Bacillus ayatagriensis sp. nov., a novel plant growth-promoting rhizobacteria strain isolated from mulberry rhizosphere

Mulberry (Morus spp. L.), belonging to the Moraceae family, has a long being cultivated in Asia for its economic significance in sericulture, food and pharmaceutical industries worldwide¹. The rhizosphere of mulberry, a complex ecosystem harboring diverse microbial communities, remains an unexplored frontier for discovering novel plant-growth-promoting rhizobacteria (PGPR). Over the years, the PGPR has gained global importance and acceptance for crops and is considered as an alternative to chemical fertilizer. Thus, undoubtedly, PGPR deserves great attention in biological control. The concept of PGPR, introduced by Kloepper and Schroth (1978)², refers to soil bacteria capable of colonizing plant roots and exerting beneficial effects. These bacteria employ multiple mode of action to facilitate plant growth, including: (i) improving nutrient access (solubilizing phosphate, potassium, oxidizing sulfur, fixing nitrogen, and binding iron and copper); (ii) helps in suppression of soil-borne pathogens by releasing antimicrobials, hydrogen cyanide and siderophores; (iii) improving plant stress tolerance to environmental stresses such as drought, metal toxicity and salinity; and (iv) promoting growth through phytohormone production like indole-3-acetic acid (IAA)³. IAA, the most common plant hormone in the auxin class, plays a vital role in regulating key physiological processes viz., cell growth, division, and tissue differentiation. Certain bacteria capable of producing IAA are particularly beneficial in agriculture, as they promote plant growth by stimulating root development, enhancing nutrient absorption, and strengthening overall plant resilience^4,5. Several soil bacterial genera identified as PGPRs, these include Azospirillum, Bacillus, Beijerinckia, Clostridium, Klebsiella, Phyllobacterium Pseudomonas, Vario–vovax, and Xanthomonas⁶.

The predominant bacterial genera present in healthy mulberry rhizosphere soil include Bacillus, Pseudomonas, Proteobacteria, and Actinobacteria⁷. Among PGPR, the Bacillus genus stands out as effective rhizobacteria with multiple beneficial traits. In addition to promoting plant growth and nutrient uptake they also play a crucial role in biocontrol as well by outcompeting harmful microbes, inducing systemic resistance (ISR), and producing antimicrobial compounds⁸. Furthermore, Bacillus species synthesize various enzymes that help plants cope with biotic stresses, making them valuable allies in sustainable agriculture. Their capability to survive in diverse soil conditions, form resilient endospores, and produce bioactive compounds makes Bacillus a promising candidate for enhancing plant growth under environmental stressors^9,10,11. This study thus aimed at isolating and characterizing PGPR strains with strong PGP traits from the mulberry rhizosphere. The plant-growth-enhancing features, along with the whole genome analysis of the most potent PGPR strain depicted as “RMG6^T,” were also initiated to explore its potential applications as biocontrol agent. Our study presents a comprehensive genome sequence analysis of RMG6^T alongside its phylogenetically related species, aiming to identify potential biosynthetic gene clusters (BGCs), predict their putative metabolic products, and classify protein-coding genes based on their functional attributes. Thus, our findings not only validate the use of Bacillus RMG6^T as a PGPR but also provide a strong foundation for future research into its wider applications in sustainable agriculture.

Sampling and isolation

The mulberry rhizosphere soil samples were taken in replicates in a sterile vial from a depth of 6 inches (15 cm) using a sterilized sampler and preserved at 4 °C for transferring to lab from the mulberry garden (25.6072° N, 88.1303° E), of Raiganj University, West Bengal, India. For each replicate, three to five soil samples were collected, thoroughly mixed, and a 10–25 g portion was selected as a representative sample and heat treated at 65 °C for 2 h to facilitate the selective enrichment of spore-forming bacteria. After that, the samples were suspended in 90 mL sterile 0.9% NaCl solutions, serially diluted, spread on nutrient agar (NA) (Himedia) medium, and incubated overnight at 35 °C¹². NA plates without any inoculum were used as a negative control to check any spontaneous growth. The bacterial colonies with distinct morphological traits were selected for further purifications. Repeated sub-culturing was performed to ensure the purity of the isolates and consistency in its characteristics. The purified colonies were picked and preserved on freshly prepared NA slants for future use.

Screening of Bacillus like isolates with biocontrol potential against Gram-positive and Gram-negative bacteria

Antimicrobial assay was performed using agar-well diffusion method on Mueller–Hinton (MH) agar. By this method, two days old liquid culture of different Bacillus like isolates were centrifuged and the supernatants were collected and added on the MH agar plates wells (6 mm diameter) having the lawn of the test organism (Escherichia coli MTCC 43 & Enterococcus faecalis MTCC 439) to screen the most potent antimicrobial producing isolates. After 24 h the zone of inhibitions was recorded and the strain exhibiting highest antimicrobial activity was selected and designated as RMG6^T. Further, RMG6^T cells were kept on NA plates consecutively for 12 generations prior to preservation at − 80 °C in 20% glycerol. To determine the changes in cellular morphology of the test organisms were examined via scanning electron microscopy (SEM; Hitachi S-3400) analysis as described earlier¹³. Briefly, the bacterial cells were fixed with a 2.5% glutaraldehyde solution, followed by dehydration through a progressive series of ethanol treatments, and subjected to examination under SEM with an accelerating voltage of 15–20 kV. Multiple fields of vision were viewed at different magnifications^13,14. Moreover, The SEM micrographs of RMG6^T cells were also captured following the previously reported protocol¹³.

Physiology and chemotaxonomy

Morphological, cultural, physiological, and biochemical characteristics were analyzed as previously described¹⁵. Colonies grown on NA plates was examined for their cultural and morphological characteristic; that includes the cell shape, colonial appearance, and endospore formation after 48 h of incubation at 35 °C. The Gram staining was done using a Gram staining kit (NICE chemicals G21950) as per manufacturer’s protocol. The pure culture of RMG6^T was biochemically characterized using Vitek-2 Compact Automated system as described earlier¹⁴. Growth in various salt concentrations (0–14% w/v), at different pH values (3–11) and at different temperatures (4, 15, 20, 25, 30, 37, 40, and 45 °C). All the tests were carried out incubating the cultures at 35 °C, except for investigations into the effect of temperature upon growth. After confirming the optimum growth conditions of RMG6^T, the doubling time (T_D) and specific growth rate (k) was also determined¹⁶. Furthermore, RMG6^T was identified using a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Microflex LT Mass Spectrometer (Bruker Daltonics) equipped with MALDI Biotyper 3.0 software (Bruker Daltonics). The samples were precisely applied onto the target plate and overlaid with 1 μL of alpha-cyano-4-hydroxycinnamic acid (HCCA) matrix before undergoing MALDI-TOF MS analysis. Identification was considered reliable at the species level for log scores of ≥ 2.0, at the genus level for scores of ≥ 1.7, while scores below 1.7 were deemed insufficient for accurate classification. Moreover, the total content of cellular fatty acid in RMG6^T was determined following the MIDI protocol (Microbial Identification). Extraction and methylation of fatty acids were done to form fatty acid methyl esters (FAME) and analyzed using Gas Chromatography (Agilent 6850 gas chromatograph) with the help of MIDI Sherlock software (MIDI Sherlock™ Microbial Identification System v.6.02 USA; http://www.midi-inc.com/) for FAME. Aerobic library (RTSBA6 6.21) was referred for the analysis^17,18.

Detection and quantification of IAA

Spectrophotometric method

The bacteria RMG6^T were incubated in L-tryptophan (2 mg/mL) containing Nutrient broth in 100 mL Erlenmeyer flasks in triplicates at 35 °C on a BOD incubator and shaker for 48 h. Initially, centrifugation of the RMG6^T culture was done at 10,000 g for 10 min, and the supernatant was mixed with Salkowski reagent (50 mL, 35% of perchloric acid, 1 mL 0.5 M FeCl₃ solution) in a 1:1 ratio. A pink color indicated IAA production, and its absorbance was measured at 530 nm. The IAA concentration was determined using a standard curve (10–100 μg/mL) from HiMedia.

Extraction of indolic derivatives

The extraction of indolic derivatives was performed from the bacterial culture supernatants obtained after 48 h of incubation in nutrient broth with tryptophan (Trp; 2 mg/mL) at 35 ± 2 °C. Following acidification to pH 2.5 with 1 N HCl, the culture supernatants underwent three sequential extractions with equal volume of C₄H₈O₂. A fraction of the C₄H₈O₂ phase was then air-dried and reconstituted in one-tenth of its original volume using methanol, in accordance with previously described earlier¹⁹. The methanolic extracts were then used for HPTLC analysis.

High-performance thin-layer chromatography (HPTLC) analysis

Thin-layer chromatography (TLC) analysis was conducted using the spray-on technique for sample loading onto TLC foils (20 × 10 cm) with a Linomat 5 applicator (CAMAG, Muttenz, Germany) equipped with a Hamilton 100 μL syringe. The application parameters included an 8 mm band length, a 150 nL/s application rate, a table speed of 10 mm/s, and an automatically set band spacing. The first track was positioned at 15 mm (X), with 11.3 mm between tracks, while scanning positions ranged from 5 to 70 mm (Y). The solvent front was set at 70 mm. A methanolic solution containing standard IAA, tryptamine, and IAM (250 µg/mL) was applied, with loaded volumes of 2 μL (500 ng), 3 μL (750 ng), 4 μL (1000 ng), and 5 μL (1250 ng), forming four tracks per standard. The TLC plate was developed using isopropanol: ammonia: water (8:1.4:0.6, v/v) as a mobile phase and pre-saturated with the same mobile phase for 10 min at 26 °C. After development of the TLC plates, those were air-dried, and visualized under a UV chamber at 254 nm. The mobile phase took 90 min to reach the solvent front (70 mm). For quantitative analysis, densitometric measurements were performed at 254 nm using a CAMAG TLC scanner 3 (D2 lamp) with a scanning speed of 20 mm/s. The slit dimensions were set at 5 × 0.45 mm, with a data resolution of 100 µm per step, using the Savitzky-Golay 7 filter for smoothing. Baseline correction was applied at the lowest slope, and display scaling was adjusted automatically. The limit of detection (LOD) and limit of quantification (LOQ) were measured via plotting a scatter graph of peak area versus the amount of standard loaded per band. These values were calculated using the formula LOD = 3.3 σ/S, where σ represents the standard deviation (SD) and S is the slope of the calibration curve. The SD (Y-axis) and slope were obtained from the standard curve (sample amount in µg on X-axis vs. peak area on Y-axis) using the LINEST function in MS Excel 2017. The recovery percentage of each standard was calculated comparing the measured quantity to the initially loaded amount, considering 100% as the reference^20,21,22,23.

Seed germination assay

Efficacy of RMG6^T was tested for any stimulating activity on the seed germination of Vigna radiata. Five viable seeds were collected and surface sterilized using a 0.1% HgCl₂ solution, followed by through rinsing in sterile distilled water to remove any residual HgCl₂. The sterilized seeds were then soaked in sterile distilled water for one hour and placed on a sterile, moistened tissue paper at the bottom of a Petri dish. The bacteria were cultured in a L-tryptophan (0.2%) containing Nutrient Broth at 35 °C for 24 h. Bacterial culture was centrifuge at 10,000 rpm for 10 min. Supernatant (containing IAA) 2 mL was utilised for seed germination assay. For the control, only sterile nutrient broth was used. The experiment was conducted in triplicate. Seed germination was observed after 24–48 h in each experimental set, following a previously reported method²⁴.

Antibiotic susceptibility testing

The Kirby-Bauer disc diffusion assay was conducted to assess the antibiotic susceptibility profile of Bacillus sp. strain RMG6T²⁵. A total of 43 antibiotics, commonly used in human and veterinary medicine, were obtained from HiMedia Laboratories Pvt. Ltd., India. The strain was cultured overnight spreading uniformly onto MH agar (HiMedia Laboratories Pvt. Ltd., India) plates. The antibiotic discs were placed on agar surface, and incubated at 37 °C for 24 h. Based on the formation of inhibition zones, the antibiotic sensitivity was determined; the presence of a halo indicated susceptibility, while the absence of a halo indicated resistance. The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints (version 14.0, https://www.eucast.org/clinical_breakpoints). The strain was classified as resistant (R), intermediate (I), or susceptible (S) based on EUCAST guidelines for Bacillus sp.

Molecular identification of isolated bacteria

The genomic DNA of the isolated bacteria was extracted using the phenol–chloroform extraction method²⁶. The purity of the extracted DNA was assessed spectrophotometrically using a NanoDrop 1000. Its quality was also verified through electrophoresis on a 1% agarose gel. Briefly, 2 µL of isolated DNA was mixed with 1 µL of 2 × loading dye (Invitrogen) and then subjected to electrophoresis at 120 V for 60 min. The results were documented using a BioRad ChemiDoc MP Imager (Model No. 12003154). For molecular identification, the 16S rDNA was amplified using universal primers 27F, 704F, and 907R²⁷. The PCR reaction (25 µL total volume) contained 12.5 µL of PCR Master Mix (Sigma Aldrich, St. Louis, MO), 2 µL of template DNA, 1 µL of each primer, and 8.5 µL of nuclease-free distilled water. The thermal cycling conditions included denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s and annealing at 55 °C for 30 s and elongation at 72 °C for 1 min, and then cycling was completed by a final elongation step for 10 min at 72 °C. A negative control with water was included in each PCR reaction. The amplified PCR products were analyzed by electrophoresis on a 1% agarose gel. The purified 16S rDNA was sequenced using the Sanger sequencing method. Both strands of the nucleotide sequences were aligned using BioEdit software (BioEdit Sequence Alignment Editor v.7.2.5; http://www.mbio.ncsu.edu/BioEdit/bioedit.html) to obtain the partial or complete sequence of the 16S rDNA. The sequence was then submitted to the EZBioCloud server for identification of the closest type strains. Multiple sequence alignment of the retrieved type strains was performed using the ClustalW program built in MEGA11 software. The neighbor-joining (NJ) method was adopted to construct the phylogenetic trees. The evolutionary distances were calculated using the Kimura 2-parameter model. Bootstrap analysis was performed with 1000 replications to validate the clades of the phylogenetic tree. The NCBI accession number for the 16S rDNA sequence of strain RMG6^T is PP494329.

Multi-locus DNA sequence based phylogenetic position analysis

Multi-Locus Sequence Analysis (MLSA) is a method that can be used for determining the evolutionary ancestry of species within a genus. In order to differentiate species from their close relatives, a phylogenetic tree based on multiple genes generates more information than one that relies exclusively on the 16S rRNA gene. The housekeeping gene sequences were used for construction of a phylogenetic tree of MLSA, and phylogenies are subsequently generated²⁸. The whole genome sequence was used to extract nine genes for the MLSA of RMG6^T, including gyrB (DNA gyrase subunit B), atpD (F₀-F₁ ATP synthase subunit β), dnaK (molecular chaperone protein), gmk (guanylate kinase), ilvD (dihydroxy-acid dehydratase), purH (bifunctional phosphoribosylaminoimidazolecarboxamide formyltransferase), pycA (pyruvate carboxylase), rpoB (DNA-directed RNA polymerase subunit beta), rpoD (RNA polymerase sigma factor) from the whole genome sequence (accession number-JBEFNT000000000). The housekeeping gene sequences and their closest relatives (according to 16S rRNA gene sequence-based analysis) were downloaded from GenBank. Following alignment using MEGA 11 software, the nucleotide sequences associated to the corresponding genes were manually trimmed at identical positions. The nine-housekeeping gene sequences were concatenated head to tail as gyrB-atpD-dnaK-gmk-ilvD-purH-pycA-rpoB-rpoD for RMG6^T and its nearest relatives. Pairwise distances were computed using the Kimura 2-parameter model²⁹. MEGA 11 software was used to generate the best model for phylogenetic analysis, and phylogenetic analysis took place using the the best model parameters. The Tamura nei (TN93) model by Gamma distributed with invariant sites (G + I) was determined to be the best fit for maximum likelihood³⁰.

Draft genome sequencing and assembly

The genome of Bacillus sp. strain RMG6^T was sequenced to identify genes involved in the biosynthesis of antimicrobial compounds and production of different types of plant growth-promoting traits like siderophore, indole acetic acid, etc.

DNA extraction was performed using the DNeasy Ultraclean Microbial Kit., followed by quantity confirmation using Nanodrop 1000. Construction of paired-end libraries was done utilizing Ultra II DNA Library Prep Kit (NEBNext #E7645S/L) and Illumina NovaSeq 6000 platform (Neuberg Diagnostics Pvt. Ltd., Ahmedabad, India) used for sequencing, producing 13,625,248 reads having 2 × 159 bp paired-end read length. The quantification of final DNA libraries was performed using a Qubit 4.0 fluorometer (Thermo Fisher #Q33238) with a DNA HS assay kit (Thermo Fisher #Q32851), as per manufacturer’s guidelines. The library insert size was analyzed using the TapeStation 4150 system (Agilent) with highly sensitive D1000 ScreenTapes (Agilent #5067–5582).

Quality assessment of the samples raw fastq reads was done and processed using FastQC v.0.11.9 (default parameters)³¹ and Fastp v.0.20.1³² (parameters: –length_required 50 –correction –trim_poly_g –qualified_quality_phred 30 –unqualified_percent_limit 30 –average_qual 30) respectively. Thereafter, the processed data were re-assessed using FastQC. The processed reads were de novo assembled using Unicycler v.0.4.4,³³ Megahit v1.2.9³⁴ and Spades v4.0³⁵ with default parameters. The completeness and contamination rate of the assembled genome were finally determined using CheckM2 v.1.0.1³⁶.

Genome sequence annotation and genomic data analysis

Prokaryotic Genome Annotation Pipeline (PGAP) v6.7 in NCBI was used to carry out the genome sequence annotation utilizing the Best-Placed Reference Protein Set and GeneMarkS-2 + methods³⁷ and later it was also compared with another annotation platform viz. BV-BRC v3.46.3. The completed genome assembly sequence was submitted to the DSMZ Type Strain Genome Server (TYGS v.391; https://tygs.dsmz.de/user_requests/new), and analyzed using the Genome to Genome Distance Calculator (GGDC v3.0; https://ggdc.dsmz.de/ggdc.php#)³⁸. Phylogenetic trees produced by TYGS were visualized using the interactive tree of life (iTOL V6; iTOL: Interactive Tree Of Life (embl.de)³⁹ with Escherichia coli DSM 30,083 selected as an outgroup⁴⁰. Average nucleotide identity (ANI) was calculated on the Ezbiocloud server (https://www.ezbiocloud.net/tools/ani) using the OrthoANIu algorithm⁴¹ against the closest relative identified by the Ezbiocloud server. Finally, a genome map of Bacillus sp. strain RMG6^T was constructed using CGView Server (https://cgview.ca/)⁴² to visualize distinct contigs, CDSs (in both forward and reverse orientations), GC content and skew. Furthermore, the assembled genomic data underwent annotation and comparative analysis using the Subsystem Technology tool kit (RASTtk). This process facilitated the prediction of genes associated with risk assessment, including virulence factors, antibiotic resistance genes (ARGs), potential drug targets, and human homologs)⁴³. Moreover, the AutoMLST web platform (autoMLST: Automated Multi-Locus Species Tree (ziemertlab.com)⁴⁴ was used to evaluate the presence BGCs and the potential of the isolate RMG6^T for secondary metabolite production. The antiSMASH v 7.1.0 (https://antismash.secondarymetabolites.org/#!/start) was used for the genome mining of secondary metabolite BGCs in strain RMG6^T45. The metabolic pathways of strain RMG6^T and other closely related Bacillus species were characterized and compared with the METABOLIC software v4.0⁴⁶. In addition, the metabolic pathway annotation and completeness of the pathway modules of strain RMG6^T and other closely related Bacillus species were determined using GhostKOALA^47,48,49,50. Heatmap analysis of differentiated pathways was performed with the complex heatmap package in the R program⁵¹. Genomes were aligned and compared with BLAST Ring Image Generator (BRIG) to generate a circular map of closely related species⁵².

Isolation, screening, 16S rDNA, and Multilocus DNA sequence based phylogenetic position analysis of RMG6^T

A total of 48 bacterial colonies were obtained from the rhizosphere soil of mulberry. Among them, only one isolate, RMG6^T exhibited significant biocontrol potential and demonstrated in vitro antagonism against Escherichia coli MTCC 43 and Enterococcus faecalis MTCC 439 Our results revealed that the culture supernatant (50 µL) of RMG6^T showed a larger zone of inhibition against Enterococcus faecalis MTCC 439 (1.92 cm) compared to Escherichia coli MTCC 43 (1.62 cm) (Fig. 1A,D; as highlighted with white color arrow). SEM micrographs also revealed RMG6^T supernatant mediated cell-lysis of the tested bacteria as evidenced by cytoplasmic leakage (as highlighted with red color arrow) (Fig. 1B,E) compared to the control (Fig. 1C,F). This study aligned with an earlier study that reported the antimicrobial activity of cell-free supernatant of Bacillus sp. against E. faecalis⁵³.

To determine the taxonomic position of the isolate, a 16S rDNA-based phylogenetic analysis was conducted. The short (1545 bp) 16S rDNA-based phylogenetic analysis of closely related type strains revealed that the isolate RMG6^T was a member of the Bacillus genus and the closest relative to the isolate was the Bacillus siamensis KCTC 13,613 (Fig. 2) with 99.93% similarity and 1 nt variation was present.

Further, The MLSA pairwise distance, calculated by employing Kimura-2 parameter model, between strain RMG6^T and its phylogenetically associated species exceeded the threshold value of 0.007, supporting its designation as a new species, as recommended by Rong and Huang (2010)⁵⁴ (Table 1).

The ML tree generated using the best fit model parameters for the MLSA analysis revealed that RMG6^T, Bacillus siamensis KCTC 13,613 and Bacillus amyloliquefaciens DSM 7, all descended from the same ancestor indicating close relativeness (Fig. 3).

Furthermore, the digital DNA-DNA hybridization (dDDH) similarity of RMG6^T with Bacillus siamensis KCTC 13,613 and Bacillus amyloliquefaciens DSM 7 was only 56.80% and 55.20%, respectively, which is much lower than the suggested threshold of 70% for species delineation⁵⁵. Thus, RMG6^T was considered as a novel species within the Bacillus genus and designated as Bacillus ayatagriensis sp. nov. RMG6^T (= MCM-B-1537).

Physiology and chemotaxonomy

The RMG6^T colonies exhibited milky white, wrinkled protuberances at the center and was opaque with an irregular edge when observed after 48 h of incubation on NA medium at 35 °C (Fig. 4A). Morphology evaluation of colonies derived from Bacillus siamensis on NA plates exhibited small, irregular, round yellowish white colonies on NA medium⁵⁶. Another study reported that the colonies of Bacillus siamensis on NA plates were round, greyish, flat and sticky with smoother edges⁵⁷. Thus, the differences in the morphology of RMG6^T colony pattern suggesting an initial clues about the bacterial classification of different species. When observed under light microscope, RMG6^T cells belong to the Gram-positive class (Fig. 4B). SEM micrograph of RMG6^T vegetative cells revealed that the isolated strain RMG-6 was rod-shaped cells with a diameter of 2.45 µm, chains or clusters with a relatively smooth surface (Fig. 4C,D); the most notable feature is the presence of endospore, which can appear as intact and cylindrical with bumpy, rough, wrinkled surfaces that possess numerous folds and creases (Fig. 4E,F), consistent with previously reported findings⁵⁸.

To further characterize the isolate, biochemical profiling of RMG6^T was conducted using Vitek-2 automated technology. Based on the biochemical characteristics, the Vitek-2 automated system was unable to identify the strain RMG6^T (Supplementary Fig. 1). Table 2 shows the main features that distinguished Bacillus sp. RMG6^T from other phenotypical and phylogenetic taxa.

The strain exhibited a broad growth range, thriving at temperatures between 20 °C and 50 °C (Fig. 5A; Table 2), with an optimum growth temperature of 35 °C (Fig. 5B; Table 2). It also demonstrated adaptability to a pH range of 4–9, with optimal growth at pH 6 and showed remarkable salt tolerance, growing in NaCl concentrations of up to 14% (Fig. 5C; Table 2). Furthermore, the growth pattern of the strain RMG6^T up to 16 h in a nutrient broth medium was also recorded and presented in Fig. 5D. The growth kinetics of RMG6^T revealed that the T_D (= doubling time) and of k (= growth rate constant) of RMG6^T cells, was 76.17 ± 1.71 min (mean ± standard deviation) and k was 0.0085 ± 0.0001 doubling times per min, respectively. These findings are in agreement with previously reported results^64,65.

Further identification of RMG6^T using MALDI-TOF MS yielded a log score of ≥ 2.0 or ≥ 1.9, confirming its classification at the genus level. One Bacillus isolate was identified with a log score of 2.04 using the Bruker system. While MALDI-TOF MS is widely used to identify Bacillus species, its accuracy can be affected by the presence of endospores, which alter the protein composition of samples. The extent of endospore formation can influence MALDI spectra, depending on culture conditions. Due to these limitations, a more precise identification of RMG6^T was pursued through additional fatty-acid profiling and genome-based analyses.

The total cellular fatty acid profile of RMG6^T revealed a high proportion of branched fatty acids; the major components (> 5.0%) were anteiso-C_15:0 (28.13%), iso-C_15:0 (20.72%), C_18:0 (10.72%), iso-C_17:0 (7.00%), iso-C_16:0 (5.88%), and anteiso-C_17:0 (5.87%) (Table 3 & Supplementary Fig. 2).

FAME analysis suggested that strain RMG6^T have highest diversity of fatty acids from the other closest related species of Bacillus. For example, C_11:0, anteiso-C_17:1ω9c, C_18:3ω6c (6, 9, 12), C_18:0, and C_20:0 exclusively present in the RMG6^T strain. However, fatty acids such as C_16:1ω7c (present only in Bacillus subtilis NCIB 3610), C_16:1ω11c (present in Bacillus velezensis NRRL B-41580, Bacillus subtilis NCIB 3610, Bacillus amyloliquefaciens DSM 7, Bacillus nakamurai NRRL B-41091), and iso-C_17:1ω10c (present only in Bacillus subtilis NCIB 3610) were not detected in the RMG6^T strain, but they were identified in other related strains. These differences further support the taxonomic uniqueness of RMG6^T.

Auxin production and plant growth-promoting potential of Bacillus ayatagriensis sp. nov. RMG6^T

Auxin plays a crucial role in plant root development, making its production an important trait for plant-associated bacteria. To further investigate if RMG6^T produces auxin, the total content of auxin was evaluated in the cell-free culture supernatant of RMG6^T. The indole-3-pyruvic acid pathway (IPA) is one of the main pathways for the biosynthesis of IAA in bacteria. The synthesis is completed in three steps. In the first step tryptophan is converted to the indole-3-pyruvic acid by aminotransferases and then indole-3-acetaldehyde is formed by pyruvate decarboxylase. In final step, aldehyde dehydrogenases form indole-3-acetic acid⁶⁷. Strain RMG6^T uses IPA pathway for the biosynthesis of IAA from tryptophan. MEQ7682189.1 protein has 83% similarity to aromatic-amino-acid aminotransferase of Bacillus subtilis. Strain RMG6^T has three pyruvate decarboxylase; MEQ7680847.1 (pdhA), MEQ7680848.1 (pdhB) and MEQ7680849.1 (pdhC) similar to Bacillus subtilis counterparts. Finally Strain RMG6^T has three aldehyde dehydrogenases protein; MEQ7681358.1, MEQ7682689.1 and MEQ7683083.1, further supporting its ability to synthesize IAA (Supplementary Table 1). The IAA production capability of the strain RMG6^T was experimentally validated in the L-tryptophan-supplemented nutrient broth. It was found that the strain could produce a maximum IAA of 73.4 μg/mL in 0.2% optimum concentration of L-tryptophan (Fig. 6B,C,E,F). HPTLC confirmed the IAA production (Fig. 6D), and the results were compared with the standard IAA (Fig. 6G–I). Moreover, the spectra comparison confirmed the presence of IAA in the strain RMG6^T culture (Fig. 6A), reinforcing its ability to produce this essential phytohormone.

Beyond its role in bacterial taxonomy, IAA production serves as an important ecological indicator, highlighting the potential interaction of RMG6^T with mulberry plants. Since IAA is critical for plant growth and development, the significant levels produced by RMG6^T suggest its potential as an effective plant growth-promoting bacterium (PGPB). Notably, RMG6^T exhibited higher IAA production compared to previously reported Bacillus species, further distinguishing it within its taxonomic group (Table 4).

We anticipate that the growth-promoting effects on other crops could be further enhanced through RMG6^T treatment, which facilitates increased IAA production. To substantiate this, Vigna radiata seed germination and rooting assays were conducted. The results demonstrated that treatment with RMG6^T cell-free supernatant, containing bacterial-derived auxin, supported 100% seed germination and promoted the development of healthy radicles with an average length of 5.4 cm, significantly longer than the 3.06 cm observed in the untreated control group (Fig. 7). These findings are in accordance with earlier studies, reinforcing the potential of RMG6^T for cross-species bioaugmentation⁷².

Antibiotic susceptibility testing

The antibiogram profiling of RMG6^T was tested against different clinically relevant antibiotics to determine its resistance profile. The antibiogram results revealed that the strain RMG6^T was mainly resistant to cell wall inhibitors like penicillin, ampicillin, and ceftazidime and protein synthesis inhibitors like piperacillin (Supplementary Table 2). Genome annotation further supported these findings, revealing the presence of a Beta-lactamase class A (penP) gene, which likely contributes to its resistance against penicillin, ampicillin, and ceftazidime. Additionally, DeepARG analysis identified ARGs related to multidrug resistance, including those for bacitracin and phenicol, highlighting the genetic basis of RMG6^T resistance (Supplementary Table 3).

Genome sequencing and biosynthetic gene clusters analysis

The genome assembly resulted in a sequence spanning 3,818,111 base pairs, distributed across 37 contigs. The assembly exhibited an N₅₀ value of 995,572 base pairs and an L₅₀ count of 2. The estimated GC content was determined to be 46.62%, with a sequencing coverage of 496.06 × . A total of 3,723 coding sequences were annotated, which include 3 rRNA genes (one 5S, one 16S, and one 23S) along with 76 tRNA genes. Additionally, according to BV-BRC v3.46.3 annotation platform, it was observed that the genome length and G + C content of RMG6^T was 3,821,630 bp long and 46.63%, respectively. N50 length, L50 count, number of t-RNA, and r-RNA was found to be consistent with the statistics of NCBI-PGAP (Table 5).

The completeness and contamination of all genome assemblies produced by Unicycler, Megahit and Spades were 100% and 0%, respectively. However, Unicycler genome assembly was produced less contigs (37 contigs) compared to Megahit (49 contigs) and Spades (288 contigs). Thus, Unicycler genome assembly was selected for further analysis (Supplementary Table 4). Figure 8 shows the genome map generated in the CGView server.

The circular map generated by comparing the genome of Bacillus ayatagriensis sp. nov. RMG6^T with other closely related Bacillus genomes showed a large number of variations with gaps or regions of low similarity (Fig. 9).

GGDC v3.0 calculated the dDDH values. In this analysis, Formula 2 was used because its calculations are independent of genome length and are specifically recommended for incompletely sequenced genomes. The distance among the genomes of strain RMG6^T and closely related Bacillus siamensis KCTC 13,613 was 56.80%. According to the previous reports, the dDDH values not exceeding 70% indicate that the tested organisms belong to different species^73,74. OrthoANIu was employed to determine the ANI between the isolated strain and its phylogenetically closest type strains. Based on this algorithm, strain RMG6^T showed a maximum average value of 94.44% with closely related Bacillus siamensis KCTC 13,613. Typically, the ANI values between genomes of the same species are above 95%⁷⁵.

Comparative metabolic analyses were performed METABOLIC and GhostKOALA. Detailed metabolic pathway annotations were presented in supplementary Table 5. Heatmap of differentiated pathways shown that B. ayatagriensis sp. nov. RMG6^T clustered with B. methylotrophicus and B. velezensis (Fig. 10).

Furthermore, a whole-genome-based phylogenetic tree was constructed via the Type (Strain) Genome Server (TYGS) to elucidate the phylogenetic placement of the strains. The constructed tree showed that the strain RMG6^T formed an individual clade in comparison with highly related Bacillus species (Fig. 11). Thus, as per the phylogenomic analysis, dDDH and ANI scores between Bacillus sp. strain RMG6^T and closely related strains reveal that the strain RMG6^T should be considered a distinct species.

The whole-genome sequence (WGS) of strain RMG6^T was submitted to the RAST server (https://rast.nmpdr.org/) for comprehensive functional annotation⁷⁶. Default parameters were applied across all software tools. The annotation process identified a total of 3,899 coding sequences and 79 predicted RNAs. RAST analysis further revealed the presence of siderophore gene clusters, as well as genes associated with the biosynthesis of indole acetic acid (IAA) and the metabolism of sulfur, phosphate, and nitrogen, key functions linked to plant growth promotion and biocontrol. A summary of the subsystem categories assigned to the genome of Bacillus ayatagriensis sp. nov. RMG6^T is presented in Fig. 12.

A phylogenetic tree of 49 Bacillus strains and representative species was also generated through In silico AutoMLST analysis using Bacillus ayatagriensis sp. nov. RMG6^T as a query sequence (Fig. 13). The biosynthetic potentials (number of BGCs detected by antiSMASH) of each Bacillus strain are reflected in the color code. Among the 49 analyzed Bacillus strains, 1 had no BGCs (grey), 34 had between 7 and 13 BGCs (blue), and 13 strains had between 14 and 20 BGCs (green). This shows that Bacillus has a high potential for producing secondary metabolites.

In silico analysis using antiSMASH v.7.1.0 (default parameters were used for all software unless otherwise noted) revealed that genome of the isolate RMG6^T harbor putatively 20 BGCs (Table 6). The most structurally related compounds (≥ 40%) were characterized chemically and cataloged in the MiBIG (Minimum Information about a Biosynthetic Gene Cluster) database, as identified directly through antiSMASH analysis.

Analysis of cluster 1.3: The genome of the strain RMG6^T was found to harbor a Trans-acyltransferase polyketide synthase (transAT-PKS) cluster which presented 100% similarity with a biosynthetic gene cluster known to code for macrolactin H (polyketide) in Bacillus velezensis FZB42^79,80,81. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0000181 (macrolactin H biosynthetic gene cluster from Bacillus velezensis FZB42) this particular BGC shown in Fig. 14A. This particular cluster consist of 88,213 nucleotides comprising of a total 44 ORFs out of which 7 are for the core biosynthetic genes, 11 are for additional biosynthetic genes, 1 transport-related gene, 1 regulatory gene, and 23 other genes. The structure of these 7 core genes (1 to 7) is consisting of 11 modules with domain (Fig. 15A). The polymer prediction by the above-mentioned cluster is (ohmal–ccmal–ccmal) + (ccmal) + (ccmal–mal) + (ccmal–mal) + (ohmal) + (ohmal–mal); and the predicted core structure for this particular cluster is shown in Fig. 15B.

Analysis of cluster 1.4: This cluster was found to be a hybrid (NRPS, T3PKS, transAT-PKS) cluster exhibiting 100% similarity with the biosynthetic gene cluster known to code for bacillaene (polyketide + NRP) in Bacillus velezensis FZB42^{79,80,82,83,84}. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0001089 (bacillaene biosynthetic gene cluster from Bacillus velezensis FZB42) this particular BGC shown in Fig. 14B. This particular BGCs is of 110,118 nucleotides length comprising 52 ORFs. Out of the 9 core biosynthetic genes, the NRPS/PKS modules analysis reveals that rest 4 of the core genes (5 to 9) is made up of 16 modules with domains (Fig. 15C). Polymer prediction by this particular cluster is (Gly–ccmal–ohmal –mal) + (ccmal–mal–ohmal–mal) + (Me–ccmal–ohmal) + (Ala–ccmal–ccmal–Me–ccmal) + (mal–mal); and the predicted core structure for this particular cluster is shown in Fig. 15D.

Analysis of cluster 1.5: This cluster was found to be hybrid (NRPS, transAT-PKS, betalactone) cluster which exhibited 80% similarity with the BGC known to code for the fengycin (NRP) in Bacillus velezensis FZB42^80,85,86. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0001095 (fengycin biosynthetic gene cluster from Bacillus velezensis FZB42) this particular BGCs shown in Fig. 14C. This particular cluster contains 87,643 nucleotides comprising of 46 ORFs. It has been observed that this cluster presents three candidate BGCs within it [i.e., Candidate Cluster 1.5.1 (location 985,466–1,073,109): neighbouring NRPS-transAT-PKS-betalactone; Candidate Cluster 1.5.2 (location 985,466–1,062,712): chemical hybrid NRPS-transAT-PKS; and Candidate Cluster 1.5.3 (location 1,045,626–1,073,109): chemical hybrid NRPS-betalactone]. The 5 core biosynthetic genes for the candidate cluster 1.5.1 is made up of 10 modules, the 3 core biosynthetic genes for the candidate cluster 1.5.2 is made up of 3 modules, and 2 core biosynthetic genes for the candidate cluster is made up of 2 modules (Fig. 15E,G,I). Polymer prediction for the cluster 1.5.1 is (D-Tyr) + (Ile) + (mal–Asn) + (D-Tyr–D-Asn–Gln–Pro) + (D-Asn–Ser); for cluster 1.5.2 is (mal–Asn) + (D-Tyr–D-Asn–Gln–Pro) + (D-Asn–Ser); and for the cluster 1.5.3 is (D-Tyr) + (Ile). The predicted core structures for these three particular candidate clusters are shown in Fig. 15F,H,J.

Analysis of cluster 2.1: BGCs was predicted to encode a protein associated with secondary metabolism that does not align with any established secondary metabolite categories identified in antiSMASH. Nonetheless, this cluster exhibited 100% similarity with the BGC of Bacillus velezensis FZB42 which has been found to encode bacilysin^87,88,89. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0001184 (bacilysin biosynthetic gene cluster from Bacillus velezensis FZB42) this particular BGC shown in Fig. 14D. This cluster was found to be of 41,419 nucleotides, having 42 ORFs, with one core biosynthetic gene, 12 additional biosynthetic genes, 4 transport-related genes, 2 regulatory genes and rest other genes. The NRPS/PKS modules, polymer structure predictions were not determined for this cluster.

Analysis of cluster 2.2: This cluster is a hybrid (RiPP-like, NRP-metallophore, NRPS) cluster which presented 100% similarity with the biosynthetic gene cluster known to encode bacillibactin (NRP) in Bacillus subtilis subsp. subtilis str. 168^90,91,92. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0000309 (bacillibactin biosynthetic gene cluster from Bacillus subtilis subsp. subtilis str. 168) this particular BGC shown in Fig. 14E. This particular cluster consists of 51,794 nucleotides comprising of 46 ORFs. It has been observed that this cluster presents three candidate BGCs within it [i.e., Candidate Cluster 2.2.1 (location 866,688–918,482): neighbouring RiPP-like-NRP-metallophore-NRPS; and Candidate Cluster 2.2.2 (location 866,746–918,482): chemical hybrid NRP-metallophore-NRPS]. Interestingly, both the candidate clusters are identically made of 2 domains (Fig. 15K,M), with both of their polymer prediction being (Gly–Thr) and their polymer structure shown in Fig. 15L,N.

Analysis of cluster 3.1: This Trans-acyltransferase polyketide synthase (transAT-PKS) cluster presented 46% similarity according to MIBiG 3.1, with a biosynthetic gene cluster known to code for BGC0000176 (difficidin biosynthetic gene cluster from Bacillus velezensis FZB42)⁷⁹, this particular BGC shown in Fig. 14F. This cluster consists of 33,927 nucleotides, comprising of 26 ORFs among which 2 is for the core biosynthetic gene, 9 are for the additional biosynthetic genes, 2 is for transport-related gene, 1 regulatory gene, and rest 12 other genes. The structure of these 2 core genes is made up of 1 module with domains (Fig. 15O). The polymer prediction by the aforementioned cluster is (Me-ccmal); and the predicted core structure for this particular cluster is shown in Fig. 15P.

Analysis of cluster 4.1: This Trans-acyltransferase polyketide synthase like (transAT-PKS-like) cluster which presented 53% similarity with a biosynthetic gene cluster known to code for BGC0000176 (difficidin biosynthetic gene cluster from Bacillus velezensis FZB42)⁷⁹, this particular BGCs shown in Fig. 14G. This cluster consists of 45,721 nucleotides, comprising of 30 ORFs among which 4 is for the core biosynthetic gene, 8 are for the additional biosynthetic genes, and rest 18 other genes. The structure of these 4 core genes comprising 6 modules with domains (Fig. 15Q). The prediction of polymers by the aforementioned cluster is (ohmal–ccmal) + (ohmal–mal) + (Me-ccmal) + (mal); and the predicted core structure for this particular cluster is shown in Fig. 15R.

Analysis of cluster 6.1: This cluster exhibits 100% similarity with the BGCs known to code for bacinapeptin in Bacillus nakamurai. Known cluster BLAST using MIBiG 3.1 showed highest similarity with BGC0002700 (bacinapeptin biosynthetic gene cluster from Bacillus nakamurai)⁹⁴, this particular BGCs shown in Fig. 14H. This cluster consists of 22,616 nucleotides, comprising of 21 ORFs among which 1 is for the core biosynthetic gene, 5 are for the additional biosynthetic genes, 1 is for transport-related gene, 4 regulatory genes, and rest 8 other genes.

Analysis of cluster 6.2: This Non-ribosomal peptide synthetase (NRPS) cluster which presented 43% similarity with a biosynthetic gene cluster known to code for BGC0000433 (surfactin biosynthetic gene cluster from Bacillus velezensis FZB42)⁸⁶, this particular BGC shown in Fig. 14I. This cluster consists of 27,706 nucleotides, comprising of 19 ORFs among which 1 is for the core biosynthetic gene, 6 are for the additional biosynthetic genes, 1 is for regulatory gene, and rest 11 other genes. The structure of this 1 core genes is made up of 2 modules with domains (Fig. 15S). The polymer prediction by the aforementioned cluster is (Glu–Leu); and the predicted core structure for this particular cluster is shown in Fig. 15T.

The genome sequence of Bacillus ayatagriensis sp. nov. RMG6^T provides information on unique biosynthetic gene clusters for secondary metabolite production suggesting that the bacterial strain may be a promising source for the discovery of new antimicrobial compounds. Moreover, deciphering the complete genome of RMG6^T will unlock deep insights into the molecular foundations of plant growth promotion and biocontrol, paving the way for groundbreaking research and transformative biotechnological advancements.

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Author notes

1. Sandip Das and Rittick Mondal equally contributed to this work.

Authors and Affiliations

1. Department of Sericulture, Raiganj University, North Dinajpur, West Bengal, 733134, India

   Sandip Das, Rittick Mondal, Pankaj Mandal, Joydeep Chakraborty, Shubhajit Shaw, Abdul Sadat, Debnirmalya Gangopadhyay & Amit Kumar Mandal

2. Department of Botany, School of Sciences, Netaji Subhas Open University, Kolkata, West Bengal, 700064, India

   Sandip Das

3. Department of Medical Biology, Hamidiye International School of Medicine, University of Health Sciences Turkey, Uskudar, Istanbul, Turkey

   Halil Kurt

4. Department of Botany, Darjeeling Government College, Darjeeling, West Bengal, 734101, India

   Md Majharul Islam

5. Faculty of Allied Health Sciences (FAHS), The ICFAI University, Tripura, Kamalghat, Mohanpur, West Tripura, 799210, India

   Biraj Sarkar

6. Central Instrumentation Facility, Odisha University of Agriculture and Technology, Bhubaneswar, Odisha, 751003, India

   Sanjeet Manna

7. Department of Biology, Faculty of Arts and Sciences, University of Çukurova, Sarıçam, Adana, Turkey

   Nihan Arabaci

8. Bioenergy Group, MACS Collection of Microorganisms, DST-Agarkar Research Institute, Gopal Ganesh Agarkar Road, Pune, Maharashtra, 411004, India

   Kamlesh Jangid

9. Department of Biotechnology, Institution of Health Sciences, University of Health Sciences Turkey, Uskudar, Istanbul, Turkey

   Ahmet Kati

Contributions

The conceptual design of the study was carried out by S.D., R.M., A.K., and A.K.M. Data analysis was performed by R.M., P.M., H.K., J.C., M.I., B.S., S.S., S.M and K.J. The analysis of findings and drafting of the manuscript was conducted by S.D., R.M., P.M., H.K., J.C., M.I., B.S., S.S., N.A., K.J., A.S., D.G., A.K., and A.K.M. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Ahmet Kati or Amit Kumar Mandal.

Competing interest

The authors declare no competing interests.

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