Bacillus megaterium HgT21: a Promising Metal Multiresistant Plant Growth-Promoting Bacteria for Soil Biorestoration

ABSTRACT

The environmental deterioration produced by heavy metals derived from anthropogenic activities has gradually increased. The worldwide dissemination of toxic metals in crop soils represents a threat for sustainability and biosafety in agriculture and requires strategies for the recovery of metal-polluted crop soils. The biorestoration of metal-polluted soils using technologies that combine plants and microorganisms has gained attention in recent decades due to the beneficial and synergistic effects produced by its biotic interactions. In this context, native and heavy metal-resistant plant growth-promoting bacteria (PGPB) play a crucial role in the development of strategies for sustainable biorestoration of metal-contaminated soils. In this study, we present a genomic analysis and characterization of the rhizospheric bacterium Bacillus megaterium HgT21 isolated from metal-polluted soil from Zacatecas, Mexico. The results reveal that this autochthonous bacterium contains an important set of genes related to a variety of operons associated with mercury, arsenic, copper, cobalt, cadmium, zinc and aluminum resistance. Additionally, halotolerance-, beta-lactam resistance-, phosphate solubilization-, and plant growth-promotion-related genes were identified. The analysis of resistance to metal ions revealed resistance to mercury (Hg^II+), arsenate [AsO₄]³^–, cobalt (Co²⁺), zinc (Zn²⁺), and copper (Cu²⁺). Moreover, the ability of the HgT21 strain to produce indole acetic acid (a phytohormone) and promote the growth of Arabidopsis thaliana seedlings in vitro was also demonstrated. The genotype and phenotype of Bacillus megaterium HgT21 reveal its potential to be used as a model of both plant growth-promoting and metal multiresistant bacteria.

IMPORTANCE Metal-polluted environments are natural sources of a wide variety of PGPB adapted to cope with toxic metal concentrations. In this work, the bacterial strain Bacillus megaterium HgT21 was isolated from metal-contaminated soil and is proposed as a model for the study of metal multiresistance in spore-forming Gram-positive bacteria due to the presence of a variety of metal resistance-associated genes similar to those encountered in the metal multiresistant Gram-negative Cupriavidus metallidurans CH34. The ability of B. megaterium HgT21 to promote the growth of plants also makes it suitable for the study of plant-bacteria interactions in metal-polluted environments, which is key for the development of techniques for the biorestoration of metal-contaminated soils used for agriculture.

INTRODUCTION

Heavy metals are present in the environment due to a variety of natural processes, such as volcanic activity, weathering of rocks, and windblown dust particles (1). However, the production of heavy metal wastes derived from mining and other anthropogenic activities has significantly increased the concentration and bioavailability of toxic metals in ecosystems (1, 2). Some metals (i.e., Zn, Fe, Co, Ni, Cu, Mg, and Mn) are essential at low concentrations for the metabolic activities of living organisms; however, the homeostasis of these metals must be tightly regulated by cells to avoid their toxic effects (3). On the other hand, heavy metals without known biological functions (i.e., Hg, Pb, Ag, Cr, Cd) are highly toxic even at low concentrations for most living organisms, particularly for microbial and plant communities in soils, and constant exposure to toxic metals alters their composition, growth, activity, and genetic variability (4, 5).

Despite the toxicity caused by metals, a variety of bacteria that have been constantly exposed to toxic metal ions have tolerance/resistance mechanisms that enable them to survive and colonize hostile environments (6). Bacterial resistance/tolerance to metal ions generally involves extracellular complexation by sorption on the cell surface, intracellular metal accumulation and complexation through intracellular binding to metallothioneins and chelation by siderophores, the intracellular redox of metal ions by enzymatic activity, and cellular exclusion by efflux systems (5, 7).

Some metal-resistant bacteria belong to well-described bacterial genera known as plant growth-promoting bacteria (PGPB), which in addition to modifying the toxic effects that metal ions produce in plants (8–10), also promote plant growth in contaminated environments through a variety of mechanisms, including nutrient mobilization, degradation of organic substances, phosphate solubilization, nitrogen fixation, antibiotic production, iron sequestration, and the production of vitamins and phytohormones such as gibberellins and auxins (11–13).

The Bacillus genus of bacteria includes a wide variety of species recognized as PGPB (14, 15); among these, Bacillus megaterium (Priestia megaterium) is one of the most studied and is a common inhabitant of soil that is frequently associated with metal-contaminated environments (16, 17). In B. megaterium, the metal tolerance associated with biosorption and siderophore production has been described for metals such as Ni (18), Cd (10, 19, 20), Pb (19, 20), Cu, and Zn (17). However, except for the mercury resistance operon (mer), which has been fully studied (21, 22), few studies of metal resistance determinants and mechanisms in B. megaterium have been reported. These studies include a description of resistance genes for Cd (17, 23, 24), Cu (25), Pb (17), and Ni (26). This work was focused on the characterization of the Hg-tolerant bacterial strain HgT21, isolated from the rhizosphere of a metal-contaminated soil. Genome analysis was carried out to determine its potential for use as a plant growth promoter in metal-contaminated soils on which extreme living conditions are prevalent. Phylogeny and genome analyses and in vitro assays indicate that the isolate HgT21 identified as B. megaterium contains a battery of genes and operons that confer resistance to a variety of heavy metals(oids) and several traits related to halotolerance and plant growth promotion. The results reveal that B. megaterium HgT21 can be used as a model for the study of metal multiresistance in Gram-positive bacteria, with application as a PGPB for phytoimmobilization and restoration of metal-contaminated sites.

RESULTS

Isolation of the mercury-tolerant HgT21 bacterial strain.

The Hg^II+ tolerance of the HgT21 strain was determined using the MIC for mercuric chloride (HgCl₂) in both agar and liquid LB medium. The MIC value in solid medium was 975 μM, whereas in liquid medium, bacterial growth was fully inhibited at 75 μM (Fig. 1).

FIG 1.

Hg tolerance of the HgT21 strain in liquid medium. HgT21 bacterial cells were aerobically grown in LB liquid medium supplemented with increasing concentrations of HgCl₂. The optical density at 600 nm (O.D._600nm) was measured, and the concentration at which bacterial growth was fully inhibited by mercury ions (MIC) was determined (black arrow).

Genome sequencing, de novo assembly, and annotation.

The whole-genome sequence of strain HgT21 was obtained using the Illumina platform with MiSeq Illumina paired-end technology with 300-bp reads. Our assembly consisted of 26 contigs spanning ~5.5 Mbp with an average GC content of 37.6% and an N₅₀ size of 1,202,273 bp (~1.2 Mb). Genome annotation by Rapid Annotation using Subsystem Technology (RAST) predicted 5,741 protein coding DNA sequences (CDSs) assigned to 351 subsystems. The subsystems feature genes including bacteriocins and antibacterial peptides (n = 9), membrane transport systems (n = 97), auxin biosynthesis (n = 5), nitrogen metabolism (n = 25), metabolism of aromatic compounds (n = 16), iron acquisition and metabolism (n = 65), phosphorus metabolism (n = 45), resistance to antibiotic and toxic compounds (n = 87), and stress response (n = 163). Additionally, the annotation process detected a total of 34 rRNA genes, 114 tRNAs, 117 ribozymes, and 6 prophage regions, 3 of which were identified as active prophages (Fig. 2). No plasmid was identified when analyzed using PLACNETw and Plasmid Finder.

FIG 2.

Bacillus megaterium HgT21 draft genome landscape. From the outside to inside, the rings correspond to the following: (1) the tile plot depicts the detected prophage regions in the genome, red tiles represent active prophages, and blue tiles indicate inactive or ambiguous regions; (2) gene density; (3) rRNAs; (4) tRNAs; (5) ncRNAs; and (6) GC content (orange, above average; light blue, below average). All the statistics are based on 10-kb nonoverlapping bins. Links between scaffolds represent colinear blocks (7). Only scaffolds with a length of >20 Kb were plotted.

Morphological and biochemical characterization of the HgT21 strain.

The morphology and biochemical characteristics, such as the bacillus shape, spore formation, positive Gram staining, acid production from glucose, and the use of mannitol and citrate as carbon sources, as well as the presence of catalase, amylase, and oxidase enzymatic activities (Table 1), identified the HgT21 strain as a member of the Bacillus genus based on characteristics reported for this genus (27, 28). Additional enzymatic activities and biochemical characteristics related to carbohydrate, polyalcohol, and xenobiotic metabolism are shown in Table 1.

TABLE 1.

Morphology and biochemical characteristics of the HgT21 strain

Characteristics	Resultsa
Morphology and general characteristics
Colony	Round, regular edge, beige, smooth, convex, 4–6 mm diam
Cell morphology and Gram stain	Rod-like long bacilli, Gram-positive
Spore formation	+
Motility	+
H2S production	−
Biochemical characteristics
Carbohydrate metabolism
Acid from glucose	+
Voges-Proskauer reaction	+
Citrate	+
d-raffinose	+
d-xylose	+
d-galactose	+
d-ribose	+
l-lactato alcalinization	−
Lactose	+
Saccharose/sucrose	+
d-maltose	+
d-trehalose	+
d-mannose	−
Cyclodextrine	−
Pullulane	−
Polyalcohol metabolism
d-sorbitol	+
Mannitol	+
Xenobiotic metabolism
d-amygdalin	−
Salicin	+
Enzymatic activity
Amylase	+
Catalase	+
Casein Hydrolysis	+
Lecithinase	−
Urease	−
Phosphatase	−
Cytochrome oxidase	+
Lysine descarboxylase	+
Ornithine descarboxylase	+
Tryptophanase	−
Arginine dihydrolase 1	+
Arginine dihydrolase 2	−
Ala-phe-pro arylamidase	−
l-aspartate arylamidase	−
Leucine arylamidase	−
l-proline arylamidase	−
l-pyrrolydonyl arylamidase	−
Alanine arylamidase	+
Tyrosine arylamidase	+
Beta galactosidase	+
Alpha galactosidase	+
Alpha glucosidase	+
Alpha-mannosidase	−
Beta glucuronidase	−
Beta galactopyranosidase resofurine	+
N-acetyl-d-glucosaminidase	+
Metyl-B-d-glucopyranosidase	−
Phosphatidylinositol phospholipase C	−

^a+, positive; −, negative.

Identification and phylogenetic analysis of HgT21.

The HgT21 strain was identified as Bacillus megaterium by whole-genome multilocus sequence typing (wgMLST) analysis. A rooted species tree was inferred for HgT21 and 28 closely related species (see Fig. S1 in the supplemental material) using 8,098 orthogroups. Phylogenetic analysis revealed the close evolutionary relationship of HgT21 with B. megaterium DSM-319 (Fig. S2). In general, the close evolutionary relationship between B. megaterium and Bacillus aryabhattai strains exhibited in the tree is in agreement with previous studies (29, 30). The tree topology groups the strains of B. aryabhattai and B. megaterium into two main clades, with B. megaterium WSH-002 as a common ancestor of these clades.

Core and pangenome of the B. megaterium-B. aryabhattai clade.

To determine the genomic plasticity and global gene reservoir of the clade B. megaterium-B. aryabhattai, a pangenome analysis was performed based on the annotated protein sequences of the 29 strains (Fig. S3). The 157,299 protein sequences present across all genomes were clustered into 15,974 orthogroups, representing the pangenome. Among them, 1,633 orthogroups (10.22%) were conserved in all 29 genomes, representing the core genome of all species analyzed (all-core), and 1,229 orthogroups (7.69%) of the core genome retained only one copy in every strain. Moreover, we identified 3,341 orthogroups (20.91%) conserved in the clade B. megaterium-B. aryabhattai (excluding B. aryabhattai PHB10) (MA-core), 3,007 of which (90%) were orthogroups of single-copy genes. On the other hand, 1,954 (12.23%) orthogroups were conserved among the outgroup clade (93.5% single copy) (out-core). To determine the relationship of core genes with some evolutionary features, we classified these genes into different functional categories using Clusters of Orthologous Genes (COG) annotation. We assigned 1,621, 1,309, and 1,166 functions for 80.36%, 66.83%, and 71.4% of proteins in the MA, out, and all-gene cores, respectively. Proteins involved in general function dominated the three cores analyzed (Fig. S4). Some of the most abundant proteins in this class were related to predicted hydrolases of the haloacid dehydrogenase (HAD) superfamily, Zn-dependent hydrolases, including glyoxylases, and predicted permease, a member of the PurR regulon. Through comparison between COG clusters, we found that transcription, secondary metabolite biosynthesis, transport, and catabolism and signal transduction mechanisms were more abundant in the MA core than in the other cores. Translation, ribosomal structure and biogenesis, replication, recombination and repair, and nucleotide transport and metabolism were the least abundant (Fig. S4).

The distribution of accessory genes also varied among strains as well as clades (Fig. 3). A total of 7,333 (45.9%) orthogroups were singletons present in only one strain, which was reduced to 490 (3.06%) when considering only the B. megaterium-B. aryabhattai clade. A total of 332 strain-specific genes were identified in B. megaterium HgT21, including 230 annotated hypothetical proteins (Table S2).

FIG 3.

Phylogenetic tree of the species closely related to HgT21 against the gene presence/absence matrix. The heatmap on the right shows the presence (dark green) or absence (light yellow) of all 15,974 orthogroups. Each row in the matrix corresponds to a branch on the tree (i.e., one species), and each column represents an orthogroup.

Relevant metal resistance operons in HgT21.

B. megaterium HgT21 genome analysis revealed the presence of several genes involved in heavy metal resistance. These genes are associated with resistance to arsenic, copper, mercury, tellurium, zinc, cadmium, and cobalt and are grouped as operons (Fig. 4). Two putative arsenic resistance operons were found (contigs 2 and 8; Fig. 4). The genes arsC, arsB, and arsR and an extra copy of arsR were found at contig 2, whereas arsA, arsD, arsC, arsB, arsR, and arsB-acr3 and an extra copy of arsR were found at contig 8. Additionally, two putative identical copper resistance-like operons (csoR-copZ-copA) were found (contigs 3 and 8; Fig. 4), as well as a putative mercury resistance operon (merRETPA) and the merB gene (contig 8; Fig. 4). A putative tellurium resistance operon that includes telA, yceG, and three tandem copies of telD was found (contig 3; Fig. 4). Finally, three genes associated with Zn-Cd-Co resistance (zntA, arsR, and czcD) were identified. Overall, these genes and operons showed gene arrangements similar to those reported in bacteria, with some differences in the cases of cadmium, arsenic, and tellurium (22, 31–33).

FIG 4.

Metal operons in B. megaterium HgT21. The locations, orientations, and products of putative operons involved in heavy metal resistance determinants. Annotated genes attributed to arsenic 1, arsenic 2, copper 1, copper 2, mercury, tellurium, and zinc-cadmium-cobalt are displayed in coral, brown, green, cyan, blue, purple and pink, respectively.

Metal resistance of the HgT21 strain.

Based on the metal-resistance operons identified in HgT21, resistance to several metal ions (Hg⁺/Zn²⁺/Cu²⁺/Co²⁺/As³⁺) was evaluated. The viability of HgT21 bacterial cells in liquid culture exposed to different metal concentrations is shown in Fig. 5. The dose-response curves reveal a gradual decrease in viability when the metal ion concentration increased. Lethal doses of Hg⁺, Zn²⁺, Cu²⁺, Co²⁺, and As³⁺ were established at 40 μM, 0.6 mM, 3.0 mM, 420 mM, and 210 mM, respectively.

FIG 5.

Lethal doses of different metal ions in HgT21 cells. (A to E) Liquid cultures of exponentially growing cells were exposed to increasing concentrations of mercury (A), zinc (B), copper (C), cobalt (D), and arsenic (E) salts to establish dose-response curves and determine the lethal dose (LD) of each metal tested (black arrows).

Plant growth promotion by the HgT21 strain.

In accordance with the plant growth promotion-associated genes in HgT21, its ability to promote the growth of Arabidopsis thaliana seedlings was evaluated. The seedlings were exposed through both direct and distant interactions with HgT21 cells for 8 days. After bacterial exposure, a highly branched root system and leafy shoot were observed compared with the control plants (Fig. 6A). Particularly, an increased number and density of lateral roots were observed in both types of interaction (contact and distant), whereas the inhibition of primary root elongation was evident when roots were in contact with bacteria (Fig. 6A and D). Accordingly, both the wet and dry weights of the plants exposed to direct and distant interactions with HgT21 cells were also increased compared to the control (Fig. 6B and C).

FIG 6.

Growth promotion of Arabidopsis thaliana by the HgT21 strain. (A) Germination and growth of A. thaliana in the absence (control), distant root interaction, and contact root interaction with B. megaterium HgT21. (B) Wet weight of A. thaliana plants after 8 days of growth in the presence of HgT21 cells. (C) Dry weight of A. thaliana plants exposed to HgT21 cells after 2, 4, 6, and 8 days of dehydration. (D) Primary root length and number and density of lateral roots of A. thaliana plants after 8 days of growth in the presence of HgT21 cells. Asterisks (*) indicate significant differences determined by ANOVA (P < 0.05).

Indoleacetic acid (IAA) production by HgT21.

Due to the observed ability of the HgT21 strain to induce qualitative and quantitative changes in the growth of A. thaliana plants, the production of IAA (a phytohormone commonly produced by PGPB) was evaluated. IAA synthesis by the HgT21 strain was demonstrated through the Salkowski assay, which revealed that 3 mg/L IAA was produced from tryptophan (Fig. 7).

FIG 7.

Indole acetic acid (IAA) production by the HgT21 strain. IAA production was observed as a pink-colored supernatant of the HgT21 culture in the presence of l-tryptophan and Salkowski reagent. Three independent experiments of quantitative IAA production were carried out spectroscopically (OD₅₃₀). The mean and standard deviation of determinations are shown.

Susceptibility of the HgT21 strain to antibiotics.

Based on the results of the genome analysis, the HgT21 strain possesses antibiotic resistance-associated genes. To determine the antibiotic multiresistance of HgT21, which could represent a risk for antibiotic resistance dissemination in natural environments, the antibiotic susceptibility of HgT21 was evaluated. The results in Table 2 reveal resistance to some beta-lactam antibiotics (ampicillin, dicloxacillin, and penicillin and the cephalosporins cefotaxime, ceftazidime, and cefuroxime) and optochin (a hydroquinine derivative). Susceptibility to beta-lactam cephalosporins (cephalothin, cefepime, and ceftriaxone) and to different groups of antibiotics, including vancomycin (glycopeptide), nitrofurantoin (sulfamide), erythromycin (macrolide), levofloxacin, pefloxacin and ciprofloxacin (quinolones), gentamicin, amikacin and netilmicin (aminoglycosides), polymyxin and bacitracin (polypeptides), novobiocin (aminocumarine), tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole, was also observed.

TABLE 2.

Key genes involved in metabolism, auxin biosynthesis, phosphate solubilization, and antibiotic resistance in HgT21

Process/activity	No. of Contig	ID	Enzyme
Carbohydrate and polyalcohol metabolism
d-Raffinose	3	4652	Alpha-galactosidase (EC 3.2.1.22)
	15	330	Alpha-galactosidase (EC 3.2.1.22)
d-xylose	3	3729	Xylose isomerase (EC 5.3.1.5)
	3	3730	Xylulose kinase (EC 2.7.1.17)
d-galactose	3	4652	Alpha-galactosidase (EC 3.2.1.22)
	15	330	Alpha-galactosidase (EC 3.2.1.22)
	3	3772	Beta-galactosidase (EC 3.2.1.23)
	3	4022	Beta-galactosidase (EC 3.2.1.23)
d-ribose	2	2867	Ribose 5-phosphate isomerase A (EC 5.3.1.6)
	2	3551	Ribose 5-phosphate isomerase B (EC 5.3.1.6)
	4	4918	Ribose 5-phosphate isomerase B (EC 5.3.1.6)
Lactose	3	3772	Beta-galactosidase (EC 3.2.1.23)
	3	4022	Beta-galactosidase (EC 3.2.1.23)
	10	16	d-Tagatose 3-epimerase
Saccharose/sucrose	5	5078	6-Phosphogluconolactonase (EC 3.1.1.31)
	1	1987	6-Phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44)
	2	2601	6-Phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44)
	2	3187	6-Phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44)
	2	2867	Ribose 5-phosphate isomerase A (EC 5.3.1.6)
	2	3551	Ribose 5-phosphate isomerase B (EC 5.3.1.6)
	4	4918	Ribose 5-phosphate isomerase B (EC 5.3.1.6)
	991	1443	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	1	1534	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	20	2473	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	21	2475	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	2	3492	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	2	3500	Sucrose-6-phosphate hydrolase (EC 3.2.1.B3)
	2	3153	Phosphoenolpyruvate-protein phosphotransferase of PTS system (EC 2.7.3.9)
d-Maltose	17	377	Maltose O-acetyltransferase (EC 2.3.1.79)
	2	2505	Maltose operon transcriptional repressor MalR, LacI family
	3	4173	Maltose O-acetyltransferase (EC 2.3.1.79)
	3	4272	Maltose/maltodextrin ABC transporter, substrate binding periplasmic protein MalE
	9	5741	Maltose O-acetyltransferase (EC 2.3.1.79)
d-Trehalose	2	2802	PTS system, trehalose-specific IIB component (EC 2.7.1.69)/PTS system, trehalose-specific IIC component (EC 2.7.1.69)
	2	2803	Trehalose-6-phosphate hydrolase (EC 3.2.1.93)
	2	2804	Trehalose operon transcriptional repressor
d-Sorbitol	3	4198	Sorbitol-6-phosphate 2-dehydrogenase (EC 1.1.1.140)
	6	5294	Glucitol/sorbitol-specific transport protein GutA
	6	5295	Sorbitol dehydrogenase (EC 1.1.1.14)
d-Mannitol	2	2862	Putative transcriptional antiterminator, BglG family/PTS system, mannitol/fructose-specific IIA component (EC 2.7.1.69)
	13	241	Mannitol-1-phosphate 5-dehydrogenase (EC 1.1.1.17)
	13	242	Mannitol operon activator, BglG family
	13	243	PTS system, mannitol-specific IIC component (EC 2.7.1.69)/PTS system, mannitol-specific IIB component (EC 2.7.1.69)/PTS system, mannitol-specific IIA component
Starch hydrolysis	3	4365	Alpha-amylase (EC 3.2.1.1)
	4	5002	Cytoplasmic alpha-amylase (EC 3.2.1.1)
Metabolism of proteins, amino acids and others
Acetoin production	3	3747	Acetoin dehydrogenase E1 component alpha-subunit (EC 1.2.4.-)
	3	3748	Acetoin dehydrogenase E1 component beta-subunit (EC 1.2.4.-)
	3	3749	Dihydrolipoamide acetyltransferase component (E2) of acetoin dehydrogenase complex (EC 2.3.1)
	3	3750	Dihydrolipoamide dehydrogenase of acetoin dehydrogenase (EC 1.8.1.4)
	3	3751	Transcriptional activator of acetoin dehydrogenase operon AcoR
	3	3752	2,3-Butanediol dehydrogenase, R-alcohol forming, (R)- and (S)-acetoin-specific (EC 1.1.1.4)
Casein hydrolysis	1	1494	ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92)
	1	2167	ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92)
	4	4812	ATP-dependent Clp protease proteolytic subunit (EC 3.4.21.92)
Cytochrome oxidase	1	1256	Cytochrome oxidase biogenesis protein Sco1/SenC/PrrC, putative copper metallochaperone
Lysine descarboxylase	14	256	Arginine decarboxylase (EC 4.1.1.19)/Lysine decarboxylase (EC 4.1.1.18)
	3	3933	Lysine decarboxylase family
Ornithine descarboxilase	2	2528	Ornithine carbamoyltransferase (EC 2.1.3.3)
	2	2507	Ornithine aminotransferase (EC 2.6.1.13)
Arginine dihydrolase 1	2	3203	Arginine decarboxylase (EC 4.1.1.19)
	14	256	Arginine decarboxylase (EC 4.1.1.19)/lysine decarboxylase (EC 4.1.1.18)
Alanine arylamidase	1	686	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	1	1212	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	1	1688	N-acetylmuramoyl-l-alanine amidase
	1	2095	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	1	2201	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	2	2903	N-acetylmuramoyl-l-alanine amidase
	3	3925	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	3	3928	N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28)
	3	4105	N-acetylmuramoyl-l-alanine amidase
Tyrosine arylamidase	1	2373	Aminopeptidase S (Leu, Val, Phe, Tyr preference) (EC 3.4.11.24)
Beta galactosidase	3	4022	Beta-galactosidase (EC 3.2.1.23)
	3	3772	Beta-galactosidase (EC 3.2.1.23)
Alpha galactosidase	15	330	Alpha-galactosidase (EC 3.2.1.22)
	3	4652	Alpha-galactosidase (EC 3.2.1.22)
Alpha glucosidase	2	3289	Alpha-glucosidase (EC 3.2.1.20)
	1	1533	Alpha-glucosidase (EC 3.2.1.20)
N-acetyl-d-glucosaminidase	18	386	Spore cortex-lytic enzyme, N-acetylglucosaminidase SleL (EC 3.2.1.-)
	2	3351	Spore peptidoglycan hydrolase (N-acetylglucosaminidase) (EC 3.2.1)
Urease	1	2274	Urease gamma subunit (EC 3.5.1.5)
	1	2275	Urease beta subunit (EC 3.5.1.5)
	1	2276	Urease alpha subunit (EC 3.5.1.5)
	1	2277	Urease accessory protein UreE
	1	2278	Urease accessory protein UreF
	1	2279	Urease accessory protein UreG
	1	2280	Urease accessory protein UreD
Auxin biosynthesis
IAA synthesis	2	3619	Acetolactate synthase large subunit (EC 2.2.1.6)
	2	2637	Alpha-acetolactate decarboxylase (EC 4.1.1.5)
	2	2638	Acetolactate synthase, catabolic (EC 2.2.1.6)
	1	614	Acetolactate synthase large subunit (EC 2.2.1.6)
	1	615	Acetolactate synthase small subunit (EC 2.2.1.6)
	3	3752	2,3-butanediol dehydrogenase, R-alcohol forming, (R)- and (S)-acetoin-specific (EC 1.1.1.4)
	1	1836	2,3-butanediol dehydrogenase, R-alcohol forming, (R)- and (S)-acetoin-specific (EC 1.1.1.4)
	3	3691	Glycerol dehydrogenase (EC 1.1.1.6)
Phosphate solubilization
	2	3621	Glucose dehydrogenase (pyrroloquinoline-quinone)
	2	3622	Glucose dehydrogenase (pyrroloquinoline-quinone)
	7	5440	l-lactate dehydrogenase (EC 1.1.1.27)
	2	3087	Predicted l-lactate dehydrogenase, Fe-S oxidoreductase subunit YkgE
	2	3088	Predicted l-lactate dehydrogenase, iron-sulfur cluster-binding subunit YkgF
	2	3089	Predicted l-lactate dehydrogenase, hypothetical protein subunit YkgG
	1	2185	Citrate synthase (si) (EC 2.3.3.1)
	1	544	Citrate synthase (si) (EC 2.3.3.1)
	3	4390	Manganese-dependent inorganic pyrophosphatase (EC 3.6.1.1)
	1	548	Alkaline phosphatase synthesis transcriptional regulatory protein PhoP
	1	2268	Alkaline phosphatase
	2	2484	Alkaline phosphatase-like protein
	2	3067	Alkaline phosphatase (EC 3.1.3.1)
	2	3367	Alkaline phosphatase (EC 3.1.3.1)
	2	3581	Alkaline phosphatase like protein
	4	5006	Alkaline phosphatase (EC 3.1.3.1)
Antibiotic resistance
	1	478	Metallo-beta-lactamase family protein
	1	1333	Metallo-beta-lactamase family protein
	1	1899	Beta-lactamase class A
	2	2615	Metallo-beta-lactamase family protein
	2	3330	Beta-lactamase (EC 3.5.2.6)
	2	3410	Beta-lactamase (EC 3.5.2.6)
	2	3473	Metallo-beta-lactamase family protein
	2	3657	Beta-lactamase class C and other penicillin binding proteins
	3	4280	Metal-dependent hydrolases of the beta-lactamase superfamily I; PhnP protein
	4	5011	Zn-dependent hydrolase (beta-lactamase superfamily)
	7	5353	Metal-dependent hydrolases of the beta-lactamase superfamily I; PhnP protein

DISCUSSION

In recent decades, sustainable agriculture for safe food production has become an important challenge for soil chemistry, plant biology, agricultural and environmental engineering, and soil microbiology due to the increased demand for healthy safe products (11). Heavy metal pollution in agricultural soils leads to the accumulation of toxic metals in plants and, consequently, their integration into the food chain. Thus, the toxicity of heavy metals for most living organisms at relatively lower concentrations (at the level of ppb or ppm) represents a threat to human health, safe food production, and maintenance of microbial diversity in soils (1, 34). The development of strategies for the recovery of degraded and metal-contaminated agricultural soils could be based on bioremediation approaches using beneficial microorganisms such as metal-resistant PGPB and natural pathogen antagonists as an alternative for metal immobilization or as a replacement for chemical fertilizers and pesticides to guarantee sustainable agriculture (12, 14, 15). In this context, the search for soil plant-associated PGPB and metal biotransformation bacteria is important for the establishment of sustainable strategies for agricultural soil restoration.

In this work, a metal-resistant PGPB was isolated and characterized. Based on 16S and complete-genome sequence analyses, the isolated HgT21 was identified as Bacillus megaterium. The phylogenetic tree based on the genome analysis shows that B. megaterium HgT21 is closely related to B. megaterium DSM319 and is grouped within the B. aryabhattai-B. megaterium clade. In addition, the average nucleotide identity (ANI) values (>95 to 96%) between the B. aryabhattai and B. megaterium strains (Fig. S1) support the proposition of Narsing Rao et al. to reclassify B. aryabhattai as the later heterotypic synonym of B. megaterium de Bary 1884 (35) due to the ANI values being higher than the recognized threshold values for bacterial species delineation (36). As in B. megaterium DSM319, no plasmid sequences were identified; however, a low GC content was observed in some contigs (6 and 8 to 11), and the presence of active prophages (contigs 10 and 11; Fig. 2) and higher coverage (>80×; contigs 6, 8, 10, and 11) suggest the integration of plasmid-borne genes into genomic DNA and/or the exchange of genes between plasmids and chromosomes. This notion is supported by studies that provide evidence of extensive gene transfer between the plasmids of B. megaterium QMB1551 and its own chromosome and those of the plasmid-less B. megaterium DSM319 (37). According to genotype identification, the morphology of the HgT21 cells correlates with the B. megaterium phenotype initially described by De Bary in 1884 (Gram-positive, approximately 8 to 10 μm size, and spore-forming), and the identification was also corroborated based on biochemical characteristics reported for B. megaterium (27).

Bacillus megaterium is widely recognized as a soil bacterium with potential industrial applications due to its ability to utilize different carbon sources, grow in a wide range of temperatures (3 to 45°C), produce proteases, promote plant growth, and antagonize plant pathogens for biocontrol (38–40). In agreement with these reports, genome annotation based on protein prediction revealed that B. megaterium HgT21 contains approximately 264 genes related to the production of bacteriocin and antibacterial peptides, resistance to antibiotic and toxic compounds, auxin biosynthesis, and stress response. Moreover, the biochemical profile of B. megaterium HgT21 reveals its ability to use diverse carbon sources (Table 1). These results are consistent with key enzyme genes encountered in its genome, which are involved in the metabolism of these carbohydrates and polyalcohols (Table 2). Additional enzymatic activities detected in B. megaterium HgT21 are also correlated with the presence of key genes involved in these enzymatic activities (Table 2), except for urease; in contrast to the biochemical data, urease was identified in the B. megaterium HgT21 genome, suggesting dysfunction of this enzyme.

According to the recognition of the Bacillus genus as PGPB, genes involved in one of the three metabolic pathways described for the synthesis of indole-3-acetic acid (IAA), a nonvolatile phytohormone involved in plant growth promotion (41), were found in B. megaterium HgT21. The production of IAA by PGPB is related to the synthesis pathway of tryptophan. In B. megaterium HgT21, the genes required for IAA synthesis through the chorismate pathway were identified (Table 2) and involve the conversion of chorismate to tryptophan (by enzymes encoded in the trpABCDEG operon) and its subsequent conversion to IAA by nitrilase activity. These results suggest that enzymes encoded by genes of the chorismate pathway could be involved in IAA production by B. megaterium HgT21. Moreover, genes involved in the synthesis of volatile organic compounds (VOCs) such as acetoin and butanediol were also identified; these genes encode enzymes that convert pyruvate to acetoin (acetolactate synthase and acetolactate decarboxylase) and acetoin to 1,3-butanediol (2,3-butanediol dehydrogenase [BDH] and glycerol dehydrogenase [GDH]) (42). Although the activity of these enzymes was not demonstrated in this work, the presence of their encoding genes in the HgT21 genome could be related to the increased growth of A. thaliana seedlings observed after its exposure to both distant and contact interactions with B. megaterium HgT21. The phenotype observed in A. thaliana after the interaction with B. megaterium HgT21 correlates with previous reports that describe the increase of lateral roots, decreasing cell elongation in primary root, and root hair density increase upon colonization (43). In relation to the phenotype observed, it has been described that some PGPB indirectly reduce the effect of ethylene production (an inhibitor of primary growth root) through the production of the ACC (1-aminocyclopropane-1-carboxylate) deaminase, which degrades ethylene, and in consequence induces primary root elongation (44). However, the gene encoding the ACC deaminase was not found in B. megaterium HgT21; this could explain the inhibition of the root growth when colonization is established.

According to reports that demonstrate that the production of VOCs by rhizobacteria induces the elongation of lateral roots and root hairs of A. thaliana seedlings (45), the production of acetoin and 2,3-butanediol in HgT21 could be responsible for the observed elongation of lateral roots and root hairs of seedlings; however, this must be experimentally demonstrated, and future experiments must be conducted to evaluate the contribution of VOCs and diffusible compounds to growth promotion. Additionally, the group of genes involved in phosphate solubilization (41, 46) that were identified in B. megaterium HgT21 reveal its potential as a phosphate solubilizer bacterium.

Bacillus megaterium is also recognized for its adaptation and resistance to stressful environmental conditions such as saline or acidic soils and soil contamination with heavy metals and other xenobiotics (40, 47, 48). Accordingly, B. megaterium HgT21 showed high tolerance to Hg^II+ ions, and the MIC values were higher in solid (975 μM HgCl₂) than in liquid (75 μM HgCl₂) medium, presumably due to the major interaction of the bacterial cell surface with metal ions in liquid medium (49, 50). This result correlates with observations in mercury-resistant Bacillus species isolated from Minamata Bay, Japan, a highly mercury-contaminated site, which have MIC values from 80 to 320 μM HgCl₂, particularly the well-characterized mercury-resistant strain B. megaterium MB1 (MIC of 80 μM HgCl₂). These values are significantly higher than those of the mercury-sensitive strain (no mer determinants) B. megaterium WH20 (MIC of 10 μM HgCl₂) (16, 22). Genomic analysis revealed that B. megaterium HgT21 contains a variety of heavy metal resistance-associated genes and/or complete putative operons, including the merRETPA operon and the merB3 gene, which confer a wide spectrum of mercury resistance (51). The mercury resistance system in HgT21 showed a gene arrangement identical to that of the mer-like determinant in Tn5083 of B. megaterium MK64-1 (Kamchatka, Russia), which has been proposed to be a derivative of mer determinants reported in Tn5084 of Bacillus cereus RC607 (Boston, USA) and TnMERI1 of B. megaterium MB1 (Minamata, Japan) (21, 22, 52–54). These results support the idea that recombination events of transposition gene exchange could be an important contribution to the evolution of Hg^II+-resistant transposons in Gram-positive bacteria and the worldwide horizontal dissemination of the class II TnMERI1-like transposons across bacterial species and geographical barriers (22, 55).

Bacillus megaterium HgT21 also contains a variety of metal(oid) resistance systems, which include well-described genes and operons for arsenic, copper, cadmium, zinc, cobalt, and tellurium resistance. Two arsenic resistance-like operons were identified in HgT21. First, the canonical operon arsRBC (contig 8), initially described in Staphylococcus aureus (pI258 plasmid), correlates with its wide distribution in the plasmids and chromosomes of Bacteria and Archaea from different origins (32). However, in Bacillus species, only variants of this operon have been reported, as well as the arsR2-orf2-arsB-arsC2 operon in the chromosome of Bacillus sp. strain CDB3 and the arsR-orf2-acr3-arsC operon in the “Tn (skin element)” of Bacillus subtilis JH642 (31, 56). The second arsenic resistance-like operon (arsRBCDA) identified (contig 2) showed an arrangement similar to that of the commonly reported operon arsRDABC (57) and was identical to those reported in the chromosome of Bacillus sp. CDB3 (31). The presence of two arsenic resistance operons, extra copies of arsR (upstream from arsRBC and arsRBCDA) and acr3 (upstream from arsRBCDA), correlates with the wide complexity and variety of gene configurations in ars clusters reported in prokaryotes (32, 58). The enzymes encoded by the arsenate resistance-like operons in HgT21 could be responsible for the observed arsenate resistance in this strain, which is similar to the most arsenic-resistant Bacillus species (56, 59, 60). The presence of the acr3 gene (arsB homolog) in Bacillus megaterium HgT21 contrasts with studies about the prevalence of acr3 in different orders of Bacteria, which revealed that acr3 is mainly present in Burkholderiales, Actinobacteria, and Alphaproteobacteria, whereas arsB is prevalent in Firmicutes and Gammaproteobacteria (61). Taken together, the results support studies that revealed that the presence of multiple and redundant ars genes in bacteria exposed to selective pressure is the result of gene duplication via horizontal transfer (32).

The arrangement of the three copper resistance-associated genes in B. megaterium HgT21 (csoR-copZ-copA) is identical to the copper resistance system described in B. subtilis (33), which includes the transcriptional regulator CsoR (a widespread copper-inducible repressor distributed in Gram-positive bacteria and Proteobacteria that regulates expression of the copZA operon), the copper chaperone CopZ, and the copper-efflux ATPase CopA (62). The presence of this copper-like resistance system in B. megaterium HgT21 correlates with the identification of this system in approximately half of the members of Firmicutes, including the Bacillales “copper users,” for which copper is essential for aerobic respiration, acting as a cofactor in terminal enzymes of the aerobic pathway (63). In copper users, the csoR-copZA system confers resistance to high levels of copper (33), as was observed in B. megaterium HgT21 (3 mM CuSO₄), compared with the copper resistance reported in both Gram-positive and Gram-negative bacteria (3.5 to 5.5 mM CuSO₄) (12, 33, 64). The presence of two identical copper resistance systems at different locations in the genome suggests duplication of this system associated with mobile elements.

Three genes associated with resistance to different metal cations (Zn²⁺, Cd²⁺, Pb²⁺, and Co²⁺) were identified in B. megaterium HgT21. cadA encodes a cadmium-translocating P-type ATPase (CadA), a homologous protein of ZntA, which is considered a multipurpose metal-exporting pump for the extrusion of Zn²⁺, Cd²⁺, Ag²⁺, and Pb²⁺ (65). The cadA gene is a component of the well-described cadmium resistance operon cadAC encountered in Gram-positive bacteria, including S. aureus (plasmid pI258), Listeria monocytogenes, B. megaterium, and other Bacillus species (24, 66–68). In the CadAC system, the expression of cadA is tightly controlled by the regulatory protein CadC, a Cd²⁺/Pb²⁺/Zn²⁺ responsive repressor encoded by cadC located downstream of cadA (67). Interestingly, although cadA was located in B. megaterium HgT21, cadC was not found; instead, a hypothetical protein was located upstream of cadA. However, in the opposite direction, near and upstream of cadA, the genes arsR (encoding an ArsR family transcriptional regulator) and czcD (encoding a membrane-bound protein member of the metal-diffusion facilitator, cation diffusion facilitator (CDF) subfamily) were found. This suggests that in the absence of CadC (a homodimeric repressor that belongs to the ArsR/SmtB family of metalloregulatory proteins), ArsR could act as a transcriptional regulator of cadA (67). With respect to czcD, it has been reported that CzcD is a heavy metal transporter involved in the regulation of the czc (cadmium, zinc, cobalt) resistance system described in Ralstonia sp. strain CH34 (Cupriavidus metallidurans CH34) (69, 70); however, the absence of additional czc genes in B. megaterium HgT21 suggests that, rather than acting as a regulator protein, CzcD could mediate resistance against Zn²⁺/Co²⁺/Ni²⁺/Cd²⁺ through an antiporter mechanism catalyzing the active efflux of divalent ions in exchange for K⁺/H⁺, as described in Bacillus subtilis (71, 72). Thus, czcD could play a physiological role related to the maintenance of metal homeostasis, as has been proposed for members of CDF family, such as CzcD in Streptococcus pneumoniae, ZRC-1 in S. cerevisiae and Znt-1 and Znt-2 in mammals (73–75). In agreement with this result, the presence of czcD without other components of the czc and cadAC operon was described in Bacillus megaterium (26) and Bacillus subtilis (71). In Bacillus paranthracis, both czcD and cadA are present; however, the gene arrangement and genome location have not been described (23). Thus, to our knowledge, this is the first report of a genic arrangement that includes cadA, arsR, and czcD, each corresponding to three different metal-resistance systems (czc, cadAC, ars). CadA and CzcD are involved in the extrusion of divalent metal ions, suggesting that this chimeric system probably arises for the resistance to and/or homeostasis maintenance of divalent metal cations (Zn²⁺, Cd²⁺, Pb²⁺, and Co²⁺) in B. megaterium HgT21, which showed Zn²⁺ resistance (0.6 mM) comparable to that reported for zinc-resistant bacteria (0.1 to 1.0 mM) (65, 71); however, this must be experimentally demonstrated. Interestingly, the Co²⁺ resistance was extremely high (420 mM) relative to the Co²⁺ resistance reported in bacteria (0.425 to 8 mM CoCl₂) (70, 76), and the mechanism involved in this unprecedented level of Co²⁺ resistance is under investigation.

A set of genes associated with tellurium resistance (Te^R) was also identified in B. megaterium HgT21. Te^R determinants are a group of ubiquitous genes distributed across phylogenetically diverse taxa in bacteria isolated from diverse environments (77). Based on the high sequence similarity, it has been proposed that Te^R genes arose from a common ancestor that evolved as the result of adaptation to ancient metal-rich environments or through horizontal transfer events (77, 78). A variety of Te^R genes have been identified in plasmids and chromosomal genomic islands (GIs) of pathogenic and extreme-environment isolated bacteria; however, the function of proteins encoded by these genes remains unclear (77, 79). The operon terZABCDE is commonly reported in bacteria; however, the minimal fragment required to confer tellurite resistance is terBCDE, whereas the complete operon also provides phage resistance and pore-forming colicins, explaining its maintenance in a variety of pathogenic bacteria for which tellurite resistance is probably not necessary (80). Moreover, comparative genomics revealed that ter genes are functionally linked to enzymes involved in DNA processing and repair and induced by H₂O₂ and superoxide, suggesting their association with the general stress response caused by ROS production or tellurite ions (81, 82). In this context, telA, yceG, and the three tandem copies of terD in B. megaterium HgT21 could be associated with different functions: (i) tellurite resistance, considering that it was isolated from a metal-polluted environment and considering the role of TelA in tellurite resistance in Rhodobacter sphaeroides (83), and/or (ii) to deal with hostile environmental conditions related to ROS generation, considering that yceG and terD have been associated with the cellular defense against oxidative stress in pathogenic bacteria (82, 84). However, the function of enzymes encoded in tellurite resistance-related genes in B. megaterium HgT21 must be experimentally demonstrated.

Due to bacterial metal resistance being associated with antibiotic resistance (85, 86), the antibiotic susceptibility in B. megaterium HgT21 was assessed. Susceptibility to most of the antibiotic families was observed, suggesting a low risk of antibiotic resistance transfer in the environment. Resistance to some beta-lactams correlates with the presence of genes associated with beta-lactam resistance. Optochin resistance could probably be produced by a lack of recognition of the target site of this antibiotic (membrane ATPase F0F1), which can be altered by mutations in the ATPase F0F1 gene (87). The resistance to beta-lactams, bacteriocins, and antibacterial peptides in B. megaterium HgT21 could be advantageous in its natural hostile environment, in which competence with other microorganisms could be crucial to survive.

The Bacillus species in the soil habitat are well recognized as “zymogenous” bacteria and ecologically have been defined as “r-strategists,” which means that they can grow quickly when the nutrient supply is abundant, as in the rhizosphere, and possess a high colonization and competitive ability. Importantly, quorum sensing-mediated processes in Bacillus species, such as endospore and biofilm formation, constitute an important survival strategy in soil under nutrient-limited conditions and hostile environments; its high adaptability to the environment is evidenced by its ubiquity in both nonextreme and extreme soils (88). In this context, the genetic information of this bacterium and the preliminary physiological trait analysis (metal multiresistance, plant growth promotion, IAA production, use of carbon sources) suggest a high adaptability of B. megaterium HgT21 to adverse environments such as metal-contaminated soils and its potential as a PGPB through phytohormone and VOC production, phosphate solubilization, and antibiotic production.

Considering the essential characteristics that help to define new PGPB as biofertilizers (high rhizosphere competence, ability to increase plant biomass, long-term survival, plant-beneficial physiological traits, a lack of risk factors for human and environmental health, and high tolerance to environmental stresses encountered in soil/plant systems) (88), the genetic information of the HgT21 strain suggests that it could be suitable for use as a biofertilizer in the promotion of plant growth in metal-contaminated environments due to its ability to produce/perform quorum sensing-related processes, endospores, flagella, siderophores, metabolism of a variety of organic compounds, IAA production, and resistance to a variety of metals. However, in vivo experiments under more realistic conditions must be conducted to demonstrate that it can be used as a good biofertilizer. Moreover, due to its sporulating, nonpathogenic, and free-endotoxin nature, B. megaterium HgT21 could be a good candidate for biotechnology applications in the food and pharmaceutical industries, among others (38).

MATERIALS AND METHODS

Isolation of the mercury-tolerant HgT21 bacterial strain.

The mercury-tolerant bacterial strain HgT21 was isolated from the rhizosphere of Fabaceae plants (Dalea bicolor) inhabiting mining tails located in Zacatecas, Mexico (22°47′01.4″ N, 102°36′21″ W and 2,426 meters above sea level [m.a.s.l.]). To test the HgT21 tolerance to mercury ions (Hg^II+) in solid medium, the MIC was determined. Briefly, LB agar was supplemented with HgCl₂ at final concentrations of 0, 1, 10, 100, 200, 400, 800, 850, 900, 925, 950, 975, and 1,000 μM and poured into petri dishes. After agar solidification, the plates were inoculated with 1 × 10⁵ colony forming units (CFU) and spread onto the agar surface and incubated at 37°C until visible growth of bacteria was detected. The concentration at which no growth was detected was reported as the MIC in solid medium. Mercury tolerance in liquid medium was also determined, by assessing the MIC value as follows: overnight cultures were diluted 1:100 in fresh LB medium supplemented with HgCl₂ at final concentrations of 0, 1, 10, 25, 50, 75, and 100 μM and grown at 37°C and 200 rpm for 8 h (when the end of exponential growth was reached in cultures not exposed to mercury ions), and then the optical density was spectroscopically determined at 600 nm (OD₆₀₀). Three independent experiments were carried out.

Morphological and biochemical characterization of the HgT21 strain.

This analysis was based on colony characteristics, Gram staining, motility, and biochemical properties such as H₂S production, acid and acetylmethylcarbinol production from glucose, use of citrate and mannitol as a carbon source, and the presence of enzymatic activities such as urease, lysine and ornithine decarboxylases, tryptophanase, cytochrome oxidase, catalase, and amylase (89). Based on this characterization, the HgT21 strain was identified according to Bergey’s Manual of Determinative Bacteriology (27). An additional test for the biochemical analysis was performed using an automated Vitek 2 system (bioMérieux) with the GP-ID card for Gram-positive bacteria.

Genome sequencing, de novo assembly, and annotation.

Genome sequencing of Bacillus megaterium HgT21 was performed at Langebio genomics services (Irapuato, Mexico City) using the 2 × 300-bp Illumina MiSeq platform. Raw reads were quality assessed using cutadapt v1.9.1 (90) and Trimmomatic v0.36 (91) to remove adapter sequences and low-quality reads, respectively. De novo assembly of the filtered reads was performed using SPAdes v3.9.1 (92) with the –careful parameter for read error correction. The assembled contigs were annotated using the Rapid Annotation using Subsystem Technology (RAST) web server (http://rast.nmpdr.org) (93) using the ClassicRAST annotation scheme, FIGfams v90, automatic error correction, and automatic frameshift correction. Finally, noncoding RNAs (ncRNAs), tRNAs, and rRNAs were searched in the HgT21 assembly using Infernal v1.1.3 (94) against the Rfam database v14.1 (95). Prophage regions were bioinformatically predicted with Prophage Hunter (96). Only regions with scores of >0.8 were classified as active prophage regions. The presence of plasmid sequences was verified using PLACNETw (97) and PlasmidFinder v2.1 (98). Colinear blocks between contigs of the B. megaterium HgT21 genome were identified using the SibeliaZ-LCB (99) algorithm with default parameters. Gene and ncRNA densities, colinearity blocks, GC content, and prophage regions were visualized in Circos (100). The gene regions were visualized using the R package gggenes v0.4.1 (https://github.com/wilkox/gggenes).

Identification of HgT21 and phylogenetic construction.

We used a whole-genome multilocus sequence typing (wgMLST) method (101) to identify the HgT21 strain. Protein sequences of 28 species phylogenetically closely related to HgT21 were used for phylogenetic analysis (Table S1). The selection of representative species was carried out according to their pairwise average nucleotide identity (ANI) values, which were determined by FastANI (102). The species tree was inferred using the STAG algorithm and rooted with the STRIDE algorithm in the OrthoFinder program (103). Bacillus cereus B4264 was used as an outgroup.

Pan- and core-genome analysis.

Gene orthogroups were derived from OrthoFinder analysis. The gene count of all orthogroups was converted to a 0/1 matrix (1 indicates the presence of the gene in the orthogroup, and 0 indicates its absence). The resulting binary matrix was used to generate the gene accumulation curves of the pan- and core genomes using PanGP software (104) with the distance guide method, sample size of 5,000, 100 sample repeats, and 100 amplification coefficients. The species tree and panmatrix were plotted together using the phytools package (105). In addition, the proteins of the pangenome were grouped into one of the three studied cores, B. megaterium-B. aryabhattai, outgroup, and all species, after they were mapped to different COGs using the WebMGA server (106) and RPS-BLAST program.

Metal resistance of the HgT21 strain.

The survival of HgT21 cells exposed to different metal salts was evaluated as a measurement of their resistance to different metal ions. Lethal doses (LDs; concentration at which the bacterial cells were dead) of metal ions were established throughout viability assays. Bacterial cells were exposed to different metal salts, including sodium arsenite, sodium arsenate, mercury chloride, zinc sulfate, copper sulfate, and cobalt chloride. Briefly, 1 mL of HgT21 cells at the mid-log phase of growth in LB medium (1 × 10⁸ CFU/mL) was deposited in 24-well polystyrene plates (Nunclon Delta surface, Nunc) and treated with metallic salts at final concentrations of 0, 2.5, 5.0, 7.5, 10, 20, 30, 40, and 50 μM HgCl₂; 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mM ZnSO₄; 0, 0.5, 1.0, 1.5, 2.0, and 3 mM CuSO₄; 0, 60, 120, 180, 240, 300, 360, and 420 mM CoCl₂; or 0, 5, 10, 30, 60, 90, 120, 150, 180, and 210 mM Na₃AsO₄. The plate was incubated for 2 h at 37°C and 120 rpm. Subsequently, 0.1 mL of each well was taken for serial dilution, and 0.1 mL of each dilution was plated in triplicate onto LB agar and incubated at 37°C for 18 h. The colonies formed were counted and graphically reported as CFU per milliliter (CFU/mL) as a measure of the bacterial viability as a function of the metal ion concentration. Finally, the LD of each metal ion was determined.

Analysis of plant growth promotion by the HgT21 strain.

Seeds of Arabidopsis thaliana ecotype Col-0 were surface-sterilized with 95% (vol/vol) ethanol solution for 5 min and bleach (20% [vol/vol]; equivalent to 0.13 M sodium hypochlorite) for 7 min. Subsequently, the seeds were washed five times with sterile distilled water and stored at 4°C for 48 h in the dark to promote and synchronize germination. The stratified A. thaliana seeds were placed and germinated on petri dishes containing 0.2× Murashige and Skoog (MS) medium, pH 5.7, and 1% agar TC (micropropagation grade, PhytoTechnology Laboratories) in a horizontal arrangement on the upper zone (eight plants in each plate with 2 cm interdistance) before bacterial inoculation (107). Plates were sealed with plastic film to avoid seed contamination and placed vertically with an angle of 65° in a growth chamber (Percival Scientific) at 22 to 20°C with a photoperiod of 16 h of light and 8 h darkness.

The plant growth promotion of the HgT21 strain on A. thaliana was carried out by in vitro direct (in contact) and indirect (distant) interactions of plant root tips with bacterial cells as previously described (108). For this analysis, 6-day-old plates with A. thaliana seedlings were inoculated by streaking with 10 μL (approximately 1 × 10⁶ CFU/mL) of a 24-h bacterial culture of the HgT21 strain using an inoculating loop to draw a straight line over the middle (contact) or the lower zone (distant) of plates containing the A. thaliana seedlings. The inoculation of the lower zone avoids the interaction of plant root tips with bacterial cells (distant), whereas the inoculation in the middle zone allows for the interaction between bacterial cells and plant roots (contact). Petri dishes without bacterial inoculation were also prepared and used as controls. After bacterial inoculation, the plates were incubated in a growing chamber at 22 to 20°C with a photoperiod of 16 h of light and 8 h of darkness. Every 2 days, until day 8 postinoculation, photographs of plates were taken to determine primary root length, number and density of lateral roots (density = number of lateral roots/length of primary root) using ImageJ software (http://rsb.info.nih.gov/nih-image/). In addition, seedlings were removed from the plates, weighed, and dried at 80°C in paper bags until they reached a constant weight. The wet and the dry biomass was determined. All experiments were replicated at least three times. The differences in the number and density of lateral roots, primary root length, and the wet and dry weights of plants exposed and not exposed (control) to direct and distant interactions with HgT21 were calculated by performing one-way analysis of variance (ANOVA) in Microsoft Excel with significance set at P < 0.05.

Indoleacetic acid (IAA) production by the HgT21 strain.

A culture of HgT21 at the mid-log phase of growth was prepared in LB medium, and 0.1 mL of this culture (containing 1 × 10⁸ CFU/mL) was inoculated in 5 mL of M9 minimal medium supplemented with 0.5% glucose and 0.1% l-tryptophan and incubated for 96 h at 37°C and 180 rpm in triplicate. The amount of IAA produced was determined by the colorimetric method described by Gordon and Weber (109).

Antibiotic resistance of the HgT21 strain.

Resistance to different antibiotics was tested by the disk diffusion method and interpreted according to the manufacturer’s instructions for BBL antibiotic SensiDiscs (Becton, Dickinson Microbiology Systems, Cockeysville, MD) or Multidiscos (Bio-Rad, Mexico).

Data availability.

The raw sequence reads have been deposited in the NCBI Sequence Read Archive (SRA; SRR17752569 and SRR17752570) under BioProject accession number PRJNA800475. The draft genome sequence was submitted to GenBank with the accession number JAKKUX000000000.