The world's population is projected to reach 9.3 billion by 2050, demanding a 60% increase in food production to ensure global food security (Fischer et al., 2014). This necessity must be met sustainably, as traditional agricultural practices relying on the overuse of chemical fertilizers and pesticides degrade soil health, harm beneficial microbial diversity, and cause environmental problems. Climate change further intensifies agricultural challenges with biotic and abiotic stresses like drought, salinity, temperature extremes, and plant pathogens (Mishra et al., 2021; Rajkumar et al., 2024). Salinity stress is a particular concern, affecting over 1 billion hectares of land globally and hampering agricultural productivity (Mishra et al., 2021). Furthermore, limited land resources and declining biodiversity complicate the achievement of demand vs. supply of food for rising population (Tilman et al., 2017). To sustainably improve agricultural production, and address these complex, intertwined challenges along with the impacts of climate change, researchers are looking for eco-friendly alternatives to conventional agrochemicals.

Plant science and biotechnology advancements offer promising strategies to counter these challenges and the application of plant growth-promoting endophytic bacteria (PGPEB) is one promising sustainable approach (Srikanth et al., 2025). By colonizing plant tissues, PGPEB boosts plant growth via phytohormone synthesis, nutrient acquisition, and producing antimicrobial metabolites. PGPEB exhibits stress resilience by accumulating compatible solutes, inducing ion channels, and upregulating antioxidant machinery (Dudeja et al., 2021; Rani et al., 2022). Studies exploring the full potential of PGPEB via a deeper understanding of the pathways and genes responsible for their beneficial and stress management traits are increasing significantly. This positions PGPEB as a valuable tool for enhancing agricultural productivity under stress conditions (Vurukonda et al., 2016; Chen et al., 2019; Kumar et al., 2020; Kushwaha et al., 2020; Bhutani et al., 2021b). Genome sequencing has emerged as a powerful tool, providing insights into beneficial microorganisms. Genome sequencing and analysis by aiding the identification and categorization of genes reveal beneficial microorganisms' adaptation strategies, metabolic and functional features for promoting plant growth and stress resilience. However, draft genomes with inaccuracies and low completeness limit comprehensive analysis in areas like phylogenomics and pan-genomics. Therefore, generating high-quality whole genome sequencing (WGS) data is essential to realize the potential of these beneficial microorganisms fully (Sharma et al., 2023). Recent studies (Chaudhary et al., 2021; Gao et al., 2022; Kumar et al., 2022) have revealed that WGS can provide abundant genomic data that can be mined for potential agricultural applications. The high-quality, well-assembled genomes are critical for robust and reliable analyses. Comparative genome analyses also have some limitations due to low-quality or incomplete assemblies, where significant gaps could lead to inaccurate gene prediction and reduce confidence in strain-level comparisons (Alkan et al., 2011).

Bacillus, a Gram-positive Firmicute, is well known for its excellent secretion systems, and production of an array of extracellular enzymes like amylases, cellulases, proteases, pectinases, etc. This makes Bacillus spp. vital in diverse sectors such as detergent, textile, food, feed, and beverage (Sansinenea, 2019; Bhutani et al., 2021a,b). Many past identifications grouped Bacillus paralicheniformis under Bacillus licheniformis due to their close relatedness. A recent genomic study revealed secondary metabolite operons i.e., fengycin and bacitracin specific to B. paralicheniformis only but absent in B. licheniformis (Olajide et al., 2021). This taxonomic clarification highlights a critical knowledge gap regarding its potential in promoting plant growth as evidenced by 232 publications listed in the Scopus database as of 12th May, 2025. The existing literature predominantly explores B. paralicheniformis for industrial and biotechnological applications, with limited attention on its potential contribution to sustainable agriculture as plant growth-promoting bacteria (Supplementary Figure 1). In previous studies, B. paralicheniformis strain MHN12 was isolated from the nodules of Vigna radiata. Based on 16S rRNA sequence similarity initially, the isolate was submitted as B. licheniformis (Bhutani et al., 2021b). The strain possessed antimicrobial activity, plant growth promotion abilities, and high salinity tolerance that indicate its role in supporting plant growth and biocontrol (Bhutani et al., 2022). In the current study, whole-genome sequencing of strain MHN12 was done followed by a comparative analysis with other B. paralicheniformis strains to ensure accurate taxonomic identification. To assess its potential for commercialization as a bioinoculant, a detailed genome analysis was performed. Genetic determinants and metabolic pathways responsible for the biosynthesis of biomolecules associated with plant growth promotion and stress tolerance in strain MHN12 were identified. In vitro potential of strain MHN12 to synthesize these compounds under saline stress conditions was also assessed. Furthermore, Gas Chromatography-Mass Spectrometry (GC-MS) was performed to compare its metabolite profiles under control and stress conditions.

Endophytes often outperform rhizospheric bacteria in promoting plant growth and stress resistance (Peng et al., 2023). The beneficial effects of these endophytes have been reported to be upregulated under abiotic stresses (Biswas et al., 2023). However, their implementation is hindered by a limited understanding of their action mechanisms (Peng et al., 2023; Zamanzadeh-Nasrabadi et al., 2023). Bacillus paralicheniformis strain MHN12 isolated from nodules of Vigna radiata has been screened for halotolerance, biocontrol property, plant-beneficial traits, and improved plant growth under lab conditions (Bhutani et al., 2021b, 2022). The current study addresses the gap in their implementation by focusing on genome sequencing, comparative analysis, and genome mining of strain MHN12.

Within the genus Bacillus, accurate differentiation between B. licheniformis and B. paralicheniformis remains a significant challenge due to their remarkable genetic similarity. 16S ribosomal RNA (rRNA) gene sequencing is insufficient for definitive identification, leading to frequent misclassifications (Olajide et al., 2021). This was also evident with strain MHN12 initially identified as B. licheniformis based on its 16S rRNA sequence. However, recent comparative genomic studies revealed the presence of specific secondary metabolite operons (fengycin, paralichenicidin, and bacitracin) exclusively in B. paralicheniformis genomes, absent in B. licheniformis. This discovery prompted us to do the whole genome sequencing of the isolate to analyze its secondary metabolite cluster genes, and perform its comparative analysis with the other reported B. paralicheniformis strains to ascertain its identity and draw the similarities and differences among them. The dDDH analysis with a 70% similarity threshold for genome boundaries and ANI and AAI analysis using a 95–96% cut-off delimiting species confirmed that all the strains belong to the same genus and species (Aliyu et al., 2016). The pan-genome analysis is a widely used approach among the currently available comparative genomic methods to evaluate the genomic diversity of entire repertoires. It helps identify core genomes (shared by all strains), accessory genomes (found in two or more strains), and specific gene pools unique to individual strains within a species (Tettelin et al., 2008). The core genome of the strains included genes responsible for the basic functioning. The accessory and unique genome encoded genes for biochemical pathways and functions that confer selective advantages, such as antibiotic resistance, drug resistance, and secondary metabolite biosynthesis. The pan-genome phylogeny revealed that strain MHN12 grouped with strains possessing accessory genes for antimicrobial and cellulase production, suggesting the acquisition of traits important for plant–microbe interactions and competitive fitness. This is further supported by the core–pan plot analysis, which indicated an ‘open' pan-genome, reflecting high genomic flexibility and a strong potential for acquiring new genes through horizontal gene transfer (Bach et al., 2022). Such genomic openness may explain how strain MHN12 and related strains gain beneficial functions such as improved environmental resilience, probiotic potential. The antiSMASH analysis identified characteristic secondary metabolite clusters (lichenysin, bacillibactin, bacitracin, and fengycin) in the genome of strain MHN12 and other strains. This finding confirmed their taxonomic classification as B. paralicheniformis, since the presence of bacitracin and fengycin biosynthetic clusters have been reported in B. paralicheniformis only when compared to B. licheniformis. Secondary metabolites synthesized by plant endophytes can improve plant resilience against different biotic and abiotic stresses (Wang et al., 2022). Secondary metabolite gene clusters lichenysin, bacillibactin, bacitracin, and fengycin, with roles in biosurfactants, iron uptake, antibacterial, and antifungal activities respectively, contribute to inhibiting pathogens and improving nutrient uptake were predicted in the genome (Harwood et al., 2018). In support of this potential, strain MHN12 demonstrated biocontrol activity against Fusarium oxysporum and Aspergillus niger (Bhutani et al., 2021b). The finding resonates with a genome mining study of B. paralicheniformis ES-1, which reported similar gene clusters associated with in vitro antibacterial activity and overall plant health improvement (Iqbal et al., 2023). Moreover, the GC-MS analysis revealed the presence of compounds such as piperidinone, 2,4-di-tert-butylphenol, and 3-trifluoroacetoxydodecane, with known antimicrobial properties (Das et al., 2025).

In line with the plant growth-promoting (PGP) traits and salt stress resilience of strain MHN12, various pathways and genes linked to stress resistance, phosphate solubilization, nitrogen metabolism and transport, phytohormone production, siderophore, acetoin, and butanediol synthesis were identified in its genome (Figure 8). Strain MHN12 genome illustrated the presence of genes for N, P, and Fe acquisition and transporters for better uptake of soil nutrients. Plant growth-promoting endophytic bacteria (PGPEBs) convert non-usable forms of nitrogen into forms usable by plants, such as ammonium. The presence of genes for nitrate and urea conversion into ammonia further validates the findings of Bhutani et al. (2021a),b on ammonia production by strain MHN12 (Bhutani et al., 2021b). Additionally, the presence of an ammonium assimilation enzyme system regulates nitrogen absorption and transformation, thereby providing nitrogen for plant growth. Polyamines such as putrescine, whose biosynthetic genes were annotated in strain MHN12, plays role in enhancing plant performance under nitrogen limitation. The transgenic Arabidopsis overexpressing gene for putrescine biosynthesis support the role of polyamines in improving plant performance under nitrogen limitation. The elevated putrescine levels were associated with enhanced nitrogen metabolism and strengthened antioxidant defenses by increased nitrate uptake and nitrate reductase activity as well as reducing oxidative damage (Recalde et al., 2025). Altogether, the data obtained depicts that strain MHN12 may modulate plant nitrogen levels directly. Additionally, the presence of genes for nitric oxide reductase and nitric oxide dioxygenase suggests a potential role for strain MHN12 in modulating nitric oxide (NO) signaling. NO serves as a crucial signaling molecule in plant growth and plant-microbe interactions (Mur et al., 2013).

Phosphate solubilization is a crucial process as phosphorus has limited bioavailability in many environments due to its complex formation with metal ions. Phosphate-solubilizing bacteria effectively convert insoluble phosphorus into plant-available forms by producing acid and alkaline phosphatases (Swain et al., 2012). Previous studies have shown that alkaline phosphatase is predominantly produced by soil microorganisms. The prokaryotic genes phoXAD encode alkaline phosphatases, with phoD acting as potential biomarker for assessing phosphate solubilization (Wan et al., 2020). The strain MHN12 genome harbored genes for alkaline phosphatase (phoABD), pyrophosphatase (ppaX), and phosphonate degradation and transport systems (pstABCS). Similar gene clusters have also been reported in plant growth-promoting phosphate solubilizing B. velezensis strains (Ercole et al., 2024).

Iron is a co-factor in many processes and activities including photosynthesis and oxidative phosphorylation thus essential for plant growth. Siderophores produced by endophytes can aid plants growing in an iron-deficient environment. Siderophores form complexes with iron to improve iron availability in plants' rhizosphere and meet plant growth's nutritional needs (Garzón-Posse et al., 2019). The strain MHN12 produced siderophores and possessed operon (dhb) involved in synthesizing bacillibactin siderophore (Bhutani et al., 2021b). Similar gene clusters for different nutrient acquisition were annotated in other plant growth-promoting strains like B. paralicheniformis RP01 and B. licheniformis TAB7 (Mpofu et al., 2019; Xu et al., 2023). Bacteria usually code for more siderophore uptake systems that enable them to efficiently sequester siderophores produced by other microorganisms (xenosiderophores). For example, Escherichia coli can assimilate ferrichrome and ferrioxamine siderophores through the fhuBCD transport system (Llamas et al., 2006; Hannauer et al., 2010; Miethke et al., 2013), and similar operons were identified in the strain MHN12 genome. Sulfur assimilation in bacteria can occur through the uptake of organic molecules, including sulfate esters, and sulfonates. The strain MHN12 exhibited a gene cluster for sulfur assimilation from sulfonates, an operon identified across diverse bacterial taxa including B. subtilis, Escherichia coli, and Pseudomonas putida (Coppée et al., 2001; Koch et al., 2005). This suggests a conserved sulfonate utilization strategy among various bacteria.

Phytohormones such as indole acetic acid (IAA), gibberellins, and cytokinins play a direct role in enhancing crop productivity (Orozco-Mosqueda et al., 2023). The strain MHN12 produced IAA in the presence of exogenous tryptophan (Bhutani et al., 2021b) and its genome analysis also revealed the presence of the genes for tryptophan and IAA biosynthesis via the indole-3-acetamide (IAM) pathway. IAA production is a common feature in around 70% of assessed halotolerant PGPB with IAM and indole-3-pyruvic acid (IPyA) pathway genes found in 60% and 80% of these bacteria, respectively (Zamanzadeh-Nasrabadi et al., 2023). Volatile organic compounds such as acetoin and 2,3-butanediol whose synthesis operons were annotated in the strain MHN12 genome play significant roles in stimulating plant growth, inducing systemic resistance, enhancing drought resilience, and influencing phytohormone profiles (Orozco-Mosqueda et al., 2023). Arabidopsis thaliana plantlets exposed to acetoin and 2,3-butanediol produced by B. amyloliquefaciens showed antagonistic activity and better growth (Wu et al., 2019).

Polyamines like putrescine and spermidine play essential roles in bacterial stress resilience and plant-microbe interactions. Under stress, plant ethylene levels increase significantly, negatively impacting growth and promoting senescence. The presence of multiple polyamine biosynthesis and transport genes in the strain MHN12 genome suggests its ability to control plant S-adenosyl methionine (SAM) levels, a precursor for ethylene, and influence ethylene biosynthesis enzymes (ACC synthase and ACC oxidase) via spermidine production. Additionally, strain MHN12 has been screened positive for ACC deaminase activity, an enzyme that degrades the immediate ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), promoting plant growth (Xie et al., 2014; Maheshwari et al., 2020; Bhutani et al., 2021b). These findings suggest that strain MHN12 utilizes a dual strategy: modulating SAM and directly degrading ACC, to regulate plant ethylene levels and consequently promote plant growth.

The beneficial plant-promoting effects of bacteria primarily lie in the successful colonization of host plants by endophytes. Bacterial colonization in plants begins with the recognition of chemical signals released by roots, followed by chemotactic responses to these signals. Bacteria then migrate toward the roots and colonize different plant parts (Kumar et al., 2020). Genomic analysis revealed genes related to chemotaxis, and flagella synthesis suggesting strong chemotactic response and motility abilities of strain MHN12. Genes involved in chemotaxis and motility are important for the colonization abilities of plant-associated bacteria, particularly endophytes (Hardoim et al., 2015). Several genes involved in exopolysaccharide synthesis were identified in the strain MHN12 genome. Exopolysaccharide (EPS) has been reported to contribute to plant salt tolerance by chelating free Na⁺ from the soil, restricting Na⁺ entry into plants, facilitating biofilm formation, and improving soil stability (Liu et al., 2022). The addition of EPS from Pseudomonas simiae MHR6 effectively improved Na⁺/K⁺ homeostasis in maize under salt stress (Liu et al., 2023).

The in vitro analysis of PGP traits as well as production of stress aiding metabolites under salinity revealed that the isolate was able to maintain its PGP traits i.e., phosphate solubilization, HCN and ammonia production even under stress. Moreover, enhanced antioxidant activity and increased EPS production may help strain MHN12 in maintaining ionic balance and thereby support its ability to cope with salinity stress. Additionally, the presence of genes for chaperones, antioxidant enzymes, and heavy metal resistance, further aids strain MHN12 to effectively combat various abiotic stresses, including thermal, oxidative, and heavy metal stress. The strain MHN12 genome harbored genes for osmolyte production and transporters crucial for cell turgor maintenance under salinity or osmotic stress. These osmolyte transporters facilitate cellular osmotic adjustments (Parmar et al., 2017). Experimentally, strain MHN12 has demonstrated the ability to tolerate and grow in high salinity, up to 15% NaCl (2.6 M) and it can produce increased amounts of proline under stress conditions, an important osmolyte (Bhutani et al., 2022).

The plasma membrane functions as the primary barrier protecting bacteria from environmental stresses. Under saline conditions, membrane function is disrupted, causing altered permeability, loss of fluidity, and impaired selective ion transport. A key adaptive response to this challenge is the regulation of membrane fluidity through the modulation of fatty acid composition (Wang et al., 2020; Rojas-Solis et al., 2020). The annotation of desaturase gene (desA) supports strain MHN12 capacity for unsaturated fatty-acid synthesis. Moreover, a decrease in saturated fatty acids (SFAs) including palmitic acid (n-hexadecanoic acid), stearic acid (octadecanoic acid), and myristic acid (tetradecanoic acid) and an increase in unsaturated fatty acids (UFAs) such as oleic acid was observed. Similarly, in other osmotolerant organisms such as Bacillus sp. CR71, Zygosaccharomyces rouxii, Tetragenococcus halophilus, and Debaryomyces hansenii, a decrease in the saturated to unsaturated fatty acid (SFA/UFA) ratio under salt stress has been observed contributing to maintain optimal membrane fluidity (He et al., 2017; Wang et al., 2020; Rojas-Solis et al., 2020). An increase in fatty acid unsaturation in membrane lipids enhances membrane fluidity, which supports the proper functioning of membrane-embedded ion transport systems (such as Na⁺/H⁺ antiporters), thereby helping maintain ion homeostasis under salt stress (Allakhverdiev et al., 2001). The significant increase in pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) under salt stress highlights its protective role. It is a non-enzymatic antioxidant capable of mitigating oxidative damage induced by stress conditions (Nas et al., 2021). This resilience and genomic potential of strain MHN12 support it to adapt to salinity. Future studies focusing on in planta assays are essential to validate efficacy of strain MHN12 in alleviating salt stress and promoting plant growth under field conditions.

The genetic proximity between Bacillus paralicheniformis and Bacillus licheniformis often leads to misclassification of the former, as evidenced by the initial identification of strain MHN12 as B. licheniformis in the present work. However, the presence of secondary metabolite operons specific to B. paralicheniformis and absent in B. licheniformis distinguishes strain MHN12 from B. licheniformis. Detailed genomic analysis of strain MHN12 revealed several pathways and a multitude of genes facilitating plant colonization, growth promotion, and stress tolerance. The genome annotation confirmed the presence of genes responsible for key plant growth-promoting and stress resilience functions, such as indole-3-acetic acid (IAA) and siderophore production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, phosphate solubilization using alkaline phosphatase, proline, and antioxidant enzyme synthesis. The presence of genes for acetoin, 2,3-butanediol, and secondary metabolite biosynthesis indicated the strain's potential for bio-control and defense. In vitro assays validated the genomic data, confirming ability of strain MHN12 to exhibit plant growth promoting traits along with production of enzymatic antioxidants and exopolysaccharides (EPS) under salinity stress. Additionally, the observed decrease in the SFA/UFA ratio under salt stress represented a vital adaptive strategy of strain MHN12 toward harsh conditions. This analysis underlines the adaptability of B. paralicheniformis MHN12 in enhancing plant growth, even in saline environments. Further research is necessary to elucidate the precise functions and regulatory mechanisms of these genes. The large-scale green house as well as field trials are essential to validate the strain's performance under diverse agro-climatic and saline conditions.