Plant growth-promoting activities and genomic analysis of the stress-resistant Bacillus megaterium STB1, a bacterium of agricultural and biotechnological interest.

In this work, the stress-resistant Bacillus megaterium STB1 is characterized and its ability to promote plant growth under normal and stress conditions is demonstrated. The genomic sequence of this bacterium, and a detailed analysis of the genes involved in facilitating its stress resistance and plant growth-promoting activities is also reported. The B. megaterium STB1 genome is rich in genetic elements involved in multiple stress resistance, xenobiotic degradation, pathogen antagonistic activities, and other traits related to soil and rhizosphere colonization. Moreover, genes participating in the biosynthesis of auxins and cytokinins, the modulation of polyamines, GABA, brassinosteroids and ethylene levels were also found. Ultimately, this study brings new insights into the role of B. megaterium as a plant growth-promoting bacterium and opens new opportunities for the development of novel strategies for agriculture and biotechnology.

Introduction

The increasing demand for food production and environmental clean-up solutions brings new challenges in agricultural and environmental biotechnology, amongst them, the recovery of degraded soils and their conversion to arable lands (bioremediation), as well as the development of sustainable agricultural products to replace the use of chemical fertilizers and pesticides that lead to unacceptable pollution levels worldwide. Beneficial soil and plant-associated bacteria can promote plant growth and stress resistance, hence, constituting a sustainable alternative to the excessive use of chemicals in agriculture [1]. These plant-growth-promoting bacteria (PGPB) can bind to or live inside of plant tissues and facilitate plant growth and protection through a variety of mechanisms, including nitrogen fixation, phosphate solubilization, modulation of plant hormone levels, production of antimicrobial compounds, among others [2].

Bacteria such as Bacillus spp., including Bacillus megaterium, are commonly found in soils and are members of the microbiome of several plant hosts worldwide [3], acting mostly as PGPB [4,5]. These strains effectively colonize soils and plant tissues, and, produce a wide range of bioactive compounds that are involved in plant-growth promotion [6,7] and antiphytopathogen activities [8]. Bacillus species are also a source for a wide range of metabolites and enzymes of biotechnological and industrial interest [9]. Nevertheless, one of the most important characteristics of Bacillus strains is the ability to form spores, thereby, increasing their capability to resist to a wide range of stress conditions [10] and enable their application as PGPB inoculants for agriculture and bioremediation processes [11]. Thus, obtaining spore-forming and stress-resistant Bacillus strains with a high level of plant-growth-promoting abilities is important for the development of novel products to be applied in agricultural/bioremediation setups, especially those subjected to stressful conditions (e.g. high salinity).

Previous studies conducted by our group have led to the isolation of several spore-forming Bacillus spp. strains from stressful environments, amongst them, Bacillus megaterium strain STB1 that was isolated from a rhizospheric soil obtained from a contaminated estuarine environment in Portugal. This strain resisted increased salinity and heavy metal concentrations and had the ability to promote the germination and early growth of several plants. Here, we report a detailed biochemical and genomic characterization of strain B. megaterium STB1, as well as it plant growth-promoting activities. The obtained results bring new insights into B. megaterium genomics and the factors involved in its ability to interact with a host plant and promote its growth.

Materials and methods

Bacillus megaterium STB1 characterization

B. megaterium STB1 was isolated from an estuarine soil collected from the Mitrena area (Setúbal, Portugal) (38°30′24″N 8°48′47″W) which contains industries producing paper and chemical fertilizers, among other pollutants, which over time has resulted in the contamination of the surface waters and soils of this area. The bacterium was maintained in 20 % glycerol stocks at -80 °C and cultured in Tryptic Soy Agar (TSA) and Broth (TSB), at 28 °C with 150 rpm shaking, whenever necessary.

The strain was tested for the ability to produce indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, protease, lipase, esterase, amylase, cellulase, catalase, siderophores, ammonia, acetoin, H2S, nitrate/nitrite reduction, motility and to solubilize phosphate as previously described [12]. Phytohormone degradation abilities were tested according to the methodology described by Nascimento et al. [13].

Tomato plant-growth promoting assay under normal and salt stress conditions

Commercial tomato (Solanum lycopersicum cv. super marmande) seeds were disinfected using 70 % ethanol (1 min), 1 % sodium hypochlorite solution (3 min) and five washes with sterile distilled water to remove any traces of the former solutions. Seeds were pre-germinated on 1 % agar plates at 25 °C. After germination, one seedling was placed in each pot (volume of 300 mL) containing a non-sterilized red clay soil (pH in H2O – 5.3; organic matter 15.62 %; P and K – 0.49 mg.kg and 0.13 cmolc.kg respectively; Ca, Mg, Al and H+Al,– 0.74, 0.43, 1.00, and 1.36 cmolc.kg, respectively), obtained from Florianópolis, Brazil. The seedlings were inoculated with 2.5 ml of a STB1 bacterial inoculum (OD600 = 0.5) obtained following the growth of strain STB1 in TSB medium at 28°ºC for 72 h with shaking at 150 rpm. The non-inoculated controls received 2.5 ml of sterile TSB medium. The experiment consisted on four treatments: normal conditions, non-inoculated control; normal conditions, inoculation with STB1; salt stress conditions, non-inoculated control; salt stress conditions inoculation with STB1. The study followed a completely randomized design with 10 replications. All pots received Hoagland and Arnon [14] nutrient solution (containing 200 mM NaCl, in the case of stress treatments) every three days. The experiment was conducted under greenhouse conditions (average temperature of 23.3 °C) in the Universidade Federal de Santa Catarina, Florianópolis, Brazil. The plants were harvested 35 days after inoculation, roots and shoots were excised from one another and dried at 60 °C for 3 days, before dry weights were measured on an analytical scale.

Statistical analysis was conducted with SPSS Statistics v.22 software (SPSS Inc., IBM Company, USA), using ANOVA and the means were compared using Tukey’s test.

Genome sequencing and analysis

Strain STB1 genome sequencing was conducted following the methodology described by Urón et al. [15]. Briefly, the genomic DNA library was constructed using Illumina TruSeq DNA Nano kit (automated), and sequenced in the Illumina MiSeq platform using the MiSeq V3 reagent kit (2 × 300 bp). A total of 2,272,938 reads were obtained and assembled using the SOAPdenovo v2.04 software [16]. The final genome sequence of B. megaterium STB1 was constructed based on a guided assembly against the complete genome sequence of Bacillus megaterium ATCC 14581 (CP009920.1) using MAUVE 2.4.0 progressive alignments [17]. A chromosome and one plasmid were assembled. The chromosome of strain STB1 (5,002,401 bp) is a scaffold of 13 contigs (N50 = 1,566,135 bp), which were joined by introducing 100 Ns in the assembly gap regions as indicated in the NCBI submission guidelines. Plasmid pSTB1A results from one contig (118,698 bp) containing plasmid replication genes and a lower GC%. The genome annotation was performed using the NCBI Prokaryotic annotation pipeline [18]. The final near-complete genome sequence of B. megaterium STB1 is available in the NCBI database under the accession number CP025700-1.

Functional genome annotation was performed using BlastKOALA [19]. Genomic islands were predicted in IslandViewer [20]. CAZyme analysis was executed in the dbCAN website using HMMER3 (hmmscan) [21]; antibiotic and secondary metabolite analysis were performed in antiSMASH [22].

Sequences described in the manuscript and supporting information were verified by BLASTp searches (default parameters) against the UniProt/SwissProt database using the Geneious software v9.1 (https://www.geneious.com). Genomic average nucleotide identity (ANI) was calculated using OrthoANiu (https://www.ezbiocloud.net/tools/ani) [23]. Genome circular views were created using CGview [24].

Results and discussion

General characterization of Bacillus megaterium STB1

Bacillus megaterium STB1 is a Gram-positive, pale yellow, sporulating bacterium, able to grow from 7° to 45 °C (maximum tested) and in the presence of up to 5 % NaCl (maximum tested). It is a catalase positive and motile bacterium, able to use glucose and lactose as carbon sources, but non-fermentative. It does not produce acetoin nor H2S, and a nitrate/nitrite reduction test was negative. This bacterium produced several extracellular enzymes including protease, cellulase and amylase, but not lipase or esterase under the tested conditions. Strain STB1 produced ammonia and siderophores, however, it was not able to solubilize phosphate or zinc. IAA or IAA-like compounds [25] were found in the supernatant of strain STB1 (17.01 ± 0.1 μg/ml) when the bacterium was grown in TSB medium supplemented with 500 μg/ml tryptophan. This strain catabolized 4-aminobutyrate (GABA) but was not able to degrade the plant hormones ACC, IAA, SA, N6-isopentenyladenine, kinetin, jasmonic acid and abscisic acid.

Bacillus megaterium STB1 promotes tomato growth under normal and salt-stress conditions

B. megaterium STB1 significantly promoted tomato plant growth under both normal and salt stress conditions, leading to increased root, shoot and leaf development (Fig. 1A–C). The inoculation of tomato seedlings with B. megaterium STB1 resulted in a significantly ∼3.5-fold increase in the root (32 vs 108 mg), and shoot dry weight (49.9 vs 187.4 mg), which resulted in an expressive increase in tomato total dry biomass (81.9 vs. 295.8 mg) when compared to the non-inoculated control, under normal conditions (Fig. 1A and C). Similar results were observed under stress conditions, where tomato seedlings inoculated with B. megaterium STB1 presented a significantly increased root (22.2 vs 83.8 mg) and shoot dry weight (50 vs 238.5 mg), and consequently, total dry biomass (72.2 vs. 268.6 mg) when compared to non-inoculated plants (Fig. 1B and C).

Fig. 1

Results obtained from the tomato plant growth promotion assay under normal and salt stress conditions (200 mM NaCl), 35 days after inoculation. A) control conditions; B) salt stress conditions; C) representative plants from each treatment.

NI- non-inoculated; STB1- inoculation with Bacillus megaterium STB1; RDW- Root Dry Weight; SDW- Shoot Dry Weight; TB- Total Biomass.

*represents significant statistical differences (p<0.05).

This data indicates a potentially significant agricultural impact of B. megaterium STB1, which is consistent with previous reports demonstrating the increased plant growth promoting abilities of several other B. megaterium strains, including induced plant protection against salt stress [[26], [27], [28]].

Bacillus megaterium STB1 genome general characteristics

The genome of Bacillus megaterium STB1 includes a single circular chromosome of approximately 5 Mbp with an average GC content of 38.2 %, and plasmid pSTB1A of approximately 118 Kb with an average GC content of 33.7 % (Fig. 2).

Fig. 2

Schematic representation of Bacillus megaterium STB1 genome.

DNA sequencing predicted a total of 5271 open reading frames (ORFs) in the genome, in which 5195 correspond to putative protein coding sequences (CDS) and 88 to ribosomal RNA and tRNA genes.

BlastKoala analysis resulted in the functional annotation of 2513 from a total of 5195 CDS (48.4 %). Environmental (609) and genetic (590) information processing functions were assigned for most of the annotated CDS, followed by carbohydrate (299) and amino acid (295) metabolism.

DbCAN analysis indicated a total of 37 proteins predicted to belong to the Glycoside Hydrolase (GH) family, 36 to Glycosyl Transferases (GT), 29 to Carbohydrate Esterases (CE), 10 to Carbohydrate Binding Modules (CBM) and 7 to Auxiliary Activities (AA).

The complete elements for sec (secretion system), tat (twin arginine) and a type VII secretion system were identified and represent the major secretion systems found in STB1 (Table S1).

Phylogenomics

Phylogenetic analysis based on the partial 16S rRNA sequences of all described Bacillus species showed that strain STB1 clusters closely to the B. megaterium ATCC 14581 type strain (data not shown). To confirm this result, genomic average nucleotide identity (ANI) analysis were conducted. The analysis showed that strain STB1 genome presents a high ANI (>95 %, which is the cut-off value for defining bacteria belonging to the same species) to the genomes of other B. megaterium strains: QM B1551 (97.23 %); DSM 329 (97.20 %); ATCC 14581 (97.12 %), hence, indicating that strain STB1 belongs to the B. megaterium species.

Genetic elements involved in B. megaterium STB1 stress resistance, soil and plant colonization abilities

Osmotic stress tolerance

B. megaterium STB1 genome analysis revealed the presence of several genes that are involved in osmotic stress tolerance, including: sodium and chloride transporters, the osmoprotectant transport system, osmolyte biosynthesis and transport (e.g. glycine-betaine, proline, glutamate, glutamine), and, membrane integrity and protection mechanisms (e.g. cardiolipin, lipoteichoic acid production, fatty acid desaturases) (Table S2). The abundance of genetic elements involved in osmotic stress tolerance in the strain STB1 genome is in agreement with the functional data showing that strain STB1 tolerates high salinity concentrations, and previous reports on the increased salinity tolerance of other B. megaterium strains, many of which are used as inoculants for increasing salt tolerance in various crops [27,28].

Temperature stress resistance and spore formation

The genome of B. megaterium STB1 contains several cold shock (csp) and heat shock (hsp) proteins, as well as other chaperone proteins involved in the response to temperature stress (Table S3). The STB1 genome harbors six cspA, three cspB, four cspC genes and a cspE gene, which are all involved in the stabilization of DNA and RNA, and, consequently, impact the transcription and translation efficiency under cold stress [29], as well as other stress conditions [30]. In addition, the genome contains several chaperones involved in heat stress response including dnaJ, dnaK, groES, groEL, as well as other several small hsp genes [31].

A total of 172 genes predicted to be involved in the several phases of spore formation and spore germination processes were detected in the STB1 genome (Table S4). This number is similar to those observed in other B. megaterium strains (165 spore-related proteins in B. megaterium QM B1551; 157 spore-related proteins in B. megaterium DSM 319; 174 spore-related proteins in B. megaterium JX285).

Heavy metals resistance

Heavy metal transport/resistance genes are abundant in the B. megaterium STB1 genome (Table S5). These include several transport genes (for zinc, cobalt, copper, cadmium, manganese, nickel, arsenate, chromate, and fluoride) as well as genes encoding arsenate and chromate reductases (three arsC genes, and one chrR gene). One of the arsenate reductase genes is present in a cluster (arsADCBRB, C0569_25700-25725) that also contains several arsenate transport/resistance genes and is found on the plasmid pSTB1A (Table S5). BLASTn analysis revealed that this cluster is rare, and close homologs were only found in the B. weihaiensis Alg07 chromosome and the B. oceanisediminis 2691 plasmid pBO1. Interestingly, B. megaterium STB1, B. weihaiensis Alg07 and B. oceanisediminis 2691 were all isolated from similar marine/estuarine environments.

The large number of heavy metal resistance genes in the B. megaterium STB1 genome suggests that this strain is able to deal with high levels of heavy metals, which is consistent with previous reports demonstrating the uptake and heavy metal resistance of several B. megaterium strains [[32], [33], [34]].

Oxidative and nitrosative stress resistance

The B. megaterium STB1 genome contains multiple genes involved in the oxidative stress response, including two Cu-Zn superoxide dismutase genes, two Fe-Mn superoxide dismutase genes, three catalase genes, two organic hydroperoxide resistance genes and single copies of genes encoding thiol peroxidase, heme-dependent peroxidase, glutathione peroxidase, peroxiredoxin and a non-heme chloroperoxidase (Table S6). This collection of genes indicates a strong adaptation and resistance to reactive oxygen species (ROS), which are commonly produced and accumulated by bacteria under stressful conditions and are also known to be involved in the plant defense response.

Furthermore, the genome contains a farnesyl diphosphate synthase (ispA) gene and three gene clusters involved in carotenoid synthesis (Table S6). The carotenoid cluster 1 (C0569_00970-00985) encodes hydroxyneurosporene desaturase (C0569_00970) and 4,4′-diaponeurosporenoate glycosyltransferase (C0569_00975) homologs, as well as an acyltransferase (C0569_00980) and bisanhydrobacterioruberin hydratase CruF-like (C0569_00985) domain-containing proteins. Cluster 2 (C0569_14990-15000) encodes a diapophytoene synthase, a diapophytoene desaturase and a diapophytofluene desaturase homolog, all presenting high identity (97.5 %, 99 %, 98.8 %) to the B. megaterium DSM319 enzymes involved in the synthesis of the C30 carotenoids, 4,4′-diapophytoene and 4,4′-diaponeurosporene [35]. Cluster 3 (C0569_21280-90) encodes a 4,4′-diapolycopene oxygenase (C0569_21280), 4,4′-diaponeurosporenoate glycosyltransferase (C0569_21285) and glycosyl-4,4′-diaponeurosporenoate acyltransferase (C0569_21290) homologs. Accordingly, the sequence data suggests that B. megaterium STB1 is able to produce 4,4′-diapophytoene and 4,4′-diaponeurosporene, as well as modified versions of these compounds, all of which play a protective role in the response against oxidative stress.

Two nitric oxide dioxygenase genes were also identified in the genome (Table S6) and may account for strain STB1 resistance to nitrosative stress.

Metabolism of xenobiotics

B. megaterium strains are known for their ability to resist and degrade several xenobiotic compounds including herbicides, pesticides, nitro compounds, azo dyes, barbiturates, epoxides, steroids, and other toxic compounds [[36], [37], [38], [39], [40], [41]]. Not surprisingly, genomic analysis revealed several genes encoding enzymes involved in xenobiotic degradation in B. megaterium STB1 (Table S7), including two nitroreductases (C0569_01050; C0569_01380) presenting high identity (99.6 %) to B. megaterium Mes11 NfrA1 and NfrA2 nitroreductases, respectively, involved in the degradation of the herbicide mesotrione and other nitro compounds [36]; an epoxide hydrolase (C0569_00825) sharing 98.3 % identity to the B. megaterium ECU1001 epoxide hydrolase involved in the degradation of α-naphthyl glycidyl ether and the production of optically active epoxides [39]; an aryldialkylphosphatase (C0569_03085) with 51 % identity to Sulfolobus solfataricus MT4 thermostable phosphotriesterase involved in the degradation of organo-phosphorus insecticides [42]; an alkyl/aryl-sulfatase (C0569_23470) participating in the degradation of sodium dodecyl sulfate and 4-nitrocatechol; a nitronate monooxygenase involved in the degradation of toxic nitronates; five azoreductases involved in the degradation of aromatic azo compounds; and a catechol-2,3-dioxygenase (C0569_05375) involved in the degradation of catechol, a common metabolite in several aromatic compound degradation pathways.

In addition, the genome of strain STB1 contains several cytochrome p450 encoding genes (Table S7), including a cytochrome P450/NADPH--P450 reductase homolog (C0569_02245) (98.1 % identity to B. megaterium ATCC 14581 CYP102A1) involved in fatty acid hydroxylation [43]; a CYP109E1 homolog (C0569_04895) (98.5 % identity to B. megaterium DSM 319 CYP109E1) involved in the hydroxylation of steroids, vitamin D3 (cholecalciferol), cholesterol, statins and terpenes [[44], [45], [46],47]; a CYP106A1 homolog (C0569_20645) (95.4 % identity to B. megaterium DSM 319 CYP106A1) involved in the hydroxylation of the pentacyclic triterpene 11-keto-β-boswellic acid and several steroids [48,49]; a CYP109A2 homolog (C0569_21565) (97.3 % identity to B. megaterium DSM 319 CYP109A2) involved in vitamin D3 hydroxylation [50]; and a unknown cytochrome P450 (C0569_21365) with 96 % identity to a B. megaterium DSM 319 WP_080514752.1 cytochrome P450.

Nitrogen, sulfur and phosphorus acquisition

B. megaterium STB1 possesses nitrate and nitrite reductase genes, as well as several genes involved in nitrate/nitrite transport (Table S8). Additionally, urea degradation (urease) and transport genes, and three ammonium transporter genes (amtB) were found in the genome (Table S8).

Genes involved in the assimilatory sulfate reduction pathway, and sulfate transport (cysPUWA, ylnA, sulP) were detected (Table S8), as well as sulfonate transport and degradation genes (ssuABCD). Three ssuD genes, encoding alkanesulfonate monooxygenase, were identified, suggesting that strain STB1 actively obtains sulfur sources (sulfite) by degrading sulfonates. The STB1 genome also contains two dimethyl-sulfide (DMS) monooxygenase (dmoA) gene homologs involved in the degradation of DMS; and a dimethylsulfone monooxygenase (sfnG) gene homolog involved in the DMS degradation pathway. Curiously, DMS is volatile compound typically found in marine and estuarine environments and is also produced by plants.

Phosphate transport system genes, as well as the alkaline phosphatase genes phoA and phoD are present in the genome of strain STB1 (Table S8), and account for its main organic phosphorus acquisition abilities.

Iron acquisition

The B. megaterium STB1 genome contains several genes involved in iron transport (Table S9), as well as in the biosynthesis of a siderophore (C0569_05785-5815), with the putative siderophore biosynthesis genes being somewhat homologous to the rhizobactin biosynthesis genes (rhaABCDEF) involved in the production of the hydroxamate-type siderophore rhizobactin from Sinohizobium meliloti 1021. Moreover, BLASTn analysis revealed that the siderophore cluster (C0569_05785-5815) is highly conserved amongst B. megaterium strains, including B. megaterium ATCC 19213 (98 % identity), that is known to produce the hydroxamate-type siderophores schizokinen and N-deoxyschizokinen [51]. This analysis also indicated that the B. megaterium ATCC 19213 genome contains only one siderophore biosynthesis gene cluster. Hence, the data suggests that the B. megaterium STB1 siderophore gene cluster (C0569_05785-5815), also found in most B. megaterium strains, is likely involved in the production of schizokinen and N-deoxyschizokinen. Additionally, a gene cluster encoding a siderophore transport system was found in B. megaterium STB1 and contains the yusV, yfhA, yfiZ and yfiY gene homologs, which have been shown to play a role in B. subtilis schizokenin transport [52].

Competition- antagonistic activities

Genomic analysis revealed the presence of several gene clusters involved in the production of antimicrobial compounds, including a gene cluster involved in phosphonate biosynthesis, a cluster containing a type III polyketide synthase gene, and a bacteriocin/lantipeptide gene cluster (Table S10).

The phosphonate biosynthesis cluster found in the B. megaterium STB1 chromosome contains at least six genes, including phosphonopyruvate mutase (pepM), phosphonopyruvate decarboxylase and 2-aminoethylphosphonate-pyruvate transaminase gene homologs. Curiously, this gene cluster also contains two genes encoding proteins related to endospore coat formation as well as peptidoglycan biosynthesis (Mur-like, catalytic domain superfamily), suggesting that it may be involved in the production of a phosphonoglycan. BLAST analysis revealed that this cluster is only found in B. megaterium strains, thus, suggesting that this is a species-specific trait.

Bacterial type III polyketide synthases have been linked to the production of phenolic lipids such as alkylresorcinols and alkylpyrones, possessing antimicrobial activity [53]. A gene encoding a type III polyketide synthase from the chalcone/stilbene synthase family (C0569_19065), and an adjacent isoprenylcysteine carboxyl methyltransferase gene (C0569_19070) were found in the genome of strain STB1. The encoded proteins are somewhat homologous to B. subtilis 168 BpsA and BpsB proteins (52.4 % and 45.3 % identity, respectively) which are involved in the production of several alkylresorcinols and alkylpyrones [54]. BLASTp analysis revealed that C0569_19065-70 homologs are commonly found in other B. megaterium strains indicating that this is a common trait in this species.

A bacteriocin/lantipeptide gene cluster was found in strain STB1 in plasmid pSTB1A. The cluster consists of an operon containing at least ten genes, three of which are involved in lantipeptide biosynthesis while the others are involved in lantipeptide/bacteriocin modification and transport (Table S10). The C0569_25480 gene encodes a type 2 lantipeptide synthetase LanM, and genes C0569_25490-95 encode Type 2 lantibiotic, SP_1948 family proteins. BLAST analysis indicates that the LanM homolog C0569_25480 is rare and close homologs are only found in five other sequenced B. megaterium strains, namely, strains AFS083949, AFS069568, AFS058476 (obtained from corn), AFS013177 (grass) and CHCC20162 (soil), hence, suggesting that this is a new class of lantipeptide. Interestingly, most of these strains were found associated with plants, suggesting a role for this product in the associative lifestyle with a plant host.

Altogether, these results are in agreement with reports indicating the antagonistic activities and production of antifungal and antibacterial compounds by B. megaterium strains [5,[55], [56], [57]].

Motility, chemotaxis and attachment to plant surfaces

The flagella biosynthesis and regulation operons were detected in the genome (Table S11), as well as, several genes involved in gas vesicle formation (gvp). Gas vesicles are intracellular hollow organelles commonly found in aquatic bacteria, which allow buoyancy and enable cells to move upwards in liquid to access oxygen [58]. Strain STB1 was isolated from an estuarine environment, so it is possible that it produces gas vesicles in order to survive a periodic aquatic environment. Nevertheless, the gvp genes are found in most B. megaterium strains [9,58], indicating that this is a species-specific trait.

Several chemotaxis genes, such as cheABYWR and motAB, five methyl-accepting chemotaxis proteins and a heme-based aerotactic transducer HemAT homolog were detected (Table S11). Moreover, The B. megaterium STB1 genome contains multiple genes involved in exopolysaccharide production, including capsule biosynthesis genes, which may facilitate bacterial attachment to plant roots. In fact, two clusters containing polysaccharide biosynthesis genes were found (Table S11). In addition, several genes involved in the production of teichoic acids, which play a role in biofilm formation and root colonization [59] were also identified (Table S3).

Utilization of root exudates

PGPB typically thrive in the rhizosphere, the portion of the soil near to plant roots that is directly influenced by plant exuded compounds. Root exudates differ amongst plants but are mainly composed by sugars, organic acids, amino acids, peptides, proteins, phenolics and flavonoids [60]. The genome of B. megaterium STB1 is rich in genetic elements involved in the catabolism/conversion of carbohydrates and responsible for the main carbon cycling pathways (Table S12). Genes encoding proteins involved in the degradation of fructose, ribose, arabinose, lactose, galactose, mannose, GDP-mannose, sucrose, trehalose, raffinose, stachyose, cellobiose and other hexoses, sugar acids (gluconate and several of its keto forms, galactonate, lactate, galacturonate, tagaturonate and altronate), sugar alcohols (myo-inositol, mannitol) and complex sugars (cellulose, starch, glycogen) were found in the genome (Table S12). Additionally, several glucosidase encoding genes are present in the genome and may account for the catabolism of several alpha and beta-glucosides (Table S12). Genes encoding proteins related to sugar transport systems, which are vital in carbohydrate acquisition, were also abundantly found in the STB1 genome (Table S13).

The genetic elements encoding the tricarboxylic acid (TCA) and glyoxylate shunt pathways, including succinate, fumarate, malate and citrate degradation/conversion genes are present in the genome (Table S14), as well as genes encoding enzymes involved in the degradation of acetate, formate, malonate and tartrate, thus, suggesting the ability of this bacterium to degrade and use a wide range of organic acids as carbon sources. Moreover, three phenolic acid decarboxylase and a polyphenol oxidase encoding gene were also detected (Table S14) and may account for this bacterium ability to catabolize several phenolic compounds. The phenolic acid decarboxylase encoded by C0569_13590 presents high identity (88.2 %) to B. subtilis 168 enzyme, PadC, involved in the decarboxylation of ferulic, p-coumaric and caffeic acids [61]. The genes C0569_24570 and C0569_24565 encode enzymes with a high homology to B. subtilis 168 BsdCD enzymes (63 % and 81.4 % identity, respectively) involved in the decarboxylation and detoxification of phenolic derivatives such as 4-hydroxybenzoate and vanillate [62].

Amino acid transport and degradation genes are abundant in the genome (Table S15) and several peptide transport and degradation genes were also identified (Table S16), including those encoding aminopeptidases, dipeptidases, oligoendopeptidases and proteases, such as bacillolysin (C0569_22935).

Genes encoding cytochrome p450 enzymes, including a CYP102A1 homolog (C0569_02245) that is known to hydroxylate resveratrol [63], are found in the genome, suggesting that strain STB1 also possesses the ability to modify/catabolize plant flavonoids.

Genetic elements involved in B. megaterium STB1 plant growth promotion activities

IAA biosynthesis

Several Bacillus strains, including B. megaterium STB1, are known to produce auxins, such as IAA, that play an important role in plant growth promotion and plant-microbe interactions [64]. Recently, Shao et al. [6] proposed that IAA production in B. amyloliquefaciens SQR9a occurs via a tryptophan-dependent indole-3-pyruvate (IPyA) pathway. Similarly, the genome sequence of B. megaterium STB1 harbors genetic elements consistent with the production of IAA via the IPyA pathway (Table S17). These include several aminotransferase genes, including patB, which may be involved in the conversion of tryptophan to IPyA; pyruvate oxidase, pyruvate decarboxylase and several phenolic acid decarboxylase genes, including a yclB homolog (C0569_24575), which encodes an enzyme involved in the conversion of IPyA to indole-3-acetaldehyde (IAAld); and aldehyde dehydrogenase genes, such as dhaS, encoding an enzyme responsible for the conversion of IAAld to IAA [6].

Curiously, the STB1 genome harbors a gene encoding an amidase and with 44.5 % identity to Arabidopsis thaliana amidase 1 enzyme, involved in the conversion of indole-3-acetamide (IAM) to IAA [65]. This suggests that B. megaterium STB1 is also able to produce IAA via the IAM pathway. In this regard, it is not unusual for bacteria that synthesize IAA to do so by more than a single pathway [64].

Cytokinin biosynthesis

Elements involved in the production of cytokinins, such as, the miaA (C0569_06005) and miaB (C0569_06035) genes encoding the tRNA dimethylallyltransferase and tRNA-2-methylthio-N6-dimethylallyladenosine synthase enzymes involved in the production of N6-(dimethylallyl)adenosine (iPR) and 2-methylthio-N6-(dimethylallyl)adenosine (2MeSiPR), respectively; the yvdD gene (C0569_21570) encoding a Lonely Guy (LOG) family protein (cytokinin riboside 5′-monophosphate phosphoribohydrolase) involved in the hydrolytic removal of ribose 5′-monophosphate from nitrogen N6-modified adenosines; and a bifunctional cytochrome P450/NADPH--cytochrome P450 reductase gene (C0569_02245) with homology to A. thaliana cytokinin hydroxylase involved in the biosynthesis of trans-zeatin, were found in the STB1 genome (Table S17). These results suggest that B. megaterium STB1 is able to synthesize several cytokinins, which is consistent with previous reports demonstrating cytokinin production by many Bacillus species and its important role in Bacillus (including B. megaterium) plant growth promotion abilities [[66], [67], [68]].

Polyamine production and modulation of ethylene levels

Polyamines such as putrescine, spermine and spermidine have an important role in the plant-growth promoting abilities of Bacillus strains, including B. megaterium [69,70]. Furthermore, spermidine production by B. subtilis has been shown to decrease the activity of the tobacco ACC oxidase gene (ACO1) responsible for ethylene production, thereby lowering plant ethylene levels that affect a range of plant-microbe interactions [69,71]. Several polyamine metabolism and transport genes are found in the genome of B. megaterium STB1, including, a speB gene encoding agmatinase (involved in putrescine production), four speE genes encoding spermidine synthase and several polyamine transport genes (potABCD, pupP) (Table S17), consistent with an important role for polyamine metabolism in B. megaterium STB1 plant growth promotion abilities.

GABA metabolism

Under stress conditions plants produce and accumulate GABA [72]. However, high concentrations of GABA can have deleterious effects on cell elongation and overall plant stress resistance [73,74]. GABA metabolism genes are abundantly found in B. megaterium STB1 (Table S17). In this sense, strain STB1 possesses the gadB gene encoding glutamate decarboxylase involved in GABA production, as well as, four gabT and three gabD genes, encoding GABA aminotransferase and succinate-semialdehyde dehydrogenase involved in GABA degradation. Moreover, strain STB1 possess two gabP genes, encoding GABA permease, involved in GABA uptake. These observations are consistent with the possibility that B. megaterium STB1 acquires and uses GABA as a nutrient source in the rhizosphere and, consequently, modulates plant GABA levels. Interestingly, plants defective in the ability to degrade GABA are often hypersensitive to salt stress [74], thus, B. megaterium STB1 GABA modulation abilities may be linked to its ability to induce salt stress tolerance in tomato.

Modulation of plant brassinosteroids levels by cytochromes p450

A recent study by Asari and colleagues [67] showed that B. amyloliquefaciens UCMB5113 inoculation of the roots of A. thaliana led to the modification of plant brassinosteroids levels. In this sense, brassinolide, homocastasterone, castasterone, and teasterone levels were all increased in UCMB5113-inoculated plants. Interestingly, B. megaterium and other Bacillus species metabolize/modify steroids via cytochrome P450 enzymes. These results are consistent with the possibility that B. megaterium STB1, possessing several cytochrome p450 (Table S7) involved in steroid metabolism, may be able to metabolize/modify brassinosteroids that are involved in plant growth regulation.

Plant growth promotion activities by modulation of VOCs

Bacillus strains are known to produce several VOCs that positively impact plant growth and defense responses [7]. The main production of bacterial VOCs results from nitrogen, sulfur, carbohydrate, amino acid, fatty acid, ketone and alcohol metabolism [75] and genes/enzymes involved in these pathways are commonly found in the B. megaterium STB1 genome (Table S8, S12, S14, S15, S18), thus, suggesting a high level of VOCs production ability by this strain.

In addition, genomic analysis revealed the presence of several genes/pathways involved in the synthesis and degradation of VOCs such as acetoin, butanediol, DMS, 1-3-propanediol and nitric oxide (Table S19), that are known to directly impact plant development [7,76].

Conclusions

B. megaterium STB1 is a versatile and stress-resistant PGPB with great potential for agriculture and biotechnology. Its genome sequence revealed the presence of multiple genes involved in important functions regarding soil and plant colonization, plant growth promotion and other relevant features of biotechnological interest (e.g. osmotic, temperature, oxidative, nitrosative and heavy metal stress resistance; carbohydrate, organic acid, amino acid, phenolic acids and xenobiotic, including steroids, terpenoids, herbicides, insecticides, nitronates, metabolism; lytic activities and antagonistic activities mediated by lytic enzymes, bacteriocins, phosphonates; VOCs modulation and multiple plant hormone biosynthesis/modulation abilities). Ultimately, the data obtained in this study brings new insights into the genomic properties governing the successful beneficial interactions between important soil microorganisms like B. megaterium and plants, and their potential use in the development of novel sustainable agricultural and biotechnological applications.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author contributions

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.