Genome-Based Characterization of Bacillus velezensis HM1 from Silver Mine Tailings Reveals Potential Metal Resistance and Sulfur Assimilation Traits

Cuaxinque-Flores, Gustavo; Gómez-Godínez, Lorena Jacqueline; Ramírez-Mosqueda, Marco A.; Cadena-Zamudio, Jorge David; Armenta-Medina, Alma; Aguirre-Noyola, José Luis

doi:10.3390/data11050119

Abstract

The genus Bacillus is widely recognized for its metabolic versatility, enabling it to colonize extreme environments, including sites contaminated with metals. In this study, we report the genome of B. velezensis strain HM1, isolated from sulfur-rich mine tailings from silver mining activities in southwestern Mexico. Isolation was performed by heat treatment followed by selective cultivation in a medium enriched with mine tailings extract (metals and sulfates), resulting in a single dominant morphotype corresponding to strain HM1. Whole-genome sequencing was carried out using the Illumina NovaSeq platform (2 × 250 bp). The assembled genome of strain HM1 has a size of 4,044,128 bp, distributed across 20 contigs, with an N50 of 700,388 bp and an L50 of 3, and an average coverage of 66.8×. The GC content was 46.31%, with an estimated completeness of 99.81% and contamination of 0.01%. Genome analyses indicate that the assembly corresponds to a single chromosome, with no evidence of plasmid replicons. Genome annotation identified 3950 coding sequences (CDSs), 83 tRNAs, 11 rRNAs, 26 ncRNAs, and 4 sORFs. Phylogenomic analysis, together with genomic similarity metrics (ANI > 98.6%, AAI > 98.8%, dDDH > 87%), confirms its classification as Bacillus velezensis. Functionally, the genome encodes multiple genes involved in resistance to metals and metalloids (including ABC transporters, efflux pumps, and biotransformation enzymes), as well as a complete pathway for sulfate assimilation. Collectively, these genomic features reveal a broad repertoire of adaptive strategies employed by strain HM1 to thrive in metal-contaminated environments.

Dataset: The assembled genome (GCA_041519775.1) presented in the study are openly available in the NCBI BioProject PRJNA1150606 (https://www.ncbi.nlm.nih.gov/bioproject/?term=+PRJNA1150606, accessed on 13 May 2026).

Dataset License: Not applicable

Keywords:

whole genome sequencing; heavy metal stress; sulfur metabolism

1. Summary

The genus Bacillus belongs to the phylum Firmicutes, class Bacilli, order Bacillales, and family Bacillaceae, and is widely recognized for its ecological and biotechnological relevance [1,2]. Although its classification was initially based on cellular phenotypic traits, such as rod shape, Gram-positive staining, and the ability to form endospores, phylogenetic and genomic studies have demonstrated that it constitutes a taxonomically heterogeneous and continuously evolving group, with more than 400 validly published species [3]. From an ecological perspective, Bacillus is a ubiquitous genus found in soils, water, air, and microbiomes associated with plants and animals [4]. Its adaptive capacity is linked to diverse physiological and genetic mechanisms that enable it to withstand nutrient limitation and extreme conditions such as high temperatures, desiccation, and exposure to radiation and chemical agents [5].

Several bacterial species exhibit resistance to heavy metals and metalloids (HMMs) through biochemical mechanisms encoded in both chromosomal and mobile genetic elements [6]. These mechanisms include active metal efflux, enzymatic biotransformation, intracellular sequestration, metal-responsive regulation, and biomineralization, all of which reduce metal toxicity and bioavailability [7]. Mine tailings, a byproduct of precious metal extraction, are highly restrictive environments characterized by acidic pH, elevated metal concentrations, nutrient limitation, and low organic matter content [8,9]. They contain diverse sulfide minerals, such as pyrite (FeS₂), chalcopyrite (CuFeS₂), and galena (PbS), which are oxidized by water and native microbiota, releasing sulfates and other oxidized sulfur compounds [10]. The generation of acid mine drainage (AMD) further increases metal solubility and toxicity, limiting biological colonization [11]. Nevertheless, some Bacillus species have successfully adapted to these conditions [12,13].

Despite this, the genomic basis underlying such adaptations remains incompletely understood. Here, we characterize the genome of Bacillus velezensis HM1, a strain isolated from sulfur-rich silver mine tailings in Taxco de Alarcón, Guerrero, Mexico. Through genome assembly, functional annotation, and targeted mining of genes involved in metal resistance and sulfur metabolism, we identify key biochemical strategies that enable this strain to colonize and persist in this extreme environment.

2. Data Description

2.1. Isolation of Bacillus velezensis HM1

The metal-resistant strain HM1 was isolated from mine tailings collected at the “El Fraile” site in Taxco de Alarcón, Guerrero, Mexico. As an initial selection step, samples were subjected to heat treatment (80 °C for 20 min) to enrich for spore-forming bacteria. Subsequently, suspensions and serial dilutions were prepared and plated on R2A medium supplemented with a metal-enriched extract, with the aim of selecting metal-tolerant strains. This second selection step yielded a single dominant colony morphotype (strain HM1), which was isolated and purified. On a rich PY medium, colonies exhibited an opaque white appearance, irregular shape, and lobulated margins. They also displayed a phenotype consistent with extracellular matrix production (Figure 1a,b), a characteristic commonly observed in species of the genus Bacillus [14]. The identification of the genus was confirmed by phylogenetic analysis of the partial sequence of the 16s rRNA gene (Figure 1c).

Figure 1. Morphology and phylogenetic placement of Bacillus velezensis HM1. (a,b) Colonial morphology of strain HM1 on PY medium, showing a rough surface, dry farinose texture, slightly umbonate elevation, and radial growth with coalescent structures. (c) Maximum likelihood 16S rRNA gene phylogeny of strain HM1 and related taxa, inferred with IQ-TREE (GTR+G+I). Node values represent ultrafast bootstrap (UFBoot) support (1000 replicates).

2.2. Genomic Insights into Bacillus velezensis HM1

Whole-genome sequencing of Bacillus velezensis HM1 generated a total of 582,787 reads, which were used for genome assembly (Table 1). The assembly resulted in a genome of 4,044,128 bp distributed across 20 contigs, with a maximum contig length of 969,499 bp. The assembly exhibited an N50 of 700,388 bp and an L50 of 3, as well as an N75 of 283,754 bp and an L75 of 5, with an average coverage of 66.8×. The GC content was 46.13%, with no ambiguous nucleotides detected (N = 0) (Figure 2). Genome completeness was estimated at 99.81%, with a contamination level of 0.01%. The predicted coding density was 89.7%, comprising 3950 coding sequences (CDSs), 83 transfer RNA genes (tRNAs), 11 ribosomal RNA genes (rRNAs), 26 non-coding RNAs (ncRNAs), and 4 small open reading frames (sORFs). No plasmid replicons were identified within the assembled contigs.

Table 1. Genomic characteristics of Bacillus velezensis HM1.

Figure 2. Circular genome representation of Bacillus velezensis HM1. From the outer to the inner rings: coding sequences (CDSs) on the forward strand, CDSs on the reverse strand, RNA genes (tRNA and rRNA), GC content, and GC skew.

Functional annotation assigned 3167 of the 3950 predicted coding sequences (80.18%) to COG functional categories (Figure 3). Metabolism was the most represented category, particularly amino acid transport and metabolism (E = 285), carbohydrate transport and metabolism (G = 264), and coenzyme transport and metabolism (H = 182), suggesting a metabolically versatile genomic repertoire in strain HM1. The “Information Storage and Processing” category was primarily represented by transcription (K = 249) and translation, ribosomal structure, and biogenesis (J = 225). Within “Cellular Processes and Signaling,” the most abundant functions included cell cycle control and chromosome partitioning (D = 217) and cell wall/membrane/envelope biogenesis (M = 194). Notably, defense mechanisms (V = 95) and signal transduction (T = 150) were also well represented.

Figure 3. Functional annotation of the genome of Bacillus velezensis HM1. Distribution of predicted proteins across COG (Clusters of Orthologous Groups) functional categories, showing the relative abundance of genes involved in metabolism, cellular processes and signaling, information storage and processing, and poorly characterized functions.

The “Poorly Characterized” category comprised 333 genes, including those assigned to general function prediction (R = 179) and unknown function (S = 154). This fraction likely represents a reservoir of unexplored functions potentially associated with environmental adaptation, highlighting the genomic novelty associated with strains inhabiting extreme, metal-impacted environments.

2.3. Phylogenetic Position of Bacillus velezensis HM1

The taxonomic assignment of the strain HM1 was established using Overall Genome Relatedness Indices (OGRIs) through comparisons with genomes of phylogenetically related strains available in the Genome Taxonomy Database (GTDB) (Table 2). Average nucleotide identity (ANI) values indicated high similarity to Bacillus velezensis NRRL B-41580^T (98.85%) and Bacillus velezensis FZB42 (98.62%). Consistently, average amino acid identity (AAI) values were 98.96% and 98.81%, respectively, while digital DNA–DNA hybridization (dDDH) yielded values of 89.4% and 87.9% relative to these strains. Pairwise comparisons for each index are provided in Tables S1–S3. Collectively, these values exceed established species delineation thresholds (ANI ≥ 95%; AAI ≥ 95%, dDDH ≥ 70%), confirming the assignment of HM1 to Bacillus velezensis.

Table 2. Genomic similarity indices of Bacillus sp. HM1 with closely related strains.

2.4. Genetic Determinants of Metal Resistance

Analysis of proteins encoded in the genome of B. velezensis HM1 identified a diverse repertoire of putative microbial metal resistance genes (MMRGs). The most common systems were those associated with iron (43.3%), followed by nickel (16.7%) and zinc (10.0%). To a lesser extent, genes related to arsenic (5.3%), as well as copper, magnesium, and tellurium (each 2.7%), and mercury (2.0%) were identified. These genes were classified into five functional categories: transport/efflux, uptake, redox biotransformation, intracellular homeostasis, and metal-associated stress response (Figure 4, Table A1).

Figure 4. Distribution and metal resistance mechanisms in B. velezensis HM1. Inner sectors represent metals (Fe, Zn, Ni, As, Cu, Hg, Mo, Te, and Multi-metal), while outer sectors correspond to grouped functional categories of resistance (ABC transport, efflux systems, redox/detoxification, binding/import, and multidrug resistance). Link widths are proportional to the number of loci associated with each metal–mechanism combination. Specific patterns were observed; for example, the ABC transport system dominated in iron (Fe), indicating a transport-based uptake strategy. In contrast, arsenic (As) exhibits a more balanced distribution between efflux and redox/detoxification mechanisms, reflecting a multifunctional resistance strategy. Nickel (Ni) is characterized by a combination of transport and binding/import systems, suggesting highly structured uptake systems.

The predominant mechanism corresponded to ABC-type transport systems and permeases (64.7%), including multiple copies of transporters associated with iron (families such as fbpC, fecE, fecD), zinc (znuABC), nickel (nikABCDE), and molybdate (modA). Efflux systems (18.0%) were also identified, including P-type ATPases involved in metal transport (Cd/Zn/Co-ATPase), cation antiporters (fieF, mneP), and specific pumps for arsenic (arsB) and copper (copZ, ycnJ), which are involved in the extrusion of toxic metals.

Within the biotransformation category (10.7%), key enzymes such as reductases and oxidases were detected, including arsenate reductase (arsC), arsenite methyltransferase (arsM), arsenite oxidase (aioA), and mercury reductases, all of which contribute to the conversion of metals into less toxic forms. Additionally, proteins associated with oxidative stress response (5.3%) were identified, including superoxide dismutases (sodA), as well as quinone-dependent systems (arsH), which contribute to mitigating metal-induced cellular damage. To a lesser extent, proteins associated with tellurium resistance (terD, klaB) and NRAMP-type divalent cation transport systems (mntH) were identified. The absence of canonical sil and pbr operons suggests that strain HM1 may rely on alternative and potentially non-specific resistance mechanisms to cope with the elevated concentrations of Pb and Ag present in this mine tailings environment.

2.5. Genetic Determinants of Sulfur Metabolism

Key genes involved in the sulfate assimilation pathway were identified, including sat (sulfate adenylyltransferase), cysC (adenylylsulfate kinase), cysH (phosphoadenylylsulfate reductase), cysI (heme-containing subunit of the NADPH-dependent assimilatory sulfite reductase), cysE (serine O-acetyltransferase), and cysK (cysteine synthase A). This gene set is consistent with a putative assimilatory sulfate reduction pathway involved in the conversion of sulfate to sulfide and its subsequent incorporation into sulfur-containing amino acids, particularly cysteine.

In conclusion, the genomic features identified in B. velezensis HM1 provide a genome-based framework for exploring potential mechanisms associated with persistence in metal- and sulfur-rich mine tailings environments. These findings provide a useful resource for future comparative and experimental studies focused on environmental Bacillus strains.

3. Methods

3.1. Isolation and Culture Conditions

Strain HM1 was isolated from mine tailings collected at the “El Fraile” site (18°31′55″ N, 99°37′43″ W). Tailings samples (pH 6.7–7.4) were subjected to heat treatment at 80 °C for 20 min, followed by cooling at room temperature. Subsequently, suspensions were prepared in sterile saline solution (0.85% NaCl), and serial dilutions were performed and spread-plated onto R2A medium (Reasoner’s 2A; yeast extract 0.5 g L⁻¹, peptone 0.5 g L⁻¹, casein hydrolysate 0.5 g L⁻¹, glucose 0.5 g L⁻¹, soluble starch 0.5 g L⁻¹, dipotassium phosphate 0.3 g L⁻¹, magnesium sulfate 0.024 g L⁻¹, and sodium pyruvate 0.3 g L⁻¹), supplemented with an aqueous tailings extract at a 1:5 ratio (tailings:water). Metal concentrations in these residues were as follows: Ag (30.50 ± 6.80 mg kg⁻¹), Cd (39.50 ± 55.60 mg kg⁻¹), Cu (225 ± 98 mg kg⁻¹), Fe (55 ± 16 g kg⁻¹), Mn (940 ± 1534 mg kg⁻¹), Pb (3677 ± 1481 mg kg⁻¹), V (16.30 ± 12.90 mg kg⁻¹), Zn (3118 ± 3329 mg kg⁻¹), and As (1324 ± 846 mg kg⁻¹) [8]. Plates were incubated at 30 °C for 72 h. Colonies displaying distinct morphologies were purified by cross-streaking on PY medium (yeast extract 3 g L⁻¹, peptone 5 g L⁻¹, agar 15 g L⁻¹, pH 7) and incubated at 30 °C for 24 h.

3.2. Whole-Genome Sequencing

Cells from a pure culture of strain HM1, grown for 24 h in PY broth, were sent to MicrobesNG (The BioHub Birmingham, Birmingham, UK) for genomic DNA sequencing. Forty microliters of a cell suspension were lysed in 120 µL of TE buffer supplemented with lysozyme and RNase A (0.1 mg mL⁻¹), followed by incubation at 37 °C for 25 min. Subsequently, proteinase K (0.1 mg mL⁻¹) and SDS (0.5% v/v) were added, and the mixture was incubated at 65 °C for 5 min.

Genomic DNA was purified using an equivalent volume of SPRI beads and resuspended in 10 mM Tris-HCl (pH 8.0). DNA quantification was performed using the Quant-iT dsDNA HS kit (Thermo Fisher Scientific, Waltham, MA, USA) on an Eppendorf AF2200 plate reader (Eppendorf, Hamburg, Germany). Library preparation was carried out on a Hamilton Microlab STAR automated system using the Nextera XT Library Prep kit (Illumina, San Diego, CA, USA), following the manufacturer’s instructions with two modifications: increased DNA input concentration and extension of PCR elongation time to 45 s. Sequencing was performed on an Illumina NovaSeq 6000 platform (paired-end, 250 bp).

3.3. Genome Assembly, Functional Annotation and Taxonomic Assignment

Raw read quality was assessed using FastQC v0.12.1, and high-quality reads (Phred > 30) were retained using TrimGalore v0.6.4 [15]. Genome assembly was performed with SPAdes v3.15.5 [16], while completeness and contamination were evaluated using CheckM v1.2.2 [17]. Gene prediction and functional annotation were conducted using Bakta v1.11.0 [18]. Genome visualization was generated using MGCplotter v1.0.1, and functional classification of genes was assigned using COGclassifier v1.0.4 (https://github.com/moshi4/MGCplotter/, accessed on 13 May 2026). Taxonomic assignment of the genome was performed using the Genome Taxonomy Database Toolkit (GTDB-Tk v2.6.1, database release 18 February 2026) [19]. In silico DNA–DNA hybridization (dDDH) analyses were conducted with phylogenetically related strains using the TYGS server (https://tygs.dsmz.de/, accessed on 13 May 2026). Average nucleotide identity (ANI) was calculated from genome alignments using MUMmer implemented in pyani v0.2.12 [20], and average amino acid identity (AAI) was estimated using CompareM v0.1.2 (https://github.com/dparks1134/CompareM/, accessed on 13 May 2026).

3.4. Genome Mining of Metal Resistance Genes and Sulfur Metabolism

Identification of microbial metal resistance genes (MMRGs) was performed using a hidden Markov model (HMM)-based approach. Predicted protein sequences were analyzed with HMMER v3.4 against the MetHMMDB database. MetHMMDB is a curated database of 254 profile Hidden Markov Models (HMMs) representing 121 microbial metal resistance genes (MMRGs) and associated functions. The database uses HMM-based functional annotation to improve the sensitivity and specificity of MMRG detection in genomic datasets compared with conventional sequence similarity-based approaches [21]. Stringent filtering criteria were applied: bit score ≥ 60, coverage ≥ 70%, and E-value ≤ 1 × 10⁻¹⁰, retaining only hits meeting all three thresholds [22]. Genes were subsequently classified according to the associated chemical element (metal or metalloid), functional mechanism, and biological role. Genes associated with sulfur metabolism were identified through BLASTp v2.17.0 searches between the HM1 proteome and a curated UniProt protein database. Representative genes from major bacterial sulfur metabolic pathways were included, including assimilatory sulfate reduction, dissimilatory sulfate reduction, and oxidation of reduced sulfur compounds. Similarity searches were conducted using stringent thresholds (E-value ≤ 1 × 10⁻¹⁵, sequence identity ≥ 30%, and alignment coverage ≥ 70%).

4. User Notes

The genome assembly and associated annotations of Bacillus velezensis HM1 are publicly available through NCBI under BioProject PRJNA1150606 (accession GCA_041519775.1). Raw sequencing reads, assembled contigs, and annotation files (including GFF, protein FASTA, and functional assignments) can be downloaded for downstream analyses. The dataset is suitable for comparative genomics, functional annotation refinement, and studies of microbial adaptation to mine tailings and acid mine drainage (AMD).

The functional traits described in this study are based on genome annotation and in silico predictions derived from bioinformatic analyses. Therefore, the inferred capacities related to metal resistance, sulfur assimilation, and metabolic versatility should be considered potential functional attributes that require future experimental validation under controlled conditions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/data11050119/s1, Table S1: Average Nucleotide Identity (ANI) values (%) among Bacillus strains used for phylogenomic comparison; Table S2: Average Amino Acid Identity (AAI) values (%) among Bacillus strains used for phylogenomic comparison; Table S3: In silico DNA–DNA hybridization (dDDH) values (%) among Bacillus strains used for species delineation.

Author Contributions

Conceptualization, J.L.A.-N. and G.C.-F.; methodology, J.L.A.-N. and G.C.-F.; software, J.L.A.-N. and G.C.-F.; formal analysis, G.C.-F. and L.J.G.-G.; investigation, G.C.-F., L.J.G.-G. and J.L.A.-N.; resources, J.L.A.-N.; data curation, M.A.R.-M., J.D.C.-Z. and A.A.-M.; writing—original draft preparation, J.L.A.-N.; writing—review and editing, J.L.A.-N., G.C.-F., M.A.R.-M., J.D.C.-Z. and A.A.-M.; funding acquisition, J.L.A.-N. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the GetGenome initiative at the Sainsbury Laboratory in Norwich, UK, with support from the Gatsby Charitable Foundation and the Biotechnology and Biological Sciences Research Council, as part of the “GetGenome Mexico 2023” project. Number Project: 20230818.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The assembled genome (GCA_041519775.1) presented in the study are openly available in NCBI BioProject PRJNA1150606 (https://www.ncbi.nlm.nih.gov/bioproject/?term=+ PRJNA1150606), accessed on 13 May 2026.

Acknowledgments

We thank UATI for their expert technical support and server maintenance at Centro de Ciencias Genómicas, UNAM.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Microbial Metal Resistance Genes (MMRGs) of Bacillus velezensis HM1.

Locus_ID	Metal	HMM_Model_Gene	Product
HM1_00044	Fe	Fe_transport_fbpC_1	ABC transporter component, ATP-binding
HM1_00063	Fe	Fe_transport_fbpC_1	ABC transporter (ATP-binding protein)
HM1_00067	Fe	Fe_transport_fbpC_1	ABC transporter (ATP-binding protein)
HM1_00071	Fe	Fe_transport_fbpC_1	Bacitracin export ATP-binding protein
HM1_00091	Zn	Zn_transport_troB_1	ABC transporter (ATP-binding protein)
HM1_00127	Multi-M	Cation_efflux_fieF_1	Cation efflux system protein
HM1_00355	Fe	Fe_transport_fbpC_1	Putative glutamine transport system ATP-binding protein
HM1_00376	As	As_oxidase_aioA_3	formate dehydrogenase subunit alpha
HM1_00474	Fe	Fe_superoxide_dismutase_1	Superoxide dismutase [Mn]
HM1_00480	Fe	Fe_transport_fbpC_1	phosphate ABC transporter ATP-binding protein PstB
HM1_00481	Fe	Fe_transport_fbpC_1	phosphate ABC transporter ATP-binding protein PstB
HM1_00499	Multi-M	MgCo_corC_1	HlyC/CorC family transporter
HM1_00573	Hg	Hg_reductase_4	dihydrolipoyl dehydrogenase
HM1_00581	Fe	Fe_transport_fbpC_1	Polar amino acid transport system ATP-binding protein
HM1_00718	As	As_methyltransferase_2	demethylmenaquinone methyltransferase
HM1_00912	Fe	Fe_superoxide_dismutase_1	superoxide dismutase
HM1_03767	Fe	Fe_transport_fbpC_1	ATP-binding cassette domain-containing protein
HM1_03898	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein
HM1_03899	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein
HM1_03965	Te	Te_protein_klaB_1	Toxic anion resistance protein
HM1_01059	Multi-M	MgCo_corC_1	Transporter associated domain protein
HM1_01083	Multi-M	Multidrug_efflux_yfmO_1	Major facilitator family transporter
HM1_01090	Fe	Fe_transport_fbpC_1	Simple sugar transport system ATP-binding protein
HM1_01147	Fe	Fe_transport_1	RBAM_029210
HM1_01182	Multi-M	Multidrug_efflux_yfmO_1	MFS transporter, DHA1 family, multidrug resistance protein
HM1_01186	Fe	Fe_transport_fbpC_1	Multiple sugar transport system ATP-binding protein
HM1_01200	As	As_methyltransferase_2	Arsenite methyltransferase
HM1_01207	Ni	Ni_Import_nikE_1	Fe-S cluster assembly ATPase SufC
HM1_01211	Ni	Ni_Import_nikE_1	methionine ABC transporter ATP-binding protein MetN
HM1_01217	As	As_reductase_2	Arsenate reductase
HM1_01230	Fe	Fe_transport_fecE_1	ABC transporter ATP-binding protein
HM1_01253	Fe	Fe_transport_fecE_1	Ferric enterobactin transport ATP-binding protein FepC
HM1_01254	Fe	Fe_transport_fecD_1	Iron complex transport system permease protein
HM1_01255	Mo	Mo_binding_molA_1	Iron complex transport system substrate-binding protein
HM1_01265	Fe	Fe_transport_fecE_1	Iron complex transport system ATP-binding protein
HM1_01266	Fe	Fe_transport_fecD_1	Iron complex transport system permease protein
HM1_01267	Fe	Fe_transport_fecD_1	Iron complex transport system permease protein
HM1_01275	Mo	Mo_binding_modA_1	molybdate ABC transporter substrate-binding protein
HM1_01279	Fe	Fe_transport_fbpC_1	Multidrug ABC transporter, ATPase
HM1_01292	Multi-M	CdCoZn_ATPase_1	Cd(2+)-exporting ATPase
HM1_01293	Cu	Cu_transport_ATPase_1	copper chaperone CopZ
HM1_01294	Cu	Cu_chaperone_copZ_1	copper chaperone CopZ
HM1_01304	Fe	Fe_transport_fbpC_1	Polar amino acid transport system ATP-binding protein
HM1_01326	Zn	Zn_import_znuC_1	ABC transporter domain-containing protein
HM1_01332	Ni	Ni_Import_nikE_1	Subtilin transport ATP-binding protein SpaT
HM1_01337	Fe	Fe_transport_fbpC_1	Quaternary amine transport ATP-binding protein
HM1_01342	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein OpuCA
HM1_01438	Fe	Fe_transport_fbpC_1	Efflux transporter (ATP-binding and permease protein)
HM1_01475	Fe	Fe_transport_fbpC_1	cell division ATP-binding protein FtsE
HM1_01545	Fe	Fe_transport_fbpC_1	heme ABC exporter ATP-binding protein CcmA
HM1_01553	As	As_pump_4	Arsenical pump membrane protein
HM1_01648	As	As_reductase_5	Protein-arginine-phosphatase
HM1_01676	Ni	Ni_Import_nikE_1	ATP-binding cassette, subfamily B, bacterial
HM1_01713	Fe	Fe_transport_fbpC_1	Fluoroquinolones export ATP-binding protein
HM1_01741	Fe	Fe_transport_fbpC_1	Antibiotic transport system ATP-binding protein
HM1_01822	Zn	Zn_transport_troB_1	Iron complex transport system ATP-binding protein
HM1_01890	Hg	Hg_reductase_4	dihydrolipoyl dehydrogenase
HM1_01918	Zn	Zn_import_znuC_1	putative ABC transporter ATP-binding protein YkpA
HM1_01925	Fe	Fe_transport_fbpC_1	Macrolide export ATP-binding/permease protein
HM1_01976	Zn	Zn_ATPase_1	Zinc-transporting ATPase
HM1_02036	Fe	Fe_transport_fecE_1	energy-coupling factor transporter ATPase
HM1_02059	Ni	Ni_Import_nikE_1	Cell wall oligopeptide ABC transporter
HM1_02064	Ni	Ni_import_nikD_1	Peptide/nickel transport system ATP-binding protein
HM1_02065	Ni	Ni_permease_nikC_1	Dipeptide transport system permease protein DppC
HM1_02066	Ni	Ni_permease_nikB_1	Peptide/nickel transport system permease protein
HM1_02134	Ni	Ni_Import_nikE_1	Putative ABC transport system ATP-binding protein
HM1_02182	Fe	Fe_transport_fbpC_1	Daunorubicin/doxorubicin resistance ATP-binding protein
HM1_02225	Ni	Ni_Import_nikE_1	Oligopeptide ABC transporter ATP-binding protein OppF
HM1_02226	Ni	Ni_import_nikD_1	Oligopeptide transport ATP-binding protein OppD
HM1_02227	Ni	Ni_permease_nikC_1	oligopeptide ABC transporter permease OppC
HM1_02228	Ni	Ni_permease_nikB_1	Oligopeptide transport system permease protein OppB
HM1_02229	Ni	Ni_periplasmic_1	Oligopeptide ABC transporter (Binding lipoprotein)
HM1_02232	Ni	Ni_permease_nikC_1	oligopeptide ABC transporter permease
HM1_02233	Ni	Ni_permease_nikB_1	Glutathione transport system permease protein GsiC
HM1_02234	Ni	Ni_periplasmic_1	Oligopeptide-binding protein AppA
HM1_02235	Ni	Ni_Import_nikE_1	Oligopeptide transport ATP-binding protein AppF
HM1_02236	Ni	Ni_import_nikD_1	Oligopeptide ABC transporter (ATP-binding protein)
HM1_02301	Multi-M	Multidrug_efflux_yfmO_1	MFS transporter, DHA1 family, multidrug resistance protein
HM1_02352	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein EcsA
HM1_02369	Fe	Fe_transport_fbpC_1	Na+-efflux ABC transporter (ATP-binding protein)
HM1_02388	Ni	Ni_Import_nikE_1	ABC transporter ATP-binding/permease protein YheH
HM1_02389	Fe	Fe_transport_1	ATP-binding cassette, subfamily B, bacterial
HM1_02402	Multi-M	MgCo_corC_1	HlyC/CorC family transporter
HM1_02406	Multi-M	MgCo_corC_1	UPF0053 protein YhdP
HM1_02424	As	Quinone_reductase_arsH_1	NAD(P)H dehydrogenase (Quinone)
HM1_02449	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein
HM1_02450	Fe	Fe_transport_fecE_1	ABC-2 type transport system ATP-binding protein
HM1_02475	Fe	Fe_transport_fbpC_1	Aliphatic sulfonate ABC transporter (Binding protein)
HM1_02478	Ni	Ni_Import_nikE_1	Spermidine/putrescine import ATP-binding protein potA
HM1_02479	Ni	Ni_import_nikD_1	Phosphate import ATP-binding protein pstB Phosphate-transporting ATPase ABC phosphate transporter
HM1_02480	Ni	Ni_permease_nikC_1	oligopeptide ABC transporter permease
HM1_02481	Ni	Ni_permease_nikB_1	Peptide/nickel transport system permease protein
HM1_02482	Ni	Ni_periplasmic_1	Oligopeptide-binding protein
HM1_02542	Fe	Fe_transport_fbpC_1	ATP-binding cassette domain-containing protein
HM1_02692	Multi-M	Multidrug_efflux_yfmO_1	Putative MFS-type transporter
HM1_02833	Fe	Fe_transport_fecD_1	Ferrichrome transport system permease protein fhuB
HM1_02834	Fe	Fe_transport_fecD_1	Ferrichrome transport system permease protein
HM1_02837	As	As_pump_4	Arsenical pump membrane protein
HM1_02849	Fe	Fe_transport_fecE_1	thiol reductant ABC exporter subunit CydC
HM1_02850	Zn	Zn_import_znuC_1	thiol reductant ABC exporter subunit CydD
HM1_02855	Fe	Fe_transport_fbpC_1	Oligosaccharides import ATP-binding protein MsmX
HM1_02908	Zn	Zn_uptake_znuC_1	ABC transporter ATP-binding protein NatA
HM1_02920	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein YxdL
HM1_02925	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein
HM1_02954	Ni	Ni_Import_nikE_1	Peptide cleavage/export ABC transporter
HM1_02997	Zn	Zn_import_znuC_1	ribosomal protection-like ABC-F family protein
HM1_02998	Multi-M	Multidrug_efflux_yfmO_1	Major facilitator superfamily (MFS)
HM1_03113	As	As_methyltransferase_2	Putative methyltransferase YcgJ
HM1_03121	Multi-M	Multidrug_efflux_yfmO_1	Bcr/CflA family efflux transporter
HM1_03126	Zn	Zn_import_znuC_1	ribosomal protection-like ABC-F family protein
HM1_03152	As	As_res_arsB_2	Arsenic resistance protein
HM1_03166	Multi-M	CdCoZn_antiport_2	Cadmium, cobalt and zinc/H(+)-K(+) antiporter
HM1_03232	Multi-M	Cation_efflux_fieF_1	Putative cation efflux system
HM1_03238	Fe	Fe_transport_fbpC_1	ABC transporter (ATP-binding protein) YdbJ
HM1_03253	Multi-M	Cation_transport_mntH_2	Nramp family divalent metal transporter
HM1_03302	Cu	Cu_transport_ycnJ_1	Putative copper resistance protein D
HM1_03315	Fe	Fe_transport_fecE_1	Iron-siderophore ABC transporter (ATP-binding protein)
HM1_03316	Fe	Fe_transport_fecD_1	Iron-siderophore ABC transporter (Permease)
HM1_03317	Fe	Fe_transport_fecD_1	Iron ABC transporter permease
HM1_03324	Fe	Fe_transport_fbpC_1	Lipoprotein-releasing system ATP-binding protein LolD
HM1_03327	Fe	Fe_transport_fbpC_1	Polar amino acid transport system ATP-binding protein
HM1_03345	Fe	Fe_transport_fbpC_1	cystine ABC transporter ATP-binding protein TcyC
HM1_03365	As	As_oxidase_aioA_1	assimilatory nitrate reductase catalytic subunit NasC
HM1_03371	Multi-M	Cation_symport_actP_1	sodium/proline symporter PutP
HM1_03394	Fe	Fe_transport_fbpC_1	ABC transporter ATP-binding protein OpuAA
HM1_03396	Te	Te_protein_klaB_1	putative protein YceH
HM1_03399	Te	Te_resistance_terD_1	putative protein YceE
HM1_03400	Te	Te_resistance_terD_1	General stress protein 16U
HM1_03401	Te	Te_resistance_terD_1	Stress response protein
HM1_03403	Zn	Zn_uptake_znuB_1	Zinc ABC transporter (Permease)
HM1_03404	Zn	Zn_uptake_znuC_1	High-affinity zinc uptake system ATP-binding protein ZnuC
HM1_03405	Fe	Fe_binding_yfeA_1	Zinc transport in via ABC system
HM1_03430	Multi-M	CdCoZn_antiport_2	Manganese efflux system protein MneP
HM1_03467	Zn	Zn_uptake_znuC_1	ABC transporter ATP-binding protein
HM1_03479	Zn	Zn_import_znuC_1	Nod factor export ATP-binding protein I
HM1_03500	Fe	Fe_transport_fecD_1	Iron complex transport system permease protein
HM1_03501	Fe	Fe_transport_fecD_1	iron ABC transporter permease FeuC
HM1_03518	Zn	Zn_import_znuC_1	ribosomal protection-like ABC-F family protein
HM1_03552	Cu	Cu_oxidase_cueO_1	Spore copper-dependent laccase
HM1_03554	Multi-M	Cation_efflux_fieF_1	Cation efflux system protein (Zinc/cadmium/cobalt)
HM1_03590	Multi-M	Cation_symport_actP_1	sodium/proline symporter PutP
HM1_03595	Multi-M	Multidrug_mdtB_1	Hydrophobic/amphiphilic exporter-1 (Mainly G-bacteria),
HM1_03599	Fe	Fe_transport_fbpC_1	Nod factor export ATP-binding protein I
HM1_03643	Zn	Zn_import_znuC_1	ribosomal protection-like ABC-F family protein
HM1_03645	Multi-M	Multidrug_efflux_yfmO_2	Multidrug efflux protein YfmO
HM1_03646	Zn	Zn_import_znuC_1	ribosomal protection-like ABC-F family protein
HM1_03717	Hg	Hg_reductase_4	dihydrolipoyl dehydrogenase
HM1_03723	Fe	Fe_transport_fbpC_1	ATP-binding cassette, subfamily B, bacterial
HM1_03724	Ni	Ni_Import_nikE_1	ATP-binding cassette, subfamily B, bacterial
HM1_03760	Fe	Fe_transport_fecE_1	ABC transporter ATP-binding protein YgaD

Multi-M = Multimetal tolerance mechanisms.

References

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Figure 1. Morphology and phylogenetic placement of Bacillus velezensis HM1. (a,b) Colonial morphology of strain HM1 on PY medium, showing a rough surface, dry farinose texture, slightly umbonate elevation, and radial growth with coalescent structures. (c) Maximum likelihood 16S rRNA gene phylogeny of strain HM1 and related taxa, inferred with IQ-TREE (GTR+G+I). Node values represent ultrafast bootstrap (UFBoot) support (1000 replicates).

Figure 2. Circular genome representation of Bacillus velezensis HM1. From the outer to the inner rings: coding sequences (CDSs) on the forward strand, CDSs on the reverse strand, RNA genes (tRNA and rRNA), GC content, and GC skew.

Figure 3. Functional annotation of the genome of Bacillus velezensis HM1. Distribution of predicted proteins across COG (Clusters of Orthologous Groups) functional categories, showing the relative abundance of genes involved in metabolism, cellular processes and signaling, information storage and processing, and poorly characterized functions.

Figure 4. Distribution and metal resistance mechanisms in B. velezensis HM1. Inner sectors represent metals (Fe, Zn, Ni, As, Cu, Hg, Mo, Te, and Multi-metal), while outer sectors correspond to grouped functional categories of resistance (ABC transport, efflux systems, redox/detoxification, binding/import, and multidrug resistance). Link widths are proportional to the number of loci associated with each metal–mechanism combination. Specific patterns were observed; for example, the ABC transport system dominated in iron (Fe), indicating a transport-based uptake strategy. In contrast, arsenic (As) exhibits a more balanced distribution between efflux and redox/detoxification mechanisms, reflecting a multifunctional resistance strategy. Nickel (Ni) is characterized by a combination of transport and binding/import systems, suggesting highly structured uptake systems.

Table 1. Genomic characteristics of Bacillus velezensis HM1.

Table 2. Genomic similarity indices of Bacillus sp. HM1 with closely related strains.

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Strain	ANI (%)	AAI (%)	dDDH (%)
Bacillus velezensis NRRL B-41580	98.85	98.96	89.4
Bacillus velezensis FZB42	98.62	98.81	87.9
Bacillus siamensis KCTC 13613	94.4	96.08	56.7
Bacillus amyloliquefaciens DSM 7	94.2	95.66	55.5
Bacillus nakamurai NRRL B-41091	87.03	90.5	30.9
Bacillus sp. ISL 51	86.93	90.42	30.6

Feature	Value
Total reads	582,787
Genome coverage (X)	66.8
Genome size (Mb)	4.04
Number of contigs	20
Contig N50 (kb)	700.4
L50	3
L75	5
GC content (%)	46.13
CDSs (total)	3950
tRNAs	83
rRNAs	11
ncRNAs	26
sORFs	4