Deciphering the ABA and GA biosynthesis approach of Bacillus pumilus, mechanistic approach, explaining the role of metabolic region as an aid in improving the stress tolerance

Abstract

Bacillus pumilus plays an essential role in agricultural applications as a beneficial microbe and for sustainable agriculture production. However, the underlying mechanisms of B. pumilus strains remain unclear as to how they are beneficial for plants as stress tolerant and growth promoters. Bacillus pumilus was isolated from the rhizosphere soil of Artemisia vulgaris. NGS (next-generation sequencing) was performed for the strain to gain new insights into the molecular mechanisms underlying plant-microbial interactions. NGS revealed 3,910 genes, 3294 genes with protein-coding, and 11 functional genomic regions related to diverse agronomic traits including stress tolerance. We identified the two possible phytohormone biosynthesis approaches from metabolic regions1(terpense→diterpense→betacarotene→xanthoxin→ABA)2(terpense→diterpense→geranyl diphosphate →C20 →GA). Several gene clusters related to the biosynthesis of phytohormones, stress tolerance, and agricultural diversification were predicted. The genome provides insights into the possible mechanisms of this bacterium for stress tolerance and its future applications. The genomic organization of B. pumilus revealed several hallmarks of its plant growth promotion and pathogen suppression activities. Our results provide detailed genomic information for the strain and reveal its potential stress tolerance mechanisms, laying the foundation for developing effective stress tolerance strategies against abiotic stress.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-78227-3.

Introduction

Host plants harbor a symbiotic relationship with the microbes. Beneficial microbes are important in enhancing the physiological traits and plant stress tolerance against several stresses¹. The agricultural industry has to face dual stress one is to meet the needs of the hungry population, and the other is environmental stress. Both biotic and abiotic stress reduce crop production negatively². However, several conventional approaches are being practiced enhancing agriculture production. Among them, the most recent approach is the application of beneficial microbes in enhancing agronomic traits and stress reticence. Beneficial microbes, especially bacteria and fungi, are known for their plant growth-promoting potential (PGP) without causing symptoms to the plant cells³. They can colonize plants and exert their beneficial effect on plants to facilitate the growth and development of the plant. The promotion of plant growth by beneficial microbes has been observed to be facilitated in several ways, such as the production of secondary metabolites and enzymes, gene modulation, and defense-related constitution in host cells. Moreover, microbes can affect the host by regulating the antioxidant defense system^4,5.

Bacillus pumilus was first isolated from the rhizosphere soil of an arid medicinal plant, Artemisia vulgaris (Pohang Beach, South Korea), and was subsequently found to increase the growth and stress tolerance of rice and soybean. It has been suggested that it can induce the production of phytohormones, organic acids, and biofilms⁶. It belongs to the genus Bacillus, and members of the Bacillus genus are rod-shaped and sprout in nature. These are gram-positive, chemoheterotrophic, and aerobic microbes. An increasing trend has been observed in the use of microbes such as Bacillus for abiotic stress tolerance⁷. B. pumilus sp. has been isolated from several different environments; however, only a few reports have described their effect on Plant abiotic stress tolerance⁸. Bacillus sp. have mostly been reported for their roles in various pollutants and the overexpression of metallothionein in host cells, which alleviates heavy metal toxicity in plants⁹. This species can also improve salinity¹⁰, drought¹¹, and hydrogen peroxide¹² stress by regulating the endogenous phytohormones and antioxidant defense system. In addition, this species can produce bioactive secondary metabolites¹³. Previous studies have suggested that the B. pumilus strain can be used for PGP¹⁴. Secondary metabolites produced by beneficial bacteria are not only significant for the producing cells but also affect the host. As bioactive substances, these metabolites have important uses in the pharmaceutics and agriculture industries. Researchers may now prioritize industrially significant secondary metabolites at the genome level and learn more about the genetic groundwork of the strain’s adaptable lifecycle. These specific metabolites are a potential source of stress-tolerant agents and sustainable crop production. For example, previous researchers revealed that B. pumilus contains several genes that promote plant development^15–18.

DNA-based investigations of the microbes are highly valuable in answering the aforementioned queries. The agronomic potential and genetic traits of beneficial microbes can be understood through metabolomics investigations of entire microbial colony genome analyses through NGS. This RNA-based research clarified the detailed mechanism of action and regulatory process of the microbes. NGS technologies are significantly possible to answer previously unanswerable issues, mostly about plant-microbial interaction¹⁹. Consequently, studying plant-microbe relationships more quickly and thoroughly than ever before is possible²⁰.

This study was performed to determine the complex biological mechanisms by characterizing and identifying the genomic regions that are responsible for stress tolerance. In addition, candidate genes (with agronomic traits that allow plant-microbial interaction) were identified via genome sequencing. It will provide a platform to understand the complex plant-microbial interaction against environmental stress.

Materials and methods

Media cultivation and isolation

The B. pumilus culture was grown from the original stock of SH-9 archived at the Crop Physiology Laboratory at Kyungpook National University, Daegu, South Korea. Previously it was isolated from the rhizospheric soil around the Artemisia plant located Pohang-si, North Gyeongsang, South Korea ( 35.84151 128.99185 36.33363 129.60108 ). Stock was streaked on L.B. agar plates and incubated at 27–30 °C until colonies appeared. After getting the single pure colony was inoculated in 5 mL of L.B. broth cultured overnight at 37 °C, agitated at 100-150 rpm which is the appropriate temperature and agitation according to standard protocol. This procedure was followed according to the method described by²¹.

DNA extraction and genomic sequencing

Genomic DNA was extracted from 1-mL culture aliquots using the Maxwell 16 DNA/RNA Extraction System (Solgent Company, South Korea). Genomic DNA was extracted for complete genome sequencing using a Qiagen kit (Qiagen, Hilden, Germany). For NGS, the single molecular real-time technique was used by Pacific Bioscience (Pac Bio, Menlo Park, CA, USA). The methodology used was followed according to that described by ADDIN EN.CITE^22–24. Sequencing was carried out at the KNU NGS Core Facility utilizing the Illumina MiSeq platform (Illumina, USA) according to the manufacturer’s instructions.

Genome annotation

The NCBI prokaryotic genome annotation pipeline (PGA) was used to perform the complete genome annotation. Rapid annotation using subsystem technology version 3.0, and an integrated microbial genome platform were used for the gene prediction. The assembled and annotated sequences were submitted to GenBank with bio project ID (PRJNA1056679), Locus tag prefix VA028 (SAMN39105494) and the information is available online in the Genome online database https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB13160900/overview.The annotation was performed as described by ADDIN EN.CITE^17,24,25.

Genome assembly statistics

NGS was performed at Kyungpook National University, South Korea, and the genome assembly statistics were performed using the build 6494 software. The NGS center at Kyungpook National University, South Korea, provided the annotation. The annotation pipeline used was the NCBI PGA. The adopted annotation methods were the best-placed reference per set and GeneMarkS-2. They provided us with the gene, coding sequence (CDS), rRNA, and tRNA data. The entire protocol was followed as described by²⁶.

Statistical data analysis

The statistical software SPSS (22.0) for Windows (SPSS, Chicago, IL, USA) was utilized to perform a one-way analysis of variance and the general linear technique on the experimental data. To compare mean data and identify treatment differences, Tukey’s test (p ≤ 0.05) was employed. Five replicates, on average, were used for each treatment value.

Results

Isolation and identification of Bacillus pumilus

The rhizosphere soil samples were collected and processed to isolate competent microbes. The objective was to decipher the mechanism of action of stress tolerance and plant-microbial interaction. After subsequent isolation, the rhizobacterial strain was identified via whole genome sequencing (Fig. 1). Moreover, the plant growth-promoting traits of the Bacillus pumilus were also analyzed by using the NGS. The results revealed that it processes multiple plant growth-promoting traits certified by whole genome sequencing technique Table 1.

Fig. 1.

Modulation of the gene expression for stress tolerance and agronomic traits of the SH-9 Bacillus pumilus genome. Outer to inner circles show the predicted protein-coding sequence on the plus and minus strands. The third circle shows the distribution of the genes related to the Clusters of Orthologous Genes (COG) categories, and the fourth and fifth circles show the G+C content and G+C skew, respectively.

Table 1.

List of genes attributed to agronomic traits in the Bacillus pumilus genome.

NRP non-ribosomal peptide, TIPKS type 1 polyketides, T3PKS type 3 polyketides, RRE Rev response element, RiPP ribosomal synthesized and post-translationally modified peptide, NA not applicable.

Identification of ABA and GA biosynthesis from precursor of terpenes

In plants ABA is synthesized in vascular tissue from there, it is transported to the targeted tissue of plants. Therefore, in the present study we detected ABA and GA biosynthesis approach from metabolic precursor terpenes Results revealed that SH-9 has an innate ability to synthesize ABA from precursor of terpenes. Interestingly, Terpenes were present at 2 metabolic region 3 and region 5. Moreover, we also detected that it also can biosynthesize GA. The entire mechanism is explained in (Fig. 2). The related genes to ABA and GA synthesis were certified in WGS of SH-9 (Table 1)0.11 metabolic regions were identified which are shown in Table 2.

Fig. 2.

Possible phytohormones biosynthesis approach from genomic region 5, terpene acts as a bio precursor of the biosynthesis and ultimately gives yield to ABA and GA.

Table 2.

Functional genomic regions of the Bacillus pumilus, genomic region, and production of secondary metabolites.

Gene prediction and annotation summary

Further analysis of the Bacillus pumilus genome revealed the total number of genes as well as the available protein genes (Fig. 3A and B, respectively). To investigate the stress tolerance mechanisms of B. pumilus, we screened the potential metabolic regions and their corresponding genes. The results revealed that it contains several coding- and pseudogenes, as shown in Fig. 3. Total genes, CDSs, and protein-coding genes were prevalent with close to 3.8 kb of data, as shown in Fig. 3A. However, the total pseudogenes and CDSs without proteins were prolific. The total RNA genes, rRNA, complete rRNA, tRNA, and ncRNAs were present in insignificant amounts in Bacillus pumilus, as shown in Fig. 3. NGS also revealed that it has metabolic regions that produce secondary metabolites. All metabolic regions (n=11) can enhance plant agronomic traits and stress tolerance via several gene modulations.

Fig. 3.

Potential genomic sequences represent corresponding genes of the Bacillus pumilus SH-9. (A) Total number of genes and (B) pseudogenes in the genome sequence.

Genomic regions associated with agronomic traits

Sequence analysis of the rhizosphere Bacillus sp. and its potential for the enhancement of plant growth. A genomic area has a biological function if it has reproducible biochemical activity, such as transcriptional activity or certain chromatin states, which can be observed. NGS has been extensively used in recent years to identify genomic regions and candidate genes for agronomic traits in microbes, and its crosstalk has several diversification qualities^27,28. Numerous candidate genes and quantitative regions are associated with agronomic traits²⁹. This study revealed that Bacillus pumilus has 11 functional genomic regions, as shown in Table 2. At genomic region 1, non-ribosomal peptides (NRPs) 64,076–139,868 and type 1 polyketides (TIPKS) were found with 22% similarity to the known cluster of Zwittermicin A. These are a class of secondary peptide metabolites. These are extraordinarily diverse and have various biological and pharmacological features. They are frequently responsible for siderophore and pigment production in plants and also act as a biocontrol agent against plant stress^30,31. Similarly, in region 11, they contain NRPs, which have 85% similarity with Lichenysin.

Figure 4 shows the functional genomic regions of the SH-9 strain i.e., SH-9 genomic region and production of secondary metabolites, and the total number of metabolic regions in the SH-9 strain genome. In terms of both structure and biological function, TIPKS represent a diverse class of chemicals. These substances, which include macrolides, polyethers, polyols, and aromatics, are frequently strongly oxygenated. They have various biological effects, including antibacterial, anticancer, antifungal, antiphrastic, and immunosuppressive^32,33. Polyketides are present in genomic region 1. The genomics region 2 contains the Rev response element (RRE) nucleotides from 323,310 to 344,032. This is a specialized and cooperative complex of Revs, and the cellular export machinery utilizes RRE as a scaffolding platform for assembly. Creating a high-affinity, export-competent complex depends on the cooperativity that is governed by the RRE structure and sequence³⁴.

Fig. 4.

Model elucidating the phytohormonal, transcriptional, and signaling modulation triggered by B. Pumilus that gives resistance to stress.

At genomic region 3, terpene NI siderophores were found from 490,298 to 518,868, which has a 60% similarity with Schizokinenn. Because of their diverse properties, terpenes are essential natural compounds that are important for pollination, fragrance, and biological control. Terpenes are also precursors of gibberellic acid (GA). This genomic region is important because GA is produced here. GA is an important phytohormone that is responsible for the elongation of cells and, ultimately, the growth of plants^35,36. Similarly, in the genomic region 5, the production of terpenes was also found. From this region, siderophores are secreted, which can induce iron chelation³⁷. At genomic regions 4 and 7, betalactone is produced with a 53% similarity to fungicide. It acts as a potent, irreversible antagonist of strigolactone receptors. When the Plant is exposed to stressful conditions, betalactone works antagonistically to inhibit branching³⁸. At genomic region 8, ribosomal synthesized and post-translationally modified peptides (RiPPs) are abundant from 2,720,609–2,730,899. These diverse peptide classes are responsible for several biological translation processes and their post-modification in response to stress^39,40. In genomic region 10, NRPS metallophores are present from 3,030,569–3,079,137, which shows an 80% similarity with bacillibactin. This diverse class of microbes can chelate metals and be used for heavy metal stress tolerance^41,42.

Metabolic regions and their potential to enhance agronomic traits

Reactive oxygen species (ROS) accumulation is enhanced in plants in response to stress conditions, such as oxidative stress. Metabolic reprogramming is performed to reduce stress^43,44. However, it is challenging to cope with stress when the duration of stress increases and metabolic reprogramming fails. In this scenario, beneficial microbes produce several secondary metabolites. In this study, SH-9 was screened by NGS to produce secondary metabolites, and the results revealed that in metabolic region one, T1PKS is produced, which can lower oxidative stress and work as an antioxidant. T1PKS also works as an antimicrobial agent. Several studies have suggested that T1PKS has the potential to lower ROS levels, and our results agree that it can lower oxidation stress. In metabolic region 6, type 3 polyketides are present that can activate the TCA cycle. One of the intermediary steps of cellular respiration is the Krebs cycle. Carbohydrates are a group of substrates in which some energy is generated by oxidizing acetyl-CoA and is also obtained by breaking down respiratory substrates such as fats and proteins.

In metabolic region 10, a metallophone is present that remediates heavy metal stress tolerance. In regions 8 and 10, RiPP and NRPS are present, and they are involved in several metabolic and enzymatic processes in plant cells. Metabolic region 5 is important because it contains terpenes, which are important secondary metabolites. Terpenes are converted into diterpenoids and triterpenoids, which are subsequently converted into xanthroin, a precursor of abscisic acid (ABA). Diterpenoid also converts into geranyl diphosphate, which produces gibberellins. ABA and GA are important phytohormones that enhance stress tolerance and improve plant growth even under stressed conditions⁴⁵.

Discussion

Higher plants are sessile and are exposed to stress throughout their lives. The plant uses several cellular events to protect itself in response to this stress (biotic and abiotic). All plants adopt nonspecific strategies, depending on the stress and duration. However, the general mechanism produces ROS and activates the antioxidant defense system⁴⁶. The ROS acts as a signaling molecule that initiates several metabolic processes in plants to combat stress, such as the production of phytohormones, secondary metabolites, activation of some genes, and modulation in physio-biochemical processes. However, plants activate their metabolic reprogramming to alleviate stress, but it is up to a certain limit⁴⁷. After a specific limit, the plant shows some symptoms of stress. Several techniques are being practiced, such as breeding techniques, agrochemicals, intercropping, and application of plant growth regulators and phytohormones. Bio stimulants are getting more attention because of their eco-friendliness and minimal side effects. The application of beneficial microbes is a promising food and energy security approach. Beneficial microbes induce the plant microbial interaction. Plant-microbial interaction facilitates plants’ biological processes and improves plant stress tolerance⁴⁸.

Several recent studies significantly impacted plant growth and stress tolerance capacities. Recently, our research team conducted experimental work on beneficial microbes (SH-9) and showed excellent results regarding plant growth promotion and stress tolerance capacities⁴⁹. The results gave us the idea to unravel the hidden mechanism of plant-microbial interaction. In the present study, we did the next-generation sequencing of the microbe SH-9. The results showed that in response to stress, the SH-9 genome encodes several genes that protect against stress. Different genomic regions are responsible for different stress conditions from different genomic regions. The NGS study revealed the presence of 3910 genes with 11 functional genomic regions. A genomic region is the presence of a functional component within the gene responsible for some specific function.

Whenever a plant is exposed to stress, there is a rapid accumulation of ROS that enhances the oxidative stress. Oxidative stress increases the oxidation burst that impedes the damage to the cellular organelles⁵⁰. In response to oxidative stress, the SH-9 genome encodes ten genes at different regions, namely cdr, rnr, resE, rapA-5, qoxb, ychF, ggt, cyPB, adhA-2, and fhs, to regulate the oxidative stress. The presence of these genes further confirms that SH-9 significantly counteracts the generation of ROS. These findings suggest that the strong antioxidative mechanisms of SH-9 alleviate the oxidative onslaught.

Four genes, namely rnr, Sa1A, SrfAB-1, and g1tA, are found in the genome of SH-9 and are responsible for abiotic stress tolerance. Abiotic stress is the most common phenomenon that limits plant production. Abiotic stress reduces the global yield of plants to 70%. The presence of metabolic regions that contain the genes that enhance abiotic stress tolerance is a vital characteristic of beneficial microbes⁵¹. The presence of these genes on the SH-9 plasmid is surprising. This is a promising approach toward stress tolerance and a potentially profitable approach to enhancing plant agronomic traits. Table 2 shows the modulation of the gene expression for stress tolerance and agronomic characteristics of the SH-9 genome.

Iron and magnesium are important micronutrients required for a plant’s physiological growth. Under nutritional or other stressed conditions, the plant faces iron and magnesium deficiency⁵². Therefore, to cope with this stress, the plasmid of the SH-9 genome encodes for the genes uxaA, dos, and FadF that can bind iron and magnesium. Therefore, it is an essential characteristic of the beneficial microbe to encode the relevant genes that enhance their solubilization. In plant-microbial interactions, biofilm plays a substantial role in facilitating the interaction and alleviating plant stress. Biofilm acts as an interacting medium for plant-microbial interaction. It enhances plant growth, promotion, and production. It also facilitates several biochemical processes^53,54. SH-9 encodes for the gene estA to facilitate this mutualistic interaction, which produces glycolipids. Phosphorus is another major molecule that is involved in plant growth and production⁵⁵. The genome of SH-9 encodes for the six genes Dxs, resE, tagF, SasA-1, CarB-1, and CarB-2 that aid in phosphate solubilization. An important plant growth-promoting trait enhances plant growth and regulation. Microbes can convert insoluble phosphorus to soluble phosphorus and improve plant phosphorus nutrition.

Secondary metabolites are important compounds that are produced in response to stress. That enhances plant growth and improves stress tolerance⁵⁶. The SH-9 genome also encodes the genes that facilitate metabolite transportation where they are needed. The SH-9 genome encodes for the six genes Csbc, YYCB-1, fruA-1, mdtc, YdfJ, and CYPB, which facilitate the metabolite transportation system. In response to stress, plant-microbial interactions produce several secondary metabolites, such as glycine, betaine, and proline³⁹. These genes are responsible for enhancing the ease of transportation. The SH-9 genome encodes BgIF-1 and mainly acts as a signaling molecule to initiate mutualistic interaction with plant microbes. Signaling molecules act as mainstream to transmit information between microbes and plant cells, activating the cascades of events such as metabolomics expression corresponding to the stress tolerance mechanism⁵⁷.

Moreover, the nuclease process is also fundamental in bringing the genetic stability that is being incorporated due to the stress, i.e., oxidation stress⁵⁸ Under oxidation stress, the entire genetic modulation impairs plant cells’ cellular function. The SH-9 genome also encodes for ten genes rnr, leus, va1S, cdr, resE, Sa1A, YhaN, addB, addA2, and ctaD that are directly involved in the nuclease process (DNA and RNA) and improve the genetic stability.

The two most important genes that are encoded by the SH-9 genome are dxs and Acsa-1, which modulate plant phytohormones. Phytohormones are essential for stress tolerance and in several biological processes in plants. Genomic regions 3 and 5 are the most important and responsible for the production of phytohormones. Phytohormones reduce the plant stress and improve the plant production. In genomic regions 3 and 5, terpene has been reported as a major precursor of GA and ABA⁴¹. ABA is an important phytohormone that acts as a signaling molecule in response to stress. In addition, it is a versatile hormone that has a role in the stomatal closure in response to stress and reserves the plant activity to combat the level of stress. Moreover, it also has several functions in the physiological process of plants. Gibberellic acid is also an important class of phytohormones that stimulates plant growth, organ development, and cell proliferation⁵⁹. Our results confirmed that the genome of SH-9 encodes the genes that are liable to respond to stress and improve plant productivity. The results of genomic sequencing and the proposed stress tolerance mechanism are given in Fig. 4.

Conclusion

In the present study, we isolated growth growth-promoting B. pumilus strain SH-9 from a rhizospheric soil field and sequenced its genome. The present study utilized NGS to obtain a comprehensive view of the mechanisms of B. pumilus and the functional diversity that is associated with stress tolerance and improving agronomic traits. The result explains that it can biosynthesis the two important phytohormones ABA and GA. The results revealed that B. pumilus genome encodes the 3910 genes at 11 different genomic regions that are responsible for agronomical traits and stress tolerance via plant-microbial interaction. This study may paw the way to the application of B. pumilus as an alternative sustainable strategy to improve crop yield under unfavorable conditions. However, further transcriptomic and metabolomic analysis is required to confirm the association of the genes highlighted here with the indicated functional potential.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

NGS

Next generation sequencing

NRP

Non-ribosomal peptide

TIPKS

Type 1 polyketides

T3PKS

Type 3 polyketides

RRE

Rev response element

RiPP

Ribosomal synthesized and post-translationally modified peptide

Author contributions

Shifa Shaffique conceptualize and write the original draft as the part of her project brain Korea BK21,4TH phase. Dr. Anis Ali Shah, Arjun Adhikari did the critical review editing.  Sang-Mo Kang, Abdul Latif Khan, Peter Odongkara, and Hosam O. Elansary did the software manipulation. Prof In-Jung Lee validated the results and supervised the entire project.

Funding

This research was funded by the Deanship of Scientific Research, King Saud University through the Vice Deanship of Scientific Research Chairs; Research Chair of Prince Sultan Bin Abdulaziz International Prize for Water.

Data availability

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.