1.- B. subtilis PTA-271 genome Properties AND comparison with other Bacillus strains
1.1.- General features of the genome
The general features of B. subtilis PTA-271 are in Table 4 and Figure 1, performed using Artemis version 16.0.0. The draft genome sequence of B. subtilis PTA-271 presented an estimated genome size of 4,001,755 bp divided in 20 contigs. The G + C content of this sequence was 1,751,999 bp, representing about 43.78% of the whole genome. Genome analysis showed that B. subtilis PTA-271 contained 4,038 genes, among which 3,945 (97.69%) were protein coding genes. This genome draft predicts 92 RNA genes among which 11 rRNA genes were identified and no CRISPR repeats. From 4,001,755 bp of the genome size, 3,550,299 bp correspond to coding genes representing 88.73% of the whole genome. From this, 3,440 genes had function prediction, 3,183 were assigned to COG categories described in Table 5, and 3,517 genes had Pfam domain descriptions.
1.2.- Insights
According to Table 5, the majority of the proteins in B. subtilis PTA-271 genome are Proteins not assigned in COG’s that represented 19.31% (762) of the whole genome, Amino acid transport & metabolism that represented 8.31% (328), Transcription (313) and Carbohydrate transport & metabolism (313) that represented 7.93% of the genome. Two biocontrol-useful-categories in B. subtilis PTA-271 genome are (1) Secondary metabolites biosynthesis, transport and catabolism, representing 2.30% (91) of the genome, and (2) Other defense mechanisms encoding proteins relevant for plant-bacteria interactions, representing 1.49% (59) of the whole genome sequence.
2.- B. subtilis PTA-271 ASSETs 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 (chemotaxis). In this sense, B. subtilis PTA-271 contains genes (Supplementary Table S1) encoding for (i) flagella maintenance, such as flhF, flhA, flhB, flgC, flgB, fliE, fliF and fliG, and (ii) chemotaxis, such as cheY, cheD, cheW, cheA and cheB. 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.
B. subtilis spp. are also described for their strong swarming motility [35]. 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 functional flagella, pili and surfactant to reduce surface tension.
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 [36]. 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 and to repress AbrB (S19-40_03988), described as a negative regulator of biofilm formation [37].
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 [4]. Some authors consider that (1) all of the microbial genera described as common inhabitants of the rhizosphere are also endophytics [38] and that (2) whatever their localization, beneficial microorganisms that successfully colonize the plant, particularly by the root system, would be advantageous both for plant growth promotion and for plant biocontrol [39].
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 non-competitive plant partner 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 [42-43]. 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 [44]. Bacterial secondary metabolites (i.e. PyrroloQuinoline Quinone, PQQ) are also known to control gluconic acid production [45], 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) [46]. 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 [47]. Iron is another very important nutrient for plant growth and development. B. subtilis PTA-271 possesses the fur gene (Supplementary Table S2) that encodes for a ferric uptake regulatory protein coordinating the homeostasis of iron uptake depending on its availability in soil [48]. B. subtilis PTA-271 appears thus as a good non-competitive plant auxiliar for iron. However, the soil contains an abundant ferric form (Fe3+) that is weakly available for plants. Fortunately, some bacteria producing siderophores with high specificity and affinity for iron, can bind, extract and transport iron near the plant roots [49]. 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 [50].
Plant root morphology is also described to impact nutrient uptake and thus plant growth thanks to the stimulation of lateral root formation and root air formation, while primary root elongation is inhibited [51-52]. Plant hormone productions (i.e. auxins, cytokinins, gibberellins) are key elements for root morphology changes. Some beneficial bacteria seem able to produce some of them, including B. subtilis PTA-271. This latter has genes encoding for tryptophan, the main precursor of the auxin IAA (indole-3-acetic acid), namely from the trp group, trpA (S19-40_02736), trpB (S19-40_02737), trpC (S19-40_02739), trpD (S19-40_02740), trpE (S19-40_02741), trpF (S19-40_02738), trpP (S19-40_02553), trpR (S19-40_03152) and trpS (S19-40_02410). Once synthesized, bacterial IAA has two main functions: (i) increase the plant root surface and length for a deeper soil prospecting capacity and nutrient acquiring capacity and (ii) release the cell walls of rootlets to facilitate molecule exudations and benefit to rhizospheric bacteria [42]. B. subtilis PTA-271 has also genes that encode for cytokinin synthesis such as yvdD (Supplementary Table S2), known as a plant growth regulator (i.e. cell division, organogenesis) in combination with IAA. Gibberellins (GA) produced by some bacteria may also affect the plant growth and survival by interfering with the plant signaling pathways through secondary metabolites changes [52]. GA pathways is not fully encoded by B. subtilis PTA-271 which only contains ispD linked to 2-C-methyl-D-erythritol 4-phosphate (MEP) and GerC3_HepT linked to geranylgeranyl diphosphate (GGPP) production (as indicated below), two successive precursors of GA and ABA synthesis in plants. But from GGPP, no genes were detected for the kaurene pathway required to complement GA synthesis in B. subtilis PTA-271 genome.
Genes encoding for some plant growth regulators are also presents in B. subtilis PTA-271 genome, such as speA (S19-40_00456) encoding for arginine decarboxylase (ADC), speB (S19-40_00673) encoding for agmatinase (leading to putrescine, Put), speG (S19-40_00166) encoding for spermidine synthase (Spd) and speE (S19-40_00672) encoding for spermine synthase (Spm). 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 [53], as delaying senescence [54].
Volatile compounds (VOCs) produced by some beneficial rhizospheric bacteria have also been identified as elicitors promoting plant growth. Those suspected to be produced by B. subtilis PTA-271 are (1) acetoin which producing pathway is known to be encoded by acuA (S19-40_01690) and acuC (S19-40_01692) among others genes encoding for acetoin utilization proteins, and (2) 2,3-butanediol known to be produced by butA and butC (encoding for S19-40_03395 and S19-40_00056, respectively) [20,55]. VOCs are especially reported to interact with some of the previously cited plant hormones (i.e. auxins, ethylene, among others) [56-58].
2.3.- Plant protection due to Induced defenses and to Microbiota preservation
PLANt induced DEFENSES upon biotic stress
Primed defenses during ISR are regulated by phytohormones, depending on either JA and ET signaling or SA signaling [7-8,10,59]. Beneficial microorganisms may thus modulate the plant hormonal balance or directly elicit the plant defenses. Literature reports that Bacillus spp. could inhibit ET synthesis and related defense responses by breaking the ET precursor ACC, using an ACC deaminase [15]. But, no gene encoding for ACC deaminase was detected in B. subtilis PTA-271 genome. In contrast, the metK gene encoding for S-adenosylmethionine (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 cited above for B. subtilis PTA-271 genome (speA, speB, speE, speG, speH), and PAs and ET biosynthetic pathways are interrelated from decarboxylated SAM [60]. Although their physiological functions are distinct and at times antagonistic, the balance between the two would enable to manipulate the plant senescence process [54]. 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).
Many other elicitors also induced host immunity, coming from microorganisms (MAMPs, microbial associated molecular patterns) but also from the plant host (DAMPs, damaged associated molecular patterns). MAMPs can act from the external surface of a beneficial microorganism (i.e. flagellin) or result from a secretion outside or inside the host (i.e. surfactin, fengycin, NO, acetoin, 2-butanone, phthalic acid methyl ester). In B. subtilis PTA-271, hag gene encodes for flagellin protein from bacterial flagellum (Supplementary Table S1) often recognized by plant pattern recognition receptors (PRRs) normally cell surface localized receptor kinases or LRR-RLP proteins, such as FLS2 and EF-Tu described to activate host defenses through mitogen-activated protein kinase cascades (MAPK) [40,61]. Lipopeptides are other elicitors encoded by genes identified in the genome of B. subtilis PTA-271, such as surfactin and fengycin. Alkalanization of host extracellular medium by surfactin provokes ions -influx and -efflux activating in turn systemic host defenses through intracellular changes of signaling compounds, then the production of antimicrobicidal phenolic compounds [62]. Fengycin encoded by fenA, fenB, fenC and fenD (S19-40_00076, S19-40_00077, S19-40_00073, S19-40_00074) in B. subtilis PTA-271 is also known to induce the production of plant phenolics compounds [62]. VOCs produced by rhizospheric bacteria, such as the previously cited 3-hydroxy 2-butanone and acetoin, are also well known to induce ISR through SA-independent pathway, but merely through the ET one that remains to be deeply investigated [57]. No other genes encoding for other VOC elicitors such as the phthalic acid methyl ester were identified in B. subtilis PTA-271, in contrast with B. subtilis IAGS174 described by Akram et al. (2015) [13]. Among inorganic volatile compound (VIC), the ubiquitous nitric oxide (NO) is a signal molecule scavenging reactive oxygen species (ROS) and regulating the level of PAs and hormonal balance (i.e. ABA versus SA) to reprogram or switch plant development upon stress [63]. Different genes related to NO metabolic pathways are found in B. subtilis PTA-271 genome, among which the gene nos encoding for a NO synthase oxygenase (S19-40_03258). Many other elicitors are additionally encoded by the genome of B. subtilis PTA-271 such as those cited above (i.e. siderophores, iron, flagella) and those cited below (i.e. N-acyl-L-homoserine lactone). Maybe their beneficial effect on plant vigor and their detrimental effect on pathogen fitness are the contributors to host protection. Exopolysaccharides (EPS) and lipopolysaccharides (LPS) are also reported as elicitors in several Bacillus genera [9-14,59]. Among the EPS encoding genes identified in B. subtilis PTA-271 are S19-40_00800, S19-40_00870, S19-40_00999, S19-40_01009, S19-40_01427. Among the LPS encoding genes identified in B. subtilis PTA-271 are lptB, lapA, lapB (S19-40_01170, S19-40_01479, S19-40_03936).
DAMP elicitors are products of lytic enzymes (i.e. chitosan, glucans, ….) from microorganisms (either beneficial or pathogenic) that may elicit plant defenses [61]. 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.
PLANt induced DEFENSES upon abiotic stress
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) [8] which precursors are encoded by genes also identified in the genome of B. subtilis PTA-271. Indeed, the identified GerC3_HepT encodes for GGPP synthase (S19-40_02907) and pcrB encodes for geranylgeranylglyceryl phosphate synthase (S19-40_03154), GGPP being a common precursor of GA- and ABA- synthesis [64]. Upstream of GGPP, MEP is another common precursor of GA- and ABA- synthesis and two ispD genes were found to encode for cytoplasmic MEP cytidylyltransferases (S19-40_00851 and S19-40_03933) in B. subtilis PTA-271 genome. Additionally, ispF encodes for a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (S19-40_03932) and ispE for a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (S19-40_03980). From GGPP, the kaurene pathway may lead to GA, while the phytoene path may lead to ABA and in the genome of B. subtilis PTA-271, yisP (a crtb KEGG gene) encodes for a 15-cis-phytoene/all-trans-phytoene synthase (S19-40_02475). Similarly, and as mentioned above, ET pathway seems not to be entirely encoded by B. subtilis PTA-271 genome from which was only identified the metK gene enabling to produce SAM, a precursor of ACC required for ET synthesis in plants. Altogether these data indicate that B. subtilis PTA-271 genome may encode for key precursors of phytohormones that may influence actively ABA and ET contents in plants. In plants, ABA, GA and ET signaling pathways may interfere altogether through different transcription factors (TF) or small proteins (i.e. GiD, DELLA, EIN, ERF, ABI, XERICO, …) that may also physically interact [65-66]. 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.
PAs such as those encoded by the genome of B. subtilis PTA-271 are also described to protect plant cells upon water deficit [67], temperature changes [68] and salinity [69]. They are known to increase the activity of various antioxidant enzymes in plants and may contribute to produce H2O2 as a signaling molecule that can activate plant antioxidant defense responses [69].
Microbiota quality and 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 [70]. Biosurfactant producers such as suspected for B. subtilis PTA-271 (i.e. surfactin), can additionally facilitate biofilm formation and the bioavailability of root exudates, which are both essential for a successful colonization of host-plants [41]. 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 [19] 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- microbiome/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 [71]. The furanosyl-borate-diester (AI-2) is described as universal for interspecies communication both in gram-positive and gram-negative bacteria [72]. 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 [73]. Genome analysis of B. subtilis PTA-271 shows that this bacterium may encode for the AIP precursors EntF (S19-40_01246) and AM373 (S19-40_03157).
When interacting with a plant, Bacillus species are also exposed to its host defenses that also include reactive oxygen species (ROS) [74]. 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.
Upon abiotic stress, a microorganism had also to stay metabolically active to exert beneficial effects. 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 [75-76]. 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.
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) [77]. B. subtilis PTA-271 genome has also genes that are involved in the degradation of organic pesticides or nitroaromatic compounds [78] 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), among others (Supplementary Table S5).
Finally, B. subtilis PTA-271 has genes to withstand the extreme environment conditions such as nutrient limitation by sporulation (turning on endospore form) [79]. Indeed, endospore is an environmentally resistant cell, metabolic dormant, able to resist extreme temperatures, desiccation and ionizing radiation for thousands of years [1]. 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, (2) the ger genes responsible for the control of the germination depending on the alleviation of stressful environmental conditions, (3) the cot genes involved in the formation of the spore over coating envelope (endospore external layer), 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 microorganism for plant profits.
2.4.- Direct confrontation with pathogens or aggressive molecules
Upon direct confrontation, Bacillus species need first to protect themselves against antimicrobial attacks from the other aggressive species that may also compete for resources [80]. As mentioned, B. subtilis PTA-271 has antimicrobial resistance genes encoding for efflux pump systems to detoxify several types of drugs such as pathogen’s antibiotic and ROS (i.e. hydroperoxide), as for the previously cited compounds accumulating in the environment (i.e. quaternary ammonium compounds, catechol and arsenate). Efflux pump systems also allow bacteria to adjust their internal environment by using diverse transporters mediating drug extrusion from the cell, whether specific to a substance or a group of substances. Some specific transporters are encoded by the genome of B. subtilis PTA-271, as for the resistance proteins against: (1) tetracyclin (S19-40_01293, S19-40_01359, S19-40_01919) encoded by tetA, tetR, tetD; (2) fosfomycin (S19-40_00125) encoded by fosB; (3) erythromycin (S19-40_03633 and S19-40_03632) encoded by msrA and msrB, respectively; (4) bacillibactin (S19-40_00235) encoded by ymfD; (5) bacitracin (S19-40_01756, S19-40_01755 and S19-40_00770) encoded by BceA, BceB and BcrC; (6) bleomycin (S19-40_01406) encoded by ble; (7) riboflavin (S19-40_03749, S19-40_01917) encoded by ribZ and rfnT, among many others. Non-specific transporters are also designated as multidrug transporters, such as those encoded in B. subtilis PTA-271 genome by mepA (S19-40_00070, S19-40_03635), ebrA (S19-40_00188) and ebrB (S19-40_00189), ykkD (S19-40_00619) and ykkC (S19-40_00620), bmrA (S19-40_00951) and bmr3 (S19-40_01151), emrY (S19-40_02033), among others.
In addition to the extruding transporters, Bacillus species may also detoxify the pathogen aggressive molecules (i.e. toxins) by the mean of antitoxins or detoxifying enzymes coming from ubiquitous multigenic families of proteins such as the transferases and CYP450 [81-82]. In B. subtilis PTA-271, the main transferase encoding genes are for glutathione-S-transferases GST, malonyl-transferases MT, glucosyl-transferases GT and many others as indicated in the Supplementary Table S5. Among B. subtilis PTA-271, CYP450 encoding genes are those for mono-oxygenases and dioxygenases as indicated in the Supplementary Table S5. By mean of such detoxifying systems, B. subtilis PTA-271 might thus contribute to decrease pathogen aggressiveness. Beneficial bacteria may also directly target pathogen aggressiveness by using quenching enzymes against the pathogen QS-dependent production of aggressive molecules [8,83]. 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. Looking at B. subtilis PTA-271 genome, genes encoding for quenching enzymes (Supplementary Table S6) may thus produce lactonases, but also β-lactamases, 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 [84-85]. Regarding B. subtilis PTA-271 genome (Supplementary Table S7), 15 genes encode for PKS and many others for acetyltransferases or share similar part of the PKS functions. By mean of PKS, B. subtilis PTA-271 might contribute to antagonize pathogens. According to antiSMASH 5.1.0, B. subtilis PTA-271 genome contains 11 secondary metabolites gene clusters, among which: 1 polyketide synthase cluster (PKS) and 1 hybrid PKS-NRPS cluster (Supplementary Table S8).
Additional genes encoding for an extensive range of beneficial molecules produced by Bacillus spp. are also identified in B. subtilis PTA-271 (Supplementary Table S3), such as those encoding for antimicrobial molecules or effectors (i.e. antibiotics, surfactants, hydrogen cyanide …), chelators (i.e. siderophores) and lytic enzymes (i.e. chitosanases, glucanases, cellulases, proteases, chitinases) able to directly alter pathogen fitness and aggressiveness [20,86]. 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 [50,87]. 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. It interferes with the integrity of biological membranes until their complete disruption at high concentrations. Fengycin causes structural deformations of the pathogen hyphae, suppressing their proliferation in plant and thus prevent phytotoxins production. (2) Surfactin is another powerful antimicrobial molecule whose encoding gene is identified in B. subtilis PTA-271.
Aside from these secondary metabolites, B. subtilis PTA-271 has also genes encoding for uncommon antimicrobials volatile compounds either inorganic (VIC) or organic (VOC), such as: (1) 1 VIC: hydrogen cyanide (HCN) encoded by hcnC (S19-40_01178) to antagonize a pathogen. As a potent inhibitor of cytochrome C oxidase and several other metalloenzymes, HCN is extremely toxic to aerobic microorganisms at very low concentrations [8]. (2) The 2 previously reported VOC as elicitors acetoin and 2,3-butanediol are also well known to work as weapons against some pathogens [20,55]. According to antiSMASH 5.1.0, B. subtilis PTA-271 genome contains 11 secondary metabolites gene clusters, among which: 3 NRPS clusters and 2 RiPPs clusters (Supplementary Table S8).
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 [88].
Lytic enzymes (CWDE) such as cellulases, proteases, chitinases, glucanases, are other important feature of Bacillus spp. that may both alter pathogen fitness and produce DAMPs. Concerning the CWDE encoding genes in B. subtilis PTA-271 genome, are found: 1 chitosanase encoded by csn, 1 β-glucanase encoded by bglS, 1 β-glucanase / cellulase (S19-40_00094) encoded by eglS, and about 80 proteases (Supplementary Table S3). These enzymes are considered as powerful fungicides since they are responsible for the degradation of key structural components of fungal cell walls [89].
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. Type-strains are living culture organisms descending from strains designated as “nomenclatural types”, according to the International Code of Nomenclature of Prokaryotes [90]. Among them are the type strains B. subtilis NCIB 3610, B. subtilis 168, B. subtilis 9407, B. amyloliquefaciens subsp. plantarum strain FZB42, and B. velezensis KTCT 13012. Among non-type strains showing ≥99% of thr 16S ribosomal gene similarity with PTA-271 are 31 distinct strains of B. subtilis and 1 Bacillus velezensis. For this genomic comparison, we used the GGDC 2.1 web server [91], the DSMZ phylogenomics pipeline to estimate DNA-DNA hybridization (DDH) [91], and the JSpecies WS web server to estimate the Average Nucleotide Identity (ANI) through pairwise comparisons [92]. The DDH value was estimated using the recommended formula (formula two) for draft genomes, at the GGDC website [93]. The ANI values were calculated using Ezbiocloud [94]. 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.
Among the type strain genomes, the closer strain to B. subtilis PTA-271 was B. subtilis 9407, with a 0.0104 distance, a DDH estimate of 91.60%, and an ANIm of 99.02%. As expected, the most distant strain was B. velezensis KTCT 13012, with a 0.2268 distance, a DDH estimate of 19.40% and a 0% probability of being the same species, corroborated with an ANIm percentage of 77.02%. Concerning the non-type strain genomes, the closer strains to PTA-271 were B. subtilis QB5413, B. subtilis SRCM 104005, and B. subtilis QB61 with distances of 0.0112, 0.0119 and 0.0119 respectively, and DDH estimates of 90.90%, 90.20% and 90.20% respectively. The most distant strains was B. velezensis strain ATR2, with a distance of 0.2144 and a DDH estimate of 20.50% corroborated with an ANIm percentage of 77.1%. The most distant B. subtilis strain to PTA-271 was B. subtilis subsp. subtilis RO-NN-1 with a distance of 0.203 and a DDH of 82.60%.