Bacillus velezensis: A Valuable Member of Bioactive Molecules within Plant Microbiomes

Bacillus velezensis is an aerobic, gram-positive, endospore-forming bacterium that promotes plant growth. Numerous strains of this species have been reported to suppress the growth of microbial pathogens, including bacteria, fungi, and nematodes. Based on recent phylogenetic analysis, several Bacillus species have been reclassified as B. velezensis. However, this information has yet to be integrated into a well-organized resource. Genomic analysis has revealed that B. velezensis possesses strain-specific clusters of genes related to the biosynthesis of secondary metabolites, which play significant roles in both pathogen suppression and plant growth promotion. More specifically, B. velezensis exhibits a high genetic capacity for synthesizing cyclic lipopeptides (i.e., surfactin, bacillomycin-D, fengycin, and bacillibactin) and polyketides (i.e., macrolactin, bacillaene, and difficidin). Secondary metabolites produced by B. velezensis can also trigger induced systemic resistance in plants, a process by which plants defend themselves against recurrent attacks by virulent microorganisms. This is the first study to integrate previously published information about the Bacillus species, newly reclassified as B. velezensis, and their beneficial metabolites (i.e., siderophore, bacteriocins, and volatile organic compounds).

Bacillus species are considered important PGPR, producing a vast array of biologically active secondary metabolites that can potentially inhibit the growth of plant pathogens and deleterious rhizospheric microorganisms [5]. Bacillus spp. are preferred in agricultural systems, due to their ability to form endospores, which can survive to heat exposure and desiccation, and capacity to be formulated into stable dry powders with long shelf lives [6]. Furthermore, because Bacillus spp. are already common inhabitants of plant root microflora, Bacillus spore-based biocontrol agents have little, if any,   The taxonomic statuses of 66 closely related B. amyloliquefaciens strains were assessed by comparing complete RNA polymerase beta-subunit (rpoB) sequences and core genome sequences [23]. The strains were clustered into a single clade, i.e., the "B. amyloliquefaciens operational group", which contains three tightly linked branches: (1) B. amyloliquefaciens subsp. amyloliquefaciens, (2) B. siamensis, and (3) B. velezensis, a taxon that includes all the strains previously classified as B. velezensis, B. methylotrophicus, and B. amyloliquefaciens subsp. plantarum (Figure 1). This review focuses on B. velezensis, which includes a variety of previously reported strains, namely, B. amyloliquefaciens subsp. plantarum FZB42 [22], B. amyloliquefaciens FR203A [24], B. amyloliquefaciens SQR9 [25], B. amyloliquefaciens NJN-6 [26], B. amyloliquefaciens SQRT3 [27], B. methylotrophicus KACC 13105 T [22], B. velezensis CR-502 T and CR-14b [16], and B. subtilis GB03 [28]. Based on rpoB gene analysis and other analyses, these taxa are synonymous with B. velezensis and are each capable of suppressing pathogens. A phylogenetic tree inferred from the type strains of species from the "B. subtilis species complex" is presented in Figure 2. According to phylogenomic analysis, Bacillus species synonymous with B. velezensis were clustered into clades consisting of B. amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, and B. methylotrophicus.  [22], B. velezensis CR-502 T and CR-14b [16], and B. subtilis GB03 [28]. Based on rpoB gene analysis and other analyses, these taxa are synonymous with B. velezensis and are each capable of suppressing pathogens. A phylogenetic tree inferred from the type strains of species from the "B. subtilis species complex" is presented in Figure 2. According to phylogenomic analysis, Bacillus species synonymous with B. velezensis were clustered into clades consisting of B. amyloliquefaciens, B. amyloliquefaciens subsp. plantarum, and B. methylotrophicus.

Bioactive Molecules Synthesized by B. velezensis
In 2007, B. amyloliquefaciens FZB42 was reported as the first gram-positive biocontrol bacteria to have its genome sequenced [15]. The strain harbors an array of nine giant gene clusters that function to produce a spectrum of bioactive secondary metabolites ( Figure 3) by modularly organized mega-enzymes, known as nonribosomal peptide synthetases and polyketide synthases ( Table 1). Five of these nine gene clusters (i.e., srf, bmy, fen, nrs, and dhb; 137 kb) are involved in synthesizing cyclic lipopeptides molecules, such as surfactin, bacillomycin-D, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. Meanwhile, three other gene clusters (i.e., mln, bae, and dfn; 199 kb) were reported to direct the synthesis of antibacterial polyketides, such as macrolactin, bacillaene, and difficidin, and the last gene cluster (bac; 6.9 kb) was reported to direct the synthesis and export of the antibacterial dipeptide bacilysin [29]. Altogether, about 10% (340 kb) of the B. amyloliquefaciens FZB42 genome is dedicated to the nonribosomal synthesis of lipopeptide and polyketide-type antimicrobial molecules, siderophores, and bacteriocins [29]. Closely related Bacillus species are also capable of synthesizing bioactive metabolites that exhibit activity against a wide range of microorganisms ( Table 2).

Bioactive Molecules Synthesized by B. velezensis
In 2007, B. amyloliquefaciens FZB42 was reported as the first gram-positive biocontrol bacteria to have its genome sequenced [15]. The strain harbors an array of nine giant gene clusters that function to produce a spectrum of bioactive secondary metabolites ( Figure 3) by modularly organized mega-enzymes, known as nonribosomal peptide synthetases and polyketide synthases ( Table 1). Five of these nine gene clusters (i.e., srf, bmy, fen, nrs, and dhb; 137 kb) are involved in synthesizing cyclic lipopeptides molecules, such as surfactin, bacillomycin-D, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. Meanwhile, three other gene clusters (i.e., mln, bae, and dfn; 199 kb) were reported to direct the synthesis of antibacterial polyketides, such as macrolactin, bacillaene, and difficidin, and the last gene cluster (bac; 6.9 kb) was reported to direct the synthesis and export of the antibacterial dipeptide bacilysin [29]. Altogether, about 10% (340 kb) of the B. amyloliquefaciens FZB42 genome is dedicated to the nonribosomal synthesis of lipopeptide and polyketide-type antimicrobial molecules, siderophores, and bacteriocins [29]. Closely related Bacillus species are also capable of synthesizing bioactive metabolites that exhibit activity against a wide range of microorganisms ( Table 2).

Antibacterial Molecules
Difficidin and bacilysin are the most effective antibacterial agents produced by B. amyloliquefaciens FZB42 [7]. Little is known about the antibacterial properties of macrolactin and bacillaene [30]. B. amyloliquefaciens FZB42 exerts biocontrol activity by synthesizing difficidin and bacilysin, which facilitates the control of several economically important rice diseases, such as bacterial blight and bacterial leaf streak, which are caused by Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola, respectively [7]. Erwinia amylovora, the causative agent of fire blight disease, can also be effectively controlled by B. amyloliquefaciens FZB42 [31]. However, a mutant strain of B. amyloliquefaciens FZB42 that produces difficidin, but not macrolactin or bacillaene, was reported to exhibit equal or slightly higher activity against E. amylovora than the wild-type FZB42 strain. Bacilysin, one of the antibacterial molecules produced by B. amyloliquefaciens FZB42, is reported to exert an inhibitory effect against E. amylovora [31]. Bacilysin, also possesses anticyanobacterial activity against the harmful alga Microcystis aeruginosa, with a killing rate of 98.78%, and can, therefore, be used as a targeted biocontrol agent [32]. Meanwhile, bacillomycin-D and fengycin from B. velezensis might play redundant roles in defense mechanisms against Ralstonia solanacearum, an etiological agent of tomato wilting [33]. The expression of lipopeptide biosynthesis genes (srfAB, ituC, and fenD for the synthesis of surfactin, iturin, and fengycin, respectively) was greatly induced in co-cultures of B. velezensis and pathogens, such as R. solanacearum [33].
Bacteriocins are ribosomally synthesized peptidic toxins that are synthesized by bacteria. In the plant rhizosphere, such toxins may be produced to kill neighboring pathogenic microbes and nematodes, usually in response to environmental stresses [34,35]. The first known bacteriocin, colicin, was isolated from Escherichia coli by Gratia in 1925 [36]. Recent genomic analysis of B. amyloliquefaciens FZB42 revealed ribosomally encoded gene clusters for plantazolicin, a novel antibacterial and nematicidal agent [37], and amylocyclicin, an antibiotic (Table 1) [38]. Plantazolicin is synthesized by a cluster of 12 genes, which span nearly 10 kb of the B. amyloliquefaciens FZB42 genome [37], and amylocyclicin is a highly hydrophobic cyclic peptide that is synthesized by a cluster of six genes, which span nearly 4.5 kb, and are involved in the compound's production, modification, exportation, and self-immunization [38].

Antifungal Molecules
The inoculation of lettuce seeds with commercial B. amyloliquefaciens FZB42 can reduce the severity of bottom rot disease caused by Rhizoctonia solani [20]. Ultra-performance liquid chromatography coupled with mass spectrometry suggested that this suppression might be due to the presence of cyclic lipopeptide molecules (i.e., surfactin, bacillomycin-D, and fengycin) in the lettuce root rhizosphere [20].
B. amyloliquefaciens FZB42 has been reported to exhibit antagonistic interactions with Fusarium graminearum, a plant-pathogenic fungus that threatens the production and quality of wheat and barley worldwide [39]. The antifungal activity exerted by B. amyloliquefaciens FZB42 has primarily been attributed to the nonribosomal synthesis of lipopeptide compounds [40]. For example, a bacillomycin-D deficient mutant strain of B. amyloliquefaciens FZB42 exhibited severely impaired antifungal activities, thereby suggesting that bacillomycin-D contributes significantly to the antifungal action of B. amyloliquefaciens FZB42 [29]. A double mutant of B. amyloliquefaciens FZB42 that was deficient in both bacillomycin-D and fengycin (∆bmyA ∆fenA) was heavily impaired in its ability to inhibit the growth of F. oxysporum, thereby indicating synergistic effects among such lipopeptides against target organisms [41]. Bacillomycin-D also induces morphological changes in the plasma membranes and cell walls of F. graminearum hyphae and conidia [39].

Nematocidal Molecules
Plant-parasitic nematodes cause serious damage to many commercially important crops throughout the world [42]. Rhizospheric microorganisms control parasitic nematodes by secreting a variety of metabolites, enzymes, and toxins that suppress nematode reproduction, hatching, and juvenile survival [43]. A variety of nematophagous microbes, including Bacillus spp., have been reported to possess nematicidal activity [44,45]. For example, treating tomato seedlings with B. amyloliquefaciens FZB42 reduced numbers of nematode eggs in tomato roots and of juvenile worms in soil and suppressed the incidence of tomato plant galls [44]. The nematicidal effect exerted by strain FZB42 has been attributed to plantazolicin, a novel compound encoded by the pzn gene cluster [45]. Culture filtrates of B. amyloliquefaciens FR203A have also been used as biocontrol agents for control of the nematode Xiphinema index, a pest of grape crops in Chile [24].

Siderophore Production
Iron is essential for growth in all living organisms, and most organisms depend on iron as a cofactor for important biochemical processes, including oxygen binding, electron transport, and catalysis [46]. In B. amyloliquefaciens FZB42, nonribosomal peptide synthetases are involved in the synthesis of siderophore bacillibactin [15], which play an important role in facilitating the acquisition of ferric ions (Fe 3+ ) from minerals and organic compounds in the rhizosphere [46]. The binding of siderophores with environmentally free ferric ion facilitates the formation of siderophore-iron complexes that are transported back into bacterial cells through specific receptors (i.e., siderophore binding proteins) in the cell membrane. In gram-positive bacteria, siderophore-binding proteins, siderophore-permeases, and ATPases are involved in the transport of siderophore-iron complexes into the cytoplasm [46], where the ferric ions are reduced to ferrous (Fe 2+ ) ions, thereby becoming available for microbial growth [47]. Rhizospheric B. amyloliquefaciens FZB42, which produces high concentrations of the siderophore bacillibactin, inhibits the growth of phytopathogenic bacterial and fungal competitors by depriving them of essential iron ions [15].

Production of Volatile Organic Compounds (VOCs)
VOCs are a complex mixture of low-molecular-weight, odorous, lipophilic compounds that are usually produced by plants and microorganisms [48]. Plant-and soil-associated microorganisms that produce VOCs have been reported to suppress virulent microbes, thereby indicating their potential as biocontrol agents against plant diseases [49]. Indeed, the VOCs released by B. amyloliquefaciens FZB42 possess antimicrobial activity and also promote plant growth and systemic resistance [50], whereas the VOCs, acetoin, and 2,3-butanediol, in particular, released by B. subtilis GB03 stimulate ISR in Arabidopsis seedlings and reduce the severity of disease in seedlings challenged with the soft rot pathogen E. carotovora subsp. carotovora [28,51].
The VOCs produced by B. amyloliquefaciens NJN-6 also inhibit fungal growth by suppressing mycelial growth and spore germination in F. oxysporum [26]. In fact, F. oxysporum growth was inhibited by approximately 30-40% [26]. In a similar study, a combination of 22 VOCs produced by B. amyloliquefaciens SQR9 were found to inhibit the growth of the tomato wilt pathogen R. solanacearum by 70% [52], and proteomic analysis indicated that the VOCs also affected pathogen virulence by downregulating catalase and superoxide dismutase activities. The SQR9 VOCs also reduced motility, biofilm formation, tomato root colonization by R. solanacearum, thereby clearly demonstrating the importance of VOCs in controlling pathogenic microbes [52].

Stimulation of Induced Systemic Resistance (ISR) by B. velezensis
The application of PGPR to seeds or seedlings can stimulate ISR in the treated plants upon recognition of pathogens (Figure 4). The term ISR was first coined by van Peer et al. [62] to describe the resistance of carnation plants, in which the root had been previously treated with the rhizobacterium Pseudomonas WCS417r, against F. oxysporum f. sp. dianthi. The process of ISR depends on the recognition of PGPR-secreted elicitors, such as lipopolysaccharides, peptidoglycans, flagellin, quorum-sensing molecules, cyclic lipopeptides, and iron chelating siderophores [63,64].
It is now recognized that PGPR induce ISR in plants through the jasmonic acid/ethylene (JA/ET) signaling pathways [5,65] (Figure 4) and that the cellular defense responses of plants include oxidative bursts, cell-wall reinforcement, the accumulation of defense-related enzymes, and the production of antimicrobial phytoalexins [66]. The well-characterized JA/ET responsive genes of Arabidopsis include LOX (lipoxygenase), VSP (vegetative storage protein), PDF1.2 (plant defensin factor 1.2), Hel (hevein), CHI (chitinase), and PAL (phenylalanine ammonia lyase) [67]. Interactions between PGPR and host plants can also activate both the JA/ET and salicylic acid signaling pathways, which are intertwined molecularly through their reliance on a functional version of NPR1 (non-expressor of PR1), a gene that encodes a defense-related regulatory protein (Figure 4). For example, the PGPR B. cereus AR156 induces the simultaneous expression of the JA/ET-responsive marker gene PDF1.2 and the salicylic acid-responsive marker genes PR1, PR2, and PR5 in the leaves of Arabidopsis thaliana when exposed to the pathogen P. syringae pv. tomato DC3000 [68]. Surfactin and other nonribosomally synthesized secondary metabolites that are produced by B. amyloliquefaciens FZB42 have been reported to enhance plant defense responses in the root rhizosphere [20]. To examine the role of surfactin in the regulation of plant defense responses against R. solani, lettuce seedlings were bacterized with B. amyloliquefaciens FZB42 and two mutant strains of B. amyloliquefaciens FZB42, namely, CH1 (surfactin deficient) and CH5 (lipopeptide and polyketide deficient). Quantitative real-time PCR analysis indicated that PDF1.2 was upregulated in the FZB42-bacterized plants in presence of R. solani but not in plants bacterized with the mutant strains [20].
In another study, a soil drench that contained both B. amyloliquefaciens SQRT3 and the pathogen R. solanacearum upregulated the expression of several defense-related marker genes (i.e., Pin2, PR-1a, It is now recognized that PGPR induce ISR in plants through the jasmonic acid/ethylene (JA/ET) signaling pathways [5,65] (Figure 4) and that the cellular defense responses of plants include oxidative bursts, cell-wall reinforcement, the accumulation of defense-related enzymes, and the production of antimicrobial phytoalexins [66]. The well-characterized JA/ET responsive genes of Arabidopsis include LOX (lipoxygenase), VSP (vegetative storage protein), PDF1.2 (plant defensin factor 1.2), Hel (hevein), CHI (chitinase), and PAL (phenylalanine ammonia lyase) [67]. Interactions between PGPR and host plants can also activate both the JA/ET and salicylic acid signaling pathways, which are intertwined molecularly through their reliance on a functional version of NPR1 (non-expressor of PR1), a gene that encodes a defense-related regulatory protein (Figure 4). For example, the PGPR B. cereus AR156 induces the simultaneous expression of the JA/ET-responsive marker gene PDF1.2 and the salicylic acid-responsive marker genes PR1, PR2, and PR5 in the leaves of Arabidopsis thaliana when exposed to the pathogen P. syringae pv. tomato DC3000 [68].
Surfactin and other nonribosomally synthesized secondary metabolites that are produced by B. amyloliquefaciens FZB42 have been reported to enhance plant defense responses in the root rhizosphere [20]. To examine the role of surfactin in the regulation of plant defense responses against R. solani, lettuce seedlings were bacterized with B. amyloliquefaciens FZB42 and two mutant strains of B. amyloliquefaciens FZB42, namely, CH1 (surfactin deficient) and CH5 (lipopeptide and polyketide deficient). Quantitative real-time PCR analysis indicated that PDF1.2 was upregulated in the FZB42-bacterized plants in presence of R. solani but not in plants bacterized with the mutant strains [20].
In another study, a soil drench that contained both B. amyloliquefaciens SQRT3 and the pathogen R. solanacearum upregulated the expression of several defense-related marker genes (i.e., Pin2, PR-1a, and Osmotin-like) in tomato leaves over that observed in the control plants (drenched with R. solanacearum only). B. amyloliquefaciens SQRT3-mediated ISR was also reported to involve JA, salicylic acid, and ET-dependent signaling pathways and to reduce tomato bacterial wilt by 68.1% [27].

Biofilm Formation by B. velezensis
In the rhizosphere, plants create environments that are nutritionally and physicochemically beneficial for root microflora by continuously releasing a variety of organic molecules. Some of these molecules function as chemical signals that attract motile bacteria to move towards the root surface (i.e., chemotaxis), thereby favoring the formation of biofilms [5], which are aggregations of cells that live on either liquid or solid surfaces in a sticky, self-produced matrix of hydrated extracellular polymeric substances. Such extracellular polymeric substances are composed of polysaccharides, proteins, nucleic acids, and lipids and mainly function to facilitate cell stability, adhesion, cohesion, interconnection, and transient immobilization of cells in a biofilm [69].
The formation of biofilms in plant rhizospheres can promote plant growth and protect plants from infectious microbes, both through the secretion of antimicrobial compounds and through systemic resistance. For example, when A. thaliana is infected with P. syringae, the plant secretes malic acid in order to recruit rhizospheric bacteria and, consequently, to enhance Bacillus biofilm formation, thereby promoting immunity against phytopathogenic microbes [70]. The formation of biofilm by B. amyloliquefaciens SQR9 in liquid culture could be regulated by maize root exudates that contained glucose, citric acid, and fumaric acid, and transcriptional profiling of the SQR9 strain revealed that the maize root exudates activated the expression of genes related to extracellular matrix production. Several genes related to fengycin, bacillibactin, and bacilysin synthesis were also upregulated as a result of biofilm formation, thereby providing further evidence of the beneficial role of SQR9 in the maize rhizosphere [25]. As the global transcription regulator, AbrB has been shown to negatively regulate chemotaxis and biofilm formation in Bacillus [25], and the disruption of abrB in B. amyloliquefaciens SQR9 was reported to significantly increase biofilm formation and biocontrol ability [71]. When compared with control seedlings, symptoms of Fusarium wilt were reduced to 50% in B. amyloliquefaciens SQR9-treated cucumber seedlings, and to as low as 20% in seedlings that had been treated with the abrB mutant of B. amyloliquefaciens SQR9 [71].
These results suggest that B. velezensis can also reduce the severity of plant diseases by forming biofilms and that the disease-controlling capacity of the strain can be improved by deleting genes that negatively regulate chemotaxis and biofilm formation.

Conclusions and Future Prospects
Increased use of chemical fertilizers and pesticides has resulted in the accumulation of residual chemical compounds in the environment, and pathogenic microorganisms are starting to develop resistance. To circumvent these undesirable effects, it is of utmost importance to use biological agents, such as bio-fertilizers and bio-pesticides. Among the closely related Bacillus species, B. velezensis is attracting attention as a valuable biocontrol agent. Accordingly, in order to develop and formulate bio-based products, it is increasingly important to understand the antimicrobial potential of biosynthesis of B. velezensis. Furthermore, the elucidation of genes responsible for bioactive secondary metabolites and the ability to control such genes are additional important steps for increasing the production of metabolites by beneficial microbes and for facilitating metabolic engineering. B. velezensis may represent a practical and powerful biocontrol agent that can be used as an effective alternative to synthetic agro-chemicals.