Genome sequence analysis of Bacillus subtilis PTA-271 isolated from a Vitis vinifera (cv. Chardonnay) rhizospheric soil: an highlight on some of its biocontrol traits

Background: Bacillus subtilis strains have been widely studied for their innumerous benefits in agriculture, including viticulture. Providing numerous assets, B. subtilis spp. are widely described as promising grapevine-protectors against a broad spectrum of pathogens, ranging from biotroph to necrotroph. B. subtilis spp. may both elicit host defenses and promote host vigor, but may also directly antagonize pathogens and detoxify their aggressive molecules. This study reports the draft genome sequence of the Bacillus subtilis PTA-271, isolated from the rhizospheric soil of healthy Vitis vinifera cv. Chardonnay at Champagne Region in France, attempting to draw outlines of its full biocontrol capacity. Results: The PTA-271 genome has a size of 4,001,755 bp, with 43.78% of G + C content and 3,945 protein coding genes. The draft genome of PTA-271 highlights (1) a functional swarming motility system hypothesizing a colonizing capacity and a strong interacting capacity, (2) strong survival capacities and (3) a set of genes encoding for bioactive substances. Bioactive compounds are known both (i) to stimulate plant growth or defenses such as hormones and elicitors, and (ii) to counteract

To date, B. subtilis species were reported both to elicit plant defenses by mean of elicitors or by interfering with phytohormone signaling and to antagonize plant pathogens and were also described as protective against a broad spectrum of pathogens ranging from biotrophs to necrotrophs [9-14, 16, 21-23, 27, 30-34, 40-49, 52]. Focusing on B. subtilis PTA-271, its protective effect was already published in grapevine against Neofusicoccum parvum and Botrytis cinerea [9][10][11], the causal agents of Botryosphaeria dieback and grey mold respectively. These beneficial characteristics of B. subtilis species, combined with the fact it was a non-pathogenic species able to sporulate in order to resist to climate changes and common disinfectants [53,54,77,121], make this microorganism suitable to control a wide spectrum of pathogens among which the most widely dangerous grapevine trunk disease (GTD) pathogens with no currently efficient control strategies [9,55]. In this study, we report the draft genome sequence of the B. subtilis strain PTA-271 and analyze and compare with other known Bacillus strains sequences, to expand our knowledge on the B. Rhizospheric samples were directly suspended in a sterile 0.85% NaCl solution (1g of soil: 10 ml of NaCl) and bacterial isolates were obtained by serial dilutions of the soil samples (10 7 , 10 3 , 10 2 cfu/g soil) in triplicate onto LB-agar (Luria-Bertani-agar), King's B-agar and glycerol-arginine-agar plates by incubating at 30°C for 24-72 h. All different colonies were then re-isolated on LB-agar, cultured in LB at 30 °C for 24 h and screened for their protective role against Botrytis cinerea by using grapevine plantlet leaf assays pretreated with bacterium [10]. Selected biocontrol microorganisms were then identified, calculated to establish the density formula and stored in a sterile 25% glycerol solution at -4 80°C for complementary purposes. The classification and general features of B. subtilis PTA-271 are in Table 2. The taxonomic information for this strain was already described by Trotel- Aziz et al. (2008) [10] and remains unaltered to this date. B. subtilis PTA-271 was designated for sequencing because of its efficient capacity to protect grapevine against several pathogens such as Botrytis cinerea or Neofusicoccum parvum, the causal agents of grey mold or Botryosphaeria dieback respectively [9][10][11]58]. As previously shown [9][10][11], this beneficial microorganism can modulate grapevine defenses, but may also directly antagonize the growth of pathogens and detoxify aggressive molecules. Such multi-target beneficial levers are adding guaranties for a wide spectrum of protection, in addition to physical and chemical tolerant characteristics (endospore-forming bacterium, large range of pH and salinity, Table 2). Altogether, there are advantages to sequence the B. subtilis PTA-271 genome to better understand its key beneficial levers and further develop the best as possible sustainable biocontrol strategies whatever the field conditions or parameters (pH, salinity, etc …).
The whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JACERQ010000000. The version described in this paper is version JACERQ010000000 and all related information is represented in Table 3.

2.2.-Genomic DNA preparation
Genomic DNA of B. subtilis PTA-271 was extracted using the Wizard® Genomic DNA Purification kit (Promega), from the pellet of a 1 mL-overnight culture incubated at 28 °C in LB medium. DNA integrity was confirmed on a 0.65% agarose gel electrophoresis in TAE buffer. DNA concentration and quality were read from 1 µL of DNA with the NanoDrop-ONE spectrophotometer (Ozyme).

2.3.-Library preparation and genome sequencing
DNA library for bacterial genome sequencing was prepared from 0.5 nanograms of high-quality genomic DNA using the Nextera XT DNA Sample Preparation Kit (Illumina, San Diego, USA) and sequenced using paired-end (PE) 2x300 bp on the MiSeq® Illumina® platform at Genoinseq (Cantanhede, Portugal). All the procedures were performed according to standard manufacturer protocols.
High-quality adapter-free reads were assembled with SPAdes version 3.9.0 [60] and contigs with size <500 bp or coverage lower 10x were removed from the assembly. Assembly metrics were calculated with Quast version 4.6.1 [61]. Contigs were checked for contamination and completeness using CheckM 1.0.9 [62]. Coding gene predictions were made with Prodigal version 2.6 [63], rRNA and tRNA genes were detected using Barrnap version 0.8 and CRISPR regions were detected by Minced version 0.2.0. Coding gene annotation was performed with Prokka version 1.12 [64] using the following repositories: SwissProt (The UniProt Consortium, 2017), HAMAP [65], TIGRFAMs [66] and Pfam [67]. Coding genes were also annotated for Pathway using KEGG [68], for peptidases using MEROPS [69] and for carbohydrate-active enzymes with dbCaN [70].

2.-B. subtilis PTA-271 MULTI-STRENGTHS FOR PLANT SUSTAINABLE BIOCONTROL
Bacillus species offer a broad range of benefits to plants, covering: (1) plant growth promotion, (2) induced systemic plant defenses and protection against pathogens, and (3) prevention of pathogen fitness or aggressiveness, by producing many compounds able to interact with the host plants, the pathogens or their tripartite intricate communication. As previously cited, these compounds include hormone and many elicitors, as well as many antimicrobial molecules, but also a range of many other substances and mechanisms contributing to increase both the plant capacity to recruit beneficial microorganisms and the tripartite communication within plant microbiota including also pathogens (i.e. surfactants, biofilm key forming-elements, quorum -sensing or -quenching molecules, among others). Considering this, the genome analysis of B. subtilis PTA-271 tried to highlight some useful characteristics directly or indirectly beneficial for a sustainable plant protection against a broad spectrum of pathogens.

2.1.-Motility, adhesion and plant root colonizing capacity
Motility of a bacterium is due to the flagellum, enabling it to move towards a vital nutrient source Once reaching a comfortable area, adhesion is due to bacterium pili, allowing the initiation of biofilm formation where both chemotaxis and gene exchanges among microorganisms of microbiota can be amplified [72]. To this end, B. subtilis PTA-271 has genes from the comG operon (Supplementary Table S1), essential for DNA binding to competent cells upon transformation of B.
B. subtilis spp. are also described for their strong swarming motility [74]. The gene swrC encoding for swarming motility protein was identified in the genome of B. subtilis PTA-271 (Supplementary Table S1). Swarming motility requires the production of both functional flagella, pili and surfactant to reduce surface tension [75].
Motility and adhesion are both considered advantageous characters for a successful host colonization and B. subtilis spp. are already described to grow in biofilm mode involved in root colonization [76]. To this end, B. subtilis PTA-271 encodes the transcription factor Spo0A (S19-40_02177, Supplementary Tables S1 and S2), described to be required for the surface-adhered cells transition to a three-dimensional biofilm structure [77] and to repress AbrB (S19-40_03988), described as a negative regulator of biofilm formation [77].
The genes identified above in B. subtilis PTA-271 support additional investigations towards (1) a tripartite communication within plant microbiota and (2) grapevine root colonization from the rhizospheric soil where it was already identified [10]. Some authors consider that (1) all of the microbial genera described as common inhabitants of the rhizosphere are also endophytics [78] and that (2) whatever their localization, beneficial microorganisms that successfully colonize the plant, particularly by the root system [79], would be advantageous both for plant growth promotion and for plant biocontrol. Indeed, the B. subtilis spp. flagellum contains flagellin proteins that are recognized as elicitors of plant defenses [80] as indicated below. Surfactin is another elicitor as indicated below too, and also a biosurfactant involved in the formation of stable biofilm essential for the successful colonization of host-plants [81].

2.2.-Plant growth promotion through trophic-and morphogenic-effects
Plant nutrition depends on the soil retention capacity of minerals and on nutrient availabilities, thus both on chelating process, on mineralization by decomposers and on the bioavailability of minerals towards the plant consumer. Upon nitrogen starvation, some bacteria are described to upregulate the ure gene cluster, since urea is an easy nitrogen source. Such ure genes are also predicted in B.
subtilis PTA-271 genome containing ureA (S19-40_00755), ureB (S19-40_00756) and ureC (S19-40_00757). This cluster of genes is known to be controlled by the global nitrogen-regulatory protein TnrA (Supplementary Table S2), also predicted in B. subtilis PTA-271 genome and consolidating this bacterium as a good plant partner as non-competitive for nitrogen. Regarding other nutrient access that also depends on soil solubilizing activity and nutrient bioavailability, it is well known that phosphate-solubilizing bacteria (PSB) may take advantage of low molecular weight molecules [51,82].
Similarly, genes of B. subtilis PTA-271 are predicted to encode for proteins involved in the production of gluconic acid and precursor of citric acid (S19-40_03830, S19-40_03828). These organic acids may lower the soil pH to solubilize phosphate and thus increase its availability to the plant [83]. Bacterial secondary metabolites (i.e. PyrroloQuinoline Quinone, PQQ) are also known to control gluconic acid production [84], and B. subtilis PTA-271 has three genes related to PQQ production pqqL, pqqF and pqqC (S19-40_00233, S19-40_00234, S19-40_00247) [85]. Additionally, as in the other Bacillus spp., B.
subtilis PTA-271 contains the phytase gene phy (S19-40_03630) encoding for phosphatases able to hydrolyze the organic complex in order to liberate phosphate and make it available for plants [86,87].  Table S2) that encodes for a ferric uptake regulatory protein coordinating the homeostasis of iron uptake depending on its availability in soil [88]. B. subtilis PTA-271 appears thus as a good plant auxiliar as non-competitive for iron. However, the soil contains an abundant ferric form (Fe 3+ ) that is weakly available for plants [89]. Fortunately, some bacteria producing siderophores with high specificity and affinity for iron, can bind, extract and transport iron near the plant roots [90]. B. subtilis PTA-271 genome also predicted the production of such siderophores, namely the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin encoded by 5 genes (dhbA, dhbB, dhbC, dhbE, dhbF: S19-40_01242, S19-40_01245, S19-40_01243, S19-40_01244, S19-40_01246, respectively). Altogether, B. subtilis PTA-271 appears as a good candidate to improve plant iron uptake. Surfactants produced by beneficial bacteria may also contribute to increase the availability of hydrophobic nutrients. In this sense, B. subtilis PTA-271 is suspected to produce surfactin from its identified genes srfAD, srfAC, srfAB and srfAA (S19-40_02068, S19-40_02069, S19-40_02070, S19-40_02071, respectively). Surfactin is a powerful biosurfactant due to its amphiphilic nature that strongly anchor with lipid layers, thus interfering with the structure of biological membranes [91].
Additionally, genes encoding for S-adenosyl-methionine (SAM) decarboxylase (speH, S19-40_01619) and putative SAM-methyltransferase (S19-40_00450) exist in B. subtilis PTA-271 genome and are needed to complete Spd and Spm synthesis from Put. These polyamines (PAs) are known to promote flowering and to play important roles in inducing cell division, promoting regeneration of plant tissues and cell cultures [95], as delaying senescence [96]. Volatile compounds (VOCs) produced by some beneficial rhizospheric bacteria have also been identified as elicitors promoting plant growth.

2.3.-Host protection due to host induced immunity and to Microbiota preservation HOST INDUCED IMMUNITY to prevent biotic stress
Primed defenses during ISR are regulated by phytohormones, depending on either JA and ET signaling or SA signaling [13-15, 21, 23, 27, 32, 111]. Beneficial microorganisms may thus modulate the plant hormonal balance or directly elicit the plant defenses [12,16,23,32]. Literature reports that Bacillus spp. could inhibit ET synthesis and related defense responses by breaking the ET precursor ACC, using an ACC deaminase [17,19,20]. But, no gene encoding for ACC deaminase was detected in B. subtilis PTA-271 genome. In contrast, the metK gene encoding for Sadenosylmethionine (SAM) synthase (S19-40_01774) leading to SAM, the ACC precursor, was identified in B. subtilis PTA-271 genome. By synthesizing the ET precursor SAM, B. subtilis PTA-271 would appear ISR-useful to plants that possess the complementary metabolic machinery for ET synthesis. Genes encoding for PAs are previously cited from B. subtilis PTA-271 genome (speA, speB, speE, speG, speH), and PAs and ET biosynthetic pathways are interrelated from decarboxylated SAM [101]. Although their physiological functions are distinct and at times antagonistic, the balance between the two would enable to manipulate the plant senescence process [102]. SA is another phytohormone for which several genes encoding its metabolic pathways (from synthesis to hydrolysis) are identified in B. subtilis PTA-271 genome, among which pchA encoding for the salicylate biosynthesis isochorismate synthase (S19-40_01801).
DAMP elicitors are products of lytic enzymes (i.e. chitosan, glucans, ….) from microorganisms (either beneficial or pathogenic) that may elicit plant defenses [27,34,111,112]. Genes encoding for lytic enzymes are identified in B. subtilis PTA-271 genome, such as those encoding for chitosanase and ß-glucanase (Supplementary Table S3). Many other genes also encode for lytic enzymes in the spore cortex (Supplementary Table S4) for which the roles remain unclear. No other genes encoding for ISR elicitors such as N-alkylated benzylamine were identified in B. subtilis PTA-271 although described in literature [27, 30-32, 34, 111].

MICROBIOTA QUALITY AND STRENGTHS PRESERVATION
Biologists showed that plant root exudates (i.e. sugars, organic acids, amino acids, lipophilic compounds, etc…), as energy and carbon sources, would enable a plant to selectively recruit some beneficial bacterial subspecies (i.e. biosurfactant producers) and then to modulate its own rhizospheric microbiota composition and its agronomic fitness in turn [113]. Biosurfactant producers such as suspected for B. subtilis PTA-271, as mentioned above, can additionally facilitate biofilm formation and the bioavailability of root exudates, which are both essential for a successful colonization of host-plants [81]. SA was also shown to mediate changes in the composition of root exudates, and in turn in the type of microorganisms recruited by the plant [114] and as indicated above B. subtilis PTA-271 has the genes to produce SA. Altogether, B. subtilis PTA-271 looks to benefit of key levers to influence actively the qualitative plant microbiota.
Bacterial auto-inducers (AI), low-molecular weight signal molecules, also activate the interactive competences of a bacterium in a quorum-sensing (QS) dependent manner. Indeed, efflux pump systems mediate QS-signals at a target concentration of AI, activating the transcription of target genes [115]. The furanosyl-borate-diester (AI-2) is described as universal for interspecies communication both in gram-positive and gram-negative bacteria [116]. Genome analysis of B.
subtilis PTA-271 shows that this bacterium contains the luxS gene (S19-40_01786) responsible for AI-2 production. Another class of AI also produced by Gram-positive bacteria for their intercellular communication is that of oligopeptides or auto-inducing peptide (AIP), consisting of 5-34 amino acids residues such as CSP, EntF, AM373, AD1, F10, PD1, OB1 and EDF [117,118]. Genome analysis of B.
When interacting with a plant, Bacillus species are also exposed to its host defenses that also include reactive oxygen species (ROS) [119]. Genes encoding for resistance to hydroperoxide such as ohrA, ohrB and ohrR (S19-40_00615, S19-40_00613, S19-40_00614) are identified in the genome of B. subtilis PTA-271, supporting a complex system of sensing, protection and regulation of ROS to ensure survival.
Additionally, B. subtilis PTA-271 has the genes to withstand to extreme environment conditions such as nutrient limitation by sporulation (turning on endospore form) [120]. Indeed, endospore is an environmentally resistant cell, metabolic dormant, able to resist extreme temperatures, desiccation and ionizing radiation for thousands of years [121]. Several genes are involved in the sporulation process of B. subtilis PTA-271 (Supplementary Table S4), among which: (1) the spo genes responsible for the control of the sporulation [122], (2) the ger genes responsible for the control of the germination depending on the alleviation of stressful environmental conditions [123], (3) the cot genes involved in the formation of the spore over coating envelope (endospore external layer) [124], and (4) the cw genes encoding for the spore cortex lytic enzymes. The sporulation capacity of B. subtilis PTA-271 represents a great asset for its survival upon extreme environmental conditions over long lasting periods, preserving then the beneficial strengths of this microorganisms for plant profits [3].

HOST INDUCED IMMUNITY to prevent abiotic stress
To exert beneficial effects, a microorganism had to stay metabolically active upon abiotic stress.
Beneficial bacteria need thus to survive abiotic stress such as dehydration, wounding, cold, heat or salinity that in turn lead to a water status regulation. For this end, bacterial species are described to control their intracellular solute pools [125,126]. In this sense, B. subtilis PTA-271 has genes encoding for two potassium uptake proteins KtrA and KtrB (S19-40_01338, S19-40_01337) enabling survival in high salinity environments [125,126]. 13 As previously described upon biotic stress conditions, some phytohormones are also useful for plant defense against abiotic stress, such as abscisic acid (ABA), gibberellins (GA) and ethylene (ET) [127] which precursors are encoded by genes also identified in the genome of B. subtilis PTA-271.
Similarly, and as already mentioned above, ET pathway seems not to be entirely encoded by B. that may also physically interact [130,131]. In the genome of B. subtilis PTA-271, many sigma factors and many TF exist, among which those encoded by ykuD, yciB, slrA, yocK, carD, infA, infB, infC, IF5B, tsf, efp, tuf and fusA genes (Supplementary Table S2). It is noteworthy to understand that useful TF upon abiotic stress could also be useful upon biotic stress. The set of genes under common regulatory controls (i.e. operons) are also listed in the same Supplementary Table S2. As mentioned above, B. subtilis PTA-271 has the genes to produce PAs, known to protect plant cells upon water deficit [132], temperature changes [133] and salinity [134]. Interestingly, the genome of B. subtilis PTA-271 also encodes for genes to detoxify compounds accumulating in the environment, such as the arsenite detoxifying system with arsR (Supplementary Table S2) [136]. B. subtilis PTA-271 genome has also genes that are involved in the degradation of organic pesticides or nitroaromatic compounds by encoding for resistance genes against quaternary ammonium compounds sugE, qacC (S19-40_00985, S19-40_01079) or else against catechol (mhqR, mhqA) (S19-40_00558, S19-40_00645) [137], among others (Supplementary Table   S5).
In addition to the extruding transporters, Bacillus species may also detoxify the pathogen aggressive molecules (i. Beneficial bacteria may also directly target pathogen aggressiveness by using quenching enzymes against the pathogen QS-dependent production of aggressive molecules [142][143]. For that, B. subtilis PTA-271 like other Bacillus species share aiiA encoding for N-acetyl homoserine lactonase hydrolyzing the lactone ring of AHLs (Acyl-homoserine lactones) that would have been useful for the QS production of pathogen virulent factors [46,151]. Looking at B. subtilis PTA-271 genome, genes encoding for quenching enzymes (Supplementary Table S6) may thus produce lactonases, but also βlactamases, deaminases, deacetylases and other (de)acylases. By mean of such quenching enzymes, B. subtilis PTA-271 might contribute to decrease pathogen aggressiveness.
Polyketide synthases (PKS) and other acetyltransferases are also described to produce polyketides (PK) as beneficial molecules. Polyketides are a large group of natural products built from acyl-coenzyme A, essential for bacterial antagonism. Many PK produced by Bacillus are bactericidal agents that play a vital role in controlling plant pathogens [152,153] Table S8).
Among the genes identified in B. subtilis PTA-271 to encode for RP (ribosomally synthesized antimicrobial peptides) and NRP (non-ribosomally synthesized peptides) antimicrobial molecules (Supplementary Table S3) are those known to produce: Baillaene (pksD), subtilosin (sboA, albG, albE, albD, albB, albA) and bacilysin (bacE, bacF, bacG). According to COG categories, 2.30% of B. subtilis PTA-271 genome is devoted to the production of such secondary metabolites, considered as one of the most important features in biocontrol activities. Genes encoding for lipopeptides, as other NRP antimicrobial molecules, are also identified in B. subtilis PTA-271 [41,156,158,160,163]. Among their products, the previously cited elicitors of plant defenses: (1) fengycin is also a powerful antifungal substance described as particularly active against filamentous fungi [157]. It interferes with the integrity of biological membranes until their complete disruption at high concentrations [158]. Fengycin causes structural deformations of the pathogen hyphae, suppressing their proliferation in plant and thus prevent phytotoxins production [159]. (2) Surfactin is another powerful antimicrobial molecule [160] whose encoding gene is identified in B. subtilis PTA-271.
As described above, B. subtilis PTA-271 has also genes encoding for siderophores such as Bacillibactin (Supplementary Table S3), known to deprive pathogen growth of iron while providing it for plant growth [163].

3-B. subtilis PTA-271 GENOME COMPARISON WITH OTHER GENOMES
To understand the magnitude of the differences between B. subtilis PTA-271 and other Bacillus strains, the PTA-271 genome has been compared to the complete genomes of 5 type-strains and 32 non-type strains, represented in Table 6 [172], the DSMZ phylogenomics pipeline to estimate DNA-DNA hybridization (DDH) [172], and the JSpecies WS web server to estimate the Average Nucleotide Identity (ANI) through pairwise comparisons [173]. The DDH value was estimated using the recommended formula (formula two) for draft genomes, at the GGDC website [174]. The ANI values were calculated using Ezbiocloud [175]. The whole data analysis enabled to obtain the intergenomic distances between genomes and their probability of belonging to the same species or subspecies. The general comparison of genomes is reported in Table 6, while the intergenomic distances (DDH estimate and ANI) are shown in Table 7  PTA-271 appears as strength to combat a broad spectrum of plant pathogens (ranging from biotrophs to necrotrophs), and looks especially as highly useful against hemibiotrophs such as those responsible of the complex grapevine trunk diseases, as reported by previous works [9].

DECLARATIONS
Ethics approval and consent to participate: Not applicable.

Consent for publication: All authors approved the final version and consent for publication.
Availability of data and material: The whole genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JACERQ010000000. The version described in this paper is version JACERQ010000000 and all related information is represented in Table 3.

Competing interests:
The authors declare that they have no competing interests.      [56]. Table 3. Bacillus subtilis PTA-271 genomic sequencing information. Table 4. Genome statistics. Table 5. Number of genes associated with general COG functional categories.   Table S1. Bacillus subtilis PTA-271 encoding genes for motility, adhesion and plant root colonizing capacity.