens de B. thuringiensis. A predição foi parcialmente validada por ensaios de esporulação sugerindo que os resíduos identificados pelo envolvimento na atividade de fosfatase em B. subtilis são conservados no grupo B. cereus, porém não são suficientes para predizer a função sobre a esporulação. Em seguida, o sistema Rap63-Phr63 codificado pelo plasmídeo pAW63 da linhagem B. thuringiensis HD73 foi caracterizado. A proteína Rap inibe moderadamente a esporulação e retarda a expressão de genes regulados por Spo0A. Rap63 é inibida por seu peptídeo cognato Phr63, cuja forma madura corresponde à extremidade carboxi-terminal do pro-peptídeo. Ensaios de esporulação em larvas de inseto sugerem uma atividade sinérgica dos sistemas Rap63

INTRODUCTION

1.1 Bacillus cereus group 1.1.1 The Bacillus genus Bacteria from the Bacillus genus are Gram positive, rod shaped, aerobic or facultative anaerobic, spore forming species (Figure 1). These spores are dormant cellular structures resistant to many adverse conditions, such as heat, radiation, disinfectants and desiccation. Bacillus bacteria can use a wide variety of nutrient sources which allows their adaptation to a broad range of ecological niches. They are ubiquitous in nature, mainly in soil, but can be also found in water, food and clinical specimens, and show medical, industrial, economic and historical relevance [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]. (Deng et al., 2014).

Bacillus genus is one of the best characterized genera and is highly represented in genomic database with almost 4000 genome sequences (complete or draft) deposited in the GenBank database until March 2019. Strains with medical or biotechnological interest are the most represented [START_REF] Hernández-González | Environmentally-driven gene content convergence and the Bacillus phylogeny[END_REF]. Different phylogenetic analyses allow to separate this genus into several groups, including the two remarking well established groups: Bacillus subtilis group and Bacillus cereus group [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF][START_REF] Bhandari | Molecular signatures for Bacillus species: demarcation of the Bacillus subtilis and Bacillus cereus clades in molecular terms and proposal to limit the placement of new species into the genus Bacillus[END_REF][START_REF] Hernández-González | Environmentally-driven gene content convergence and the Bacillus phylogeny[END_REF][START_REF] Maughan | Bacillus taxonomy in the genomic era finds phenotypes to be essential though often misleading[END_REF][START_REF] Schmidt | Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group[END_REF].

B. subtilis group includes B. subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus atrophaeus, and Bacillus mojaviensis. B.

subtilis is the type species of Bacillus genus and Gram positive bacteria. B. subtilis strain 168 is the model strain and has been extensively used to investigate important microbiological characteristics as spore structure and the sporulation process.

Furthermore, B. subtilis and its close relatives have great biotechnology relevance and are frequently used for industrial production of several components as enzymes and antibiotics [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF][START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF].

The B. cereus group is a very diverse group, which is actually composed by eight species: Bacillus cereus (sensu stricto), Bacillus thuringiensis, Bacillus anthracis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus weihenstephanensis, Bacillus cytotoxicus and Bacillus toyonensis. This group shows the largest genome, the higher number of proteins predicted and the lower GC content within the Bacillus genus [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF][START_REF] Anderson | Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis[END_REF].

However, the B. cereus group composition is often modified and some novel species have been proposed: Bacillus gaemokensis* [START_REF] Jung | Bacillus gaemokensis sp. nov., isolated from foreshore tidal flat sediment from the Yellow Sea[END_REF], Bacillus manliponensis* [START_REF] Jung | Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment[END_REF], Bacillus bingmayongensis* (Liu et al., 2014a),

Bacillus wiedmannii [START_REF] Miller | Bacillus wiedmannii sp. nov., a psychrotolerant and cytotoxic Bacillus cereus group species isolated from dairy foods and dairy environments[END_REF], Bacillus paranthracis, Bacillus pacificus, Bacillus tropicus, Bacillus albus, Bacillus mobilis, Bacillus luti, Bacillus proteolyticus, Bacillus nitratireducens and Bacillus paramycoides [START_REF] Liu | Proposal of nine novel species of the Bacillus cereus group[END_REF]) (species names with * are not validly published yet). The validation of all these bacteria at the species level will totalize 21 species in the B. cereus group.

The Bacillus cereus group species

Bacteria from the B. cereus group were described in the end of the 19 th century or on the beginning of the 20 th century and show a long history of medical or biotechnological relevance [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF][START_REF] Sanchis | From microbial sprays to insect-resistant transgenic plants: history of the biospesticide Bacillus thuringiensis . A review[END_REF]Vilas-Bôas et al., 2007). B. cereus sensu stricto is a ubiquitous bacterium and some strains are considered as opportunistic pathogens that have been involved in food poisoning outbreaks with emetic and diarrheic symptoms, pneumonia, and endophthalmitis [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]. B. thuringiensis is an entomopathogenic bacterium used as alternative insect pest control for more than 60 years [START_REF] Sanchis | From microbial sprays to insect-resistant transgenic plants: history of the biospesticide Bacillus thuringiensis . A review[END_REF]. B. anthracis is the etiological agent of anthrax [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]. B. mycoides is characterized by the rhizoidal morphology of its colonies, lack of motility, and have been considered as a plant growth-promoting bacterium [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]. B.

pseucomycoides is isolated mainly from soil and distinguishable from B. mycoides by their composition of fatty acid [START_REF] Nakamura | Bacillus pseudomycoides sp. nov[END_REF]. B. weihenstephanensis comprise psychrotolerant strains that represent potential risk of food poisoning [START_REF] Lapidus | Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity[END_REF][START_REF] Lechner | Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group[END_REF]. B. cytotoxicus is also associated to severe food poisoning outbreaks and produces a high amount of cytotoxin [START_REF] Guinebretiere | Bacillus cytotoxicus sp. nov. is a novel thermotolerant species of the Bacillus cereus Group occasionally associated with food poisoning[END_REF] and B. toyonensis is used as probiotic in animal nutrition [START_REF] Jiménez | Description of Bacillus toyonensis sp. nov., a novel species of the Bacillus cereus group, and pairwise genome comparisons of the species of the group by means of ANI calculations[END_REF]. Even if the group presents overall phenotypical heterogeneity, these different species may be genetically closely related, essentially at the chromosomal level.

B. cereus group species have a large range of ecological lifestyle, varying according to the species, toxins or virulence factor content and the host (Figure 2) [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF][START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF].

Figure 2. Ecology of B. cereus group bacteria. Species of the group could develop different lifestyles and colonize different hosts in a symbiotic or pathogenic way [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF].

As the soil is the primarily environmental reservoir of B. cereus group spores, the common ancestor of these species was suggested to be a soil bacterium.

However, genetic content analyses of the B. cereus group genomes contradict this hypothesis, since they encode a lower number of genes related to carbohydrate catabolic pathways, numerous in soil bacteria such as B. subtilis [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF][START_REF] Ivanova | Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis[END_REF]Raymond, 2017). Moreover, genomes of this group are rich in genes encoding enzymes involved in peptide and amino acids processing.

This nutrient source preference could suggest that the intestine of invertebrates was the habitat of the common ancestor of the group [START_REF] Ivanova | Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis[END_REF]. B. cereus (sensu stricto) is described as a soil saprophyte and an opportunistic vertebrate pathogen, causing foodborne outbreaks and other diseases in humans [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF][START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF]. Formerly, B. thuringiensis was also considered as a soil saprophytic with incidental insecticide activity. Nowadays, B. thuringiensis is considered as a specialized invertebrate pathogen that could use plants as vectors for transmission [START_REF] Monnerat | Translocation and insecticidal activity of Bacillus thuringiensis living inside of plants[END_REF]Raymond, 2017;Raymond & Federici, 2017;Raymond et al., 2010a) and the soil as reservoir (Raymond et al., 2010b). On the other hand, B. anthracis is an obligate vertebrate pathogen that is unable to grow in soil [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF][START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF].

The three most frequently studied and well characterized species of the B.

cereus group are B. cereus (sensu stricto), B. thuringiensis, and B. anthracis that will be further describe below.

Bacillus cereus sensu stricto

Bacillus cereus sensu stricto is a ubiquitous bacterium. It can complete its saprophytic lifestyle in organic rich soil conditions besides colonizing invertebrate guts as symbionts. Some strains of this species are also involved in various opportunistic and nosocomial systemic or local infections, especially in immunocompromised patients, such as periodontitis (Helgason et al., 2000a), pneumonia resembling inhalation anthrax [START_REF] Hoffmaster | Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes[END_REF] and endophthalmitis [START_REF] Callegan | Relationship of plcR-Regulated Factors to Bacillus Endophthalmitis Virulence[END_REF]. Furthermore, B. cereus strains are implicated in two types of food-poisoning syndromes: emetic and diarrheic types. The ability of B. cereus strains to induce disease is highly variable, depending on a wide variety of factors, including the bacteria dose, the presence of toxin genes and their level of expression and cellular form of bacteria (vegetative cell or spore). In addition, the physiology of the host and the properties of the food, as nutritional composition, processing methods and storage conditions are also important [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF].

The emetic syndrome is caused by cereulide toxin (Figure 3), a small nonribosomal synthesized dodecadepsipeptide non antigenic, highly resistant to heat treatments, extreme pH values and proteolysis. The cereulide synthetase gene cluster (cesHPTABCD) is carried by a pXO1-like plasmid (270 kb) and its expression does not depend of sporulation (Ehling-Schulz et al., 2006;[START_REF] Hoton | The cereulide genetic determinants of emetic Bacillus cereus are plasmid-borne[END_REF]. The emetic illness is the result of the ingestion of preformed cereulide toxins, produced by bacteria in food and not degraded by food processing or gastrointestinal environment. Consequently, the ingestion of viable bacteria or spores is not required to emetic food-poisoning. This type of disease is usually related to food products with high starch content, such as rice, pasta, and mashed potatoes (although the majority of emetic B. cereus strains are unable to hydrolyze starch) and, occasionally pasteurized cream or milk pudding [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF][START_REF] Jensen | The hidden lifestyles of Bacillus cereus and relatives[END_REF]Stenfors Arnesen et al., 2008;Vilas-Bôas et al., 2007). The diarrheic syndrome is the consequence of the production of enterotoxins with hemolytic and pore-forming cytotoxic activity as haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K (CytK). Their activities are enhanced by other virulence factors that are not directly cytotoxic, such as phospholipases C, cereolysin and sphingomyelinase. In contrast to the cereulide toxin and spores, enterotoxins and the vegetative cells are inactivated by the gastrointestinal passage. Thus, the diarrheal food poisoning is the consequence of the ingestion of spores and their subsequent germination to vegetative cells with the production of toxins in the small intestine of the host. Most exotoxin genes related with diarrheic syndrome are located on the chromosome and are regulated by the pleiotropic activator PlcR, which regulates the transcription of virulence genes in bacteria of the B. cereus group (further description below). Diarrheic cases caused by B. cereus strains occur with a wide variety of food commodities. As the food heattreatments (such as pasteurization) are not able to efficiently kill them, spores of B.

cereus are found in food of plant or animal origin [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF]Jensen et al., 2003;Stenfors Arnesen et al., 2008;Vilas-Bôas et al., 2007).

Taken together, both types of foodborne illness related to B. cereus correspond to an important number of outbreaks worldwide. Between 2007 and 2014, the European Food and Safety Authority (EFSA) reported 413 cases where B.

cereus was the causative agent, totalizing more than 6,600 human cases [START_REF] Efsa | Risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs[END_REF]. Moreover, in France, the B. cereus cases correspond to 16% of foodborne outbreaks with confirmed pathogenic agent or 28% from the cases with a suspected cause in 2017 (Santé Publique France, 2019). Regarding foodborne outbreaks occurred in Brazil, B. cereus was described to cause 2.3% of outbreaks that the causative agents were laboratorial confirmed from 2009 to 2018 (Ministério da Saúde, 2018). Furthermore, the Centers for Disease Control and Prevention from the USA reported that B. cereus is suspect to an average of 37.6 outbreaks per year, affecting more than 1,000 people [START_REF] Food | Bad Bug Book Handbook: Foodborne Pathogenic Microorganisms and Natural Toxins[END_REF]. Despite the expressive number, the number of cases may be underestimated because of unreported or misdiagnosed events; for example, in 64% of the Brazilian outbreaks in the last ten years the cause was not identified.

Bacillus thuringiensis

Bacillus thuringiensis strains differ from B. cereus sensu stricto by the production of parasporal crystalline proteins (Figure 1D) during the stationary or sporulation phase of the growth cycle. These proteins are toxic to a wide range of insect larvae, mainly belonging to the orders Lepidoptera (butterflies and moths), Diptera (flies) and Coleoptera (beetles), but also to other insect orders (Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and even some nematodes, mites, and protozoa (Deng et al., 2014;[START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]Schnepf et al., 1998;Vilas-Bôas et al., 2007).

Crystal inclusions may display different forms, such as bi-pyramidal, cuboid, spherical or ovoid, and could represent up to 30% of the dry weight of the sporangia [START_REF] De Souza | Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription[END_REF]. The crystal proteins are also known as δ-endotoxins and consist predominantly of Cry proteins. These proteins are encoded by cry genes, mainly located on large conjugative plasmids and frequently associated with transposable elements such as insertion sequences and transposons, which facilitate their transfer to other cells (Schnepf et al., 1998;Vilas-Bôas et al., 2007).

Cry toxins have their own nomenclature and classification based on amino acid identity, which allows related toxins with similar host specificity to be ranked together [START_REF] Crickmore | Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins[END_REF][START_REF] Crickmore | Bacillus thuringiensis toxin nomenclature[END_REF]. These proteins are composed by three domains: Domain I (perforating domain) is involved in the insertion of the toxin in the cellular membrane and pore formation in the intestinal epithelium of the susceptible insect; Domain II (central or middle domain) is involved in toxin-receptor interactions;

and Domain III (galactose-binding domain) also participate in receptor binding and pore formation [START_REF] Palma | Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity[END_REF]Schnepf et al., 1998). The mode of action of Cry proteins is variable, depending on the type of Cry toxin and susceptible insect. The classical and first described mode of action (Figure 4, top panel) starts with the ingestion and solubilization of the crystal in the alkaline midgut of insect larvae, followed by proteolytic activation of protoxins that are able to bind to specific receptors (such as cadherins) on the apical brush border cells of the midgut. The insertion of the toxin in the membrane cell gives rise to the formation of pores permeable to inorganic ions and small molecules, resulting in leakage intracellular contents and cell lyses. The release of cellular content could activate the germination of spores and consequent septicemia, followed by death of the insect [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF][START_REF] Palma | Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity[END_REF][START_REF] Soberón | Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?[END_REF]. [START_REF] Bravo | How to cope with insect resistance to Bt toxins?[END_REF].

Other proposed action mechanism of Cry toxins is the signal transduction model (Figure 4, bottom panel). In this model the mechanism begins in the same way that the pore formation model with solubilization and proteolytic activation. However, the interaction with a specific receptor, named cadherin, activates a Mg-dependent signal cascade, via G protein, intracellular adenylyl cyclase (cAMP) and protein kinase, activating cellular process that result in cell death [START_REF] Soberón | Signaling versus punching hole: How do Bacillus thuringiensis toxins kill insect midgut cells?[END_REF][START_REF] Zhang | A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis[END_REF].

In addition to the Cry proteins, some B. thuringiensis strains also produce parasporal cytotoxic (Cyt) proteins, with cytolytic activity predominantly against dipteran larvae [START_REF] Bravo | Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control[END_REF]. Furthermore, B. thuringiensis bacteria may produce other insecticidal proteins, such as vegetative insecticidal protein (Vip) and secreted insecticidal protein (Sip) [START_REF] Palma | Bacillus thuringiensis Toxins: An Overview of Their Biocidal Activity[END_REF]. Highly toxic B. thuringiensis strains contain higher amount of insecticidal-toxicity related genes and higher expression of these virulence genes than the less virulent strains [START_REF] Zhu | Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis[END_REF].

Moreover, the accumulation of multiple virulence factors, that acts against a specific insect order, allows B. thuringiensis strains to have an host specialization, which is valuable to increase pathogenicity and overcome insect resistance or expand insecticidal spectrum [START_REF] Zheng | Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts[END_REF].

Due to its insecticidal properties and its extremely specific mechanism of action, B. thuringiensis has been used as biopesticides for over half century as alternative or supplement to synthetic chemical pesticides (Raymond & Federici, 2017;Vilas-Bôas et al., 2007). B. thuringiensis products are widely used in the control of insect pests in agricultural areas and forest management, as well as for the control of mosquito disease vectors. Furthermore, in the last decades, cry genes have been introduced in plants, developing the so-called Bt transgenic crops, resistant to target insects [START_REF] Bravo | How to cope with insect resistance to Bt toxins?[END_REF]Schnepf et al., 1998).

Bacillus anthracis

Bacillus anthracis is the etiological agent of anthrax, an acute disease that affects mammals, primarily herbivores. Anthrax in human is rare, despite of the human susceptibility to natural infections. B. anthracis is also a potential agent for bioterrorism and has an important role in the history of microbiology. This bacterium was used by Robert Koch for the development of Koch postulates, which relates infectious agent and specific disease and also for the development of the first antibacterial vaccine by Pasteur [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]Vilas-Bôas et al., 2007).

There are three forms of anthrax disease, defined by the route of infection: cutaneous (most common of human infections), gastrointestinal or inhalational. All forms may progress to the systemic disease. The disease begins with the entrance of the spores in the body. Afterwards, spores are phagocytosed by macrophages, which carry them to lymph nodes, where they germinate and multiply in vegetative form, producing toxins and capsule [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]Vilas-Bôas et al., 2007).

The main virulence factors of B. anthracis are the tripartite toxins and the capsule, encoded by genes located in the large extra chromosomal plasmids pXO1

(182 kb) and pXO2 (95kb) and required for B. anthracis full virulence. The plasmid pXO1 carries genes pag, lef, and cya that encode protective antigen (PA), lethal factor (LF) and edema factor (EF), respectively. These genes are organized within a 44.8-kb pathogenic island, flanked by insertion sequences. None of these proteins are toxic separately but active in binary combination: lethal toxin (PA and LF) and edema toxin (PA and EF). The role of PA component is to bind to the host cellular receptor whereas LF and EF are the catalytic fraction of the toxins (Liu et al., 2014b;[START_REF] Okinaka | Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes[END_REF].

The other characteristic virulence factor of B. anthracis is the poly-D-γglutamic acid capsule which confers resistance to phagocytosis. The genes involved in the capsule biosynthesis (operon capBCADE) are carried on the plasmid pXO2.

This capsule enables the vegetative bacteria to evade the host immune system, leading to systemic infection (Kolstø et al., 2009;Van der Auwera et al., 2005).

Toxins and capsule production is regulated by the transcriptional activator AtxA, located in pXO1. AtxA is functionally incompatible with the pleiotropic regulator PlcR. The acquisition of pXO1 plasmid, harboring the atxA gene is suggested to have led to the selection for the plcR nonsense mutation found in B. anthracis strains (Kolstø et al., 2009;[START_REF] Mignot | The incompatibility between the PlcR-and AtxA-controlled regulons may have selected a nonsense mutation in Bacillus anthracis[END_REF].

Bacillus anthracis can be differentiated from B. cereus and B. thuringiensis through some phenotypic and genotypic features. The microbiological and biochemical traits that characterize the B. anthracis isolates are non-hemolytic, nonmotile, penicillin-sensitive, susceptible to γ-phage and the poly-D-γ-glutamic acid capsule (Kolstø et al., 2009;Rasko et al., 2005). Concerning genomic traits, B.

anthracis have the particular nonsense point mutation in the plcR gene and four chromosome regions unique to B. anthracis that correspond to lambda prophages (Kolstø et al., 2009). [START_REF] Helgason | Multilocus sequence typing scheme for bacteria of the Bacillus cereus group[END_REF]Ko et al., 2004;[START_REF] Priest | Population Structure and Evolution of the Bacillus cereus Group[END_REF][START_REF] Sorokin | Multiple-Locus Sequence Typing Analysis of Bacillus cereus and Bacillus thuringiensis Reveals Separate Clustering and a Distinct Population Structure of Psychrotrophic Strains[END_REF]Tourasse et al., 2006). Their results lead to two phylogenetic grouping interpretations in which strains were allocated into three clades or seven groups (Figure 5, Table 1). [START_REF] Ceuppens | Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles[END_REF]Guinebretière et al., 2008;[START_REF] Patiño-Navarrete | Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria[END_REF]Tourasse et al., 2011). (Bazinet, 2017).

The three clade categorization was first described by [START_REF] Priest | Population Structure and Evolution of the Bacillus cereus Group[END_REF]. evidence that recombination occurs mainly among bacteria of a given clade than between strains from different clade [START_REF] Didelot | Evolution of pathogenicity in the Bacillus cereus group[END_REF][START_REF] Okinaka | The Phylogeny of Bacillus cereus sensu lato[END_REF][START_REF] Priest | Population Structure and Evolution of the Bacillus cereus Group[END_REF][START_REF] Zwick | Genomic characterization of the Bacillus cereus sensu lato species: Backdrop to the evolution of Bacillus anthracis[END_REF].

The seven groups classification described by Guinebretière et al. (2008) was developed taken into account the ecological diversification of the group by using molecular and phenotypic data (Figure 5, Table 1 (Guinebretière et al., 2008;[START_REF] Patiño-Navarrete | Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria[END_REF]Tourasse et al., 2011).

The advancement of whole genome sequencing has reported the genomic analyses to a new statement. For the study of population structure the up to date evaluation is the pan-genome phylogeny, in which genes were divided into three categories: genes shared by all isolates (core genome), partially shared genes, i.e.

non-core genes present in several strains (accessory genes) and strain-specific genes (unique genes) (Bazinet, 2017;[START_REF] Tettelin | Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial -pan-genome[END_REF]. The first attempt to build a B. cereus group pan-genome identified around 3,000 core genes and the pangenome was estimated to be about 20-25,000 protein-coding genes [START_REF] Lapidus | Extending the Bacillus cereus group genomics to putative food-borne pathogens of different toxicity[END_REF]. Considering 45 genome sequences of B. cereus group strains, [START_REF] Zwick | Genomic characterization of the Bacillus cereus sensu lato species: Backdrop to the evolution of Bacillus anthracis[END_REF] found around 1,750 core and 2,150 accessory genes. As new isolates are sampled, the number of core genes reduces and the number of unique genes, and consequently the whole pan-genome, increase, which means that the B. cereus pan-

genome is still open. The most recent study based on 114 complete genomes, recognized a pan-genome of 60,000 genes and almost 600 core genes (1% of the total pan-genome) suggesting that the environmentally diversity and adaptability of bacteria from this group may be due to the abundance of strain specific genes (about 50% of the pan-genome). Moreover, the phylogenetic analyses based on the pangenome was largely concordant with the MLST's interpretations (Bazinet, 2017).

The overall evaluation of the B. cereus group population structure, with all these methods, indicates that B. anthracis strains have a monophyletic origin. B.

anthracis have been characterized as a clonal species, with very low genetic diversity, as a result of its life cycle. The acute nature of anthrax infections led to a rapid period as vegetative cells, a reproductive stage that allows genetic variation (mutation, genetic transfer…) and a long period as dormant spores in the environment (Helgason et al., 2000b;Kolstø et al., 2009;Rasko et al., 2005). B.

thuringiensis was considered as polyphyletic species having been found in more than one group of the tree. Additionally, this species was also considered as paraphyletic, since clades containing B. thuringiensis are also composed of B. cereus. B.

thuringiensis could not be distinguished from B. cereus if the bacteria lost the plasmid containing cry genes, showing the great relevance of plasmids for these species (Raymond, 2017).

Another example of taxonomic mixed species is the recently proposition to reclassify B. weihenstephanensis strains as later heterotypic synonym of B. mycoides based on the inability to distinguish both species by signature sequences of 16S rDNA, cspA genes, digital DNA-DNA hybridization and average nucleotide identity values (Liu et al., 2018). Even if the authors do not consider the determinant characteristic of B. mycoides strains, their rhizoidal format of colony, this new classification is already used in NCBI Genome Database.

After all, the taxonomy of B. cereus group is still in debate. Currently species definitions within the group consider in advance their phenotypes (virulence, physiology and morphology) than the phylogenetic relatedness. New taxonomic organizations of the group have been suggested, considering a polyphasic taxonomic approach, which includes genotypic and phenotypic characterization. For instance, one method determined the different taxa using the whole-genome sequence-based Genome BLAST Distance Phylogeny (GBDP) approach. This approach divided the group into 30 clusters, each representing independent species (Liu et al., 2015).

Another proposition suggests a new taxonomic rearrangement, but in a more moderate view of splitting the group. Taking into account the three major clades, it clearly separates strains responsible for acute vertebrate infections from entomopathogenic lineages (Raymond, 2017). causes the emetic syndrome harbor the pCER270 plasmid, that contain the genetic apparatus to the production of the cereulide toxin (Rasko et al., 2005;Vilas-Bôas et al., 2007).

The B. cereus group strains possess a wide range of plasmids, especially in B.

thuringiensis strains (Patiño-Navarrete [START_REF] Patiño-Navarrete | Evolutionary processes and environmental factors underlying the genetic diversity and lifestyles of Bacillus cereus group bacteria[END_REF][START_REF] Zhu | Genomic and transcriptomic insights into the efficient entomopathogenicity of Bacillus thuringiensis[END_REF], varying in number (up to 14 different plasmids) and size (from 2kb to 500kb). Considering that each plasmid could be present in multiple copies, the total amount of plasmid DNA (sum of plasmid sizes by its copy number) could be greater than the chromosomal DNA amount (Zhong et al., 2011). Frequently the copy numbers of a plasmid is inversely proportional to plasmid size, such as B. thuringiensis YBT-1520 strain plasmid pBMB8513 (8,5 kb), identical to the pHT8_1 from B. thuringiensis HD73 (Fazion et al., 2018), with approximately 75 copies and pBMB293 (293 kb) with only 3 copies (Zhong et al., 2011).

The presence of mobile genetic elements (MGE) in the genome of B. cereus group bacteria allows the occurrence of genetic exchanges between chromosomes and plasmids. As consequence, chromosomes and plasmids share genes of all functional categories, differing in proportion and in regulatory components (Zheng et al., 2015). The presence of mobile elements and plasmids promote horizontal gene transfer among different strains, improving diversification and adaptation to different conditions.

The main question is that the genes encoding the specific traits used to taxonomic definition are carried on plasmids, which could be lost or transferred to bacteria that belong to other species of the group [START_REF] Santos | Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains[END_REF]. However, genomic analyses linking chromosomal properties with plasmid content evidenced the co-evolutionary interactions of both replicons and that this association resulted in proliferation of successful plasmid and chromosome combinations and that are important to adaptation of specialized pathogens to characteristic niches (Méric et al., 2018;[START_REF] Zheng | Comparative Genomics of Bacillus thuringiensis Reveals a Path to Specialized Exploitation of Multiple Invertebrate Hosts[END_REF].

Quorum sensing systems -RNPP Family

For a long time, bacteria had been described as simple single-cell organisms that live in an autonomous and individual manner. Findings in the last 30 years have changed this concept and nowadays, the multicellularity social behavior is considered as a general trait of bacteria species [START_REF] Shapiro | Bacteria as Multicellular Organisms[END_REF][START_REF] Shapiro | Thinking about bacterial populations as multicellular organisms[END_REF]. One of the remarks that leads to this change of thinking was the complex architecture of colonies from some species, such as B. subtilis [START_REF] Aguilar | Thinking about Bacillus subtilis as a multicellular organism[END_REF]. Multicellular cooperation and labor division provide countless benefits for bacteria, such as more effective access to resources and protection against antagonists or environmental variations.

To coordinate their multicellular functions, bacteria perform a cell-cell communication known as quorum sensing. This communication allows bacteria to coordinate their gene expression and behavior with population density by producing, releasing, detecting and responding to autoinducers (AIs), small extracellular signaling molecules. The concentration of AIs in the bacterial environment increases in relation to the bacterial population density until it reaches a threshold level that is detected by cells and result in modification on gene expression. Quorum sensing enable bacteria to regulate key processes such as bioluminescence, sporulation, competence, antibiotic production, biofilm formation, production of virulence factors and gene transfer [START_REF] Miller | Quorum Sensing in Bacteria[END_REF]Rutherford & Bassler, 2012;[START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. In pathogenic bacteria for example, release of virulence factors when in a small number of cells may not be relevant because bacteria could be detected and destroyed by the immunity system of the host. Thus, it is more beneficial for these bacteria to activate the expression of the virulence genes in conditions where the population density can efficiently attack the host and run over the immune system [START_REF] Shapiro | Thinking about bacterial populations as multicellular organisms[END_REF].

Quorum sensing in Gram negative and Gram positive bacteria

The quorum sensing systems were first described in the Gram negative bioluminescent marine bacterium Vibrio fischeri. This bacterium colonizes the light organ of the Hawaiian squid Euprymna scolopes, which is rich in nutrients. Inside the light organ, the bacteria are able to grow and reach a high population density, resulting in activation of expression of the luciferase operon (luxCDABE) by quorum sensing. This light provides a counter-illumination to the host, avoiding shadow formation and, consequently, predator's evasion. When the bacteria are in the ocean, outside the light organ, the autoinducers released by them are spread in water and are not able to activate the luciferase expression. This circuit is controlled by the LuxI-LuxR system (Figure 6). LuxI synthesize the autoinducer, an acyl-homoserine lactone (AHL) and LuxR is the cytoplasmic receptor that acts as a transcriptional activator. The LuxR-AHL complex binds to the luxICDABE promoter and activates its transcription [START_REF] Miller | Quorum Sensing in Bacteria[END_REF][START_REF] Ng | Bacterial Quorum-Sensing Network Architectures[END_REF][START_REF] Papenfort | Quorum sensing signal-response systems in Gramnegative bacteria[END_REF][START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. The LuxI-LuxR system is the paradigm for most Gram negative quorum sensing. The Gram negative autoinducers are generally AHL (Figure 7), composed by a core N-acylated homoserine-lactone ring carrying acyl chains of 4-18 carbon in length with occasional modifications or unsaturated double bonds. The AHL molecules are able to diffuse through the membrane, resulting in equal intra-and extracellular concentrations. When the autoinducer achieves a threshold concentration it binds to a LuxR-like receptor and activate gene expression, whose products allow bacteria to react to a given stimuli [START_REF] Miller | Quorum Sensing in Bacteria[END_REF][START_REF] Ng | Bacterial Quorum-Sensing Network Architectures[END_REF][START_REF] Papenfort | Quorum sensing signal-response systems in Gramnegative bacteria[END_REF][START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF].

Figure 7. Core molecule of acyl-homoserine lactone autoinducers [START_REF] Ng | Bacterial Quorum-Sensing Network Architectures[END_REF].

Gram positive bacteria use oligopeptides as autoinducers. These signaling molecules are encoded as a precursor with a signal sequence for secretion. Unlike the AHL autoinducers, membrane is not permeable to oligopeptides that need to be actively exported. In the extracellular environment, the peptides can also undergo processing and modifications to acquire their active form. These pheromones are detected by two different mechanisms (Figure 8): (i) by binding to receptors in the membrane surface (middle panel) or (ii) by being re-imported within the bacteria to act over cytoplasmic receptors (left panel) [START_REF] Hoch | Two-component and phosphorelay signal transduction[END_REF][START_REF] Kalamara | Social behaviours by Bacillus subtilis : quorum sensing, kin discrimination and beyond[END_REF][START_REF] Perego | Two-Component Systems, Phosphorelays, and Regulation of Their Activities by Phosphatases[END_REF]Rutherford & Bassler, 2012). The first mechanism is known as two component signal transduction systems, composed by a membrane histidine kinase sensor and a cytoplasmic response regulator (Figure 9). The oligopeptide is recognized by the sensor domain (Signal Input) of the histidine kinase in the extracellular surface of the membrane and activates the autophosphorylation of the catalytic domain of this protein (Autokinase).

The phosphoryl group is, then, transferred to the regulatory domain of the cytoplasmic response regulator (Regulator). The phosphorylation of the response regulator activates the effector domain of this protein (Output), generally a DNA binding domain that allows it to function as a transcription factor, regulating gene expression and consequently, the bacterial response to that specific signal.

Figure 9. The two-component signal transduction system. Detailed description is found in the text [START_REF] Perego | Two-Component Systems, Phosphorelays, and Regulation of Their Activities by Phosphatases[END_REF].

The signal transduction system for competence in B. subtilis, ComP-ComA system (or ComXQPA system), is an example of two-component systems (Figure 10). [START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF].

In this pathway, the autoinducer precursor of ComX is secreted and processed by ComQ. The ComX active decapeptide binds to the ComP membrane bound sensor histine kinase resulting in its autophosphorylation. ComP transfers the phosphate to the response regulator ComA enabling it to bind to DNA and activating expression of genes related to bacterial competence, and also production of surfactin [START_REF] Kalamara | Social behaviours by Bacillus subtilis : quorum sensing, kin discrimination and beyond[END_REF][START_REF] Lazazzera | Quorum sensing and starvation: signals for entry into stationary phase[END_REF][START_REF] Miller | Quorum Sensing in Bacteria[END_REF].

The two-component systems may have evolved to a more elaborated signal Spo0A, that needs to be phosphorylate to be active. Additionally, this cascade is also regulated by two phosphatase families: Rap and Spo0E, which dephosphorylate Spo0F and Spo0A, respectively. The higher complexity of this signal transduction pathway enables the bacteria to accurately regulate the cell fate, as the sporulation, which is a complex and irreversible process (Burbulys et al., 1991;[START_REF] Hoch | Two-component and phosphorelay signal transduction[END_REF]Jiang et al., 2000b;[START_REF] Perego | Two-Component Systems, Phosphorelays, and Regulation of Their Activities by Phosphatases[END_REF][START_REF] Trach | Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727[END_REF].

RNPP family

The second quorum sensing mechanism of Gram positive bacteria is based on the direct binding of the oligopeptide to the cytoplasmic response regulator in the responder cell, particularly in bacteria from the Firmicutes phylum. These systems belong to the RNPP familynamed from the key regulator members -Rap, NprR, PlcR and PrgX. Even if these proteins regulate various processes in different bacterial species, they share two main features: the intracellular interaction with a linear processed oligopeptide (Phr, NprX, PapR, cCF10, respectively) that is reimported by oligopeptide permeases (Opp), and a similar structure of the regulators, which contain tetratricopeptide repeat (TPR) motifs (Figure 12) (Declerck et al., 2007;[START_REF] Perez-Pascual | Bacterial Cell-Cell Communication in the Host via RRNPP Peptide-Binding Regulators[END_REF][START_REF] Rocha-Estrada | The RNPP family of quorum-sensing proteins in Gram-positive bacteria[END_REF]. The TPR domain is a structural motif present in a wide range of proteins, identified in diverse organisms from bacteria to humans and involved in different biological processes such as cell cycle regulation, transcriptional control and protein folding. The TPR motifs comprise three to sixteen tandem repeats of degenerated sequences of 34 amino acids residues and adopt a structural arrangement of twoantiparallel α-helices. This arrangement is essential to its role in mediating proteinprotein or protein-peptide interactions or in assembling multiprotein complex (D'Andrea & Regan, 2003).

In addition to these main features, the genes coding for the precursor of the signaling oligopeptide is generally located downstream from genes coding for the response regulator and can be found on the chromosome or plasmids. When the oligopeptide binds to its cognate receptor it induces a conformational change, activating or inhibiting its activity. Except from the Rap phosphatase, the RNPP regulators possess a helix-turn-helix (HTH) DNA binding domain in the N-terminal region (Figure 12), allowing them to act as transcriptional factors. The similar characteristics of the RNPP systems suggest that they derive from a same ancestor (Figure 13) (Declerck et al., 2007;[START_REF] Do | Structural Mechanisms of Peptide Recognition and Allosteric Modulation of Gene Regulation by the RRNPP Family of Quorum-Sensing Regulators[END_REF]Perchat et al., 2016b;[START_REF] Perez-Pascual | Bacterial Cell-Cell Communication in the Host via RRNPP Peptide-Binding Regulators[END_REF][START_REF] Rocha-Estrada | The RNPP family of quorum-sensing proteins in Gram-positive bacteria[END_REF].

Figure 13. Evolution of RNPP regulators. The disposition of all Bacillus RNPP systems in signaling cassettes suggest that the ancestor system was encoded by a unique gene that was separated during evolutionary time. Another evolutionary process was the acquisition of the HTH motif, which enable the regulators to directly control the expression of genes related to its pathway. nTPR: number of TPR repeats; +/-: if the signaling molecule inhibits (-) or activates (+) the regulator activity: HTH: DNA-binding domain (Perchat et al., 2016b).

Rap-Phr systems

Response regulator aspartate phosphatases (Rap) are found in genome of Bacillus species. These proteins were extensively studied in B. subtilis, in which they were shown to regulate sporulation, competence, transfer of genetic mobile elements (plasmids or insertion sequence), production of extracellular proteases and biofilm formation (Perego, 2013). The main described roles of Rap are in sporulation and competence with two distinct modes of action: (i) as phosphatase, by dephosphorylating the intermediate response regulator of the phosphorelay, Spo0F, thus inhibiting indirectly the phosphorylation of Spo0A and consequently, the sporulation process (Perego et al., 1994); or (ii) by binding to a cytoplasmic regulator, such as the ComA response regulator, in which Rap binds to the HTH DNA-binding domain, inhibiting the transcriptional activator activity of ComA over genes from its regulon, related to competence (Bongiorni et al., 2005;Core & Perego, 2003). The Rap proteins are inhibited by the Phr oligopeptide (Perego & Brannigan, 2001;Perego & Hoch, 1996). In the B. cereus group species, Rap-Phr systems were already shown to regulate sporulation in B. anthracis (Bongiorni et al., 2006) and B.

thuringiensis (Fazion et al., 2018).

Rap proteins are structurally organized in an N-terminal three helix bundle and a C-terminal domain containing TPR motifs (six canonical and one non-canonical).

Phr binding induces a conformational change where the entire protein consists of one single domain containing nine TPR-like folds (Figure 12) (Parashar et al., 2011;Perego, 2013). Due to the lack of the HTH domain (Figure 12) and the highly diversity among the RNPP regulators, Rap proteins are suggested as the most ancestral system of the RNPP family (Figure 13) (Declerck et al., 2007).

NprR-NprX system

In bacteria from the B. cereus group, the neutral protease regulator (NprR)

was primarily described to regulate the expression of the metalloprotease NprA (also known as NprB or Npr599), which is the main constituent of the secretome of these bacteria during the stationary phase in sporulation medium [START_REF] Chitlaru | Differential Proteomic Analysis of the Bacillus anthracis Secretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities[END_REF]Perchat et al., 2011). NprR is active as a transcriptional regulator in the presence of its cognate oligopeptide NprX (Perchat et al., 2011). The gene encoding the NprX precursor is located downstream from the nprR gene, and both genes are cotranscribed. The expression from the nprR promoter is repressed by the global regulator CodY during the exponential growth and activated by the PlcR quorum sensor (described below) at the onset of the stationary phase. Moreover, the transcription of the nprX gene is also independently activated by two promoters, located upstream the nprX gene, within the nprR gene and related to the sporulation specific sigma factors σ H and σ E . These independent expressions result in higher concentration of active NprX in the late exponential phase. Differently from some RNPP systems, the transcription of the nprR-nprX cassette is not auto-regulated [START_REF] Dubois | Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation[END_REF].

In fact, NprR is a bifunctional regulator (Figure 14), depending on the presence of NprX that modify its conformational structure (Perchat et al., 2016a;[START_REF] Zouhir | Peptide-binding dependent conformational changes regulate the transcriptional activity of the quorum-sensor NprR[END_REF].

Figure 14. Bifunctional activity of NprR. When in its dimeric form, NprR (R) inhibits the sporulation phosphorelay. Binding of the NprX signaling peptide (R/X), activates the transcriptional regulator function of NprR. The switch between the two forms is regulated in a quorum sensing manner (Perchat et al., 2016a).

In the absence of NprX, NprR is in a dimeric form structurally closed to Rap protein, with an additional HTH domain (Figure 12). NprR inhibit the initiation of the sporulation process by binding and dephosphorylating Spo0F from the sporulation phosphorelay, in a Rap phosphatase-like manner. Residues described to be involved in the activity of Rap over Spo0F are conserved in NprR. The binding of the NprX switches the structure of NprR from the dimeric to the tetrameric form and activates its function of transcriptional regulator (Perchat et al., 2016a). The NprR regulon includes the nprA gene and at least 40 other genes, some of which encoding degradative enzymes (proteases, lipases and chitinases). The main role of the NprR regulon is the survival of B. thuringiensis in the insect host cadaver (Dubois et al., 2012). Thus, the NprR-NprX system seems to play a role in the tight control of the transition between the necrotrophic lifestyle and sporulation (Perchat et al., 2016a(Perchat et al., , 2016b)). Furthermore, due to its bifunctional characteristic, the NprR regulator is thought to be the evolutionary intermediate of the RNPP family proteins, between the Rap phosphatase and the transcriptional regulators PlcR and PrgX (Figure 13) (Declerck et al., 2007;Perchat et al., 2016b).

The active form of NprX is located within the C-terminal portion of the 43 amino acids precursor peptide, and a heptapeptide is suggested to be its active form.

Phylogenetic analyses revealed a coevolution of the two cognate components of this system and that NprR-NprX and housekeeping genes have a different evolutionary history. In addition, seven different NprR-NprX clusters were detected, which correspond to different pherotypes. Comparison between groups indicates that these pherotypes are strain-specific with the possibility of cross-talk between some groups (Perchat et al., 2011).

PlcR-PapR system

The phospholipase C regulator (PlcR) was first described to positively regulate the expression of phosphatidyl inositol-specific phospholipase C (PI-PLC). The expression of this regulator starts at the end of vegetative growth and reaches the peak two hours after the onset of stationary phase [START_REF] Lereclus | Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase[END_REF]. PlcR is a 34 kDa pleiotropic regulator and the PlcR regulon contains at least 45 genes that represent 80% of the secretome of B. cereus or B. thuringiensis at the onset of the stationary phase [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF][START_REF] Gohar | The PlcR Virulence Regulon of Bacillus cereus[END_REF][START_REF] Gohar | Twodimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon[END_REF]. Genes activated by

PlcR have in their promoter region a PlcR-box, a highly conserved palindromic sequence, which is the binding site of the PlcR-PapR complex [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF].

The most strongly PlcR-induced genes encode for enterotoxins (hbl and nhe), cytotoxins (cytK) and hemolysins (clO) [START_REF] Gohar | The PlcR Virulence Regulon of Bacillus cereus[END_REF]. The PlcR regulon is involved in the virulence with three main functions: (i) food supply, with phospholipases, proteases and toxins; (ii) cell protection, such as bacteriocins, toxins, transporters and cell wall biogenesis; and (iii) environment sensing, as twocomponent sensors or chemotaxis proteins [START_REF] Gohar | The PlcR Virulence Regulon of Bacillus cereus[END_REF]. Genes belonging to the PlcR regulon are spread in the bacterial chromosome, not forming a pathogenic island [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF].

PlcR is activated by the signaling peptide PapR (from Peptide activating PlcR) (Figure 15) (Slamti & Lereclus, 2002). PapR active form is a heptapeptide and its precursor peptide (48 amino acids) is encoded by the papR gene located 70bp downstream from the plcR gene and belongs to the PlcR regulon (Bouillaut et al., 2008;Slamti & Lereclus, 2002). The PlcR-PapR system positively regulates its own transcription [START_REF] Lereclus | Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase[END_REF]. The expression of plcR-papR is also negatively controlled by Spo0A, leading to the absence of expression of this system in sporulation-specific medium [START_REF] Lereclus | Regulation of toxin and virulence gene transcription in Bacillus thuringiensis[END_REF]. This system is indirectly controlled by CodY, as this regulator controls the transcription of genes encoding for the Opp permease required for the re-import of the PapR oligopeptide (Gominet et al., 2001;[START_REF] Slamti | CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis[END_REF].

Figure 15. The PlcR-PapR quorum sensing system. PapR is transcribed in a premature form of 48 amino acids that is secreted, processed and re-imported as a heptapeptide. Inside the cell, the PapR7 binds to PlcR and activates the transcription of virulence factor genes that belong to the PlcR regulon (Slamti et al., 2014).

The PlcR structure is composed by an N-terminal HTH DNA binding domain and a regulatory domain that consist of five degenerated TPR motifs and a capping helix at the C-terminal end of the protein (Figure 12) (Declerck et al., 2007). PlcR is a dimer and the binding of PapR result in a slight conformational change of the regulatory domain, enabling PlcR to bind to DNA (Declerck et al., 2007). PlcR-PapR system seems to be specific from species of the B. cereus group. However, as already cited, plcR genes from B. anthracis strains bear a nonsense mutation and, consequently this regulator is inactive in this species [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF]. This singular variation could explain some phenotypic differences between B. anthracis strains from B. cereus or B. thuringiensis, such as the absence of hemolysis.

Similarly to the NprR-NprX systems, PlcR-PapR exhibit strain-specific properties, with four distinct pherotypes, in which a given PlcR is more strongly activated by its cognate PapR than heterologous molecules (Bouillaut et al., 2008;Slamti & Lereclus, 2005). Moreover, plcR and papR genes seem to have co-evolved but have a different evolutionary history when compared to the housekeeping genes (Ko et al., 2004;Slamti & Lereclus, 2005).

The PlcR, and consequently its regulon, play an important role in virulence and thus in adaptation of bacteria from the B. cereus group to its host environment.

Due to the ability of B. cereus group strains, especially B. cereus sensu stricto, to cause human infections, new studies rely on mechanisms to disrupt bacterial quorum-sensing by interfering in cell-cell communication to affect their capacity to produce virulence factors, a processes called quorum quenching [START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. Recently, [START_REF] Yehuda | Turning off Bacillus cereus quorum sensing system with peptidic analogs[END_REF] design synthetic PapR peptide analogs that prevent the activation of PlcR by endogenous PapR, leading to the loss of virulence factors production.

PrgX & cCF10/iCF10

PrgX is a sex pheromone receptor of Enterococcus faecalis, which is a commensal bacterium but also act as opportunistic human pathogen, associated to nosocomial infections [START_REF] Bae | Analysis of expression of prgX, a key negative regulator of the transfer of the Enterococcus faecalis pheromone-inducible plasmid pCF10[END_REF][START_REF] Perez-Pascual | Bacterial Cell-Cell Communication in the Host via RRNPP Peptide-Binding Regulators[END_REF]. The prgX gene is encoded in the tetracycline resistant plasmid pCF10 and participates in the transcription regulation of conjugative transfer gene (prgQ operon), which controls the conjugation capacity of pCF10. The repressor activity of PrgX is controlled by two antagonistic peptides (Figure 16): the inhibitor iCF10, located in the plasmid pCF10, and the activator cCF10, encode by a chromosomal gene [START_REF] Do | Structural Mechanisms of Peptide Recognition and Allosteric Modulation of Gene Regulation by the RRNPP Family of Quorum-Sensing Regulators[END_REF]. Both cCF10 and iCF10 are heptapeptides corresponding to the C-terminal end of their precursor lipoprotein [START_REF] Mori | Structure of cCF10, a peptide sex pheromone which induces conjugative transfer of the Streptococcus faecalis tetracycline resistance plasmid, pCF10[END_REF][START_REF] Nakayama | The prgQ gene of the Enterococcus faecalis tetracycline resistance plasmid pCF10 encodes a peptide inhibitor, iCF10[END_REF].

Moreover, both peptides bind to the same pocket but induce different conformations in C-terminal part of PrgX. The two PrgX-peptide complexes adopt a tetrameric form that induce a particular conformation (Figure 16) [START_REF] Chen | Mechanisms of peptide sex pheromone regulation of conjugation in Enterococcus faecalis[END_REF]. When iCF10 binds to PrgX regulator, they attach to two sites of the DNA sequence and form a loop that prevents activity of RNA polymerase on the prgQ operon. By contrast, when bonded to cCF10, this repression is disrupted and the transcription of conjugation genes could take place [START_REF] Neiditch | Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria[END_REF]. Donor and recipient cells express cCF10, which is neutralized by iCF10 in cells containing pCF10 plasmid [START_REF] Rocha-Estrada | The RNPP family of quorum-sensing proteins in Gram-positive bacteria[END_REF]. When the density of recipient cells increases, concentration of cCF10 overcome iCF10 concentration and release the transcription of conjugation genes repressed by PrgX, enabling conjugation.

Expansion of the RNPP family

New findings in Gram positive bacteria quorum sensing systems had led to the description of new RNPP-like systems, as the Rgg-SHP system from Streptococci, given rise to the new RRNPP family [START_REF] Monnet | Quorum-sensing regulators in Gram-positive bacteria: ‗cherchez le peptide[END_REF][START_REF] Parashar | Rgg protein structurefunction and inhibition by cyclic peptide compounds[END_REF].

Rgg (regulator gene of glucosyltransferase) transcription factors are widespread in

Firmicutes, but it is particularly associated to a signaling peptide in Streptococcus species. Rgg proteins represents a large family of receptors that regulate genes with diverse functions, such as commensalism and production of virulence factors, and are regulated by small hydrophobic peptides (SHP) [START_REF] Neiditch | Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria[END_REF][START_REF] Parashar | Rgg protein structurefunction and inhibition by cyclic peptide compounds[END_REF][START_REF] Perez-Pascual | Bacterial Cell-Cell Communication in the Host via RRNPP Peptide-Binding Regulators[END_REF]. Rgg protein, as well as PrgX, do not contain detectable TPR motif but adopt TPR-like folds [START_REF] Neiditch | Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria[END_REF]. The ComR-XIP system, initially described as member of the Rgg family, was shown to be another new member of the RNPP family [START_REF] Talagas | Structural Insights into Streptococcal Competence Regulation by the Cell-to-Cell Communication System ComRS[END_REF]. ComR is a transcription factor that positively controls competence in Streptococci and is directly activated by XIP (the active form of the ComS peptide).

Regulation of the infectious cycle in B. thuringiensis

The three RNPP systems present in the B. cereus group -PlcR, NprR and

Rap-Phr Systems

Sporulation in Bacillus

Spores play an important role in survival and dispersion of Bacillus species and the sporulation process was well studied in B. subtilis. The capacity of bacterium to sense its own metabolic state and the environmental changes contribute to regulate the commitment to sporulation. This decision is driven by the major sporulation response regulator Spo0A that is active when it reaches a phosphorylation threshold (Burbulys et al., 1991;Fujita & Losick, 2005). The phosphorylation state of Spo0A is controlled by the phosphorelay (Figure 11) which is a signal transduction system allowing the bacteria to sense different types of environmental signals: the cell density by Rap-Phr quorum sensing systems and nutrient limitation (such as source of carbon, nitrogen and phosphorus) and other environmental stresses sensed by kinases (Sonenshein, 2000). In B. subtilis, there are five different kinases related to the phosphorelay (KinA to KinE) and they are all able to phosphorylate Spo0F (the intermediate response regulator). KinA and KinB are the most active of these sensor kinases, being required to initiate sporulation, and seem to depend on signals present on early stationary phase (Jiang et al., 2000b). Whereas KinC and KinD may respond to signals present during growth and were shown to control biofilm formation, while KinE did not have a sufficient effect to lead bacteria to sporulation [START_REF] Aguilar | KinD Is a Checkpoint Protein Linking Spore Formation to Extracellular-Matrix Production in Bacillus subtilis Biofilms[END_REF]Fujita & Losick, 2005;[START_REF] Wu | Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF]. Spo0F~P is used as source of phosphate by the phosphotransferase Spo0B to phosphorylate the Spo0A transcription factor (Burbulys et al., 1991).

The Spo0A regulon comprise at least 121 genes, responding to a low or a high threshold of Spo0A~P, that are repressed or activated (Fujita et al., 2005;Molle et al., 2003). This dose-dependent response is related to the binding constant of the DNA binding domain of Spo0A~P to the 0A box at the promoter region of target genes (Fujita et al., 2005). Spo0A~P auto regulate its own transcription directly, by four different 0A box target sites [START_REF] Chastanet | Just-in-Time Control of Spo0A Synthesis in Bacillus subtilis by Multiple Regulatory Mechanisms[END_REF][START_REF] Strauch | SpoOA activates and represses its own synthesis by binding at its dual promoters[END_REF] or indirectly by the activation of the stationary phase sigma factor σ H , which directs transcription of spo0A and other sporulation related genes, as spo0F, spoIIAA-AB-sigF and some phr [START_REF] Britton | Genome-Wide Analysis of the Stationary-Phase Sigma Factor (Sigma-H) Regulon of Bacillus subtilis[END_REF]. Genes regulated by low level of Spo0A~P

show a high binding constant or are under indirect control of Spo0A, via repression of abrB gene. AbrB is a pleiotropic transcription repressor and its inhibition by Spo0A release the expression of genes related to the stationary phase and early stage of sporulation [START_REF] Strauch | SpoOA activates and represses its own synthesis by binding at its dual promoters[END_REF]. Low level of Spo0A~P regulates genes that are related to other processes during stationary development, such as cannibalism, competence and biofilm formation. During the time that the activity of kinases and phosphatases (Rap and Spo0E) are counter-balanced, Spo0A~P are maintained at low levels (Sonenshein, 2000). Some factors, as the activity of Phr peptides inhibiting Rap phosphatases, the increase of kinases activity and the expression induced by σ H , allow to accumulate Spo0A~P up to a critical threshold leading to commitment of sporulation. Thus, increasing amount of Spo0A~P controls the expression of several genes directly involved in the sporulation process, such as spoIIA, spoIIE and spoIIG (Fujita et al., 2005;Fujita & Losick, 2005). In fact, the phosphorelay generates different levels of Spo0A~P in the bacteria from a same population, resulting in a heterogenous triggering of the sporulation [START_REF] Chastanet | Broadly heterogeneous activation of the master regulator for sporulation in Bacillus subtilis[END_REF].

Sporulation begins with an asymmetrical cell division triggered by Spo0A~P.

This asymmetrical division originates two distinct cell types, genetically identical but with different fates: a larger mother cell and a smaller forespore (also known as prespore). The sporulation process is driven by differential and sequential activation of sigma factors (Figure 18), subunits of RNA polymerase, that direct transcription of different set of genes in the two bacterial compartments. These sigma factors are activated at specific times and in a particular compartment allowing changes in gene regulation to be coupled with morphological states. Just after the septum formation (asymmetric division) the sigma factor σ F is activated in the forespore and shortly after the σ E become active in the mother cell. σ E is encoded in a pro-σ E precursor form and its proteolytic activation is controlled by proteins regulated by σ F . σ E and σ F regulate the expression of genes involved in the engulfment of the forespore in the mother cell, a phagocytic-like process that results in a double membrane forespore included in the mother cell cytosol. After the engulfment, σ G is activated in the forespore, the sigG gene is under control of σ F and its sigma factor product regulate the transcription of genes related to the protection of chromosome and the arrangement of the spore for germination, when in an appropriate condition. σ G also induces the cleavage of the pro-σ K (regulated by σ E ) in the mother cell. σ E and σ K are required for the synthesis of the cortex (a peptidoglycan layer) and the coat (a proteinaceous layer) that encase the mature spore. σ K also regulate genes which products lead to the mother cell lysis and delivery of the mature spores to the environment [START_REF] Fimlaid | Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes[END_REF][START_REF] Kroos | Control of sigma factor activity during Bacillus subtilis sporulation[END_REF][START_REF] Piggot | Sporulation of Bacillus subtilis[END_REF][START_REF] Tan | Spore formation in Bacillus subtilis[END_REF]. Although analogous sporulation process are described in other species of Bacillus and genes involved in sporulation was initially expected to be found among the core genes in their pan genome, a study on Bacillus genus found a great intraspecific and interspecific variation in these genes [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF]. From the 185 sporulation related genes from B. subtilis considered in this pan genomic analysis, only 52 were categorized as core genes. Among them were found the major regulators of sporulation, required for temporal and spatial regulation of this process (spo0A, genes coding for sporulation sigma factors, spoIIE…). It is in agreement with [START_REF] Stephenson | Evolution of signalling in the sporulation phosphorelay[END_REF] showing that the main components of the phosphorelay -Spo0F, Spo0B and Spo0Aare highly similar between B. subtilis and B. anthracis.

In contrast, the divergence could be found, for example, in the number of sensor histidine kinases: five in B. subtilis against nine in B. anthracis, and the sensor domain are not well conserved [START_REF] Brunsing | Characterization of Sporulation Histidine Kinases of Bacillus anthracis[END_REF]. Another important difference concerns Rap-Phr systems, which shows divergence in sequence and in number.

rap-phr systems were described to be present in all strains of B. subtilis and B.

cereus group species and also in more distant Bacillus species such as Bacillus halodurans and Bacillus clausii (Even-Tov et al., 2016). Phylogenetic analysis of Rap sequences from B. subtilis and B. cereus groups showed that Rap proteins of each group clustered separately (Figure 19B) (Even-Tov et al., 2016). Moreover, in the pan genomic study, rap genes from the B. cereus group species were not sufficiently conserved to be recognized as core genes, using B. subtilis genome as reference [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF]. In addition to sequence divergence, the difference in number of these quorum sensing systems have already been described for these two most important groups of Bacillus genus: B. subtilis group strains possess a higher number of rap genes (11 ± 2) than the B. cereus group strains (6 ± 3) (Figure 19A) [START_REF] Anderson | Comparative genome analysis of Bacillus cereus group genomes with Bacillus subtilis[END_REF]Even-Tov et al., 2016). These results suggest that the regulatory components of the phosphorelay cascade may be species or strain-specific and have separately evolved to respond to particular environments [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF].

Rap-Phr systems in B. subtilis

The first studied Rap protein was described as a phosphatase that acts on the sporulation phosphorelay by dephosphorylating Spo0F. This activity gives its name: response regulator aspartate phosphatase (Perego et al., 1994). Rap relevance as phosphatase relies on the fact that it represents some additional regulatory points to the signal transduction system that regulates the sporulation, and contribute to the phosphorelay complexity (Perego & Brannigan, 2001). Thereafter, it was shown that some Rap proteins regulate other developmental pathway of the stationary phase, such as the competence (ability to DNA uptake) via the ComA response regulator.

The release of the B. subtilis complete genome sequence reveals that its type strain (str. 168) possesses 11 rap genes (rapA to rapK) and eight of which are followed by a downstream short coding sequence, the phr gene [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF]. The phr genes slightly overlap the rap gene sequences forming transcript units: the rap-phr signaling cassettes (Perego & Hoch, 1996). The mature form of the Phr peptide inhibits its cognate Rap activity, excepted for RapB, RapD and RapJ that are orphans. However, the PhrC, also known as competence and sporulation factor (CSF), is able to inhibit RapB and RapJ phosphatases (Parashar et al., 2013a;Perego, 1997).

RapA, RapB, RapE, RapI and RapJ regulates sporulation by specifically acting on Spo0F (Figure 20, Table 2) (Jiang et al., 2000a;Parashar et al., 2011;Perego et al., 1994;Singh et al., 2013). Likewise, RapC, RapD, RapF and RapK inhibit competence by binding to the response regulator ComA and preventing its functions as transcription factor (Auchtung et al., 2006;Bongiorni et al., 2005;Ogura & Fujita, 2007;[START_REF] Solomon | Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis[END_REF]. RapH is the unique protein of the Rap family that was demonstrated to have dual specificity on Spo0F and on ComA (Smits et al., 2007).

RapH was shown to play a role in the temporal separation of competence and sporulation pathways which are mutually exclusive. Thus, RapH prevents sporulation initiation in competent cells and later enables the cell to escape from the competent state to enter into the sporulation pathway (Smits et al., 2007). In addition, the other B. subtilis chromosomal Rap proteins were shown to act on other bacterial pathways.

RapG regulates negatively the activity of the response regulator DegU which activates the expression of extracellular alkaline protease encoded by aprE (Ogura et al., 2003). RapI is encoded on the mobile genetic element ICEBs1 which is an integrative and conjugative element (ICE) (Auchtung et al., 2005). These conjugative transposons are able to excise, transfer to recipient cells through conjugation and integrate to the chromosome. RapI regulate the ICEBs1 expression, excision and transfer via the inhibition of the immunity repressor ImmR activity. When the cell is surrounded by bacteria that also harbor ICEBs1, RapI is inhibited by the PhrI produced by these bacteria, preventing the expression of ICEBs1 when there are no potential recipient cells (Auchtung et al., 2005).

Table 2. Rap-Phr systems already functionally analyzed.

Rap Phr active Species/replicon Target Pathway

Reference RapA ARNQT B. subtilis chromosome Sporulation (Perego et al., 1994;Perego & Hoch, 1996) Figure in the text (Perego, 2013).

The involvement of multiple Rap-Phr systems in a given developmental processes (sporulation or competence) could represent different environmental cues that are integrated for a strict regulation of bacterial development (Auchtung et al., 2006;Mirouze et al., 2011). Analogous Rap-Phr systems could act synergistically, being all required for effective regulation of sporulation or competence. For example, PhrC, PhrF and PhrK activity was shown to happen in different times during growth and in a distinct level but all of them are indispensable to the full expression of ComA regulated genes (Auchtung et al., 2006). Alternatively, this redundancy could promote communication of subpopulations that form an isogenic community based on spatiotemporal structure, namely in biofilms [START_REF] Bischofs | Complexity in bacterial cell-cell communication: Quorum signal integration and subpopulation signaling in the Bacillus subtilis phosphorelay[END_REF].

Rap characteristics

The transcription of the rap genes (coupled to phr genes) is regulated by different factors. Expression of rapA, rapC, rapE and rapF genes is controlled by

ComA (Perego et al., 1996(Perego et al., , 1994)). ComA~P may activates expression of sporulation-Rap proteins in order to prevent commitment to sporulation in bacteria that are on a competent state (Perego et al., 1994). Whereas, the occurrence of a ComA-box in the promoter region of competence-Rap proteins may serve as an auto regulatory circuit modulating ComA activity (Bongiorni et al., 2005). rapB expression is under control of AbrB and is induced in vegetative growth (Perego et al., 1994). AbrB also inhibits the transcription of rapI (Auchtung et al., 2005). rapC is repressed by CodY and rapK is suggested to be indirectly activated by Spo0A (Auchtung et al., 2006).

Moreover, the transcription factor RghR represses the transcription of rapD, rapG and rapH [START_REF] Hayashi | Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon[END_REF]Ogura & Fujita, 2007). Hence, the expression of Rap proteins is generally regulated by conditions antithetical to the sporulation process (Perego & Hoch, 1996).

The chromosomal Rap proteins of B. subtilis are about 380 amino acids long and share around 45% of identity among their sequence and this high level of homology indicates a similar overall structure arrangement [START_REF] Diaz | Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide[END_REF]Perego, 2013). Rap proteins were generally found to be dimers in their native state (Bongiorni et al., 2005;[START_REF] Ishikawa | Biochemical Characterization of Aspartyl Phosphate Phosphatase Interaction with a Phosphorylated Response Regulator and Its Inhibition by a Pentapeptide[END_REF]Parashar et al., 2011). For example, RapA forms a stable complex with Spo0F in which a RapA dimer is associated with 2 molecules of Spo0F (Figure 21A) [START_REF] Ishikawa | Biochemical Characterization of Aspartyl Phosphate Phosphatase Interaction with a Phosphorylated Response Regulator and Its Inhibition by a Pentapeptide[END_REF]. In contrast, RapF was found as a monomer, alone or in complex with ComA (Baker & Neiditch, 2011). Early insights about Rap protein structures had recognize TPR motifs in the protein sequences (Perego & Brannigan, 2001). The TPR repeats are described to mediate protein-protein interaction and the six putative motifs of the Rap proteins were predicted to shape in a right-handed super helical structure forming a suitable binding environment to the Phr peptide (Perego & Brannigan, 2001). Later X-ray crystal structure of Rap proteins alone and in complex with Spo0F (RapH-Spo0F),

ComA (RapF-ComA) or with Phr (RapF-PhrF or RapJ-PhrC) had contributed to the comprehension about Rap structure and interaction mechanisms (Baker & Neiditch, 2011;[START_REF] Del Sol | Structural Basis of Rap Phosphatase Inhibition by Phr Peptides[END_REF]Parashar et al., 2013aParashar et al., , 2011)). The Rap proteins are formed by an N-terminal 3-helix bundle separated from the C-terminal TPR domain by a flexible linker and a short helix (Figure 21B) (Parashar et al., 2011).

The TPR domain is composed of 6 genuine TPR folds and a helix-turn-helix TPR-like fold that is located between the fifth and sixth TPR motifs.

The N-terminal module of Rap proteins contains the determinants for substrate binding and catalytic activity [START_REF] Diaz | Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide[END_REF]. In fact, the enzymatic active site for dephosphorylation of Spo0F~P is a glutamine (Q47) located within the 3-helix bundle (Parashar et al., 2011). Besides the catalytic role, binding of Rap to Spo0F also inhibits the transfer of phosphate to and from the response regulator by steric hindrance limiting the access of other phosphorelay components, kinases and Spo0B, to the aspartyl phosphate active site of Spo0F (Parashar et al., 2011).

Regarding Rap interaction with Spo0F, some residues of the TPR domain also play a role in placing both proteins in a correct position to enable their interaction. Alignment of sporulation-Rap sequences showed that the residues required for Rap activity on Spo0F were conserved among them. This conservation is to such an extent that the sporulation activity of a given Rap could be predicted using this data (Parashar et al., 2011).

Initially, the regulation of ComA by the Rap protein was supposed to be based on its phosphatase activity. Then, it was demonstrated that the competence-Rap proteins did not dephosphorylate ComA~P but bind to the DNA-binding domain of the response regulator to prevent its activity as transcription factor (Baker & Neiditch, 2011;Core & Perego, 2003). Even if the N-terminal of Rap proteins is the responsible module for both response regulators, Spo0F and ComA bind to distinct non-overlapping sites within the 3-helix bundle (Baker & Neiditch, 2011;[START_REF] Diaz | Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide[END_REF]Parashar et al., 2011).

The Phr oligopeptide was shown to bind to the Rap protein in an extend conformation along the groove of the TPR domain, specifically to the region from the TPR3 to TPR5 [START_REF] Diaz | Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide[END_REF] 21B) (Parashar et al., 2013a). Therefore, the inhibitor peptide and the target substrate interact with Rap at distinct sites. The N-terminal part is generally the effector domain related to the activity on the target response regulator, while the Cterminal part is the regulatory domain involved in the binding module of the quorum sensor [START_REF] Diaz | Bacillus subtilis RapA Phosphatase Domain Interaction with Its Substrate, Phosphorylated Spo0F, and Its Inhibitor, the PhrA Peptide[END_REF][START_REF] Del Sol | Structural Basis of Rap Phosphatase Inhibition by Phr Peptides[END_REF][START_REF] Reizer | Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases[END_REF]. (Pottathil & Lazazzera, 2003) B) Phr molecules are transcribed in a premature form, which is secreted through the Sec export system and then by proteases. An additional protease cleavage seems to occur in Phr peptides that the active form is found inside the C-terminal region (Pottathil & Lazazzera, 2003).

phrC, phrE, phrF, phrG, phrI and phrK genes are controlled by the alternative sigma factor σ H which may ensure a higher expression of the signaling molecules during the early stationary phase (McQuade et al., 2001). For instance, Spo0A~P induces the sigH transcription which promote the production of sufficient Phr peptide to prevent activity of Rap phosphatase on the phosphorelay after sporulation had begun.

Unlike Rap sequences, Phr precursors show little homology in amino acid sequences but a similar structure. Phr sequences are arranged in a hydrophobic Nterminal part linked to an hydrophilic C-terminal part by a potential signal peptidase cleavage site (Perego & Hoch, 1996). To achieve its active form, the Phr precursor undergoes two maturation processes (Figure 22B). Primarily, the pre-proPhr is probably processed by a SecA dependent system for export (Pottathil & Lazazzera, 2003). However, the five known B. subtilis peptidases were not indispensable to this activity [START_REF] Stephenson | Molecular Analysis of Phr Peptide Processing in Bacillus subtilis[END_REF]. Nonetheless, all the Phr peptides do not seem to be processed in the same manner.

Indeed, the mature form can be found in the C-terminal end of the precursor peptide (PhrA, PhrC, PhrG, PhrI, and PhrF) or within the C-terminal region (PhrE, PhrH, and PhrK) (Jiang et al., 2000a;Pottathil & Lazazzera, 2003). In the latter case, the proteases from the subtilisin family are not able to process these signaling peptides, at least not alone, as two cleavage reactions are required.

After the maturation, the Phr oligopeptide is reimported into the bacterial cell by oligopeptide permeases, as the other signaling peptides of the RNPP family. The active Phr were firstly described to be pentapeptides (Perego, 1997) but lately, it was demonstrated that these inhibitors could also have six amino acids long, as in the case of PhrH and PhrI (Mirouze et al., 2011). Altogether, these characteristics difficult the prediction of the active signaling peptide (Pottathil & Lazazzera, 2003). However, the active Phr have a conserved positively charged amino acid (arginine, histidine or lysine) in the second position when it is a pentapeptide. This export-import circuit is typical of the oligopeptides way of life in cell-cell communication. In addition to its role as quorum sensors, this circuit is also supposed to be a mechanism to regulate the timing of the phosphatase activity (Mirouze et al., 2011;Perego, 1997).

Except the regulation of RapB and RapJ by PhrC, the Phr oligopeptides act specifically on their cognate Rap proteins (Pottathil & Lazazzera, 2003). Indeed, no cross reactivity was shown among RapA-PhrA and RapC-PhrC neither between these systems and RapE-PhrE (Jiang, Grau, et al., 2000;Perego, 1997). Therefore, it is suggested that both components had co-evolved and the presence of multiple Rap-Phr systems in B. subtilis was due to recent gene duplication event (Perego, 2013;Pottathil & Lazazzera, 2003;[START_REF] Reizer | Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases[END_REF].

Plasmid-borne Rap-Phr (QS) systems

Plasmids and other genetic mobile elements (such as ICE) represent important elements for genes spreading and diversification. Furthermore, rap-phr genes were found in various plasmids and some of them were further evaluated with functional studies which have shown the relevance of these plasmid Rap-Phr systems for bacterial development.

The first described plasmid Rap-Phr system from B. subtilis was the Rap60-Phr60, harbor on plasmid pTA1060. This system regulate the production of extracellular proteolytic enzymes (Koetje et al., 2003). The quorum sensing control of the production of protease allows the bacteria to better exploit the nutrients. When in just a small number of bacteria, in which there are sufficient resources to their development, Rap60 inhibit protease production, since it is not required. Once in a high cell density, when the nutrients were already depleted, the Phr60 binds to Rap60 and activates expression of the extracellular protease to improve energy sources. Lately, the Rap60-Phr60 couple was demonstrated to also control the phosphorelay and ComA activity by a non-canonical mechanisms, resulting in modification of diverse process, such as sporulation, cannibalism, biofilm formation and genetic competence [START_REF] Boguslawski | Novel mechanisms of controlling the activities of the transcription factors Spo0A and ComA by the plasmid-encoded quorum sensing regulators Rap60-Phr60 in Bacillus subtilis[END_REF]. In addition to the usual role as phosphatase on Spo0F, Rap60 inhibit the autophosphorylation of kinase KinA.

Concerning ComA, Rap60 inhibits its activity as transcription regulator by forming a ternary complex with ComA and the DNA promoter region of its target.

Encoded by plasmid pBSG3 from B. amyloliquefaciens, the RapQ-PhrQ system shows great similarity with Rap60-Phr60. In fact, RapQ presents 64% of identity to Rap60 sequence and was also described to inhibit ComA activity through the establishment of a ternary complex (Qiao et al., 2011;Yang et al., 2015). When expressed in a heterologous host (B. subtilis OKB105), RapQ-PhrQ was shown to control protease production, sporulation and genetic competence.

The RapP-PhrP from B. subtilis NCIB3610 plasmid pBS32 regulates biofilm formation, sporulation and competence (Parashar et al., 2013b). Biofilm formation were shown to be also regulated by sporulation-Rap proteins, via low level Spo0A~P activity through AbrB and SinI-SinR. Generally, most B. subtilis studies were based in domesticated plasmid-free strains that had lose their ability to form dense biofilms, then this post exponential process was not evaluated in B. subtilis chromosomal Rap-Phr systems. However, considering the role of Spo0A in biofilm formation all Rap proteins with sporulation effect would also regulate biofilm. RapP inhibits Spo0F and an additional target seems to be required for its strong effect on biofilm (Omer [START_REF] Bendori | The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP[END_REF]. In contrast to all known Rap-Phr system, RapP is not inhibited by its cognate PhrP, due to a rare mutation in the RapP sequence. Moreover, the catalytic residue of RapP related to phosphatase activity is a glutamate instead of the well conserved glutamine (Q47).

In another case of plasmid-borne Rap-Phr system, Rap LS20 -Phr LS20 controls the mobility of its conjugative plasmid pLS20 from B. subtilis natto strain IFO3335 [START_REF] Rösch | Induction of Plasmid Conjugation in Bacillus subtilis Is Bistable and Driven by a Direct Interaction of a Rap/Phr Quorum-sensing System with a Master Repressor[END_REF]Singh et al., 2013). This control occurs in the same manner that of the ICEBs1 regulation by RapI-PhrI. The expression of genes essential for the plasmid conjugation is controlled by the master repressor Rco LS20 .

Rap LS20 act as conjugation antirepressor over Rco LS20 , binding to its DNA-binding domain [START_REF] Rösch | Induction of Plasmid Conjugation in Bacillus subtilis Is Bistable and Driven by a Direct Interaction of a Rap/Phr Quorum-sensing System with a Master Repressor[END_REF]. When surrounded by recipient cells that do not express Phr LS20 , Rap LS20 inhibit Rco LS20 and the conjugation apparatus is expressed.

When the majority of the population carries the pLS20 plasmid, hence the concentration of the anti-antirepressor Phr is high and the conjugation is inhibited (Singh et al., 2013).

Regarding the B. cereus group, the genome of B. anthracis bear six rap-phr modules, five on chromosome and one in the pathogenic plasmid pXO1 (Bongiorni et al., 2006). These quorum sensing systems were identified using the B. subtilis RapA as query in a search through the B. anthracis A2012 genome. The six identified systems share, in average, 24% identical residues with RapA. One chromosomal (BA3790) and the plasmid (BXA0205) Rap proteins were shown to inhibit sporulation via phosphatase activity on Spo0F (Bongiorni et al., 2006). The C-terminal end pentapeptide of BXA0205Phr was able to counteract its cognate Rap activity; however, it was not possible to outline the active form of the Phr Ba3791. A strict regulation of sporulation is especially relevant to the pathogenicity of B. anthracis bacteria (Bongiorni et al., 2006;[START_REF] Brunsing | Characterization of Sporulation Histidine Kinases of Bacillus anthracis[END_REF]. When inside the host, it is valuable to maintain the vegetative state that is able to produce toxins and escape from the immunity system. Thus, during growth in the blood stream, the Phr produced are dissipated and not able to neutralize Rap phosphatase, inhibiting commitment to sporulation and maintaining cell growth. However, once the infection is established in an organ, bacteria proliferate; increasing cellular density and, consequently, the amount of Phr molecules. Thus, together with the decrease of nutrient availability,

Phr peptides are able to inhibit Rap proteins and bacteria sporulate.

Another plasmid-borne Rap-Phr system studied in the B. cereus group was the Rap8-Phr8, encoded by the small cryptic plasmid pHT8_1 from B. thuringiensis var. kurstaki HD73 strain (Fazion et al., 2018). Rap8 acts on the phosphorelay inhibiting sporulation and biofilm formation. The mature form of Phr8 is the C-terminal end heptapeptide of the precursor Phr peptide, similarly to the other RNPP family signaling peptides from the B. cereus group. Moreover, this Rap-Phr system was demonstrated to regulate sporulation in the insect larvae, the ecological niche of B.

thuringiensis. Likewise in B. anthracis, the Rap8-Phr8 system may allow a precise control of the sporulation process, improving the bacterial survival and dissemination.

OBJECTIVES

The main objective of this study was to perform an overview of the Rap-Phr quorum sensing systems in the B. cereus group concerning their number, their location and their diversity. Moreover, we aimed to predict the activity of Rap proteins on sporulation by using in silico analyzes based on data described for RapH in B.

subtilis. Thereafter, the validation of prediction was made by functional analysis. The second objective of this study was the characterization of the plasmid-borne Rap63-Phr63 system in relation to the sporulation function. The determination of the active Phr form and the possible interaction with the co-existent Rap8-Phr8 in the B.

thuringiensis HD73 strain were also investigated.

ARTICLE 1

Diversity of the Rap-Phr quorum-sensing systems in the Bacillus cereus group Priscilla de F. Cardoso (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 [START_REF] Callegan | Relationship of plcR-Regulated Factors to Bacillus Endophthalmitis Virulence[END_REF]Ehlingschulz et al. 2006;Stenfors Arnesen et al. 2008). B. thuringiensis is the world's most used biopesticide due to its production of insecticidal toxins (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 causative agent of anthrax (Liu et al. 2014). Although phenotypically different, these species are closely related genetically (Rasko et al. 2005) These regulators are formed by tetratricopeptide repeat (TPR) domains that are structural motifs of degenerated residues that mediate protein-protein and proteinpeptide interactions (D'Andrea and Regan 2003). The activity of these cytoplasmic regulators is activated (NprR and PlcR) or inhibited (Rap) by secreted, matured, and re-imported peptides that function as signaling molecules (Perchat et al. 2011, Perego andHoch 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 Sporulation is essential for survival and dispersion of a wide variety of organisms [START_REF] Huang | Sporulation: how to survive on planet Earth (and beyond)[END_REF]. In Bacillus subtilis, this differentiation process is regulated by a complex pathway (Sonenshein 2000), in which Spo0A is the major regulator of sporulation that must be phosphorylated to be active. External signals, such as starvation, are detected by different sporulation kinases (KinA to KinE), which phosphorylate the Spo0F response regulator (Burbulys et al. 1991). The phosphoryl group is then transferred through the phosphorelay from Spo0F to the phosphotransferase Spo0B, and then to Spo0A (Jiang et al. 2000a). Certain 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;Parashar et al. 2011Parashar et al. , 2013a;;Perego et al. 1996;Smits et al. 2007). RapC, RapD, RapF, RapH,

Materials and Methods

Bacterial genomes

The B. cereus group strains with a complete genome sequence available in the NCBI short open reading frames (encoding 35 to 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 20kb region around the rap genes (10kb upstream and 10Kb downstream)

was analyzed using the ISfinder database (Siguier et al. 2006) to verify the presence of mobile elements within these genomic regions.

Rap proteins 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 [START_REF] Xia | DAMBE5: A Comprehensive Software Package for Data Analysis in Molecular Biology and Evolution[END_REF]) was used to gather sequences with 100% identity. Unique Rap sequences were aligned using the MUSCLE algorithm, and MEGA 6 [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]) 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 [START_REF] Letunic | Interactive Tree Of Life (iTOL) v4: recent updates and new developments[END_REF]. For Rap clustering delimitation, a value of 0.8 was used as the cut-off 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 xylA , a multicopy vector with xylose-inducible promoter [START_REF] Grandvalet | Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation[END_REF]. All genes were amplified by PCR using primers listed in Online Resource Table 2 and ligated to the plasmid pHT315-P xylA using the appropriate restriction sites. For cloning steps, these plasmids were transformed in Escherichia coli K-12 strain TG1 and then in the 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 Miniprep 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 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 identified (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 two 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 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 rapphr 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 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 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). 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 generally 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) profile and similar plasmid profile. These three strains might derive from a same parental 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 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 xylA 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-(rap6-BtHD1, rap6-Bt407, and rap7-BtHD73) did not inhibit the sporulation efficiency compared to the control strain, confirming their prediction. Among the Rap Spo+ analyzed, the expression of rap8-Bt407, rap10-BtHD1, rap7-Bt407, and rap5-HD73 strongly prevent the sporulation (P<0.001) while the expression of rap1-BcATCC14579, and rap2-BcATCC14579 slightly inhibited the sporulation efficiency compared to the control strain (P<0.01) (Fig. 4b, Online Resource Table 6). However, the rap8-BtHD1, predicted Rap Spo+ does not display a role in sporulation in our B.

thuringiensis HD73 model strain in this growth condition. Hence, the sporulation results allow us to confirm the predicted 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 (Bongiorni 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- The role of these QS systems in the production of extracellular proteases or sporulation 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 (Perchat et al. 2016a). This hypothesis could also explain the difference in the number of rap-phr genes between B. subtilis and

B. cereus.

Our analysis revealed that 30% of the identified rap-phr genes were plasmidborn. The plasmid rap-phr genes have been described in diverse Bacillus species (Koetje et al. 2003;Parashar et al. 2013b;Singh et al. 2013;Yang et al. 2015), including the B. cereus group (Bongiorni et al. 2006;[START_REF] Chao | Complete nucleotide sequence of pBMB67, a 67-kb plasmid from Bacillus thuringiensis strain YBT-1520[END_REF]Fazion et Identical Rap protein sequences are located on the same type of replicon: i) in the chromosome as a consequence of common ancestor, or ii) in plasmids as a consequence of conjugation events. The proportion of identical Phr is higher than that of identical Rap. This characteristic might allow the Phr peptides to act cooperatively on various Rap proteins from different strains. The mature Phr can be located inside or at the C-terminal extremity of the pro-peptide (Pottathil and Lazazzera 2003), hampering the identification of the active form of some Phr. Due to this difficulty and to the wide variability of pro-Phr sequences, the Phr phylogenetic tree was not estimated and the evolutive correlation between Rap and Phr was not determined. Nevertheless, this coevolution was described for the NprR-NprX and PlcR-PapR systems (Perchat et al. 2011;Slamti and Lereclus, 2005).

The correlation between the Rap-Phr system distribution and the MLST tree based on housekeeping genes revealed that closely related strains harbor a similar Rap-Phr system pattern, suggesting a similar evolutionary history of both genetic characters. This correlation was not observed for the PlcR and NprR regulators from the RNPP family (Ko et al. 2004;Perchat et al. 2011). Also, the closest strains have a similar profile, even for plasmid genes, suggesting a beneficial association of particular plasmid-chromosome combinations that leads to the maintenance and propagation of these proficient combinations (Méric et al. 2018).

One-third of the Rap proteins of the B. cereus group are predicted to have phosphatase activity on Spo0F. However, some strains did not have any predicted functions leading to the maintenance of these plasmids in the bacterial cell. Recently, the plasmid pHT8_1 from the B. thuringiensis HD73 strain has been characterized, and the role of its Rap-Phr system in the regulation of the sporulation process in insect larvae has been demonstrated (Fazion et al. 2018).

Sporulation assays validated the in silico prediction except for one Spo+ Rap.

The residues involved in RapH-Spo0F interaction are highly conserved in B. subtilis and B. cereus, and small differences are sufficient to lose this activity. However, these key residues are relatively well-conserved in Rap proteins that do not regulate the sporulation pathway, suggesting that the Rap activity on sporulation was the ancestral role of these proteins (Even-Tov et al. 2016). Thus, the presence of these residues is a good indicator but is not sufficient to predict the sporulation activity. The role of the Rap Spo-remains the main unsolved question. Given the large amount of plasmid Rap-Phr and of the role of these systems in plasmid conjugation in B. subtilis (Singh et al., 2013), it will be interesting to study this phenotype.

Cell-cell communication systems enable a fine regulation of important processes in bacteria. Indeed, some Rap-Phr systems regulate sporulation that allows bacteria to adapt, survive and disseminate. This work highlights the importance of Rap-Phr systems linked to genetic mobile elements in the B. cereus group, especially in B. thuringiensis. This location on mobile elements could increase the spreading of these genes in bacteria of the B. cereus group.

Tables

Rap6HD1-F CGCGGATCCAATTGGTATTGAGGGGGTAGATTATG BamHI Rap6HD1-R CCCAAGCTTCTATTTTTTTCATTTTAAAGCTCCTTCC HindIII Rap8HD1-F CCCAAGCTTGCCTTTTTCATTTTAATGCCTCC HindIII Rap8HD1-R CGCGGATCCGGATGGGGGAATATATAATGATGG BamHI Rap10HD1-F CCCAAGCTTGATTTTTTTCATTATTTTAACGCTCC HindIII Rap10HD1-R CGCGGATCCGGAAAAGGGGAAGATTCTATGAATG BamHI Rap6-Bt407-F CGCGGATCCGGGAAGAGGGGATTTTATGAACAC BamHI Rap6-Bt407-R AACTGCAGAATTTTTTCATCACTGTAACGCTCC PstI Rap7-Bt407-F AACTGCAGTTTTCTTCATTTTAATGCCTCCTTATC PstI Rap7-Bt407-R CGCGGATCCAGATGAGGAGGGATTTTATGTATACAGC BamHI Rap8-Bt407-F AACTGCAGGATTGTTTTTTTCATTTTAAAGCCC PstI Rap8-Bt407-R CGCGGATCCTCGTAAAAAAGGGGGCTATTTTG BamHI Rap5-BtHD73-F AACTGCAGCTTTAACTTTTTCATCACACTAACGCC PstI Rap5-Bt HD73-R CGCGGATCCGAAAGAAGGAAGTGATTTTATGAATGC BamHI Rap7061-For CGCGGATCCGAAAGAAGGGGGATATTATGAGC BamHI Rap7061-Rev CCCAAGCTTTCATTATTTCAACGCCCCTTTTC HindIII Rap1-Bc14579-F CGCGGATCCGAGGAGGACGTAGTGATATGAGCAC BamHI Rap1-Bc14579-R AACTGCAGCAAGCTTCTTCATTATTTTAACGCC PstI Rap2-Bc14579-F CGCGGATCCGGAGGGGTTTCATGATTACATCTAC BamHI
Rap2-Bc14579-F AACTGCAGCCACTTTTTTCATTTTAAAGCCC PstI Rap7061 corresponds to Rap7 -Bt HD73. Nucleotides in bold represent the restriction enzyme sites. Table 5 Consensus of the key residues involved in the sporulation activity of the Rap proteins already described to act as a phosphatase over Spo0F. Rap proteins used to construct the consensus: RapA, RapB, RapE, RapH, RapJ, and Rap60 from B. subtilis (Koetje et al. 2003;Parashar et al. 2011); RapQ from B. amyloliquefaciens (Yang et al. 2015); RapBXA0205 and RapBA3790 from B. anthracis (Bongiorni et al. 2006); and Rap8 from B. thuringiensis (Fazion et al. 2018). Profile of chromosomal and plasmid Rap proteins of the B. cereus group. Orange patterns are predicted as Spo+ and green patterns as Spo-; residues divergent from the consensus are typed in white. Residues typed in green do not differ from the consensus but are not found in this exact order in Rap proteins validated as Spo+, as consequence, Rap proteins with these residues were predicted as Spo-. The number of proteins is the sum of Rap corresponding to each profile in each group.

E D Q L F L D E Y RapA/RapQ E D Q L F L D E Y RapE E D Q L F M D E Y RapBXA0205 E D Q L F F D E Y Rap8 E D Q L L F D E Y RapB E D Q L Y L D E Y Rap60 E N Q L F M D E Y RapJ E N Q L F L D E Y RapBA3790 Q D Q L F F D E Y Consensus Spo+ E, Q D, N Q L F, L, Y L, F, M D E Y Bc group -Chromosome Rap Group 1 V D Q L F V D E Y 10 100% V D Q L F F D E Y V D Q L F L N E Y V D Q S F F H E C L D E L F Y N E C E D Q A F L S E Y E E K S F V N E Y Group 2 T D Q S F F D E Y 13 100% T D Q L F F D E Y A D H S F F D E Y T N Q S F F D E Y Group 3 Q D Q L F Y D E Y 16% 5-BtHD73 E D K L F F N E Y 84% E D K L F F A E Y E D T Q F L D E C E D E L F F T E Y E D E S F F N E Y K D T L F C E E Y Group 4 S D V S F S D E Y 100% S D I S F S D E Y G D I L F L D E H Group 5 E D E A F P D E Y 100% E D E A F P D E F

Rap proteins Amino acid residues Nb of proteins % in the group

Tested Rap

E D E A F Q D E Y E D G W F R D E Q E D G W F R E E Q K D Q L F T D E H K D R L F T D E H E D P L F S D E Y L N S H F Q S E C T I P Q F H S E C T F P Y F H S E C Group 6 Q D Q L F F D E Y 25 68% E N Q L L L D E Y 1-Bc14579 E D Q L F L N E Y 32% A D Q S F D G E Y E N Q L F F N E C Q N Q L F L D E Y Group 7 E D Q L F L D E Y 32% E D Q L L L D E Y E N Q L F L D E Y Q D Q L F L D E Y 2-Bc14579 E D Q L F L D E C 68% E D Q F F L D E Y D N S L F L D E N A D S L F L D E Y E D K L F L D E Y D D K L F L D E Y N N P Q F L D E F Q K Q L F L D E Y P D Q L F L D E Y Q N Q L F L D E Y Group 8 Q N Q L Y Y N E Y 100% Q E Q L F F E E Y Q D Q L Y Y N E Y Group 9 E D S S F F D E Y 100% E D Q L A F D E Y 10 Group 10 E C D L W R D E F 100% Group 11 E N T F F L D E Y 100% K H T F F F N E Y Group 12 E N Q T F S D E Y 100% E D R T F S D E Y

Bc group -Plasmid Rap

Group 1

E D Q L F F D E Y 11 95% E D Q L F L D E Y 8-Bt407 E N Q L F L D E Y N D Q L F M D E Y 5% Group 2 E D Q L F F D E Y 65% 8-BtHD1 Q D Q L F F D E Y 7-Bt407 E D Q L F T D E Y 35% K D Q L F T D E Y 6-BtHD1 S D Q H F I D E H Group 3 Q D Q L F F D E Y 40% Q N Q L F L D E Y 60% Q H Q L F L D E Y D D K L F L D E C

Rap proteins Amino acid residues Nb of proteins % in the group

Tested Rap

Group 4

L S E Q F V D E N 100% 6-Bt407 Group 5 K D S L F T D E Y 100% Group 6 Q D Q L F M D E Y 100% Group 7 E N Q L F F D E Y 18% 10-BtHD1 Q D Q L F Y D E Y 82% K D E S F F N E Y T D P S F F D E Y T D P S F F N E Y T D Q S F F D E Y 7-BtHD3 Group 8 Q D Q L Y Y N E Y 100% Group 9 E D Q L F F D E Y 93% E D Q L L F D E Y E N Q L F L D E Y E D Q L A F D E Y 7%
Rap proteins used to construct the consensus: RapA, RapB, RapE, RapH, RapJ, and Rap60 from B. subtilis (Koetje et al. 2003;Parashar et al. 2011); RapQ from B. amyloliquefaciens (Yang et al. 2015); RapBXA0205 and RapBA3790 from B. anthracis (Bongiorni et al. 2006); and Rap8 from B. thuringiensis (Fazion et al. 2018). The viable cells and heat-resistant spores were counted after 48 h in HCT medium at 30°C. Results are given as mean ± standard error of the mean (SEM). The percentages of spores were calculated as 100 × the ratio between heat-resistant spores per milliliter and total viable cells per milliliter. Email: didier.lereclus@inra.fr

Introduction

Bacillus thuringiensis belongs to the Bacillus cereus group of Gram positive rodshaped spore-forming bacteria and distinguishes from the other seven closely related species of this group by the production of a crystal inclusion that is toxic to larvae of various insects (Vilas-Bôas et al., 2007). Due to its entomopathogenic properties, B.

thuringiensis is widely used as biopesticide to control agricultural pests or human disease vectors (Schnepf et al., 1998). Crystal inclusions are encoded by cry (and cyt) genes mainly harbored on plasmids and the production of Cry toxins are generally regulated by sporulation sigma factors (Deng et al., 2014). B. thuringiensis strains were shown to carry a complex pattern of plasmids, up to 17 different extrachromosomal elements with different size, from a cryptic plasmid of 2Kb to megaplasmids of 600Kb (Cardoso et al., in press;Lereclus et al., 1982;[START_REF] Reyes-Ramirez | Plasmid Patterns of Bacillus thuringiensis Type Strains[END_REF]Zhong et al., 2011). As result of its biotechnological relevance, plasmids encoding cry genes are the most studied. Some of the other plasmids have also been investigated because of their conjugation properties, such as pAW63 [START_REF] Van Der Auwera | Transcriptional analysis of the conjugative plasmid pAW63 from Bacillus thuringiensis[END_REF]Wilcks et al., 1998) and pXO16 [START_REF] Jensen | The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16[END_REF][START_REF] Makart | pXO16 from Bacillus thuringiensis serovar israelensis: Almost 350 kb of terra incognita[END_REF] or for the role of plasmid-borne Rap-Phr system, as pHT8_1 (Fazion et al., 2018).

B. thuringiensis plasmids carry a great amount of Rap-Phr systems with 38% of its plasmids harboring at least one Rap-Phr system (Cardoso et al., in press). Rap-Phr quorum sensing systems were first described and extensively studied in B.

subtilis (Perego, 2013;Perego et al., 1994). In this species, they regulate various processes such as sporulation, competence, transfer of mobile genetic elements, production of proteases and biofilm formation (Perego, 2013). Firstly identified on the chromosome, rap-phr genes were also described to be carried on plasmids of Bacillus species (Bongiorni et al., 2006;Fazion et al., 2018;Koetje et al., 2003;Parashar et al., 2013;Qiao et al., 2011;Singh et al., 2013).

Rap-Phr systems belong to the RNPP family of quorum sensing systems from Gram positive bacteria, which consist in a response regulator with tetratricopeptide repeat (TPR) domains and a signaling oligopeptide that modulates the regulator activities (Declerck et al., 2007). The infectious cycle of B. thuringiensis in the insect is regulated by the sequential activation of these RNPP systems (Slamti et al., 2014).

Firstly, PlcR-PapR regulates the virulence stage [START_REF] Agaisse | PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis[END_REF]; next, NprR-NprX controls the necrotrophic stage and the transition to sporulation (Dubois et al., 2012;Perchat et al., 2016); finally, Rap-Phr also controls the initiation of the sporulation process (Fazion et al., 2018).

Commitment to sporulation is regulated by the phosphorylation state of the major response regulator Spo0A (Molle et al., 2003;Sonenshein, 2000). Different signals, such as nutritional deprivation, are recognized by sporulation kinases which are able to autophosphorylate (Jiang et al., 2000b). Next, kinases phosphorylate Spo0F, which is used as substrate by the phosphotransferase Spo0B to phosphorylate Spo0A (Burbulys et al., 1991). Response regulator aspartate phosphatases (Rap) are able to hamper this signal transduction pathway by dephosphorylating the Spo0F response regulator (Perego et al., 1994). Rap protein activity is counteract by its cognate Phr peptide, which is translated in a premature form that needs to be secreted, processed and reimported by oligopeptide permeases to be active (Perego & Hoch, 1996;Pottathil & Lazazzera, 2003).

Rap-Phr systems are largely found in bacteria of the B. cereus group, with a relevant amount in B. thuringiensis plasmids, whose systems were rather predicted to have an effect on sporulation (Cardoso et al., in press;Even-Tov et al., 2016). The pXO1 pathogenicity-plasmid from B. anthracis (Bongiorni et al., 2006) and the pHT8_1 cryptic plasmid of B. thuringiensis HD73 (Fazion et al., 2018) carry a Rap-Phr system that regulate the sporulation process. Moreover, Fazion et al. (2018) demonstrated that the Rap8-Phr8 system control the sporulation of B. thuringiensis in its ecological niche, the insect larvae. Sequencing of the plasmid pAW63 (Van der Auwera et al., 2005), also revealed the presence of rap-phr alleles. pAW63 and pHT8_1 are found in the B. thuringiensis serovar kurstaki HD73 strain with another five plasmids [START_REF] Liu | Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73[END_REF], among them the pHT77 also harbors a Rap-Phr system which does not have a role in sporulation (Cardoso et al., in press).

pAW63 is a theta-replicating conjugative plasmid of 72Kb (Wilcks et al., 1998[START_REF] Wilcks | Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis[END_REF] that efficiently conjugates and mobilizes non-conjugative plasmids even in food matrices [START_REF] Modrie | Differential transfer dynamics of pAW63 plasmid among members of the Bacillus cereus group in food microcosms[END_REF][START_REF] Van Der Auwera | Plasmid exchanges among members of the Bacillus cereus group in foodstuffs[END_REF] and in adverse conditions [START_REF] Beuls | Bacillus thuringiensis Conjugation in Simulated Microgravity[END_REF][START_REF] Beuls | High-salt stress conditions increase the pAW63 transfer frequency in Bacillus thuringiensis[END_REF]. pAW63 also harbors a toxin-antitoxin system that promotes plasmid stabilization during vegetative growth or sporulation [START_REF] Short | A type III protein-RNA toxinantitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development[END_REF], and two Group II introns: B.th. I1 and B.th.I2 (Van der Auwera et al., 2005).

These RNA based mobile elements were found to interrupt genes of the transfer module. Furthermore, pAW63 shares a common backbone with the second pathogenic plasmid of B. anthracis pXO2 and with pBT9727 from the pathogenic B.

thuringiensis serovar konkukian strain 97-27, including replication and transfer modules (Van der Auwera et al., 2005).

In this study, we characterized the Rap63-Phr63 quorum sensing system from the pAW63 plasmid. Although this system presents a moderate effect on sporulation, we show a synergistic sporulation effect of this system together with the Rap8-Phr8 from the pHT8_1 in vitro and in vivo. However, no cross-talk between these close related Rap-Phr systems was detected in this study, revealing a high specificity of the Phr peptides for their cognate Rap proteins. These results reinforce the relevance of plasmid-borne Rap-Phr systems to bacteria development in its naturalistic conditions.

Materials and Methods

Bacterial strains and growth conditions

All strains used in this study derived from the B. thuringiensis var. kurstaki HD73 acrystalliferous (Cry-, cured of the pHT73 plasmid) strain (Wilcks et al., 1998).

Escherichia coli K-12 strains TG1 was used as host strain for plasmid construction.

E. coli strain ET12567 (Dam-Dcm-) was used to prepare plasmids to transform B.

thuringiensis strains by electroporation (Lereclus et al., 1989). E. coli strains were transformed by thermal shock and were cultivated in Luria Bertani (LB) medium at 37°C. B. thuringiensis strains were grown in LB or in the sporulation-specific medium HCT [START_REF] Lecadet | Generalized transduction in Bacillus thuringiensis var. berliner 1715 using bacteriophage CP-54Ber[END_REF]Lereclus et al., 1982) at 30°C or 37°C. Liquid cultures were maintained with shaking at 175 r.p.m. For bacterial selection, the antibiotics were used at the following concentrations: ampicillin 100 µg/ml and spectinomycin 50 µg/ml for E. coli and erythromycin 10 µg/ml, spectinomycin 200 µg/ml and kanamycin 200 µg/ml for B. thuringiensis. LB plates with 100 µg/ml of X-gal were used for colorimetric screening during pMAD mutagenesis. When required, xylose was used at a concentration of 20 mM.

DNA manipulations

Genomic DNA from B. thuringiensis strains was extracted using the Puregene Yeast/Bact. Kit B (Qiagen, France) and plasmid DNA from E. coli was extracted with the QIAprep Spin Miniprep Kit (Qiagen, France). Phusion High-Fidelity DNA polymerase, Standard Taq DNA Polymerase, restriction enzymes and T4 DNA ligase were used following the manufacturer's recommendations (New England Biolabs, USA). PCRs were performed in an Applied Biosystems 2720 Thermal cycler using oligonucleotides listed in Table S1, synthesized by Eurofins Genomics (Germany).

The amplified DNA fragments were purified using QIAquick PCR Purification Kit (Qiagen, France) and the QIAquick Gel Extraction Kit (Qiagen, France) was used to purify digested DNA fragments separated on 1% agarose gels. All constructs were verified by DNA sequencing (GATC Biotech, Germany).

Plasmid and strains constructions

The plasmid pHT315-PxylA [START_REF] Grandvalet | Identification of genes involved in the activation of the Bacillus thuringiensis inhA metalloprotease gene at the onset of sporulation[END_REF], a multi-copy vector with xyloseinducible promoter, was used to express rap63 and/or phr63. Promoter regions of rap63 or phr63 genes were inserted in the plasmid pHT304.18'yfp [START_REF] Fagerlund | SinR controls enterotoxin expression in Bacillus thuringiensis biofilms[END_REF] for evaluation of their expression. The plasmid pMAD [START_REF] Arnaud | New Vector for Efficient Allelic Replacement in Naturally Gram-Positive Bacteria[END_REF] was used for homologous recombination.

The rap63-phr63 or phr63 genes were disrupted in the wild-type HD73-strain and on its derivative HD73 Δrap8-phr8 or Δphr8 (Fazion et al., 2018) by using pMADΩrap63-phr63::specR and pMADΩphr63::specR. These genes were replaced by the spectinomycin resistance gene with its own promoter by homologous recombination [START_REF] Lereclus | spbA locus ensures the segregational stability of pTH1030, a novel type of Gram-positive replicon[END_REF]. The recombinant strains were Lacˉ, erythromycin sensible and spectinomycin resistant. All the constructed plasmids used in this study are described in Table S2.

RT-PCR experiment

This experiment was performed as described by Fazion et al. (2018), concerning growth conditions, RNA extraction and reverse transcriptase reaction. Three different fragments were amplified by PCR reactions with oligonucleotides PromRapF7557 and RT7557-2 for the rap63 gene and its upstream region, RT7557-3 and RT7557-4 for the rap63 and phr63 genes and RT7557-5 and RT7557-7 for the phr63 gene and its downstream region. Sequences of the oligonucleotides are given in Table S1.

Synthetic oligopeptides

Several Phr peptides, corresponding to the C-terminal end of the phr63 gene product, were synthesized, purified and identified by mass spectrophotometry by GenScript (USA). To determine the active oligopeptide, the synthetic peptides were tested in sporulation assays (see below) at final concentrations of 50 µM.

In vitro sporulation assays

In vitro sporulation efficiency tests were carried in the sporulation-specific medium HCT. B. thuringiensis strains were grown at 30°C for 48h and serial dilutions were plated before and after heat treatment (12 min at 80°C). When required, xylose and synthetic peptides were added to the culture at the beginning of stationary growth phase (t0). The sporulation percentage was calculated as 100 × the ratio between heat-resistant spores per milliliter and total viable cells per milliliter. Experiments and plating were done at least in triplicate and mean values were calculated. Results

were analyzed by statistical test ANOVA followed by Tukey (p<0.05).

In vivo sporulation assays

Experiments with insect larvae were performed as described in Fazion et al. (2018).

Essentially, larvae of Galleria mellonella were infected with intrahemocoelic injection of vegetative bacteria and were maintained at 30°C. Dead larvae were crushed after 96h of treatment and diluted in 0.9% NaCl solution. Sporulation efficiencies of the harvested B. thuringiensis cells were calculated as described for the in vitro assays.

Results were statistically analyzed by unpaired t test with Welch correction (p<0.05).

Fluorescence analysis

Yfp fluorescence produced from different promoter regions was measured from B.

thuringiensis strains cultures grown in HCT medium at 37°C. Cells were harvest at determined time points and were fixed as described by [START_REF] Fagerlund | SinR controls enterotoxin expression in Bacillus thuringiensis biofilms[END_REF]

Aggregation kinetics

The aggregation curve was determined in HCT medium with xylose at 30°C. Cultures were sampled each hour from the onset of stationary phase. The optical densities at 600nm (OD 600 ) of each sample were measured once they were collected and after 15 minutes. The percentage of aggregation was calculated as the formula: % aggregation = [1-(DO 600 t15min / DO 600 t0)]*100.

Microscopy

The cultures used for analysis of aggregation kinetics were also sampled for microscopy at t4, t8, t14 and t32h after the onset of the stationary phase. The cells were observed with a Zeiss AxioObserver.Z1 inverted microscope in phase contrast.

Pictures were taken with a Zeiss AxioCam MRm CCD camera connected to the microscope.

Results

Transcriptional analysis of the rap and phr genes rap and phr genes were shown to form transcriptional units with a promoter located upstream the rap gene (Perego & Hoch, 1996). As generally described for these genes, phr63 gene slightly overlaps the rap63 gene (by just one bp). To verify if rap63 and phr63 genes are co-transcribed, a reverse transcription PCR reaction (RT-PCR) was performed from RNA extracted 3 hours after the onset of the stationary phase. The result demonstrates that rap63 and phr63 genes are co-transcribed (Figure1A and 1B) and that the transcription unit is only formed by these two genes.

Transcription of most phr genes are also regulated by an additional specific promoter situated upstream the phr gene and inside the rap gene, generally controlled by the alternate sigma factor sigmaH (σ H or sigH) (McQuade et al., 2001).

To measure the expression of the rap63-phr63 signaling cassette, DNA regions upstream from the rap63 and the phr63 genes were coupled to the yfp fluorescent reporter gene on the plasmid pHT304.18'yfp. Transcription of Prap63 starts one hour after the entry on stationary phase (t1) and extended at least until t5 (Figure 1C). The expression of Pphr63 begins at t2 and lasts until t5 (Figure 1D) demonstrating that the phr63 transcription is also controlled by its own promoter. Interestingly, the expression from Pphr63 is one hour delayed in a sigH-deficient strain (ΔsigH) suggesting a role of SigH in phr expression activation (Figure 1D). However, as the Pphr63 transcription is not abolished in the ΔsigH mutant strain, this result shows that another transcription factor is involved on the regulation of this promoter.

Promoter prediction using different tools were not capable to figure out a concise result about the prediction of transcription factors binding sites. Considering the timing of the Pphr63 expression in the sigH-deficient strain which starts at t3 and the role of SigE in the expression of the Pphr8 in the same strain (Fazion et al., 2018), transcription from Pphr63 was assessed in the sigE-deficient strain (ΔsigE). As shown in Figure 1D, the expression of Pphr63 on the ΔsigE strain is similar to the wild-type strain suggesting that SigE is not required for Pphr63 transcription.

Rap63 inhibits sporulation

Our previous Rap-Phr systems analyses in the B. cereus group predicted that the Rap63 could affect the sporulation (Cardoso et al., in press). To verify this activity, rap63, rap63-phr63 and phr63 genes were introduced into the pHT315-PxylA, a multicopy plasmid in which the expression of these genes is under regulation of a xylose inducible promoter. These constructions were transformed into the B.

thuringiensis HD73-wild-type strain. The control strain, bearing the empty pHT315-PxylA plasmid, sporulates efficiently (82% of spores) upon 48h culture at 30°C in a HCT medium supplemented with xylose (Figure 2; Table S3). In sharp contrast, the strain expressing rap63 gene presented less sporulation efficiency (38% of spores) with a 5-fold reduction of heat-resistant spore production in the same conditions

(5.80E+07 versus 3.25E+08 in the control strain). The sporulation efficiencies of strains expressing rap63-phr63 or phr63 genes present a similar sporulation profile that the control strain (84% and 85%, respectively). Therefore, these results confirm that Rap63 inhibits sporulation, in a moderate manner, and that the Rap63 activity is counteracted by its cognate Phr63.

Rap63 delays expression of Spo0A-regulated genes Rap proteins affect sporulation by acting as phosphates on Spo0F~P resulting in phosphorylation inhibition of the major response regulator Spo0A (Perego et al., 1994). The phosphorylated form of Spo0A regulates the expression of several genes related to sporulation. The spoIIE gene is regulated by Spo0A-P in B. subtilis (Molle et al., 2003) and in B. thuringiensis (Perchat et al., 2016). The promoter region of spoIIE gene (PspoIIE) was coupled to the yfp fluorescent reporter gene and inserted in the pHT315-PxylA-rap63 or rap63-phr63 plasmids (xylΩrap63-PspoIIE'yfp and xylΩrap63-phr63-PspoIIE'yfp, respectively). In the control strain (harboring plasmid xyl-PspoIIE'yfp) the transcription of spoIIE starts two hours after the onset of the stationary phase (Figure 3). When the transcriptional fusion is carried together with the rap63 gene, the expression from PspoIIE is delayed, beginning around T5. When both Rap63 and Phr63 are produced, expression from the PspoIIE is restored to the same kinetics and level found with the control strain. Therefore, these results demonstrate that the Rap63 delays the expression of Spo0A-regulated genes and that Phr63 inhibits Rap63 activity. This is in accordance with the sporulation test, confirming the moderate role of the Rap63 on the control of the sporulation process.

Auto-aggregation phenotype linked to sporulation At the end of the sporulation assays, after 48h of culture, the HD73 strain expressing Rap63 displayed a remarkable aggregative phenotype. To a better understanding of this phenomenon, we realized an aggregation kinetic in HCT medium with shaking at 30°C with the three HD73-derivated strains tested (315xyl, xylΩrap63 and xylΩrap-phr63). The results showed that all the strains presented a transitory aggregation phenotype which starts at t4 with a maximum at t8 and the disaggregation beginning between t10 and t12 (Figure 4A). Interestingly, the decrease in the aggregation is coincident to spore liberation, as revealed by the microscopic observations (Figure 4B). In the strain expressing rap63, the disaggregation is postponed and does not reach a null aggregation as observed with the control strain and with the strain expressing rap63-phr63. This result shows that this aggregation phenotype is not specifically dependent on Rap63. Moreover, the defect in the disaggregation phenotype could be correlated to the regulation profile of Rap63 delaying the Spo0Aregulated genes expression and thus the sporulation process.

The ΔPhr8ΔPhr63 mutant strain negatively affects the commitment to sporulation

The complete infectious cyclepathogenesis, necrotrophism and sporulationof B.

thuringiensis in insect larvae was shown to be controlled by quorum sensing systems of the RNPP family (Slamti et al., 2014). Hence, we analyzed the role of Rap63-Phr63 in a naturalistic condition, on death larvae of G. mellonella. For this purpose, HD73-mutant strains with rap63-phr63 or phr63 deletions were constructed. Since the ΔPhr8 strain was already shown to present a reduction on sporulation efficiency in this environment (Fazion et al., 2018), a double Phr mutant (ΔPhr8ΔPhr63) was also built and the mutant strains with rap8-phr8 or phr8 deletions from Fazion et al.

(2018) study were added to allow comparisons among the two systems. These strains were evaluated for their sporulation efficiencies in insect 96h after intra hemocelic injection (Figure 5A, Table S3). The wild type strain (HD73) and its derivatives mutant strains ΔRap63-Phr63 and ΔPhr63 presented a similar sporulation efficiency (22%, 24% and 16% of spores, respectively). However, sporulation efficiency was strongly reduced in the ΔPhr8ΔPhr63 mutant strain (0.23%), in a greater extent than the ΔPhr8 strain (3,1%). The results obtained in vitro, in HCT medium, showed that the ΔRap-Phr63 and ΔPhr63 strains sporulate as the wild type strain whereas the sporulation was slightly but significantly affected in the ΔPhr8ΔPhr63 strain. These results suggest a synergic effect of the Rap8-Phr8 and Rap63-Phr63 systems to regulate the sporulation process.

Determination of the Phr63 active form

Rap proteins are inhibited by Phr oligopeptides, which active form commonly correspond to the C-terminal end of the phr gene product and were already described to contain five, six or seven amino acids (Fazion et al., 2018;Mirouze et al., 2011;Perego, 1997;Pottathil & Lazazzera, 2003). To determine whether the mature form of Phr63 correspond to the C-terminal end of the pro-peptide, we constructed a plasmid expressing the rap63 gene with a truncated phr gene at the 3' end (xyl_rap63-phr63_R3'), missing codons related to the six C-terminal amino-acids of the phr gene product (Figure 6A). The sporulation efficiency of the strain harboring this plasmid (32%) is similar to the strain expressing the Rap63 (Figure 6B, Table S3). This result suggests that the active form of the Phr63 is included in the Cterminal end of the premature Phr.

To define the Phr63 active form, various Phr63 peptides corresponding to the C-terminal end were synthetized: GETI (Phr63-4); HGETI (Phr63-5); AHGETI (Phr63-6); YAHGETI (Phr63-7) and QYAHGETI (Phr63-8) (Figure 6A). To evaluate the capacity of the synthetic peptides to inhibit Rap63 activity, they were separately added in the culture medium of the strain expressing rap63 (xyl_rap63). Phr63-4 was not able to inhibit Rap63 activity (30% of spores), while Phr63-5 (77%), Phr63-6 (84%), Phr63-7 (87%), and Phr63-8 (87%) efficiently counteracted Rap63 effect on sporulation (Figure 6B, Table S3). These results demonstrated that the active form is well in the C-terminal end of the Phr63 peptide but did not allow precise identification the mature sequence.

A crosstalk between Rap-Phr63 and Rap8-Phr8 systems?

B. thuringiensis HD73 strain possess eight Rap-Phr systems, five on the chromosome and three on plasmids (pHT8_1, pAW63, and pHT77). Two of them were more deeply studied: Rap8-Phr8 from pHT8_1 (Fazion et al., 2018) and Rap63-Phr63 from pAW63 (this study). The C-terminal region of both Phr peptides including the mature form shows a high similarity (Figure 7A), especially considering the eight last amino acids where just two residues are divergent. Due to these sequence similarities and the results of the ΔPhr8ΔPhr63 mutant strain, we investigated the possibility of cross talk between Rap8-Phr8 and Rap63-Phr63 systems. Hence, we constructed the ΔRap8-Phr8ΔRap63-Phr63 mutant strain to avoid the effect of intrinsic systems. This mutant strain was transformed with pHT315-PxylA, pHT315-PxylA-rap8 and pHT315-PxylA-rap63 plasmids and the sporulation of the resultant strains was assessed (Figure 7B, Table S3). The control strain (ΔΔ 315xyl)

sporulates efficiently (84% of spores). Likewise the sporulation test with the wild-type strain, the strain expressing rap63 presented reduced sporulation efficiency (32%). In addition, in the presence of the Phr63-7 peptides this strain showed sporulation efficiency similar to the control (82%). However, when the strain expressing rap63 was cultured in the presence of Phr8-6, Phr8-7 or Phr8-8 no effect on sporulation was recorded (31%, 35% and 29%, respectively). Then, none of the Phr8 peptides used was able to counteract the Rap63 activity. Furthermore, the ΔΔ 315xyl strain expressing rap8 is strongly affected in sporulation (1.3%) and the Phr8-7 peptides inhibited Rap8 activity (80%) as already described in the wild-type strain by Fazion et al. (2018) (Figure 7B, Table S3). Unfortunately, none of the Phr63 peptides tested was able to prevent the Rap8 sporulation effect (Figure 7B). Altogether, these results

showed that there is no crosstalk between Rap8-Phr8 and Rap63-Phr63 systems.

Discussion

In agreement with the rich plasmid profile of the B. thuringiensis species (Reyes-Ramirez & Ibarra, 2008), the complete genome sequence of the HD73 strain [START_REF] Liu | Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73[END_REF] includes seven plasmids (pHT7, pHT8_1, pHT8_2, pHT11, pAW63, pHT73, pHT77). Among them, three harbor a Rap-Phr system. The Rap8-Phr8 from the pHT8_1 is involved in biofilm formation and regulation of the sporulation (Fazion et al., 2018) and the Rap-Phr from the pHT77 did not have a sporulation activity (Cardoso et al., in press). In this study, we described the Rap63-Phr63 system harbored in the pAW63. Plasmid pAW63 is a broad range conjugative plasmid able to mobilize other plasmids (Wilcks et al., 1998). It shows high conjugation rates in various conditions such as in food matrices [START_REF] Modrie | Differential transfer dynamics of pAW63 plasmid among members of the Bacillus cereus group in food microcosms[END_REF][START_REF] Van Der Auwera | Plasmid exchanges among members of the Bacillus cereus group in foodstuffs[END_REF], simulated microgravity [START_REF] Beuls | Bacillus thuringiensis Conjugation in Simulated Microgravity[END_REF] and salt stress [START_REF] Beuls | High-salt stress conditions increase the pAW63 transfer frequency in Bacillus thuringiensis[END_REF]. This plasmid also carries a Type III toxin-antitoxin system, based on a RNA antitoxin and an endoribonuclease toxin, that were shown to play a role in plasmid maintenance in vegetative growth, sporulation and germination [START_REF] Short | A type III protein-RNA toxinantitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development[END_REF]. However, the Phr signal also presented a high level in the cured strain suggesting that the probe used to detect phr63 gene is not specific and reveals the presence of another phr gene in the B. thuringiensis HD73 genome. The transcription of rap63-phr63 genes happen concomitantly with this of rap8-phr8 genes from pHT8_1 plasmid (Fazion et al., 2018). Interestingly, both Rap proteins are able to inhibit the sporulation process but with a different extent. While Rap8 strongly inhibits sporulation by preventing expression of Spo0A-regulated genes (Fazion et al., 2018),

Rap63 has a moderate inhibition of sporulation and delays the expression of the spoIIE gene controlled by Spo0A. Moreover, the Rap63 presents the same profile concerning the key residues involved in sporulation activity than the Rap-BXA0205 from the pXO1 plasmid that strongly inhibits sporulation (Bongiorni et al., 2006;Cardoso et al., in press). This suggests that other residues play an important role in Rap-Spo0F interaction resulting in a modulation in sporulation inhibition.

Rap proteins are inhibited by their cognate Phr oligopeptides (Perego & Hoch, 1996). In accordance, we showed that the expression of the phr63 gene prevent

Rap63 activity on sporulation. The ΔPhr63 mutant strain did not show significant effect on sporulation but the deletion of both phr genes in the ΔPhr8ΔPhr63 mutant strain strongly prevents the commitment to sporulation in vivo. In insect larvae, the ecological niche of B. thuringiensis, sporulation of the ΔPhr8 strain showed a repression about 4-fold (Fazion et al., 2018) while in the double mutant strain the inhibition was of almost 100-fold, suggesting a synergistic activity of both quorum sensing systems. Generally, the mature peptide corresponds to the C-terminal end of the Phr peptide sequence and contains a positively charged residue (Pottathil & Lazazzera, 2003). We demonstrated that the active form of Phr63 is part of the Cterminal end. However, it was not possible to determine the exact size of the mature Phr63 by using synthetic peptides. Indeed, synthetic oligopeptides Phr63-5, Phr63-6, Phr63-7, and Phr63-8 effectively inhibit Rap63 activity on sporulation. This result suggests that the Phr-binding pocket in the Rap63 could be more versatile to Phr63 binding than Rap8 that was inhibited only by Phr8-7 (Fazion et al., 2018); or, yet, that the longer peptides might undergo further processing to achieve its active form. In B.

subtilis, the mature Phr are penta or hexapeptides (Mirouze et al., 2011;Pottathil & Lazazzera, 2003) while mature peptides of the RNPP regulators in B. cereus group bacteria are commonly heptapeptides, such as NprX (Perchat et al., 2011), PapR (Bouillaut et al., 2008) and Phr8 (Fazion et al., 2018). RapA-PhrA, RapC-PhrC and RapE-PhrE has been also demonstrated (Jiang et al., 2000a;Perego, 1997). Therefore, this absence of crosstalk demonstrates the high specificity of the Rap and Phr interactions supporting the hypothesis of the coevolution of Rap-Phr system components (Perego, 2013). In the other B.

thuringiensis RNPP systems, a cross-activation have been described among different pherotypes but not within one given strain, since PlcR-PapR and NprR-NprX are present only once in each strain (Perchat et al., 2011;Slamti & Lereclus, 2005).

In this study, we described an auto-aggregation phenotype that seems to be linked to sporulation. This aggregation phenotype is a transitory phenomenon extending from 4 to 14 hours after the onset of the stationary phase. During this time, bacteria develop their sporulation process and the disaggregation is concomitant with the spore release. In the strain expressing rap63, we observed a prolonged aggregation phenotype that could be due to the delay that the Rap63 causes on Spo0A-regulated genes. For example, the expression of CwlB and CwlC autolysins described to play a role in spore release are regulated by the late sporulation sigma factor σ K [START_REF] Chen | Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis[END_REF]Yang et al., 2013), thus, indirectly regulated by Spo0A.

The aggregation observed during the sporulation process differs from the known aggregation involved in conjugation induced by pXO16 plasmid in B. thuringiensis israelensis [START_REF] Andrup | Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation[END_REF][START_REF] Andrup | Mobilization of -Nonmobilizable‖ Plasmids by the Aggregation-Mediated Conjugation System of Bacillus thuringiensis[END_REF][START_REF] Jensen | The genetic basis of the aggregation system in Bacillus thuringiensis subsp. israelensis is located on the large conjugative plasmid pXO16[END_REF]. Indeed, these two phenomena occur at different growth phases. The aggregation system related to pXO16 conjugation arises during exponential growth, beginning about 15-20 minutes after the combination of the strains and disappearing at yet a low optic density (OD 600 > 1) [START_REF] Andrup | Mobilization of -Nonmobilizable‖ Plasmids by the Aggregation-Mediated Conjugation System of Bacillus thuringiensis[END_REF]. Moreover, while the conjugation aggregation is formed between donor and recipient strains in a mixed culture [START_REF] Andrup | Mobilization of small plasmids in Bacillus thuringiensis subsp. israelensis is accompanied by specific aggregation[END_REF], here we observe an auto-aggregation phenotype. Recently, a cell-cell aggregation related to the expression of c-di-GMP second messenger was described in the B. thuringiensis BMB171 strain [START_REF] Fu | c-di-GMP regulates various phenotypes and insecticidal activity of gram-positive Bacillus thuringiensis[END_REF][START_REF] Tang | Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitchregulated collagen adhesion protein[END_REF]. In this strain, a c-di-GMP-binding riboswitch controls the expression of a collagen binding protein that was shown to modulate different bacterial physiological process, such as motility, biofilm formation, aggregation and virulence [START_REF] Tang | Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitchregulated collagen adhesion protein[END_REF]. Moreover, an aggregation phenotype related to sporulation was described for the Gram negative bacteria Myxococcus xanthus. In this species, the aggregation is the first step for fruiting body formation, within which the bacteria sporulate [START_REF] Kruse | C-signal: A cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus[END_REF]. However, this process occurs in a solid surface while the aggregation observed on this work arises in liquid medium and with vigorous shaking. Several questions remain unsolved to better understand this phenotype. It would be interesting to characterize the occurrence of this phenotype within the B. cereus group and its importance in the developmental pathway of bacteria, particularly in the sporulation process. This auto-aggregation might play a key role in quorum sensing, for example by ensuring a high local concentration of Phr peptides within the aggregates in order to optimize the sporulation process. In Vibrio harveyi, cell aggregates were demonstrated to display stronger cell-cell communication than a dispersed population with the same number of cells [START_REF] Gao | A crucial role for spatial distribution in bacterial quorum sensing[END_REF].

Horizontal gene transfer can increase the variability of the genetic set of a bacterial species allowing the bacteria to better adapt to environmental changes. The B. thuringiensis plasmids had been widely studied, mainly due to the cry and cyt genes coding for the insecticidal toxin genes and preferably located on large conjugative plasmids. However, B. thuringiensis strains harbor a wide and diverse amount of plasmids, many of which contain Rap-Phr systems predicted to regulate sporulation. For the first time, the integrative activity of two plasmid-borne Rap-Phr systems in a given strain was tested. The Rap8-Phr8 and the Rap63-Phr63 systems from B. thuringiensis HD73 strain act synergistically to control the commitment to sporulation in insect larvae. Since B. thuringiensis is an invertebrate specialized pathogen, it is expected that this species presented a rich repertoire of mechanism that facilitate its development and survival in its environmental niche. Our results reinforce the relevance of plasmid Rap-Phr quorum sensing systems and highlight that plasmids other than the cry-harboring plasmids play an important role in the B.

thuringiensis infectious cycle regulation and bacterial survival. S3. Different letters correspond to significant differences in the main values (p<0.001). S3. Different letters correspond to significant differences in the main values (p<0.001). Amino acid sequences of Phr63 and Phr8 peptides. C-terminal ends similarities are highlighted in red and the two divergent residues are in black bold. Phr8-6, Phr8-7 and Phr8-8 correspond to the Phr8 synthetic peptides described by Fazion et al. (2018). B) Sporulation efficiency of the HD73 Δrap8-phr8Δrap63-phr63 control strain (ΔΔ 315xyl), and expressing rap63 (ΔΔ rap63) or rap8 (ΔΔ rap8) strains in HCT medium at 30°C in the presence of xylose 20 mM. The strain expressing rap63 was complemented with Phr8-6, Phr8-7 and Phr8-8 synthetic peptides and the strain expressing rap8 with Phr63-6, Phr63-7 and Phr63-8 synthetic peptides. Peptides were added independently 1h after the onset of the stationary phase at 50 mM final concentrations. The percentages of spores were calculated as 100 × the ratio between heat-resistant spores per ml and total viable cells per ml. Error bars represent the Standard Error of the Mean (SEM). Experimental values are detailed in Table S3. Different letters correspond to significant differences in the main values (p<0.001).

HD73 ΔPhr8

Insect larvae 1,57E+07 (±5,68E+06) 5,63E+05 (±2,91E+05) 3,10 (±1,71)

HD73 ΔPhr8 ΔPhr63

Insect larvae 1.12E+07 (±2.68E+06) 2.95E+04 (±1.90E+04) 0.23 (±0.11)

For the in vitro sporulation assays, the viable cells and heat-resistant spores were counted after 48 hours of growth in HCT medium at 30°C. For in vivo sporulation assays, the viable cells and heat-resistant spores were counted in dead larvae of Galleria mellonella 96 hours after injection at 30°C. Results are given as mean ± standard error of the mean (SEM). The percentages of spores were calculated as 100x the ratio between heatresistant spores per ml and total viable cells per ml. Phr63-4: GETI. Phr63-5: HGETI. Phr63-6: AHGETI. Phr63-7: YAHGETI. Phr63-8: QYAHGETI. Phr8-6: AHGKDI. Phr8-7: YAHGKDI. Phr8-8: QYAHGKDI.

DISCUSSION

Rap-Phr systems were shown to regulate relevant pathways on Bacillus species, such as sporulation, competence, genetic mobile element transfer and production of proteases. The residues involved in their phosphatase activity on Spo0F are generally conserved even in Rap proteins with another function suggesting that the ancestor Rap protein regulated the sporulation phosphorelay (Even-Tov et al., 2016). The phosphorelay is a cascade of phosphoryl group transfer that modulates the phosphorylate state of Spo0A, the master regulator of commitment to sporulation (Burbulys et al., 1991). The spore formation is an important developmental process of bacteria from the Firmicutes phylum, namely

Bacillus and Clostridium genera [START_REF] Fimlaid | Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes[END_REF]. Spore is a metabolic dormant cell-type that is highly resistant to adverse conditions, which enable survival and dispersion of the bacteria in unfavorable environments.

Rap-Phr systems belong to the RNPP family of quorum sensing systems from Gram positive bacteria. RNPP cell-cell signaling is characterized by a regulator containing TPR domain that is regulated by its signaling peptide intracellularly (Declerck et al., 2007). The infectious cycle of B. thuringiensis in insect larvae is regulated by the sequential activation of RNPP systems: PlcR-PapR controls the virulence stage, NprR-NprX regulates the saprophytic stage and sporulation, and Rap-Phr also plays a role in the initiation of sporulation (Slamti et al., 2014). Among these three systems, the Rap-Phr quorum sensing system was the less studied in the B. cereus group.

Overview of Rap-Phr systems in B. cereus group

In B. subtilis type strain 168, 11 chromosomal Rap-Phr systems have been described [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF], a number corresponding to the average of Rap-Phr systems by strain in this species (Even-Tov et al., 2016). Moreover, some plasmidborne rap-phr signaling cassettes were further studied in B. subtilis strains and related bacteria (Koetje et al., 2003;Parashar et al., 2013b;Qiao et al., 2011;Singh et al., 2013). Sequencing projects have revealed the presence of rap-phr alleles on chromosome and plasmids of B. cereus group genomes. The construction of a Rap-

Sporulation activity prediction

Rap proteins from each group were aligned with RapH of B. subtilis, in which the residues involved in the phosphatase function on Spo0F were described (Parashar et al., 2011). Only 32% of the Rap regulators from the B. cereus group were predicted to inhibit the phosphorelay (Spo+). The predicted sporulation function was not related to the groups established by phylogenetic analysis, as three from 12 chromosome groups and five from nine plasmid groups are composed by proteins with different predicted effect on sporulation. Moreover, the Spo+ Rap proteins are mainly located on plasmids and thus in B. thuringiensis strains. The higher amount of Rap-Phr systems with effect on sporulation enables B. thuringiensis to better regulate its development, preventing the commitment to sporulation when enough nutrients are available.

To validate the sporulation activity prediction, ten Rap proteins were tested in sporulation assays. All Rap predicted as Spo-did not affect sporulation efficiency whereas among the seven Rap proteins predicted as Spo+, three did not inhibit spore formation. This absence of the expected activity could be due to the conditions of culture used, since the HCT medium is extremely favorable to sporulation, hindering the effect of Rap proteins that may act in a slight extent. Or yet, variation in other residues than the depicted ones could lead to conformational changes that impair the interaction with Spo0F. Hence, the residues assigned as implicated in the phosphatase activity of Rap proteins seems to be required to the sporulation activity but not sufficient to an accurate prediction. Consequently, the estimation of Spo+ Rap in the B. cereus group must be overestimated.

Although 14% of the selected strains did not carry any Spo+ Rap, this absence may be supplanted by the Rap-like phosphatase activity of NprR, which was recently demonstrated to also regulate sporulation (Perchat et al., 2016a). The high ratio of Rap proteins predicted as Spo-raise the question of the role of these proteins. Unlike B. subtilis bacteria, in which the ComA competence response regulator is the second main target pathway of Rap proteins, B. cereus group strains do not bear ComA homologs (Even-Tov et al., 2016). Considering the great amount of plasmid-borne systems and the role of Rap LS20 -Phr LS20 in the conjugation of the pLS20 plasmid (Singh et al., 2013), the effect of these systems on their encoding plasmid transfer could be an attractive subject to future analyses.

Rap63-Phr63 regulates sporulation

Since sporulation is the major process known to be regulated by Rap-Phr systems in B. cereus group bacteria and considering the interesting amount of plasmid-borne systems, the Rap63-Phr63 system from the well-studied pAW63 plasmid was further assessed with regard to sporulation activity. rap63 gene is cotranscribed with the phr63 gene, as largely described for the rap-phr signaling cassette (Perego & Hoch, 1996;Pottathil & Lazazzera, 2003). The expression from the rap63 promoter begins one hour after the onset of the stationary phase.

Furthermore, the phr63 gene is transcribed by its own promoter, partially controlled by the sporulation specific sigma factor δ H , likewise phr genes from B. subtilis McQuade et al., 2001).

(
Contrasting to the strong effect on sporulation already described for the plasmid-borne Rap8 (Fazion et al., 2018) and Rap-BXA0205 (Bongiorni et al., 2006), the Rap63 moderately inhibits sporulation. Rap63 and Rap-BXA0205 (Rap5 -B.

anthracis Ames Ancestor) display the same profile of residues implicate in sporulation activity but they are positioned in opposite extremity groups of the plasmid Rap phylogenetic tree (Group 9 and Group 1, respectively). These data reinforce the idea that other residues could affect the interaction between Rap and Spo0F. Moreover, while the Rap8 completely inhibits the expression of Spo0Adependent genes (Fazion et al., 2018), Rap63 delays the expression of these genes.

In sharp contrast, no effect was observed in the Δphr63 mutant strain, as already reported for other Rap-Phr systems (Auchtung et al., 2006;Fazion et al., 2018;[START_REF] Bendori | The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP[END_REF].

The strain expressing Rap63 present an auto-aggregation phenotype at the end of sporulation assays. Aggregation kinetics and microscope observation revealed a transient phenomenon that initiates four hours after the entry into stationary growth phase and dissipates with spore release. The peptidoglycan hydrolases that allow spores to release are under indirect control of Spo0A transcription regulator, as the autolysins CwlB and CwlC which expression are regulated by the sporulation sigma factor σ K [START_REF] Chen | Novel Cell Wall Hydrolase CwlC from Bacillus thuringiensis Is Essential for Mother Cell Lysis[END_REF]Yang et al., 2013).

Since Spo0A-regulated genes expression is delayed by Rap63 protein, the autolysins are also impacted leading to prevention of the disaggregation. One hypothesis for the occurrence of the aggregation phenotype is that the bunch of cells favors cell-cell communication and then ensures an amount of Phr enough to achieve the sporulation process. Several questions remain unsolved about the aggregation phenotype, especially its regulation and the aggregation factor. Preliminary analysis has shown that most of strains of the B. cereus group present this phenotype, except for B. cereus ATCC 10987.

Synergistic activity of Rap63-Phr63 and Rap8-Phr8 on regulation of the commitment to sporulation

As already demonstrated for multiple sporulation [START_REF] Bischofs | Complexity in bacterial cell-cell communication: Quorum signal integration and subpopulation signaling in the Bacillus subtilis phosphorelay[END_REF] and

ComA-related Rap regulation (Auchtung et al., 2006;Bongiorni et al., 2005) on B.

subtilis, Rap proteins cohabitating in a given strain may act synergistically on the target process. The B. thuringiensis var. kurstaki HD73 strain contains eight rap-phr signaling cassettes, five on the chromosome and three on plasmids (pAW63; pHT77; pHT8_1) with half of these systems predicted to be Spo+, among them Rap8-Phr8 system was already described (Fazion et al., 2018).

Interestingly, the Δphr8Δphr63 mutant strain exhibits an inhibition of sporulation efficiency both in vitro and in vivo. Indeed, in death larvae of G. mellonella, the sporulation was strongly affected with reduction of almost 100-fold compared to the wild-type strain. This effect is also more important than in the Δphr8 mutant strain that reduced four-times the sporulation efficiency (Fazion et al., 2018).

These results suggest a synergistic action of Rap8-Phr8 and Rap63-Phr63 systems on the commitment to sporulation in the B. thuringiensis HD73 strain. Consequently, this regulation reinforces the relevance of the rich plasmid-pattern of B. thuringiensis strains for adaptation and survival in its ecological niche.

The Phr63 peptide and the specificity of Rap-Phr systems

The mature form of the Phr oligopeptides are generally found in the C-terminal end of the premature peptide (Pottathil & Lazazzera, 2003). The Phr63 active form is part of the C-terminal end of the pro-Phr63 peptide. However, the exact size of the mature peptide was not unveiled, since synthetic peptides corresponding to five, six, seven or eight last amino acids of the C-terminal end of Phr63 were able to inhibit the Rap63 activity. Similarly, the active form of Phr-BA3791 encoded on B. anthracis chromosome is included in the C-terminal region of the peptide sequence but the exact mature peptide was not known (Bongiorni et al., 2006).

The C-terminal part of the Phr63 sequence is highly similar to that of Phr8. In addition to the synergic effect of these two systems, this similarity suggests a crosstalk between Rap63-Phr63 and Rap8-Phr8. Nevertheless, the Phr from one system was not able to inhibit the Rap regulator of the other. Similarly, the absence of crossreactivity between RapA-PhrA, RapC-PhrC and RapE-PhrE was demonstrated in B.

subtilis (Jiang et al., 2000a;Perego, 1997). Thus, the absence of cross-talk between the Rap63-Phr63 and Rap8-Phr8 systems strengthen the hypothesis of the specificity of the Rap-Phr signaling pair.

CONCLUSION

Cell-cell communication is now regarded as a general trait of bacteria species.

Indeed, the quorum sensing enables bacteria to coordinate their metabolic processes Les systèmes Rap-Phr ont été décrits chez B. subtilis où ils régulent la sporulation, la conjugaison, la production de protéases et le transfert d'éléments génétiques mobiles. L'engagement dans la sporulation est contrôlé par un système de transfert de phosphate connu sous le nom de ‗phosphorelay' [START_REF] Trach | Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727[END_REF].

Certains signaux, comme le manque de nutriments, provoquent l'autophosphorylation des kinases de sporulation puis le phosphate est transféré jusqu'à Spo0A, le régulateur majeur de la sporulation, via les protéines de transfert Spo0F et Spo0B. Certaines protéines Rap sont capables de déphosphoryler Spo0F-P interrompant ainsi le transfert du phosphate ce qui va bloquer l'entrée en sporulation de la bactérie (Perego et al., 1994). L'activité phosphatase de la Rap est inhibée par la fixation de son peptide Phr, ce qui permet de restaurer le transfert du phosphate et de déclencher la sporulation. La souche type 168 de B. subtilis possède 11 gènes rap chromosomiques dont 8 sont suivis par un gène phr en aval, l'ensemble constituant une seule unité transcriptionnelle [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF].

Figure 1 .

 1 Figure 1. Bacteria from the Bacillus genus .............................................................................

Figure 2 .

 2 Figure 2. Ecology of B. cereus group bacteria. .......................................................................

Figure 3 .

 3 Figure 3. Chemical structure of the cereulide toxin ................................................................

Figure 4 .

 4 Figure 4. Mode of action of Cry proteins .................................................................................

Figure 5 .

 5 Figure 5. Phylogeny of B. cereus group bacteria ....................................................................

Figure 6 .

 6 Figure 6. Signaling circuit of LuxI-LuxR quorum sensing system in V. fisheri .......................

Figure 7 .

 7 Figure 7. Core molecule of acyl-homoserine lactone autoinducers .......................................

Figure 8 .

 8 Figure 8. Quorum sensing in Gram positive bacteria .............................................................

Figure 9 .

 9 Figure 9. The two-component signal transduction system .....................................................

Figure 10 .

 10 Figure 10. Competence quorum sensing system of B. subtilis...............................................

Figure 11 .

 11 Figure 11. Sporulation phosphorelay ......................................................................................

Figure 12 .

 12 Figure 12. Structure of RNPP protein .....................................................................................

Figure 13 .

 13 Figure 13. Evolution of RNPP regulators ................................................................................

Figure 14 .

 14 Figure 14. Bifunctional activity of NprR. ..................................................................................

Figure 15 .

 15 Figure 15. The PlcR-PapR quorum sensing system ...............................................................

Figure 16 .

 16 Figure 16. PrgX is regulated by two types of signaling peptides ............................................

Figure 17 .

 17 Figure 17. The infectious cycle of B. thuringiensis in the insect is regulated by quorum sensing systems .......................................................................................................................

Figure 18 .

 18 Figure 18. Sequential and alternative activation of sporulation-specific sigma factors control the spore formation process .....................................................................................................

Figure 19 .

 19 Figure 19. Differences between B. cereus and B. subtilis groups concerning number and sequence of rap genes .............................................................................................................

Figure 20 .

 20 Figure 20. Activity of chromosomal Rap-Phr systems from B. subtilis ...................................

Figure 21 .

 21 Figure 21. Structure of Rap proteins .......................................................................................

Figure 1 .

 1 Figure 1. Bacteria from the Bacillus genus. A) Colonies of B. anthracis on blood agar (Logan & De Vos, 2009); B) Vegetative cells of B. subtilis; C) Sporulating cells of B. cereus; D) Sporulating cell of B. thuringiensis containing the spore and the parasporal crystal(Deng et al., 2014).

Figure 3 .

 3 Figure 3. Chemical structure of the cereulide toxin (Ehling-Schulz et al., 2006).

Figure 4 .

 4 Figure 4. Mode of action of Cry proteins. Initially, the crystal is solubilized and activated in the insect gut. Following steps could arise in two different mechanism: the pore formation model (top) or the signal transduction model (bottom)(Bravo &

Figure 5 .

 5 Figure 5. Phylogeny of B. cereus group bacteria. Different analyses lead to two interpretations of genetic structure classification, clustering strains in three Clades or seven Groups(Bazinet, 2017).

Clade 1 (

 1 Figure 5, red) was mainly composed by B. cereus strains (as emetic cluster), all B. anthracis strains and some B. thuringiensis strains; Clade 2 (Figure 5, blue) consisted predominantly by B. thuringiensis strains (kurstaki, sotto, thuringiensis, tolworthi subspecies) with some B. cereus isolates; Clade 3 (Figure 5, green) gathered together all other species and B. cereus and B. thuringiensis isolates, forming a more heterogeneous clade. Using seven housekeeping alleles, this MLST scheme unveiled that the B. cereus group was largely clonal, with

Figure 6 .

 6 Figure 6. Signaling circuit of LuxI-LuxR quorum sensing system in V. fisheri. Red triangles: autoinducer (produced by LuxI); IM: inner membrane; OM: outer membrane (Waters & Bassler, 2005).

Figure 8 .

 8 Figure 8. Quorum sensing in Gram positive bacteria. The signaling molecules of both types of systems are autoinducer peptides (AIP). AIPs could be detected by membrane receptors (middle paneltwo components system) or imported to act intracellularly (left panel -RNPP family). Dark grey panel: common steps for both mechanisms. RR: response regulator (Slamti et al., 2014).

Figure 10 .

 10 Figure 10. Competence quorum sensing system of B. subtilis. Detailed description is found in the text[START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF].

  transduction pathway: the phosphorelay, also activated by the transfer of phosphoryl groups. The phosphorelay is a cascade of phosphate transfer, with additional components (domains or proteins) that allow a precise modulation with several points for regulation. The most known bacterial representative is the sporulation phosphorelay (Figure11), well studied in B. subtilis.

Figure 11 .

 11 Figure 11. Sporulation phosphorelay. Transfer of phosphoryl groups are indicated by arrows.Signal are detected by kinases that autophosphorylate and transfer the phosphoryl groups to the response regulator Spo0A through two phosphotransfer proteins, Spo0F and Spo0B[START_REF] Wu | Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase, KinD[END_REF].

Figure 12 .

 12 Figure 12. Structure of RNPP protein. Regulators of the RNPP family contain multiple TPR repeats. TPR: tetratricopeptide repeat; HTH: helix-turn-helix, a DNA binding-domain (Perchat et al., 2011).

Figure 16 .

 16 Figure 16. PrgX is regulated by two types of signaling peptides. When bind to the plasmid-encoded iCF10 (i), PrgX binds to DNA, inhibiting the access of RNA polymerase to the conjugation genes. Alternatively, when PrgX is bound to cCF10 (c), the regulator achieves another conformation that liberates the transcription of the prgQ operon (adapted from Chen et al., 2017).

  Raphave been shown to regulate the infectious cycle of B. thuringiensis in the insect model Galleria mellonella. These quorum sensing systems are sequentially activated during the lifestyle of B. thuringiensis throughout the different phases of infectious process, namely: pathogenic, necrotrophic and spore formation (Figure17)(Slamti et al., 2014).

Figure 17 .

 17 Figure 17. The infectious cycle of B. thuringiensis in the insect is regulated by quorum sensing systems. In larvae of G. mellonella, the infectious process of B. thuringiensis was shown to be controlled by the sequential activity of PlcR-PapR; NprR-NprX and Rap-Phr. Solid lines: transcriptional regulation; Dotted lines: peptide-or proteinprotein interaction (modified from Slamti et al., 2014).

Figure 18 .

 18 Figure 18. Sequential and alternative activation of sporulation-specific sigma factors control the spore formation process. The crisscross activity of these sigma factors allows the tight regulation between morphogenesis and gene expression. Green arrows: transcriptional regulation; Black arrows: post-translational activation. Complete description is found in the text (modified from Fimlaid & Shen, 2015).

Figure 19 .

 19 Figure 19. Differences between B. cereus and B. subtilis groups concerning number and sequence of rap genes. A) Distribution of rap genes among strain genomes. Most part of B. cereus group strains possess from four to eight rap genes, while the majority of B. subtilis strains have at least nine rap genes. B) Phylogenetic tree of Rap proteins. Sequences of regulators from B. subtilis and B. cereus groups cluster separately (Even-Tov et al., 2016).

  Figure 20. Activity of chromosomal Rap-Phr systems from B. subtilis. The schematic representation illustrates the two main process regulated by Rap-Phr systems, competence (left side) and sporulation (right side). RapD and RapK were also demonstrated to have a role in the competence regulation. Complete description is found

Figure 21 .

 21 Figure 21. Structure of Rap proteins. A) Rap proteins are normally found in a dimeric form. In this representation a RapH dimer interact with two molecules of Spo0F (Parashar et al., 2011); B) Schematic representation of RapI crystal structure.

  1.3.4 Phr: sequence and maturationAside from being transcriptionally regulated by the promoter upstream from the rap gene, most of phr genes are also independently transcribed from a promoter located inside rap gene and upstream from the phr gene (Figure22A)(McQuade et al., 2001).

Figure 22 .

 22 Figure 22. Transcription regulation and maturation of Phr signaling molecules. A) Chromosome rap-phr signaling cassettes of B. subtilis. phr-orphan rap genes are not represented; likewise phrA, expression of phrH gene is not regulated by the sigma factor σ H . Black arrows: transcriptional start sites.(Pottathil & Lazazzera, 2003) B) Phr molecules are transcribed in a premature form, which is secreted through the Sec export system and then by proteases. An additional protease cleavage seems to occur in Phr peptides that the active form is found inside the C-terminal region(Pottathil & Lazazzera,

  The second cleavage occurs extracellularly to release the mature oligopeptide from the proPhr peptide. In B. subtilis, this step is performed by the redundant proteases subtilisin, Epr and Vpr that are widespread in bacteria[START_REF] Lanigan-Gerdes | Identification of subtilisin, Epr and Vpr as enzymes that produce CSF, an extracellular signalling peptide of Bacillus subtilis[END_REF]. The five residues that precede the mature peptide (cleavage site) were shown to be important to cleavage reaction and relatively conserved in B. subtilis Phr sequences[START_REF] Lanigan-Gerdes | Identification of Residues Important for Cleavage of the Extracellular Signaling Peptide CSF of Bacillus subtilis from Its Precursor Protein[END_REF].

  , and the species determinants are encoded by plasmid genes[START_REF] Vilas-Boas | Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis[END_REF]). 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. thuringiensis 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 toxicity 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 formation, sporulation, motility, genetic exchange (competence and conjugation), and virulence factor production, are regulated by quorum sensing (QS), a cell-cell communication process that allows bacteria and eukaryotic microorganisms to coordinate their biological processes based on the population density[START_REF] Polke | Quorum sensing by farnesol revisited[END_REF] 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).

  proteins, RNPP regulators 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 successively activated(Slamti et al. 2014): i) PlcR-PapR regulates the virulence stage by controlling the expression of virulence genes; ii) NprR-NprX regulates the necrotrophic stage, allowing bacteria to survive and to sporulate in the insect cadaver; and iii) Rap-Phr regulates the initiation of the sporulation process.

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 extracellular protease production by inhibiting DegU(Ogura et al. 2003), and RapI also regulates the mobility of the ICBs1 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 conjugation(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 internalized 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 identification, 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 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.

  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.

FiguresFig. 1 Fig. 2

 12 Figures

Fig. 3

 3 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. (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.

Fig. 4

 4 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 total viable cells per milliliter. The data were analyzed by 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).

Fig. 1

 1 Fig.1Rap protein sequence groups (refer to Fig.2): a) Chromosomal Rap sequences distributed within 12 groups; b) Plasmid Rap sequences distributed within nine groups. Phylogenetic trees were inferred by Neighbor-Joining method and the evolutionary distances were computed using the JTT matrix-based method and are in the units of the number of amino acid substitutions per site. Evolutionary analyses were conducted in MEGA6.

  The pAW63 possesses B.th.I1 and B.th.I2 introns that interrupt genes whose products are related to conjugation function. Interestingly, the B.th.I2 was demonstrated to excise during transcription allowing expression of host gene(Van der Auwera et al., 2005;[START_REF] Van Der Auwera | Transcriptional analysis of the conjugative plasmid pAW63 from Bacillus thuringiensis[END_REF]. The pAW63 shares a common backbone with pXO2 (the second toxin-plasmid of B. anthracis) and pBT9727 from the pathogenic B. thuringiensis strain konkukian 97-27 (Van derAuwera et al., 2005). Even if these similar plasmids share 42 CDSs, more than half of pAW63 CDSs, pAW63 is the only one harboring a regulatory Rap-Phr system(Cardoso et al., in press).Our data show that the rap63-phr63 genes are co-transcribed from a promoter activated in the beginning of the stationary phase in sporulation medium. The phr63 gene is also independently transcribed from a promoter located in the rap63 gene. Its transcription starts 2h after the onset of the stationary phase and is partially activated by the σ H factor. In B. subtilis, most of the phr genes are controlled by σ H ensuring Phr accumulation to trigger sporulation(McQuade 2001). Transcriptional analyses of pAW63 coding-sequences detected the expression of rap63 (CDS 57) and phr63 (CDS 57b) at mid exponential growth but in different growth conditions[START_REF] Van Der Auwera | Transcriptional analysis of the conjugative plasmid pAW63 from Bacillus thuringiensis[END_REF]. They compared the expression pattern of a wild type HD73 strain and the same strain cured of the pAW63 plasmid by a microarray analysis. Their results showed that the rap63 gene is expressed at a moderate level while the phr63 gene was found in high level[START_REF] Van Der Auwera | Transcriptional analysis of the conjugative plasmid pAW63 from Bacillus thuringiensis[END_REF].

  The C-terminal ends of the Phr8 and Phr63 present a high sequence similarity with six out eight amino acids identical and a histidine residue (positively charged) at the -5 position from the C-terminal end of the precursor peptides. Consequently, we hypothesize a possible crosstalk between the Rap63-Phr63 and Rap8-Phr8 plasmid systems. Unfortunately, ours results show that neither Phr8 is able to inhibit Rap63 nor Phr63 prevents Rap8 activity. In B. subtilis, the absence of crosstalk between

Figure 1 .Figure 2 .

 12 Figures

Figure 3 .Figure 4 .AFigure 5 .

 345 Figure 3. Rap63 delays expression of Spo0A-regulated genes. Kinetics of spoIIE expression in the HD73 wild-type strain carrying the pHT315xyl-PspoIIE'yfp, the pHT315xylΩrap63_PspoIIE'yfp, or the pHT315xylΩrap63-phr63_PspoIIE'yfp plasmids. The YFP fluorescence was measured during growth in HCT medium at 30°C in the presence of xylose 20 mM added at t0 (entry into stationary phase). The results are expressed in arbitrary units per OD 600 unit.

Figure 6 .

 6 Figure6. Characterization of Phr63 active form. A) Amino acid sequence of the pre-Phr63 peptide and of the five different synthetic peptides used in complementation experiments. Cterminal end sequence truncated in the xyl_rap63-phr63_R3 is colored in red, and the positively charged residue in blue. B) Sporulation efficiencies of the HD73 control strain (315xyl), HD73 expressing rap63 (xyl_rap63) and expressing rap63-phr63_R3 producing the C-terminal truncated Phr63. The strain expressing rap63 was complemented with the synthetic peptides Phr63-4, Phr63-5, Phr63-6, Phr63-7 and Phr63-8 added independently 1 h after the onset of the stationary phase at 50 mM final concentrations. The percentages of spores were calculated as 100 × the ratio between heat-resistant spores per ml and total viable cells per ml after 48h in HCT medium at 30°C in the presence of xylose 20 mM. Error bars represent the Standard Error of the Mean (SEM). Experimental values are detailed in TableS3. Different letters correspond to significant differences in the main values (p<0.001).

Figure 7 .

 7 Figure7. Evaluation of cross-talk between the Rap8-Phr8 and Rap63-Phr63 systems. A) Amino acid sequences of Phr63 and Phr8 peptides. C-terminal ends similarities are highlighted in red and the two divergent residues are in black bold. Phr8-6, Phr8-7 and Phr8-8 correspond to the Phr8 synthetic peptides described byFazion et al. (2018). B) Sporulation efficiency of the HD73 Δrap8-phr8Δrap63-phr63 control strain (ΔΔ 315xyl), and expressing rap63 (ΔΔ rap63) or rap8 (ΔΔ rap8) strains in HCT medium at 30°C in the presence of xylose 20 mM. The strain expressing rap63 was complemented with Phr8-6, Phr8-7 and Phr8-8 synthetic peptides and the strain expressing rap8 with Phr63-6, Phr63-7 and Phr63-8 synthetic peptides. Peptides were added independently 1h after the onset of the stationary phase at 50 mM final concentrations. The percentages of spores were calculated as 100 × the ratio between heat-resistant spores per ml and total viable cells per ml. Error bars represent the Standard Error of the Mean (SEM). Experimental values are detailed in TableS3. Different letters correspond to significant differences in the main values (p<0.001).

  regarding to the population density. Gram positive bacteria utilize peptides as signaling molecules and the quorum sensing systems from the RNPP family play a key role in the infectious cycle of B. thuringiensis in its naturalistic environment. Rap-Phr systems are crucial for the coordination of bacterial process in B. cereus group species, specifically sporulation, as wide described for B. subtilis systems. The precise control of sporulation allows the bacteria to survive and to better adapt in their ecological niche, taking into account the modifications of the environment. The great number of Rap-Phr systems encoded by plasmid reinforces the importance of the wide plasmid pattern of B. cereus group, especially in B. thuringiensis. The fact that the main virulence gene and regulator of bacterial development (such as Rap-Phr systems) are encoded on plasmids emphasize that these replicative extrachromosomal elements play an important role in adaptation of their host bacteria.notamment de contrôler le développement de plusieurs processus biologiques importants comme la formation de biofilm, la sporulation ou la production des facteurs de virulence. Parmi les systèmes de quorum sensing des bactéries à Gram positive se trouvent les systèmes de la famille RNPP. Les systèmes de cette famille sont composés d'un régulateur cytoplasmique structuré en motifs TPR (tetratricopeptide repeats), impliqués dans les interactions protéine-protéine ou protéine-peptide, et par un oligopeptide sécrété qui contrôle l'activité du régulateur(Declerck et al., 2007). Le cycle infectieux de B. thuringiensis dans l'insecte hôte est contrôlé par l'activation séquentielle de systèmes de la famille RNPP(Slamti et al., 2014). Premièrement, PlcR-PapR régule la phase de virulence puis, NprR-NprX contrôle la phase de nécrotrophisme et la sporulation, et enfin, Rap-Phr régule également la sporulation. Parmi ces systèmes, Rap-Phr est le moins étudié chez les bactéries du groupe B. cereus.

  De plus, plusieurs gènes phr sont également transcrits à partir de leur propre promoteur contrôlé par le facteur sigma de transcription SigH(McQuade et al., 2001). Le peptide Phr est produit sous une forme pré-mature puis il est sécrété, maturé dans le milieu extracellulaire et réimporté sous sa forme active pour inhiber l'activité de sa protéine Rap. La forme active des peptides Phr peut avoir 5, 6 ou 7 acide aminés et L'activité sur la sporulation des protéines Rap a été prédite en fonction de la présence des résidus clés décrits chez la protéine RapH de B. subtilis(Parashar et al., 2011). Un tiers des protéines Rap du groupe B. cereus sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Pour valider cette prédiction, l'activité sur la sporulation de 10 protéines Rap, 3 chromosomiques et 7 plasmidiques, a été mesurée. Les Rap prédites pour n'avoir aucun rôle sur la sporulation (Rap6 -BtHD1, Rap6 -Bt407 et Rap7 -BtHD73) n'affectent pas la sporulation validant ainsi la prédiction. Parmi les Rap prédites pour inhiber la sporulation, trois résultats différents ont été obtenus: 4 ont un effet majeur (Rap10 -BtHD1, Rap7 -Bt407, Rap8 -Bt407 et Rap5 -BtHD73), 2 ont un effet modéré (Rap1 -BcATCC14579 et Rap2 -BcATCC14579) et une seule n'a pas l'effet prédit sur la sporulation (Rap8 -BtHD1). Par conséquent, les résidus décrits chez RapH de B. subtilis sont conservés et nécessaires pour l'activité sur la sporulation des Rap du groupe B. cereus cependant ils ne sont pas suffisant pour garantir la prédiction de cette fonction. Les bactéries du groupe B. cereus possèdent de multiples systèmes Rap-Phr dont une grande partie est portée par des plasmides. Le fait que la plupart des protéines Rap prédites pour réguler la sporulation soient localisées sur des plasmides peut aider à la dispersion de ces gènes parmi les bactéries du groupe et améliorer leur adaptation aux changements environnementaux en modulant de leur capacité à sporuler. Le système Rap63-Phr63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. Ce système est porté par le plasmide pAW63, beaucoup étudié en lien avec la conjugaison, et est présent dans la même souche que le système plasmidique Rap8-Phr8 déjà décrit (Fazion et al., 2018). Les gènes rap63 et phr63 sont co-transcrits à partir d'un promoteur localisé en amont du gène rap63 et le gène phr63 est également exprimé indépendamment à partir de son propre promoteur. L'expression du promoteur Prap63 démarre à T1 (une heure après l'entrée en phase stationnaire de croissance) et celle à partir du Pphr63 commence une heure plus tard. La Rap63 réduit la sporulation sans l'inhiber totalement et retarde l'expression d'un gène de sporulation contrôlé par Spo0A. L'activité de la Rap63 sur la sporulation est inhibée par la présence du Phr63. La forme active du Phr63 est localisée dans l'extrémité C-terminal du Phr63 pré-mature. La taille exacte du Phr

Diversité et analyse fonctionnelle des systèmes Rap-Phr du groupe Bacillus cereus. 165p

  CARDOSO, Priscilla de Freitas. . Thèse (Pós-Graduação em Genética e Biologia Molecular) -Universidade Estadual de Londrina & Université Paris-Saclay, Londrina, 2019. RÉSUMÉ Le groupe Bacillus cereus est composé de huit espèces de bactéries à Gram positif sporulantes qui peuvent coloniser plusieurs niches écologiques. Les espèces les plus importantes sont B. cereus, une bactérie ubiquitaire du sol et un pathogène opportuniste; B. thuringiensis, un entomopathogène très utilisé comme biopesticide; et B. anthracis l'agent de la maladie du charbon. Bien que ces espèces présentent différents phénotypes, elles sont étroitement liées génétiquement et leurs facteurs de virulences principaux sont portés par des plasmides. Le cycle infectieux de B. thuringiensis dans la larve d'insecte est régulé par l'activation séquentielle de systèmes de quorum sensing de la famille RNPP. Parmi eux, les systèmes Rap-Phr, caractérisés chez B. subtilis, ont très peu été étudiés dans le groupe B. cereus. Ces systèmes régulent divers processus bactériens importants dont la sporulation. La Rap63 est inhibée par son peptide Phr63, dont la forme mature correspond à l'extrémité C-terminale du pro-peptide. Les résultats de sporulation dans l'insecte suggèrent une activité synergique des systèmes Rap63-Phr63 et Rap8-Phr8 (porté par le pHT8_1) dans la régulation de la sporulation. Malgré la similarité entre les Phr63 et Phr8 aucun cross-talk n'a pu être mis en évidence, ce qui confirme la spécificité de ces systèmes de communication cellulaire. L'ensemble de ces résultats démontre la grande diversité des systèmes Rap-Phr dans le groupe B. cereus et souligne l'impact des systèmes plasmidiques dans le développement de ces bactéries. Par conséquent, les plasmides sont des éléments importants pour l'adaptation et la survie de ces bactéries et particulièrement pour B. thuringiensis.

L'objectif de cette étude est d'analyser les systèmes Rap-Phr dans le groupe B. cereus, pour connaitre leur distribution, leur localisation et leur diversité afin d'obtenir une vue globale de ces systèmes chez ces bactéries. De plus, leur possible implication dans la régulation du processus de sporulation a été prédite sur la base de données structurales décrites chez RapH de B. subtilis. Les gènes rap, toujours associés à un gène phr, sont présents dans toutes les souches étudiées avec une moyenne de six gènes rap-phr par souche et avec 30% de ces systèmes qui sont portés par des plasmides. Les souches de B. thuringiensis portent six fois plus de systèmes Rap-Phr plasmidiques que les souches de B. cereus. Par ailleurs, les souches phylogénétiquement proches possèdent un profil de gènes rap-phr similaire. Un tiers des protéines Rap sont prédites pour inhiber la sporulation et ces protéines sont préférentiellement localisées sur les plasmides et donc plus fréquemment présentes chez B. thuringiensis que chez B. cereus. Cette prédiction a été partiellement validée par des tests de sporulation suggérant que les résidus impliqués dans cette activité chez B. subtilis sont conservés mais insuffisants pour prédire cette fonction. Le système Rap63-Phr63 porté par le plasmide pAW63 de la souche B. thuringiensis HD73 a ensuite été caractérisé. La protéine Rap63 a un effet modéré sur la sporulation et retarde l'expression des gènes régulés par Spo0A. Mots-clés: Bacillus thuringiensis, sporulation, quorum sensing, famille RNPP, plasmide

Table 1 .

 1 Phylogenetic classification of the B. cereus group species

	Clade	Group Species	Growth temperature Features
	3	I	B. pseudomycoides	10°C to 43°C
		II	B. cereus B. thuringiensis	7°C to 40°C
			B. anthracis		Mainly pathogenic to mammals
	1	III	B. cereus	15°C to 45°C	Strains related to Anthrax, Cereulide and
			B. thuringiensis		Periodontitis
	2	IV	B. thuringiensis B. cereus	10°C to 45°C	Environmental strains Pathogenic to invertebrates Strains used in biopesticide products
			B. cereus
		V	B. thuringiensis	8°C to 40°C
			B. toyonensis
	3	VI	B. weihenstephanensis B. mycoides B. thuringiensis	5°C to 37°C	Psychrotrophic strains Isolated from soil and food
		VII	B. cytotoxicus	15°C to 55°C	Some strains display citotoxicity

  1.1.4 Plasmids Plasmids play an important role in the ecology of B. cereus group species. The main virulence factors of B. anthracis, B. thuringiensis and B. cereus are encoded by genes located on plasmids. B. anthracis is mainly characterized by the presence of the plasmids pXO1 and pXO2, which carries the toxin genes and capsule biosynthesis genes, respectively. Strains are characterized as B. thuringiensis when produce δ-endotoxins, the primary factor for its entomopathogenic features, encoded by cry genes predominantly harbored in plasmids. Moreover, B. cereus strains that

  . Binding of Phr induces a constriction in Rap structure which is propagated from the TPR domains to the 3-helix bundle region disrupting the association of Rap protein with ComA or Spo0F (Gallego del Sol & Marina, 2013; Parashar et al., 2013a). In this rearranged structure, the Rap protein is a single continuous super helical structure formed by 9 TPR-like folds (Figures 12 and

  1,2 , Stéphane Perchat 2 , Laurival A. Vilas-Boas 1 , Didier Lereclus 2 , Gislayne T. Vilas-Bôas 1* 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

	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

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.

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 pseudomycoides and Bacillus cytotoxicus) of rod-shaped, spore-forming, Gram-positive bacteria that are found in diverse ecological niches and able to colonize different hosts

  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 Table1 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

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, B. cereus group genomes. All protein sequences were analyzed using InterProScan (http://www.ebi.ac.uk/interpro/sequence-search) and SMART (http://smart.emblheidelberg.de) servers for the identification of domains and motifs

[START_REF] Jones | InterProScan 5: genome-scale protein function classification[END_REF][START_REF] Letunic | SMART: recent updates, new developments and status in 2015[END_REF]

. The detection of TPR domains and the absence of HTH DNAbinding 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)

  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 contingence table were used to confront both species, the Fisher's Exact test was used. The data obtained with sporulation assay was analyzed by 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 of these species. All B. cereus and B. thuringiensis available 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. anthracis 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.

Table 3 )

 3

	cereus and B. thuringiensis (4.04 and 4.45, respectively). However, the average
	number of plasmid rap-phr genes is 6-fold higher in B. thuringiensis than in B. 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

. 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. chromosomal and plasmid rap-phr genes, while in the B. cereus strains the number of chromosomal rap-phr genes is almost 7 times higher (P<0.001) than plasmid genes (Table

2

).

Table 5

 5

	). Interestingly, the amount of Rap

thuringiensis YBT1518 strain is the only one B. thuringiensis to have no predicted Rap Spo+ at all.

  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. Bacillus groups is related to the phr gene occurrence. While 27% of the rap genes from B. subtilis strain 168 do not have an associated phr gene(Perego

	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

Tov et al. 2016

). The rap-phr genes are located in all chromosomes and numerous plasmids. 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 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. Interestingly, 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 subjected to genetic variations that also evolved this quorum sensing system to the other RNPP family systems, like PlcR-PapR and NprR-NprX.

  which has only twice as many plasmids as B. cereus and sixtimes as many rap-phr plasmid genes. Moreover, this difference might be explained by the higher presence of mobile elements close to plasmid rap genes in B. thuringiensis than in B. cereus. We have shown that plasmids larger than 200 kb can host multiple rap-phr genes, and it is also remarkable that rap-phr genes were found on virulence plasmids, such as pXO1 in B. anthracis and pCER270 in B. cereus, as well as in some Cry plasmids in B. thuringiensis. However, we did not find specific rap-phr genes associated to the Cry plasmids. The multiplicity of the rap-phr genes in the B. cereus group creates appropriate conditions for their diversity and evolution,

[START_REF]plus de systèmes plasmidiques que B. cereus alors que B. thuringiensis présente seulement deux fois plus de plasmides que B. cereus. L'arbre phylogénétique construit avec l'ensemble des protéines Rap montre une grande diversité de cette famille de protéines dans le groupe B. cereus. De plus, la plupart des Rap plasmidiques sont regroupées en fonction du type de plasmides qui les portent. La comparaison de la distribution des Rap avec l'analyse phylogénétique des souches avec les séquences du schéma MLST montre que les souches phylogénétiquement proches présentent un profil de systèmes Rap-Phr similaire[END_REF] Slamti et al. 2014; Van der Auwera et al. 2005)

. These plasmid rap-phr genes are carried by a wide range of plasmids but are mainly located in conjugative plasmids greater than 70 kb and are more abundant in B. thuringiensis than in B. cereus. This difference is not only the consequence of a higher number of plasmids in B. thuringiensis, as demonstrated by

Even-Tov et al. (2016)

. Thus, different rap-phr genes can respond to various signals and be regulated differently at transcriptional level.

  This absence of Rap Spo+ could be compensated by NprR, which has a Rap-like activity on the sporulation phosphorelay and which is conserved in all the strains of the B. cereus group(Perchat et al. 2016b). In these strains, Rap proteins might have undergone genetic variations leading to the loss of sporulation function and the acquisition of new undetermined functions. Interestingly, Rap proteins predicted to regulate sporulation are mainly encoded by plasmid genes and, therefore, are more abundant in B. thuringiensis. These plasmid Rap-Phr systems could help B. thuringiensis to adapt and survive in its complex ecological niche, the insect. Moreover, many Rap-Phr plasmid systems are located on cryptic plasmids less than 16 kb in size. These plasmid genes could regulate different beneficial

Rap Spo+ (four B. cereus strains, one B thuringiensis strain, B. mycoides and B. pseucomycoides).

Table 1 .

 1 Main features of the 49 selected genomes from the B. cereus group strains. 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). * This strain was first annotated as B. cereus[START_REF] Johnson | Complete Genome Sequences for 35 Biothreat Assay-Relevant Bacillus Species[END_REF], and is now classified as B. pseudomycoides (strain BTZ). # Nowadays, B. weihenstephanensis strains are considered as heterotypic synonym of B. mycoides(Liu et al. 2018).

	Strain (named as in NCBI database)	Release date	Assembly	Chromosome Size (Mb) %GC	Nb	Plasmids Size (Mb) genome % ↑	STs (MLST)
		ATCC 10987	2002	GCA_000008005.1	5.22	35.6	1	0.21	4%	2
		ATCC 14579	2003	GCA_000007825.1	5.41	35.3	1	0.01	0%	33
		E33L	2004	GCA_000011625.1	5.30	35.4	5	0.55	10%	57
		AH187	2008	GCA_000021225.1	5.27	35.6	4	0.33	6%	3
		B4264	2008	GCA_000021205.1	5.42	35.3	0	-	-	-2
		G9842	2008	GCA_000021305.1	5.39	35.3	2	0.35	6%	120
		AH820	2008	GCA_000021785.1	5.3	35.4	3	0.28	5%	39
		Q1	2009	GCA_000013065.1	5.21	35.6	2	0.29	6%	40
		03BB102	2009	GCA_000022505.1	5.27	35.4	1	0.18	3%	122
		anthracis CI	2010	GCA_000143605.1	5.2	35.4	3	0.28	5%	153
	B. cereus	NC7401 F837/76 FRI-35	2011 2011 2012	GCA_000283675.1 GCA_000239195.1 GCA_000292415.1	5.2 5.22 5.08	35.6 35.4 35.6	5 2 4	0.33 0.07 0.3	6% 1% 6%	3 182 188
		FT9	2014	GCA_000724585.1	5.22	35.5	0	-	-	191
		03BB87	2014	GCA_000789315.1	5.46	35.3	2	0.26	5%	58
		D17	2015	GCA_000832385.1	5.38	35.4	1	0.21	4%	179
		FM1	2015	GCA_000832525.1	5.3	35.5	1	0.40	8%	186
		3a	2015	GCA_000832765.1	5.27	35.4	3	0.37	7%	124
		G9241	2015	GCA_000832805.1	5.27	35.4	3	0.45	9%	58
		ATCC 4342	2015	GCA_000832845.1	5.27	35.4	1	0.04	1%	4
		03BB108	2015	GCA_000832865.1	5.34	35.3	7	0.73	14%	119
		Al Hakam *	2015	GCA_000832885.1	5,23	35,8	6	0.45	9%	173
		S2-8	2015	GCA_000835185.1	5.27	35.4	2	0.37	7%	124

Table 2 .

 2 Comparison between B. cereus and B. thuringiensis concerning the Rap-Phr systems.

	B. cereus	B. thuringiensis	Significance

Table 2 .

 2 Primers used in this study.

	Primer	Sequence	Restriction site

Table 3 .

 3 List of plasmids from all strains studied.

	Strain	Plasmid	%GC	Size (Kb)	Nb of rap-phr genes	Special traits
	Bc ATCC 10987	pBc10987	33.4	208	1	-
	Bc ATCC 14579	pBClin15	38.0	15	-	-
		pE33L5	30.9	5.1	-	-
		pE33L8	31.9	8.2	-	-
	Bc E33L	pE33L9	31.0	9.1	-	-
		pE33L54	31.9	53	-	-
		pE33L466	33.1	466	-	-
		pAH187_3	34.9	3.1	-	-
		pAH187_12	31.1	12	-	-
	Bc AH187	pAH187_45	35.5	45	-	-
		pAH187_270 (pCER270)	34.2	270	1	Cereulide genes
	Bc B4264	-				-
	Bc G9842	pG9842_140 pG9842_209	32.9 30.0	140 209	-1	--
		pAH820_3	34.9	3.1	-	-
	Bc AH820	pAH820_10	33.6	11	-	-
		pAH820_272	33.6	272	-	-
	Bc Q1	pBc53 pBc239	35.1 33.5	53 239	-2	--
	Bc 03BB102	p03BB102_179	32.2	180	-	anthrax capsule genes anthrax toxins genes
	Bc biovar anthracis CI	pBAslCI14 pCIXO2 pCIXO1	37.9 33.1 32.5	14 182 94	--1	-anthrax capsule genes anthrax toxins genes
		pNC4	34.9	3.1	-	-
		pNC3	34.7	3.9	-	-
	Bc NC7401	pNC2	34.1	5.4	-	-
		pNC1	36.5	48	-	-
		pNCcld	34.2	270	1	Cereulide genes
	Bc F837/76	pF837_10 pF837_55	31.1 36.2	10 55	--	--
		p04	34.9	3.1	-	-
	Bc FRI-35	p03 p02	30.7 32.5	36 41	-1	--
		p01	33.4	219	1	-
	Bc FT9	-				-
	Bc 03BB87	pBCN pBCX01	36.5 31.7	52 209	--	--
	Bc D17	plasmid unnamed 33.2	211	1	-
	Bc FM1	plasmid unnamed 32.6	403	-	-
		pBFC_1	35.7	7.3	-	-
	Bc 3a	pBFC_2	36.6	51	-	-
		pBFC_3	32.7	312	-	-
		pBFH_1	36.5	52	-	-
	Bc G9241	pBC210	31.7	209	-	-
		pBCX01	32.6	190	1	anthrax toxins genes
	Bc ATCC 4342	pBGM	30.7	37	-	-
		pBFI_7	33.0	4.9	-	-
		pBFI_6	31.8	9.8	1	-
		pBFI_2	31.9	239	1	anthrax capsule genes
	Bc 03BB108	pBFI_5	36.1	42	-	-
		pBFI_4	35.5	62	-	-
		pBFI_1	30.8	282	-	-
		pBFI_3	32.5	86	1	-
	Bc Al Hakam	pBTZ_5	33.0	4.9	-	-

Table 4 .

 4 Distribution of the rap-phr genes by strain, with gene sizes, location in the genome, and DNA strand. Rap proteins highlighted in green were predicted as Spoand those in orange were predicted as Spo+.

	Strains	rap-phr	replicon	DNA strand	size (bp)	rap genes Location (bp)	size (bp)	phr genes Location (bp)
		1		+	1110	1.078.652-1.079.761	138 1.079.758-1.079.895
	Bc ATCC 10987	2 3	chromosome	+ +	1095 1095	2.886.506-2.887.600 3.278.356-3.279.450	147 2.887.600-2.887.746 189 3.279.447-3.279.635
		4	pBc10987	-	1095	130.663-131.757	141	130.526-130.666
		1		+	1110	971.287-972.396	* 177	972.349-972.525
		2		+	1113	1.007.124-1.008.236 * 132 1.008.234-1.008.365
	Bc ATCC 14579	3	chromosome	+	1074	2.091.417-2.092.490	126 2.092.487-2.092.612
		4		-	1098	3.457.151-3.458.248	132 3.456.965-3.457.096
		5		+	1116	3.475.275-3.476.390	162 3.476.390-3.476.551
		1		+	1095	1.040.836-1.041.930	309 1.041.930-1.042.238
	Bc E33L	2 3	chromosome	-+	1113 1095	2.393.749-2.394.861 3.345.257-3.346.351	159 2.393.591-2.393.749 189 3.346.348-3.346.536
		4		+	1095	4.927.987-4.929.081	306 4.929.081-4.929.386
		1		+	1095	1.861.946-1.863.040	144 1.863.037-1.863.180
	Bc AH187	2 3	chromosome	+ +	1104 1095	3.062.411-3.063.514 3.279.233-3.280.327	288 3.063.511-3.063.798 189 3.280.324-3.280.512
		4	pAH187_270	+	1095	204.553-205.647	141	205.644-205.784
		1		+	1095	1.030.971-1.032.065	324 1.032.065-1.032.388
	Bc B4264	2 3	chromosome	+ +	1011 1095	2.069.164-2.070.174 3.390.307-3.391.401	216 2.070.171-2.070.386 189 3.391.398-3.391.586
		4		+	1116	3.409.737-3.410.852	162 3.410.852-3.411.013
		1		+	1095	1.009.685-1.010.779	201 1.010.779-1.010.979
		2		-	1095	1.652.488-1.653.582	138 1.652.351-1.652.488
	Bc G9842	3 4	chromosome	+ -	1074 1101	2.038.586-2.039.659 2.364.416-2.365.516	126 2.039.656-2.039.781 159 2.364.258-2.364.416
		5		-	1095	3.387.052-3.388.146	132	3.386.866-3.386.997
		6		+	1116	3.429.984-3.431.099	162 3.431.099-3.431.260
		7	pG9842_209	-	1095	89.302-90.396	138	89.168-89.305
		1		+	1089	1.050.152-1.051.240	204 1.051.240-1.051.443
	Bc AH820	2 3	chromosome	-+	1101 1116	2.410.483-2.411.583 3.118.600-3.119.715	159 2.410.325-2.410.483 162 3.119.715-3.119.876
		4		+	1095	3.353.248-3.354.342	189	3.354.339-3.354.527
		1		-	1095	581.509-582.603	258	581.252-581.509
		2	chromosome	+	1095	1.863.400-1.864.494	144 1.864.491-1.864.634
	Bc Q1	3		+	1098	3.249.705-3.250.802	189	3.250.805-3.250.993
		4 5	pBc239	+ -	1095 1119	101.157-102.251 155.498-156.616	141 132	102.248-102.388 155.370-155.501
		1		+	1110	959.479-960.588	138	960.585-960.722
		2		+	1095	996.508-997.602	246	997.602-997.847
	Bc 03BB102	3	chromosome	-	1101	2.428.174-2.429.274	159 2.428.016-2.428.174
		4		+	1116	3.109.162-3.110.277	162 3.110.277-3.110.438
		5		+	1095	3.344.266-3.345.360	138 3.345.360-3.345.497
		1		+	1089	998.747-999.835	204	999.835-1.000.038
	Bc anthracis CI	2 3	chromosome	-+	1101 1113	2.349.164-2.350.264 3.262.482-3.263.594 * 135 3.263.592-3.263.726 159 2.349.006-2.349.164
		4		+	1170	4.218.902-4.220.071	123 4.220.068-4.220.190
		5	pCI-XO1	+	1095	172.539-173.633	* 141	173.631-173.771
		1		+	1095	1.820.769-1.821.863 * 144 1.821.861-1.822.004
	Bc NC7401	2 3	chromosome	+ +	1104 1095	3.016.626-3.017.729 3.233.393-3.234.487	288 3.017.726-3.018.013 189 3.234.484-3.234.672
		4	pNCcld	+	1095	259.831-260.925	141	260.922-261.062
		1		+	1110	951.743-952.852	138	952.849-952.986
		2		+	1095	990.203-991.297	330	991.298-991.627
	Bc F837/76	3	chromosome	-	1101	2.389.042-2.390.142	159 2.388.884-2.389.042
		4		+	1116	3.040.092-3.041.207	162 3.041.207-3.041.368
		5		+	1095	3.269.648-3.270.742	138	3.270.742-3.270.879

Table 6 .

 6 Efficiency of sporulation of strains expressing rap genes.

	Strains	Viable cells	Heat-resistant spores	% spores	Prediction
	315xyl	1.21E+08 (±7.58E+07) 1.21E+08 (±5.23E+07)	95 (±10)	-
	Rap6-HD1	1.21E+08 (±4.54E+07) 1.30E+08 (±5.01E+07)	93 (±10)	Spo-
	Rap8-HD1	1.41E+08 (±6.55E+07) 1.57E+08 (±4.62E+07)	99 (±2)	Spo+
	Rap10-HD1	7.13E+07 (±1.20E+07) 1.33E+05 (±3.51E+04) 0.2 (±0.04)	Spo+
	Rap6-Bt407	1.45E+08 (±7.35E+07) 1.47E+08 (±4.58E+07)	91 (±9)	Spo-
	Rap7-Bt407	1.01E+08 (±1.04E+07) 1.59E+05 (±1.26E+04) 0.2 (±0.02)	Spo+
	Rap8-Bt407	6.13E+07 (±2.10E+07) 7.15E+06 (±1.56E+06)	13 (±8)	Spo+
	Rap5-Bt HD73	4.03E+07 (±6.48E+06) 2.07E+05 (±1.26E+05) 0.51 (±0.3)	Spo+
	Rap7-Bt HD73	3.08E+08 (±2.93E+07) 2.35E+08 (±2.25E+07)	77 (±3.7)	Spo-
	Rap1-BcATCC14579	3.92E+08 (±4.73E+07) 2.80E+08 (±3.28E+07)	71.5 (±2)	Spo+
	Rap2-BcATCC14579	3.51E+08 (±3.89E+07) 2.47E+08 (±9.29E+06)	70.8 (±6)	Spo+

  The viable cells and spores were counted in death larvae 4 days after treatment maintained at 30°C. B) In HCT medium culture (in vitro) the viable cells and spores were counted after 48h of culture at 30°C. The percentages of spores were calculated as 100 × the ratio between heat-resistant spores per ml and total viable cells per ml. Error bars represent the Standard Error of the Mean (SEM). Experimental values are detailed in TableS3. Significant differences to the WT values are presented as * if P<0.05 or ** if P<0.01.

Table S3 .

 S3 Efficiency of sporulation of B. thuringiensis HD73 derivative strains.

	Strain	Medium	Viable cells	Heat-resistent spore	% spore
	HD73 pHT315xyl (xyl)	HCT	3.97E+08 (±1.53E+07)	3.25E+08 (±1.46E+07)	81.69 (±1.33)
	xyl_rap63	HCT	1.60E+08 (±2.31E+07)	5.80E+07 (±6.89E+06)	38.11 (±2.26)
	xyl_rap63-phr63	HCT	4.10E+08 (±1.68E+07)	3.44E+08 (±1.47E+07)	83.99 (±1.37)
	xyl_phr63	HCT	3.37E+08 (±1.25E+07)	2.86E+08 (±1.53E+07)	84.79 (±2.17)
	xyl_rap-phr63_R3'	HCT	2.31E+08 (±4.37E+07)	7.38E+07 (±1.44E+07)	32.06 (±1.96)
	xyl_rap63 + Phr63-4	HCT	2.57E+08 (±3.76E+06)	7.73E+07 (±6.38E+06)	30.12 (±2.38)
	xyl_rap63 + Phr63-5	HCT	4.00E+08 (±2.19E+07)	3.07E+08 (±7.13E+06)	76.90 (±2.77)
	xyl_rap63 + Phr63-6	HCT	2.86E+08 (±4.46E+07)	2.42E+08 (±4.11E+07)	84.33 (±1.04)
	xyl_rap63 + Phr63-7	HCT	3.57E+08 (±1.92E+07)	3.09E+08 (±1.72E+07)	86.69 (±1.63)
	xyl_rap63 + Phr63-8	HCT	3.20E+08 (±2.94E+07)	2.79E+08 (±2.99E+07)	86.84 (±1.87)
	HD73	HCT	5.46E+08 (±4.52E+07)	4.54E+08 (±3.89E+07)	83.31 (±2.82)
	HD73 ΔRap-Phr63	HCT	6.20E+08 (±9.51E+07)	5.50E+08 (±9.76E+07)	88.68 (±9.86)
	HD73 ΔPhr63	HCT	5.70E+08 (±6.19E+07)	5.45E+08 (±7.03E+07)	95.29 (±3.78)
	HD73 ΔPhr63 ΔPhr8	HCT	4.62E+08 (±2.73E+06)	2.51E+08 (±1.44E+07)	54.41 (±2.82)
	HD73 Δrap63-phr63 Δrap8-phr8 (ΔΔ) xyl	HCT	2.56E+08 (±2.42E+07)	2.16E+08 (±2.48E+07)	83.83 (±3.11)
	ΔΔ xyl_rap63	HCT	1.25E+08 (±7.88E+06)	4.07E+07 (±5.70E+06)	32.40 (±2.87)
	ΔΔ xyl_rap63-phr63	HCT	2.78E+08 (±2.10E+07)	2.44E+08 (±2.09E+07)	87.68 (±2.24)
	ΔΔ xyl_rap63 + Phr63-6	HCT	2.57E+08 (±2.08E+07)	2.03E+08 (±1.66E+07)	79.15 (±2.35)
	ΔΔ xyl_rap63 + Phr63-7	HCT	2.79E+08 (±1.47E+07)	2.30E+08 (±1.40E+07)	82.41 (±2.31)
	ΔΔ xyl_rap63 + Phr63-8	HCT	2.78E+08 (±2.58E+07)	2.39E+08 (±2.14E+07 )	86.03 (±0.46)
	ΔΔ xyl_rap63 + Phr8-6	HCT	1.46E+08 (±2.08E+06)	4.52E+07 (±4.41E+05)	30.95 (±0.64)
	ΔΔ xyl_rap63 + Phr8-7	HCT	1.78E+08 (±1.05E+07)	6.18E+07 (±4.41E+05)	34.97 (±1.94)
	ΔΔ xyl_rap63+ Phr8-8	HCT	2.25E+08 (±1.15E+06)	6.60E+07 (±1.56E+06)	29.33 (±0.54)
	ΔΔ xyl_rap8	HCT	4.45E+07 (±6.55E+06)	6.08E+05 (±1.59E+05)	1.34 (±0.29)
	ΔΔ xyl_rap8-phr8	HCT	3.21E+08 (±2.01E+07)	2.66E+08 (±9.40E+06)	83.10 (±3.27)
	ΔΔ xyl_rap8 + Phr8-6	HCT	6.17E+07 (±7.78E+06)	6.99E+06 (±4.66E+05)	11.48 (±0.64)
	ΔΔ xyl_rap8 + Phr8-7	HCT	2.53E+08 (±1.39E+07)	2.03E+08 (±1.28E+07)	80.42 (±1.32)
	ΔΔ xyl_rap8 + Phr8-8	HCT	2.32E+08 (±1.27E+07)	3.04E+07 (±3.26E+06)	13.09 (±1.30)
	ΔΔ xyl_rap8 + Phr63-6	HCT	9.83E+07 (±2.10E+07)	7.61E+05 (±1.40E+04)	0.84 (±0.16)
	ΔΔ xyl_rap8 + Phr63-7	HCT	1.42E+08 (±6.15E+07)	1.27E+06 (±4.40E+05)	0.96 (±0.08)
	ΔΔ xyl_rap8 + Phr63-8	HCT	1.18E+08 (±3.61E+07)	1.49E+06 (±5.12E+05)	1.23 (±0.07)
	HD73	Insect larvae	1.84E+08 (±1.08E+08)	3.58E+07 (±1.41E+07)	22.30 (±6.89)
	HD73 ΔRap63-Phr63	Insect larvae	1.52E+08 (±8.91E+07)	2.16E+07 (±6.11E+06)	24.53 (±11.06)
	HD73 ΔPhr63	Insect larvae	1.04E+08 (±4.55E+07)	2.25E+07 (±1.13E+07)	16.69 (±6.88)
	HD73 ΔRap8-Phr8	Insect larvae	1,88E+08 (±1,14E+08)	2,83E+07 (±1,09E+07)	23,56 (±7,09)

Cylinders: α-helices; N: amino terminal end; C: carboxyl terminal end; HTH: TPR-like helix-turn-helix(Parashar et al., 2013a).

Acknowledgements

We gratefully acknowledge Jacques Mahillon for the B. thuringiensis var. kurstaki strain HD73 Cry-and Fernanda A. P. Fazion for the plasmid pHT315xyl_rap7061. The project was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, contract nº: 2065/2014, 23038.001776/2014-29) and 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. Acknowledgements We gratefully acknowledge Jacques Mahillon for the B. thuringiensis var. kurstaki strain HD73 Cry-. The project was supported by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and COFECUB (Comité Français d'Evaluation de la Coopération Universitaire et Scientifique avec le Brésil). P.F.C. was supported by fellowship from CAPES/Brazil.

RapB

PhrC B. subtilis chromosome Sporulation (Perego, 1997;Perego et al., 1994) RapC ERGMT B. subtilis chromosome Competence (Auchtung et al., 2006;[START_REF] Lazazzera | An autoregulatory circuit affecting peptide signaling in Bacillus subtilis[END_REF][START_REF] Solomon | Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis[END_REF] RapD -B. subtilis chromosome Competence (Ogura & Fujita, 2007) RapE SRNVT B. subtilis chromosome Sporulation (Jiang et al., 2000a) RapF QRGMI B. subtilis chromosome Competence (Auchtung et al., 2006;Bongiorni et al., 2005) RapG EKMIG B. subtilis chromosome Protease production [START_REF] Hayashi | Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon[END_REF]Ogura et al., 2003) RapH TDRNTT B. subtilis chromosome Competence; Sporulation [START_REF] Hayashi | Bacillus subtilis RghR (YvaN) represses rapG and rapH, which encode inhibitors of expression of the srfA operon[END_REF]Mirouze et al., 2011;Smits et al., 2007) RapI ADRVGA B. subtilis chromosome Sporulation; Transfer of ICEBs1 (Auchtung et al., 2005;Mirouze et al., 2011;Singh et al., 2013) RapJ PhrC B. subtilis chromosome Sporulation (Parashar et al., 2013a(Parashar et al., , 2011) ) RapK ERPVG B. subtilis chromosome Competence (Auchtung et al., 2006)

Rap60

SRNAT or ASRNAT B. subtilis pTA1060 Protease production [START_REF] Boguslawski | Novel mechanisms of controlling the activities of the transcription factors Spo0A and ComA by the plasmid-encoded quorum sensing regulators Rap60-Phr60 in Bacillus subtilis[END_REF]Koetje et al., 2003)

RapQ

SRNAT B. amyloliquefaciens pBSG3

Competence; Surfactin production; Sporulation (Qiao et al., 2011;Yang et al., 2015)

RapP

RapP is insensitive to PhrP

B. subtilis pBS32

Biofilm formation; Sporulation ; Competence [START_REF] Bendori | The RapP-PhrP Quorum-Sensing System of Bacillus subtilis Strain NCIB3610 Affects Biofilm Formation through Multiple Targets, Due to an Atypical Signal-Insensitive Allele of RapP[END_REF]Parashar et al., 2013b) Rap LS20 QKGMY B. subtilis pLS20 Conjugation [START_REF] Rösch | Induction of Plasmid Conjugation in Bacillus subtilis Is Bistable and Driven by a Direct Interaction of a Rap/Phr Quorum-sensing System with a Master Repressor[END_REF][START_REF] Parashar | [END_REF] BXA0205 GHTGG B. anthracis pXO1 Sporulation (Bongiorni et al., 2006) BA3790 Not defined B. anthracis chromosome Sporulation (Bongiorni et al., 2006)

Rap8

YAHGKDI B. thuringiensis pHT8_1

Sporulation; Biofilm formation (Fazion et al., 2018) PhrC (ERGMT) was demonstrated to regulate the non-cognate RapB and RapJ proteins. [START_REF] Kalamara | Social behaviours by Bacillus subtilis : quorum sensing, kin discrimination and beyond[END_REF][START_REF] Neiditch | Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria[END_REF]Pottathil & Lazazzera, 2003).

Online Resources

Online Resources -Table 1. Source and main features of the selected strains. The two components of this signaling cassette are co-transcribed and the phr63 gene is also transcribed by an additional promoter depending partially of the σH factor.

Interestingly, we show that the ΔPhr8ΔPhr63 mutant strain strongly prevent sporulation in the insect larvae. Despite the Phr8 and Phr63 similarities, there is no cross-talk between the Rap63-Phr63 and Rap8-Phr8 systems. Our results suggest a synergistic activity of these two Rap-Phr systems in the regulation of the sporulation process ensuring the survival and the dissemination of the B. thuringiensis at the end of the infectious cycle. Our results also pointed out the relevance of the plasmid network to the fitness of B. thuringiensis bacteria.

B Supporting Information

Table S1. Primers used in this study

pHT315xylΩphr63 (xyl_phr63)

phr63 gene was amplified using primers Phr7557-F/Phr7557-R and the B. thuringiensis HD73 genomic DNA as template. The fragment was inserted between the BamHI and HindIII sites of pHT315xyl plasmid.

pHT315xylΩrap63-phr63 (xyl_rap63-phr63)

rap-phr63 genes was amplified using primers Rap7557-F/Phr7557-R and the B. thuringiensis HD73 genomic DNA as template. The fragment was inserted between the BamHI and HindIII sites of pHT315xyl plasmid.

pHT315xylΩrap63-phr63_R3 (xyl_rap63-phr63_R3)

rap63 gene together with phr63 truncated gene was amplified using primers Rap7557-F/Phr7557R3 and the B. thuringiensis HD73 genomic DNA as template. The fragment was inserted between the BamHI and HindIII sites of pHT315xyl plasmid.

pHT304.18-Prap63'yfp

Primers Prom7557-F and Prom7557-R were used to amplify the promoter region of rap63 gene, using the B. thuringiensis HD73 genomic DNA as template. The fragment was inserted between the HindIII and BamHI sites of pHT304-18YFP.

pHT304.18-Pphr63'yfp

Primers Prom7557Phr-F and Prom7557Phr-R were used to amplify the promoter region of phr63 gene, using the B. thuringiensis HD73 genome as template. The fragment was inserted between the HindIII and BamHI sites of pHT304-18YFP.

pHT315xyl-PspoIIE'yfp

Plasmid described by Fazion et al. (2018).

pHT315xylΩrap63_PspoIIE'yfp

The PspoIIE'yfp fragment was amplified using primers PU-EcoRI /YFP-R and pHT315xyl-PspoIIE'yfp plasmid as template; and the fragment was inserted into the EcoRI site of pHT315xylΩrap63 plasmid. To avoid influence of PxylA promoter, the orientation of the inserted fragment was verified by PCR using primers PspoIIE-F/xylRout3'.

pHT315xylΩrap63-phr63_ PspoIIE'yfp

The PspoIIE'yfp fragment was amplified using primers PU-EcoRI /YFP-R and pHT315xyl-PspoIIE'yfp plasmid as template; and the fragment was inserted into the EcoRI site of pHT315xylΩrap63-phr63 plasmid. To avoid influence of PxylA promoter, the orientation of the inserted fragment was verified by PCR using primers PspoIIE-F/xylRout3'.

pMADΩrap-phr63::spec 5' and 3' regions of rap63-phr63 genes were amplified using primers 7557Amont1-F/7557Amont1-R and 7557Aval-F/Aval7557-R, respectively, and B. thuringiensis HD73 genomic DNA as template. The 5' end was purified as an NcoI/KpnI fragment and the 3' end as an XbaI/EcoRI fragment. The spectinomycin resistance gene was purified as a KpnI/XbaI fragment from the pUC18Ωspec and inserted together with the 5' and 3' regions of rap63-phr63 between the NcoI and EcoRI sites of pMAD.

pMADΩphr63::spec 5' and 3' regions of phr63 genes were amplified using primers 7557Amont2-F/7557Amont2-R and 7557Aval-F/Aval7557-R, respectively, and B. thuringiensis HD73 genomic DNA as template. The 5' end was purified as an NcoI/KpnI fragment and the 3' end as an XbaI/EcoRI fragment. The spectinomycin resistance cassette was purified as a KpnI/XbaI fragment from the pUC18Ωspec and inserted together with the 5' and 3' regions of phr63 between the NcoI and EcoRI sites of pMAD.

Phr database from B. cereus group allowed to a more complete description of the distribution and diversity of these systems among bacteria of this group. rap genes were found in all studied strains and a phr gene was detected downstream from each rap gene, diverging from B. subtilis in which there are nearly three orphans rap genes per strain (Even-Tov et al., 2016).

Compare to B. subtilis, strains from the B. cereus group possess less rap-phr alleles, six in average. Furthermore, Rap proteins from the B. cereus and B. subtilis groups form two distinct clusters suggesting that these proteins have differentiated after the evolutionary separation of the two species (Even-Tov et al., 2016). This variation in the mechanism for sensing and responding to environmental changes enable these groups to occupy different niches [START_REF] Alcaraz | Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics[END_REF]. Considering that the Rap protein is described as the most ancestor regulator from the RNPP family (Declerck et al., 2007), it could have undergone modifications that give rise to the other regulators of the family, such as NprR and then PlcR. The fact that, in B.

cereus group, NprR and PlcR are described to control pathways, namely protease production (Dubois et al., 2012;[START_REF] Gohar | The PlcR Virulence Regulon of Bacillus cereus[END_REF] and sporulation (Perchat et al., 2016a), that are normally associated to Rap proteins in B. subtilis reinforces this hypothesis.

Plasmids were shown to play an important role in the adaptation of the bacteria from the B. cereus group to their ecological niche (Fazion et al., 2018;Vilas-Bôas et al., 2007) [START_REF] Logan | Genus I. Bacillus Cohn 1872, 174AL[END_REF]Schnepf et al., 1998;Vilas-Bôas et al., 2007). Les protéines Cry sont codées par des gènes plasmidiques (gènes cry) et sont généralement produits pendant la phase stationnaire de croissance et la sporulation (Deng et al., 2014).

Grace à cette propriété, B. thuringiensis est utilisé comme biopesticide dans le monde entier. B. anthracis est l'agent étiologique de la maladie du charbon qui affecte les mammifères et notamment les herbivores. Ses facteurs principaux de virulencetoxines et capsulesont portés respectivement par les plasmides pXO1 et pXO2 (Kolstø et al., 2009;[START_REF] Okinaka | Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes[END_REF]. Bien que les bactéries du groupe B. cereus aient des caractéristiques phénotypiques différentes, elles sont étroitement liées génétiquement. Par conséquent, les plasmides, qui portent les facteurs de virulence majeurs, jouent un rôle essentiel dans l'écologie des bactéries du groupe (Vilas-Bôas et al., 2007).

En plus des facteurs de virulence spécifiques portés par des plasmides, les bactéries du groupe B. cereus produisent un certain nombre de facteurs impliqués dans la virulence et l'adaptation régulés par des systèmes de quorum sensing. Le quorum sensing est un processus de communication cellulaire qui permet aux bactéries de réguler l'expression des gènes en fonction de la densité de la population [START_REF] Miller | Quorum Sensing in Bacteria[END_REF][START_REF] Waters | Quorum sensing: cell-to-cell communication in bacteria[END_REF]. Cette communication permet actif n'a pas pu être déterminée cependant, la forme minimale active est un pentapeptide de séquence HGETI. L'efficacité de sporulation des souches mutantes Δrap63-phr63 et Δphr63 a été mesurée dans la larve d'insecte de Galleria mellonella.

Les deux mutants présentent une sporulation comparable à celle du témoin. Par contre, le double mutant Δphr63Δphr8 inhibe fortement ce processus bactérien avec une différence de 100 fois par rapport à la souche sauvage. Ce résultat est plus important que celui obtenu avec le simple mutant Δphr8 qui présente une différence de 5 fois avec la souche sauvage (Fazion et al., 2018)