To withstand saline stress and metal stress, some bacteria have adaptive metabolic strategies to maintain osmotic equilibrium between the intracellular and the environment thanks to a system of membrane proteins that export cations such as Na⁺/metal efflux pumps or K⁺ and organic solutes (sugars, amino acids) accumulation pumps inside the cell (Uma et al. 2020, Yu et al. 2022). Moreover, certain bacteria synthesise EPS, polymeric substances that contribute to cation adsorption, promote soil aggregation, form complexes with metals and serve as a protective barrier — enhancing bacterial and plant tolerance to saline and metal stress (Zadeh et al. 2023). EPS are key extracellular polysaccharides that account for a large proportion of extracellular macromolecules and are the most intensively studied due to their notable structural and compositional diversity, which confer unique properties to each type of EPS (Costa et al. 2018). EPS are an essential part of biofilms, aiding microorganisms in surviving adverse environmental conditions such as drought, salinity, heavy metals etc. (Qurashi and Sabri 2012, Lian et al. 2022). In addition, EPS participate in a crucial role in various biological processes, including water retention (Vu et al. 2021), metal complexing (Mahgoub et al. 2018) and antimicrobial activities such as antibacterial (Alharbi et al. 2023), antiviral, immunomodulatory (Netrusov et al. 2023) and anti-tumour properties (Mahgoub et al. 2018, Chirakkara and Abraham 2023). Additionally, EPS exhibits antioxidant activity. Furthermore, the production of biofilms by microorganisms in soil is vital for holding on to water and nutrients, minimising the impact of environmental toxic factors on plants, thus benefitting plant health and enhancing their resistance to abiotic stresses (Paul et al. 2024).

EPS biosynthesis in bacteria is a complex process influenced not only by genetic factors, but also by environmental conditions, which significantly impact both yield and structural composition. EPS can be synthesised extracellularly to produce glucan (dextran) or fructan (levan) type homopolymers and uses a single sucrase protein. However, EPS is mostly synthesised intracellularly, including many complex steps in which nucleotide sugars undergo many enzymatic transformations inside the cell before being assembled, transported and elongated into the polymer chain and exported to the extracellular environment (Schmid et al. 2015). Therefore, a variety of EPS types with monosaccharide composition and complex branching are created. Exploiting the genetic traits, as well as environmental factors affecting EPS yield and structure, is essential to predict and adjust culture conditions to obtain EPS types suitable for application orientation.

The coral islands of Vietnam are mainly composed of calcium carbonate (CaCO₃) and are characterised by high alkalinity, loose texture, low fertility, poor water retention, high salinity, low cation exchange capacity and strong exposure to heat and solar radiation. These harsh conditions hinder cultivation, making soil improvement essential. EPS-producing bacteria contribute to soil remediation through multiple mechanisms: (i) their EPS facilitate the adsorption and immobilisation of toxic cations such as Na⁺ and heavy metals, thereby reducing their bioavailability (Paul et al. 2024); (ii) due to their high molecular weight, negative charge and abundance of hydrophilic functional groups, EPS enhance soil aggregation and water retention, improving the physical structure of the soil (Zadeh et al. 2023); and (iii) EPS-mediated biofilm formation creates a protective matrix that enhances microbial survival and root colonisation under adverse environmental conditions (Lian et al. 2022). These synergistic effects highlight the potential application of EPS-producing bacterial strains in the rehabilitation of degraded, arid and saline soils and in promoting sustainable agriculture, especially in coastal and coral island regions.

Bacillus species are widely recognised as soil-dwelling bacteria that promote plant growth and enhance stress tolerance under adverse environmental conditions (Vardharajula 2014). Several Bacillus strains have demonstrated significant potential in soil remediation, stress alleviation in plants and biorestoration of contaminated environments. Notably, the Cd-tolerant strain Bacillus sp. M6 has been reported to reduce the bioavailability of heavy metals in soil and enhance the effectiveness of plant-assisted remediation when combined with biochar and rhamnolipid, achieving up to 16.47% Cd removal, while stimulating the growth of microbial communities involved in nitrogen cycling and metal transport (Abid et al. 2022). In saffron cultivation, Bacillus sp. strain D5, isolated from saffron corms, has demonstrated both plant growth-promoting and antifungal activities, effectively reducing corm rot caused by Fusarium oxysporum R1 and enhancing plant performance under both pot and field conditions (Magotra et al. 2021). Similarly, two native Bacillus strains were reported to induce systemic resistance and reduce disease severity in Crocus sativus through activation of plant defence mechanisms, supporting their potential as biocontrol agents (Ali et al. 2024). In addition, some Bacillus strains produce EPS, which contribute to heavy metal immobiliation by forming stable precipitates such as Fe₂Pb(PO₄)₂ and Cd₂(PO₄)₂ and enhancing soil structure through microaggregate formation. These EPS-mediated effects support beneficial microbial communities and help reduce the uptake of toxic metals by plants such as lettuce (Zhang et al. 2024).

Amongst Bacillus species, B. velezensis is recognised as a probiotic for both animals and plants (Ercole et al. 2024). Several studies have reported that B. velezensis strains are capable of producing EPS and have investigated the biological activities of both the EPS and these strains (Mahgoub et al. 2018,Vu et al. 2021,Alharbi et al. 2023). Additionally, with the help of whole genome sequencing (WGS) studies, genetic characteristics of EPS-producing B. velezensis strains have become clearer. WGS analysis has identified eps gene clusters related to EPS biosynthesis that are relatively conserved amongst Bacillus species (Wu et al. 2020), genes encoding for the production of secondary metabolites, as well as beneficial nutrients or proteins associated with stress resistance and adhesion (Raj et al. 2023). Here, we present a comprehensive analysis of the salt-tolerant, EPS-producing B. velezensis DTA1 strain, which was isolated from a soil sample collected on a Vietnamese coral island. This study investigates its ability to utilise various nutrient sources, its EPS biosynthesis capacity and its tolerance to environmental stress, using biochemical tests and WGS analysis. Although several B. velezensis strains have been reported for their ability to produce EPS and support bioremediation, most were isolated from non-stressful or moderately controlled environments rather than from naturally extreme habitats. Meanwhile, this study focuses on the native B. velezensis strain DTA1, isolated from offshore coral island soil in Vietnam, an environment characterised by high salinity, drought, nutrient scarcity and intense solar radiation. These island ecosystems represent the frontline of climate change impacts, where agricultural development is severely limited by poor water and nutrient retention and widespread salinisation. Therefore, our research not only characterises the genetic and physiological traits of this uniquely halotolerant strain, but also aims to contribute to the development of microbe-based strategies for improving the structure and fertility of degraded coral sand soils, thereby supporting sustainable agriculture in Vietnam’s coastal and island regions.

Bacterial isolation and biochemical tests on carbohydrate utilisation

The EPS-producing strain DTA1 was isolated from a coral island soil in Khanh Hoa Province, Vietnam. This strain grew on Luria-Bertani (LB) agar plates supplemented with 3% sodium chloride (NaCl) for 24 hours at 30°C and its EPS-producing ability was assessed by the String test (Ramya et al. 2020). The strain was preserved at -85°C in LB medium supplemented with 38% (v/v) glycerol at the Department of Biotechnology, Joint Vietnam-Russia Tropical Science and Technology Research Center. The ability of the DTA1 strain to ferment 49 different carbohydrates was characterised using API® 50CHB medium (BioMérieux, France) following the manufacturer's guidelines.

Determination of salt and heavy metal tolerance

To evaluate salt tolerance, NaCl/MgSO₄ salts were diluted with LB medium to the final concentration after adding strain DTA1 to 96-well microplates to reach the following values: 0, 2.5, 5, 7.5, 10, 12.5, 15 and 17.5%. Each sample was repeated 3 times; the negative control samples contained no bacterial inoculum. The culture medium of strain DTA1 after 16–18 hours in LB medium was diluted and added to the 96-well plates so that the bacterial density in each well reached 5×10⁵ CFU/ml. The plates were shaken at 30^oC, 150 rpm for 48 hours and then evaluated based on visible growth. The salt tolerance was assessed by the ability of strain DTA1 to grow (cloudy culture fluid or appearance of opaque white biofilm on the well surface) at different salt concentrations.

Similarly, the heavy metal tolerance of strain DTA1 was tested by diluting Cd(NO₃)₂, Hg(NO₃)₂, Cr(NO₃)₂, Co(NO₃)₂, Pb(CH₃COO)₂, FeSO₄ and ZnCl₂ with LB medium so that the final concentrations after adding strain DTA1 to 96-well microplates reached the following values: 1, 2, 4, 8, 16, 32, 64 and 128 µg/ml. The test was continued at heavy metal concentrations of 150–1050 µg/ml if the bacterial strain could grow at 128 µg/ml. Each sample was repeated 3 times; the control samples were LB medium without added bacteria (negative control) and with added bacteria (positive control).

Characterisation of EPS under stress conditions

The strain DTA1 was cultured in Terrific Broth (TB) medium, pH 8.46, 72.6 g/l sucrose under four conditions:

1. without NaCl,
2. with 3% NaCl,
3. with 1% NaCl, 1 µg/ml Co(NO₃)₂, 1 µg/ml Cd(NO₃)₂ and
4. with 1% NaCl and FeSO₄, CuSO₄, Ag₂SO₄, ZnCl₂, MgCl₂, and MnSO₄ salts all at 1 µg/ml concentration.

The samples were cultured at 30^oC and shaken at 150 rpm. After 48 hours of culture, EPS was extracted and its sugar, protein and elemental content were determined according to Le et al. (2025).

Determining the monosaccharide composition of EPS

Strain DTA1 was inoculated into TB medium, pH 8.46, supplemented with 3.27% NaCl and selected sugar sources. Liquid cultures were collected after 72 h of fermentation at 30^oC, shaking at 150 rpm. EPS was extracted and its sugar content, protein content and monosaccharide composition were determined according to the method described by Le et al. (2025).

Extraction of genomic DNA and whole-genome sequencing

The DTA1 strain was grown in LB medium at 32^oC and 200 rpm for 16–18 hours. Following centrifugation at 6000 rpm and 4^oC for 20 minutes, the cell pellet was gathered and subjected to DNA extraction using the TracePure™ DNA extraction kit (LabNova) following the manufacturer's instructions. The A260/A280 ratio of the extracted DNA was 1.84. In addition, the DNA integrity number (DIN) score of the extracted DNA exceeded 7.2, corresponding to the presence of a single, clear band without any breakage, meeting the criteria for WGS. The purified DNA sample of strain DTA1 was transferred to Novogen AIT Company (Singapore) for WGS on the Illumina Novaseq 6000 platform with 150 bp paired-end reads.

Bioinformatics analysis

Processing raw reads and de novo assembly: Fastp v.0.23.2 (Chen et al. 2018) was used to trim raw reads with a quality score below 20. The retained clean reads were assembled de novo using SPAdes v.3.15.5 (Prjibelski et al. 2020). The quality of the assembled genome was evaluated with QUAST v.5.0.2 (Gurevich et al. 2013). All the scaffolds below 500 bp were discarded from the assembled genome.

Functional annotation and gene cluster comparison: Functional annotation of the study genome was determined with Bakta v.1.8.1 (Schwengers et al. 2021) and Prokka v.1.14.6 (Seemann 2014). Metabolic pathways and carbohydrate metabolism were further identified by COGclassifier v.1.0.5 (Shimoyama 2022) and BlastKOALA (Kanehisa et al. 2016) against Cluster of Orthologous Genes (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (Kanehisa and Goto 2000). The EPS cluster of the study strain and several Bacillus reference strains were compared and visualised with the CAGECAT web server (van den Belt et al. 2023).

Statistical Analysis

Each experiment was repeated three times. Statistical differences were determined by one-way ANOVA followed by the Tukey HSD test for pairwise post-hoc testing using Minitab Statistical Software. Statistically significant differences were considered when the p-value was less than 0.05.

B. velezensis DTA1 sequencing data is accessible on NCBI's SRA database, with accession number BioProject ID - PRJNA1222916.

Morphological characteristics and capability of carbohydrate utilisation of B. velezensis DTA1

Screening results showed that strain DTA1 exhibits salt tolerance at 3% NaCl (with colony formation). The colonies were opaque white and round, with shiny surfaces and slimy edges. The formation of 30 mm-long strings in the String test confirmed this strain’s ability to produce EPS (Fig. 1A). The cells were observed under electron microscopy, which occurred as rod-shaped, 2-3 µm in size, gram-positive and have the ability to produce endospores (Fig. 1B).

The results of carbon source testing showed that the study strain was capable of using 28 out of 49 carbon sources, of which D-glucose, D-fructose, D-mannose, esculin, D-cellobiose and D-saccharose sources turned yellow as the most suitable carbon source. The other positive reactions were obtained with D-mannitol, D-maltose, amidon, glycerol, D-arabinose, L-arabinose, L-rhamnose, D-ribose, D-xylose, inositol, D-mannitol, D-sorbitol, methyl-b-D-glucopyranside, N-acetyl-glucosamine, amygdalin, arbutin, salicin, D-trehalose, D-raffinose, glycogen and gentiobiose. Conversely, 14 carbon sources were shown in negative reactions, including erythritol, L-sorbose, dulcitol, methyl-b-D-manopyranoside, D-turanose, xylitol, D-lyxose, D-fucose, D-arabitol, L-fucose, L-arabitol, potassium gluconate, potassium 5-ketogluconate and potassium 2-ketogluconate. Besides, seven carbon sources that were not defined were D-galactose, D-adonitol, L- xylose, methyl-β-D-xylopyranoside, inulin, D-melezitose and D-tagatose (Suppl. material 1).

S alt and h eavy metal tolerance of B. velezensis DTA1

According to tolerance tests, strain DTA1 was able to grow in media containing 0–12.5% NaCl (w/v), as indicated by the observed turbidity, which reflects bacterial growth under these NaCl conditions. The turbidity levels decreased as the NaCl concentration increased. However, no turbidity (indicating no bacterial growth) was observed under NaCl concentrations of 15% and 17.5% (w/v). A similar evaluation with MgSO₄ showed that strain DTA1 grew at MgSO₄ concentrations ranging from 0% to 10% (Fig. 2A).

The tolerance of strain DTA1 to cadmium (Cd), cobalt (Co), mercury (Hg), chromium (Cr) and arsenic (As) was evaluated across a concentration range of 1–128 µg/ml. Specifically, DTA1 demonstrated tolerance to high concentrations of Cr and As up to 64 µg/ml and Cd up to 32 µg/ml, while its resistance to Hg and Co was limited to concentrations of 2 and 4 µg/ml, respectively (Fig. 2B). In addition, lead (Pb), zinc (Zn) and iron (Fe) were tested at concentrations ranging from 150–1050 µg/ml, as the DTA1 strain could tolerate up to 128 µg/ml of these metals. The study strain could withstand up to 900 µg/ml of Pb, Zn and 600 µg/ml of Fe (Fig. 2C).

Characteristics of EPS under salt/heavy metal stress conditions

Although DTA1 grew better under non-stress conditions, EPS content increased significantly under NaCl and/or heavy metal stress conditions (p < 0.05). Furthermore, total sugar and total protein contents in EPS under salt and/or heavy metal stress conditions were also twice as high as those under no-stress conditions (Table 1).

The EDS spectrum showed changes in the elemental composition of EPS, especially in the amount of Na and Cl, which accounted for only 0.73% and 0.52%, respectively, in the NaCl-free environment (Fig. 3A), but increased to 9.03% and 2.04% under NaCl stress conditions (Fig. 3B). Noteworthy is also the presence of cations in EPS; in addition to Ca, Mg, Na and K, there was also the appearance of Co, Cd, Mn, Fe, Cu, Zn and Ag only in EPS from the environment supplemented with these metal sources (Fig. 3C and Fig. 3D).

Effect of sugar sources on the monosaccharide composition of EPS of B. velezensis DTA1

The addition of either glucose or sucrose to the culture medium of strain DTA1 significantly increased EPS production compared to the control, with EPS levels in the medium reaching only 16.35 g/l (p < 0.05) (Table 2). At the same concentration, sucrose was more effective than glucose in stimulating EPS production (glucose 10 g/l: 21.42 g/l vs. sucrose 10 g/l: 23.93 g/l, glucose 50 g/l: 23.29 g/l vs. sucrose 50 g/l: 30.39 g/l). Additionally, increasing the concentrations of either glucose or sucrose further enhanced EPS yield, with EPS concentrations of 23.29 g/l and 30.39 g/l achieved when 50 g/l glucose and sucrose were added, respectively. This result surpasses the EPS yields reported for other B. velezensis strains, such as TSD5, AG6, HY23, KY471306, MHM3 and OM03, but remains lower than the yield of the VTX20 strain isolated in Vietnam, which reached 75.5 g/l (Table 2).

To examine the composition of monosaccharides in EPS from the above samples, the retention time of EPS hydrolysate samples was compared with the standard monosaccharides (Suppl. material 2). The EPS from strain DTA1 are heteropolysaccharides with varying compositions and monosaccharide ratios, depending on the sugar source used (Table 1 and Fig. S2). When no sugar source was added (control medium) or when glucose was added, the EPSs had monosaccharides including glucose, rhamnose and mannose (Suppl. material 2A and C), similar to those in strain TSD5; however, the proportions of monosaccharides were different. When sucrose was added, in addition to the above monosaccharides, fructose and N-acetylglucosamine were also present (Suppl. material 2B). As expected, the sugar concentration also influenced the ratio of monosaccharides in the EPS. In the control sample, EPS contained mainly rhamnose (Suppl. material 2C), while mannose was the major component when glucose was added, but accounted for different amounts of 85.02% and 99.37% when 10 g/l and 50 g/l glucose were added, respectively (Suppl. material 2A). Similarly, N-acetylglucosamine and rhamnose were the major components when 10 g/l sucrose was added. However, with 50 g/l sucrose, the EPS primarily consisted of rhamnose, N-acetylglucosamine and fructose (Suppl. material 2B).

General genomic characteristics of B. velezensis DTA1

The size of the assembled genome is 3,898,926 bp with 46.5% GC content, which matches well with the reference genome, B. velezensis FZB42 (genome size: 3,918,596 bp; GC content: 46.5%). Functional annotation with Bakta v.1.9.2 detected 3,784 coding sequences (CDSs), 62 tRNA and 11 rRNA within the genome of the DTA1 strain (Table 3 and Suppl. material 3). Amongst these CDSs, 81.79% (3,095 out of 3,784) of them were classified into 22 COG functional categories. The majority of CDSs were associated with amino acid transport and metabolism (E, 9.28%), transcription (K, 8.27%), carbohydrate transport and metabolism (G, 8.18%) and translation, ribosomal structure and biogenesis (J, 7.18%) (Suppl. material 4). This suggests the DTA1 strain is capable of degrading a broad range of carbohydrates and proteins. Notably, the EPS biosynthesis pathway was potentially identified in this strain as 2.75% of CDSs were assigned to the biosynthesis, transport and catabolism of secondary metabolites (Q).

Sugar transport systems of B. velezensis DTA1

A total of 22 phosphotransferase system (PTS) transport proteins for various types of sugars were detected in the genome of strain DTA1 (Suppl. material 5). These transporters were categorised into 11 PTS systems with the following distribution: alpha-glucoside, cellobiose PTS, fructose PTS, glucose PTS, lactose PTS, mannitol PTS, mannose PTS, N-acetylglucosamine PTS, sucrose PTS, sugar PTS and trehalose PTS, based on the transporter database. These corresponding pathways are amino sugar and nucleotide sugar metabolism, fructose and mannose metabolism, galactose metabolism, gluconeogenesis/starch and sucrose metabolism, glycolysis/gluconeogenesis, phosphotransferase system and starch and sucrose metabolism.

For the sugar-specific permease transport system of the strain DTA1, proteins specific for arabinose, maltose and multi-sugar permease transport proteins were found. Maltose and maltodextrin ABC transporter subunit (ATP-binding protein) that facilitates maltose/maltodextrin import was also detected (Suppl. material 6). According to these results, B. velezensis DTA1 can use cellobiose, glucosides, glucose, mannose/mannitol, fructose, sucrose, arabinose and N-acetylglucosamine. Furthermore, data from Suppl. materials 5, 6 revealed multiple copies of genes encoding transport systems for cellobiose, sucrose and arabinose in the genome of strain DTA1, which could enhance the ability to utilise these carbon sources of this strain.

Nucleotide sugars biosynthetic pathway of B. velezensis DTA1

We discovered 29 genes in the DTA1 genome that encode key enzymes facilitating the conversion of various carbon sources into their corresponding nucleotide sugars. The metabolisms of glucose, sucrose, fructose, maltose, cellobiose, mannose, galactose and arabinose were aided by these genes. Some, including galU, sacA, bglA, galE and MPI, exist in multiple copies (Suppl. material 7). Even though there are PTS of lactose and trehalose, the assembled genome lacked the enzymes required for the metabolism of these two sugars.

Based on these detected genes, the nucleotide biosynthetic pathway of B. velezensis DTA1 can be simplified as in Fig. 4. Mannose, fructose, sucrose, cellobiose and glucose are amongst the sugars that are transported into the cell by the PTS system in the first step. Once inside, these sugars undergo phosphorylation to form mannose-6-phosphate, fructose-1-phosphate, sucrose-6-phosphate, cellobiose-6-phosphate and glucose-6-phosphate, respectively. Similarly, arabinose and galactose are transported into the cell by permease enzymes. Maltose is also absorbed into the cell through the ATP-binding cassette (ABC) transport system. They are then converted into fructose 6-phosphate, glucose 6-phosphate and galactose 1-phosphate by the relevant enzymes (MBI: mannose; araA, araB, araD, tktA: arabinose; fruK, FBP: fructose; sac: sucrose; bglA: cellobiose; glk: glucose; glvA: maltose; galM, galK: galactose). Thus, this study strain has the ability to synthesise eight types of nucleotide sugars, including UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, UDP-N-acetylmannosamine, dTDP-glucose, dTDP-rhamnose, UDP-glucose, UDP-glucuronate and UDP-galactose.

EPS biosynthetic pathway in B. velezensis DTA1

Two gene clusters involved in EPS biosynthetic pathways, the Wzx/Wzy-dependent pathway and the extracellular synthesis by the enzyme levansucrase, were detected in the DTA1 genome (Suppl. material 8). In the Wzx/Wzy-dependent pathway, EPS can be synthesised by a complex system of proteins with multiple functions such as regulation, synthesis, transport, polymerisation and export, encoded by the epsA-O operon. We found an epsA-O cluster encoded by a 15.7-kb fragment containing 15 eps genes in the DTA1 genome using the BlastKOALA annotation server. Amongst these genes, epsE encodes a membrane-bound priming glycosyltransferase (GT) enzyme that does not catalyse glycosidic bonds. This GT facilitates the transfer of sugar-1-phosphate and activates the undecaprenyl-phosphate-lipid carrier on the cytoplasmic side of the membrane (Wu et al. 2020). This first step allows other sugar nucleotides to be sequentially attached to assemble repeating units catalysed by other soluble or membrane-bound GTs. Specifically, epsL encodes GTs that transport UDP-glucose or UDP-galactose, while epsD transports UDPGlcA, epsH and epsJ transport amino sugars. EpsF, epsG, epsH, epsI, epsJ and epsK can transfer various types of nucleotide sugars. Furthermore, the genes epsC, epsI, epsO, the acyltransferase gene epsM and the aminotransferase gene epsN all participate in the modification of the repeating units of the polysaccharide chain. The repeat units are transported across the inner membrane into the periplasm by eps K (Wzx flippase) (Vu et al. 2021). Meanwhile, epsG functions to polymerise the repeat units (Wzy polymerase) by creating new glycosidic bonds at the reducing end of the growing polymer, forming the extracellular polysaccharide structure. Two other important genes are epsA and epsB, which encode for protein-tyrosine kinase and its modulator, which appeared to be essential for maximal production of EPS (Dogsa et al. 2024). In addition, these two genes are known to function in determining polysaccharide chain length, epsA b also being responsible for polysaccharide export (Vu et al. 2021). The epsA-O operon was highly conserved amongst B. velezensis as well as other Bacillus species such as B. amyloliquefaciens and B. subtilis (Fig. 5A).

The second EPS biosynthetic pathway, involving a single sucrase protein encoded by a levan biosynthetic gene cluster was also detected on the DTA1 chromosome (Fig. 5B). The 6.1-kb fragment encodes genes for manganese-dependent inorganic pyrophosphatase (ppaC), phosphate acetyltransferase (pta), levansucrase (sacB), levanbiose-producing levanase (levB) and HTH-type transcriptional repressor RspR (rspR). The levansucrase SacB from DTA1, which is known for synthesising levan, shared 96.61% and 90.47% sequence identity with the SacB protein from Bacillus amyloliquefaciens (Uniprot ID: P21130) and Bacillus subtilis strain 168 (Uniprot ID: P05655), respectively. Besides, the LevB gene, which encodes for levanbiose-producing levanase, is located next to SacB and is responsible for levan degradation.

Genes and pathways in response to stress responses in B. velezensis DTA1

The strain DTA1 contains multiple genes involved in osmotic stress, heavy metal stress, oxidative stress and antibiotic resistance (Table 4). The presence of genes related to the accumulation of solutes helps regulate osmosis, such as opuCD, opuCC, opuCB, opuCA, opuAB, opuAA, gbsB and putP. Genes related to Na⁺ transport proteins across the membrane help regulate osmotic balance, such as nhaC, nhaK, nha, mnhC and phaD. The presence of genes khtU, khtT and khtS in the genome of strain DTA1 are one-way K⁺ transport channels into the cell, helping to balance osmosis with the extracellular environment under salt stress conditions. In addition, genes, such as degU, degS, sodA, dnaK etc. help transmit signals for cells to respond to environmental stress conditions (Ayaz et al. 2022). The bacterial strain carries czcD, cadA, chrA, chrB, ydpP, mneP, mntP, mgtE, corA and asrB that help bacteria increase their resistance to cadmium, zinc, cobalt, chromate, manganese and arsenic. Several genes, such as nsrR, tpx, bcp and hmpA, are related to the oxidative stress defence of the study strain.

Salinity is a critical factor affecting soil health and crop productivity. Under the influence of climate change and unsustainable agricultural practices, saline soil areas are expanding rapidly and are projected to affect up to 50% of cultivated land by 2050 (Bhagat et al. 2021). Vietnam, with its long coastline and many islands, is highly vulnerable to climate change (Buys et al. 2006). Soils on offshore coral islands are dry, porous and saline, with poor water and nutrient retention due to harsh weather, salt-laden winds and freshwater scarcity. In some cases, irrigation relies on brackish water, making cultivation extremely difficult. EPS-producing bacteria have been reported to reduce salinity and improve saline soils (Bhagat et al. 2021), improve soil structure (Awasthi et al. 2017), restore arsenic-contaminated soils (Mukherjee et al. 2019), reduce heavy metal stress caused by Cd, Pb and Fe and enrich the lettuce rhizosphere microbiota (Zhang et al. 2024). Amongst EPS-producing bacteria, B. velezensis is recognised as a safe microorganism and is being applied in microbial fertilisers as a sustainable alternative to chemical fertilisers (Zhong et al. 2024). In this study, the indigenous microorganism strain B. velezensis DTA1 exhibited both heavy metal adsorption and stress tolerance through EPS production and multiple stress response systems were identified. Combined with other beneficial traits of B. velezensis, such as water retention, soil aggregation, phosphorus solubilisation and plant growth promotion, this strain demonstrates strong potential for improving arid, saline and contaminated soils, while enhancing plant resilience to abiotic stresses and pathogens (Zhong et al. 2024, Xie et al. 2024). These characteristics make strain DTA1 an ideal candidate for soil improvement and the remediation of heavy metal-contaminated environments, especially in Vietnam.

The salt tolerance of strain DTA1 is higher than that of strains from other species, such as B. amyloliquefaciens, B. paramycoides and B. pumilus (NaCl tolerance from 2–10%) (Sharma et al. 2021). Nevertheless, its salt tolerance is lower than that of B. subtilis LR-1, which can survive in environments with NaCl concentrations of up to 16% (Zhao et al. 2022). The ability of strain DTA1 to tolerate high salt concentrations can be explained by three main factors. First, the strain carries many genes related to the accumulation of osmotic solutes such as glycine/proline betaine to balance osmotic pressure with the extracellular environment (Uma et al. 2020). Second, the bacterial strain possesses many genes encoding proteins that transport Na⁺ (Na⁺/H⁺ antiporters) to the extracellular environment and pump K⁺ unidirectionally into the intracellular environment under osmotic stress conditions to help balance osmotic pressure (Yu et al. 2022). In addition, genes encoding signaling proteins (e.g. degU, degS, sodA, dnaK) also contribute to DTA1's rapid response to stress conditions (Ayaz et al. 2022). Na⁺/H⁺ antiporters are common in a variety of organisms from bacteria to animals and plants. They help balance pH and Na⁺ for cells (Padan et al. 2001). The main role of Na⁺/H⁺ antiporter is to pump Na⁺ out of the cell (Munns and Tester 2008). Finally, bacterial strains that produce EPS help adsorb cations from the environment, helping to reduce the concentration of free Na⁺ in the environment, preventing them from adversely affecting cells (Qurashi and Sabri 2012).

Certain metal stress factors have been shown to stimulate an increase in extracellular polysaccharide and protein content in various bacterial strains, such as Pb with Phanerochaete chrysosporium strain (Li et al. 2020); Cd and Pb in Chlamydomonas reinhardtii (Li et al. 2021); Cd in Pseudomonas aeruginosa and Alcaligenes faecalis (Lian et al. 2022) etc. We observed that the strain DTA1 is tolerant to several heavy metals, including Cd, Hg, Cr, Co, As, Pb, Zn and Fe. This can be explained by the presence of genes encoding heavy metal efflux pumps, such as czcD and cadA which confer resistance to Co, Zn and Cd (Silver 1996) and arsB, which confers resistance to As (Sato and Kobayashi 1998). Another mechanism involves an increase in EPS as well as extracellular polysaccharide and protein content, which play an important role in reducing heavy metal toxicity by helping cells resist metal stress (Lian et al. 2022).

The ability of EPS to adsorb cations is attributed its large molecular size and polyanionic nature with a highly negative zeta potential (Le et al. 2025) and the presence of negatively charged functional groups such as amine, thiol, carboxyl, hydroxyl, phosphate etc. (Li et al. 2020) that exhibit varying affinities for metal ions and help reduce the concentration of free metal ions. Sulphur (S) and phosphorus (P) present in EPS in strain DTA1 are anionic functional groups in EPS that facilitate the binding of cations (Andrew and Jayaraman 2022). The mechanisms underlying cation adsorption by EPS include complexation, biosorption-induced precipitation, ion exchange and redox reactions (Zhang et al. 2024). Notably, under salt and/or heavy metal stress conditions, strain DTA1 produces a significantly higher amount of EPS, total sugar and total protein contents in EPS compared to non-stress conditions. Elevated EPS production under salt stress enhances cell survival by forming a protective biofilm barrier that shields cells from salt and heavy metal toxicity. In addition to enhancing the resilience of EPS-producing cells, biofilm production also contributes to soil aggregation, which, in turn, improves the absorption of water and nutrients. This process helps both microorganisms within the biofilm and plants to better resist adverse environmental conditions (Qurashi and Sabri 2012). Due to this ability to absorb cations, especially heavy metals, EPS from strain DTA1 holds promise for applications in the remediation of saline soils as well as the treatment of environments contaminated with heavy metals.

WGS analysis revealed that the strain DTA1 can produce two types of EPS: levan, which is synthesised extracellularly via a single sucrase enzyme called levansucrase and another type of EPS synthesised by the epsA-O operon. A levan cluster containing the sacB and levB genes was detected in the genome of the strain DTA1. As a homopolysaccharide, levan consists of β-2,6-linked D-fructose units. It may also contain additional glucose branches with terminal fructose through α-glycosidic bonds. It is synthesised extracellularly by levansucrase in sucrose-rich media (Dahech et al. 2011, Vu et al. 2021). Levansucrase is encoded by the sacB gene and is strongly induced by sucrose. In contrast, levanbiose-producing levanase, which is encoded by levB, is responsible for levan degradation (Pereira et al. 2001). The expression of this gene has been proven to enhance the expression of levansucrase in Bacillus subtilis (Daguer et al. 2004). We also found that the DTA1 strain contains an epsA-O cluster consisting of 15 genes involved in the Wzx/Wzy-dependent pathway. The EPS biosynthesis process via this pathway is highly complex, requiring the involvement of regulatory, synthesis, transport, polymerisation, modification and export genes, which have been well described in previous studies (Wu et al. 2020, Dogsa et al. 2024). In addition, this cluster shows high similarity to those in other B. velezensis strains and Bacillus species, in line with previously published results (Wu et al. 2020, Vu et al. 2021).

The production of EPS requires nucleotide sugars as precursor molecules (Zhao et al. 2023). The DTA1 strain harbours multiple genes encoding 11 PTS and permease transport systems, suggesting that the strain can utilise a broad range of sugar sources available in its environment. Indeed, nine sugars, including cellobiose, glucoside, glucose, mannose, mannitol, fructose, sucrose, arabinose and N-acetylglucosamine, predicted to be utilised by strain DTA1 through WGS analysis, yielded positive results in the API^® 50CHB test. Furthermore, the DTA1 strain was stimulated to grow and produce EPS by sucrose, soluble starch, mannose, D-maltose, D-galactose, cellobiose, L-arabinose, D-glucose, L-rhamnose and D-fructose, with sucrose, soluble starch and cellobiose being particularly effective. This can be explained by the presence of multiple copies of genes encoding the transport systems for these sugars. The presence of PTS systems has been reported in a variety of species, such as B. subtilis, B. amyloliquefaciens, E. coli and Staphylococcus aureus (Hengstenberg et al. 1969, Chen et al. 2007) highlighting their essential role in carbohydrate uptake in microorganisms. Apart from this function, Morabbi Heravi and Altenbuchner (2018) reported that PTS transporters in the genome of B. subtilis are also involved in sensing nutrient fluctuations in the medium. As predicted from previous studies, EPS yield and structure depend on many factors, including C source, which significantly affect the EPS production process (Netrusov et al. 2023). EPS from strain DTA1 contained 3-5 monosaccharides with the composition and ratio of monosaccharides not only different in the media supplemented with different sugar sources, but also different from previous publications in B. velezensis (Moghannem et al. 2018,Zou et al. 2024). Notably, fructose in EPS only appeared when the medium was supplemented with sucrose, most clearly at 50 g/l sucrose. This was explained through the results of WGS analysis; strain DTA1, in addition to the ability to biosynthesise heteropolysaccharide (according to the Wzx/Wzy-dependent pathway), also had the ability to biosynthesise levan (with a monosaccharide component of fructose) according to the extracellular synthesis pathway. Levan synthesis was only induced in sucrose-rich media (Vu et al. 2021); therefore, in sucrose-containing media, especially 50 g/l sucrose, levan was produced, resulting in EPS-containing fructose. This is the first time that there is a clear publication on the influence of glucose and sucrose content on EPS content and structure, especially the presence of five monosaccharides glucose, rhamnose, fructose, mannose and N-acetylglucosamine in EPS obtained from sucrose-rich medium. As expected, we detected the presence of monomers including glucose, rhamnose, fructose and N-acetylglucosamine, because the strain DTA1 possesses several genes encoding for enzymes related to the ability to biosynthesise these types of nucleotide sugars. Unlikely, the presence of mannose in EPS, as well as the ability of strain DTA1 to adsorb and preferentially use this sugar source and mannose to stimulate EPS production, proves that this strain has the ability to produce mannose-type nucleotide sugars. However. the biosynthetic pathway of mannose-type nucleotide sugars appears to be incomplete, as the full set of genes responsible has not yet been identified.

In short, this work demonstrated the remarkable salt and heavy metal tolerance of the strain DTA1. This was explained by the presence of genes involved in cation pumps and osmotic accumulation, as well as the ability to produce EPS under stress conditions to help absorb salts and heavy metals, preventing them from adversely affecting the cells. The WGS of this strain has provided valuable insights into its genetic characteristics related to stress tolerance, nucleotide sugars biosynthetic pathway and EPS biosynthetic pathway. In addition, nutrient sources also showed significant effects on EPS structure and production, especially the sucrose source, which revealed interesting discoveries about the monosaccharide composition of EPS. This study enriches the microbial and genetic resources of B. velezensis species, highlighting its potential applications in environmental bioremediation and biotechnology, particularly for the remediation of saline soils and heavy metal pollution.

This work was supported by the Department of Biotechnology, Joint Vietnam-Russia Tropical Science and Technology Research Center. We thank our colleagues for their contributions to this study.

This research is funded by the Joint Vietnam-Russia Tropical Science and Technology Research Center under project code: SH.Đ2.06/25.

HTL and TTTT conceived the study. HTL conducted experiments. TTTT and STN conducted bioinformatics analyses. HTL, TTTT, STN, NDV, HML and HTN interpreted the data. HTL, TTTT, DDL and STN wrote the original draft. All authors reviewed and approved the final manuscript.

The authors have declared that no competing interests exist.