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Article

Genome Sequence and Characterization of Bacillus cereus Endophytes Isolated from the Alectra sessiliflora and Their Biotechnological Potential

by
Khuthadzo Tshishonga
and
Mahloro Hope Serepa-Dlamini
*
Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, Doornfontein Campus, Johannesburg 2028, South Africa
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(9), 198; https://doi.org/10.3390/microbiolres16090198
Submission received: 30 June 2025 / Revised: 25 August 2025 / Accepted: 28 August 2025 / Published: 1 September 2025
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Abstract

Bacillus cereus AS_3 and Bacillus cereus AS_5 are bacterial endophytes isolated from sterilized leaves of the medical plant Alectra sessiliflora, which were previously identified using 16S rRNA sequencing. Here, we present the whole-genome sequencing and annotation of strains AS_3 and AS_5, the first genome report of Bacillus cereus strains from A. sessiliflora. The genome of strain AS_3 has 59 contigs, 5 503 542 bp draft circular chromosome, an N50 of 211,274 bp, and an average G+C content of 35.2%; whereas strain AS_5 has 38 contigs, 5,510,121 bp draft circular chromosome, an N50 of 536,033 bp, and an average G+C content of 35.2%. A total of 5679 protein-coding genes, 62 genes coding for RNAs, and 122 pseudogenes in the strain AS_3 genome were identified by the National Center for Biotechnology Information Prokaryotic Annotation pipeline, whereas a total of 5688 gene protein-coding genes were identified in AS_5, with 60 genes coding for RNAs and 120 pseudogenes. Phenotypic analysis and whole-genome sequencing analysis showed that AS_3 and AS_5 share similar characteristics, including Gram-positive, motile, rod-shaped, and endospore-forming have shown a high sequence similarity with Bacillus cereus, type strain ATCC 14579T. Strains AS_3 and AS_5 had genomic digital DNA–DNA hybridization (dDDH) with the type strain Bacillus cereus ATCC 14579T of 85.8% and 86%, respectively, and average nucleotide identities (ANIs) of 98% and 98.01%, respectively. Phylogenomic analysis confirmed that strains AS_3 and AS_5 share very similar genomic and phenotypic characteristics, and are closely related to the type strain Bacillus cereus type strain ATCC 14579T, supporting their classification within the Bacillus cereus species. A total of 10 secondary metabolite gene clusters, including siderophore type petrobactin, terpene type molybdenum cofactor, non-ribosomal peptide synthetase (NRPS) type bacillibactin, and β-lactone type fengycin, were predicted using AntiSMASH software (version 5.0). Putative genes potentially involved in bioremediation and endophytic lifestyle were identified in the genome analysis. Genome sequencing of Bacillus cereus AS_3 and Bacillus cereus AS_5 has provided genomic information and demonstrated potential biotechnological applications.

1. Introduction

Endophytes are a highly diverse group of symbiotic microorganisms, mainly fungi or bacteria, that colonize internal plant tissues without posing any threat or causing harm to the host plant’s health [1,2]. Several factors, such as environmental conditions and the host plant’s organs, contribute to their diversity [1]. The community of endophytes can spend part or all of its life cycle within a host plant and establish various interactions, such as antagonism and mutualism, but rarely parasitism [3]. In their relationship with plants, these species exhibit plant growth and development properties by producing a range of unique biochemical compounds and enhancing plant absorption of nutrients such as potassium, phosphate, and nitrogen [4,5,6,7]. This study focuses on bacterial endophytes, which are known to be present in all plants [8], and have also been identified as a potential source of microbial bioproducts such as enzymes, antibiotics, and secondary metabolites with applications across various industrial sectors such as agriculture, food, cosmetics, and pharmaceuticals [9].
Bacterial endophytes associated with medicinal plants are known to produce bioactive secondary metabolites similar to those of their host plant, with enhanced therapeutic potential in the pharmaceutical sector [10]. Secondary metabolites produced by bacterial endophytes include antimicrobial, antifungal, and anticancer compounds that may be used in the treatment of multiple diseases [8]. Bacterial endophytes associated with medicinal plants have gained increasing research interest due to their ability to produce therapeutic compounds such as antimicrobial peptides, polyketides, and other pharmacologically active metabolites; however, little is known about other attributes of endophytes, such as their functional roles during symbiosis with medicinal plants and the mechanisms underlying their interaction with hosts [11].
Bacillus species are widely found in diverse ecological environments such as soil, water, animals, and plants [12]. Bacillus is a genus of spore-forming, Gram-positive, motile, rod-shaped bacteria. Their ability to survive in harsh environmental conditions is attributed to endospore production [13]. These species form a symbiotic relationship with plants by protecting them against microbial infection through the production of various bioactive substances, including antibiotics, enzymes, and other volatile organic compounds [14,15,16,17]. Numerous Bacillus species have been previously isolated from medicinal plant Alectra sessiliflora and demonstrated antibacterial activity against pathogenic microbes [18].
Alectra sessiliflora is a widespread weedy medicinal plant from the family Orobanchaceae that grows throughout Sub-Saharan Africa, India, China, and the Philippines [19,20,21]. In South Africa, it is commonly found across the eastern summer rainfall region and the Southern Cape to the Cape Peninsula. A. sessiliflora is reported to have healing properties and is traditionally used to treat oral thrush, diarrhea, gastrointestinal illness, and scabies [19,20]. In Central Africa, the leaves are used as a galactogen by pregnant women, and in several other African countries, such as Nigeria, the plant is used to treat tuberculosis [22,23]. Previously published studies have shown that A. sessiliflora harbors bacterial endophytes with potential biotechnology applications, including antibacterial and antitumor activities [18]. Endophytes, especially those in the genus Bacillus, are known for producing bioactive compounds similar to those made by their host plants. Thus, the genomic analysis of the Bacillus cereus strain associated with A. sessiliflora could provide valuable insights into potential sources of novel antimicrobial and therapeutic compounds. Its relatively underexplored status makes it an ideal candidate for discovering novel endophytic bacteria with potential biotechnology applications.
The objective of this study was to sequence, assemble, annotate, and characterize the whole genome of the endophytic bacteria Bacillus cereus AS_3 and Bacillus cereus AS_5, isolated from A. sessiliflora, with a focus on identifying gene clusters associated with plant growth promotion, antimicrobial compound biosynthesis, and environmental resilience, thereby highlighting their potential applications in agriculture, bioremediation, and pharmaceuticals. To the best of our knowledge, this is the first genome-based characterization of Bacillus endophytes isolated from the medicinal plant A. sessiliflora. Bacillus cereus AS_3 and Bacillus cereus AS_5 were previously isolated and identified using the 16S rRNA sequence by [18], and they demonstrated significant antibacterial and antitumor activities against human clinical pathogens and cancer cells [18]. Comprehensive genomic data on bacterial endophytes can provide valuable genomic information and further enable exploration of their biotechnological applications.

2. Materials and Methods

2.1. Isolation, Maintenance, and Growth of Bacterial Strains

Several bacterial endophytes, including Bacillus cereus AS_3 and Bacillus cereus AS_5, were previously isolated from sterilized medicinal plant leaves A. sessiliflora, harvested from Eisleben, Botlokwa (23°31′49.5″ S 29°49′27.1″ E) in Limpopo province, South Africa, as described by [18]. The bacterial endophytes were isolated from fresh leaves (approximately 10–15 leaves) of a single plant following the protocol described by [24]. Sequencing of the 16S ribosomal RNA gene (GenBank accession numbers MZ976848 and MZ976850) was previously used to identify the isolate strains by [18]. A glycerol stock (30% v/v) of strains AS_3 and AS_5 was plated on a nutrient agar (NA) plate and incubated for 24 to 48 h at 28 °C. For routine culture maintenance, strains AS_3 and AS_5 were regularly subcultured, incubated at 28 °C, and stored for long-term preservation in 30% glycerol stocks at −80 °C. Bacillus cereus AS_3 and Bacillus cereus AS_5 were selected for whole genome analysis due to their dominant growth, sporulation ability, and suitability for high-quality genomic DNA extraction for downstream sequencing.

2.2. Phenotypic Characterization of Bacillus cereus AS_3 and Bacillus cereus AS_5

To differentiate Bacillus cereus AS_3 and Bacillus cereus AS_5 from other closely related Bacillus species recommended characteristics by [25] were applied. The Gram stain of AS_3 and AS_5 was determined using a Gram stain kit (Merck KGaA, Darmstadt, Germany) following the manufacturer’s protocol. The optimal growth conditions of strains AS_3 and AS_5 were investigated by growing the bacterial culture in tryptone soy broth (TSB) medium at different temperature ranges (4, 15, 20, 25, 30, 37, 40, 45, 50, and 55 °C) for 72 h. Sodium chloride (NaCl) tolerance was determined by growing a bacterial culture on TSB medium at different NaCl concentrations ranging from 0 to 15% (w/v) with 1% intervals for 72 h at 30 °C. The pH range for bacterial growth was investigated in TSB medium at pH 3–11 with increments of 1 pH unit. Bacterial growth under different temperature, NaCl, and pH conditions was determined by measuring optical density at 600 nm (OD600) after 24 h of incubation in TSB medium. The cell motility was evaluated using light microscopy by applying the hanging drop method after the cells were grown for 24 h. Different colonies’ characteristics, such as size, form, and color of strains AS_3 and AS_5, were studied by growing the cells on the tryptone soy agar (TSA) for 48 h at 30 °C. Furthermore, the anaerobic growth of cells was investigated by inoculating the strain on TSA for 48 h in a GasPak AnaeroGen (Thermofisher Scientific, Waltham, MA, USA) at 30 °C. Catalase and oxidase activity were determined using standard biochemical assays as described by [26]. Casein hydrolysis was determined on modified nutrient agar supplemented with 1% skimmed milk to assess protease activity. Starch hydrolysis was determined on nutrient agar supplemented with 1% soluble starch.

2.3. Total DNA Extraction, Library Preparation, and Sequence

The total DNA of strains AS_3 and AS_5 was extracted from pure solid colonies using the Nucleospin microbial DNA extraction kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following the instructions of the manufacturer. The quantification of the DNA was determined using the Implen Nanophotometer N60 (Implen GmbH, Munich, Germany). The complete genome sequence of strains AS_3 and AS_5 was sequenced by a commercial service provider, Biotechnology Platform, Agricultural Research Council (ARC), Onderstepoort, South Africa. Paired-end libraries (2×150 bp) were prepared using the Nextera DNA library preparation kit (Illumina, San Diego, CA, USA) as per the manufacturer’s protocol, and sequencing was performed using the Illumina MiSeq instrument v3.

2.4. Genome De Novo Assembly and Annotation

The raw genome sequence reads were uploaded to the Galaxy web platform accessible from https://usegalaxy.org, where all pre-annotation analyses were performed [27]. The raw reads quality was examined with FastQC (version 0.72.0) [28]. The de novo assembly was carried out using Unicycler (version 0.4.8.0) [29], followed by assessing the quality of the assembled genome using Quest (version 5.0.2) [30]. The draft genome’s annotation was performed using the National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline (NCBI-PGAP) [31] and the Rapid Annotation using Subsystems Technology (RAST) server [32].

2.5. Phylogenome Analysis

The phylogenomic analysis of strain AS_3 and AS_5 draft genomes was carried out from the bioinformatics platform Type Strain Genome Server (TYGS) accessible from https://tygs.dsmz.de (accessed on 7 November 2024) [33]. The average nucleotide identity (ANI) value between strains AS_3 and AS_5 with other closely related Bacillus species was determined using the Orthologous Average Nucleotide Identity Tool (OrthiANI) [34]. The NCBI-PGAP annotation file was used to identify the genomic islands (GIs) on the IslandViewer 4 online database (version 4.0) accessible from https://www.pathogenomics.sfu.ca/islandviewer/ (accessed on 4 November 2024) [35]. The shared and unique gene clusters of strains AS_3 and AS_5 were identified by comparing them to other closely related Bacillus species using the EDGAR 2.0 online platform accessible from EDGAR 3.5: Login (accessed on 31 July 2025) [36]. The cluster regularly interspaced short palindromic repeats (CRISPR) were predict ed using CRISPRCasFinder software (version 2.2) accessible at https://crisprcas.i2bc.paris-saclay.fr/ (accessed on 4 November 2024) [37,38,39]. Secondary metabolite gene clusters were predicted using the antiSMASH software tool (version 5.0) accessible at https://antismash.secondarymetabolites.org (accessed on 23 October 2024) [40] and PRISM 4 (prediction informatics for secondary metabolomes) accessible at http://prism.adapsyn.com. The NCBI-PGAP annotation file was used to screen potential putative genes from previous literature that may be used in biotechnology applications.

3. Results and Discussion

3.1. Basic Genomic Characteristics of Bacillus cereus AS_3 and Bacillus cereus AS_5

The de novo assembly of Bacillus cereus AS_3 and Bacillus cereus AS_5 resulted in draft genome sequences with 59 and 38 contigs, respectively. The genome size of strain AS_3 was 5,503,542, and strain AS_5 was 5510,121, with a similar G+C content of 35.2% and N50 of 2112,74 and 5360.33, respectively. The observed difference in N50 values between AS_3 and AS_5 may be attributed to variation in DNA quality and sequencing depth, which influenced the continuity and completeness of their respective genome assemblies. The DNA G+C and genomic size of AS_3 and AS_5 were comparable and were within the range of other Bacillus strain genomes (Tables S3 and S4). A total of 5679 genes were predicted on strain AS_3, of which 5495 were protein-coding genes (CDS); and strain AS_5 had a total of 5688 genes, of which 5508 were protein-coding genes. The PGAP annotation identified a total of 62 RNAs, 51 tRNAs, 5 rRNA genes comprising three operons (5S, 16S, 23S), 5 non-coding RNAs (ncRNAs), and 122 pseudogenes in strain AS_3. Strain AS_5 has a total of 60 RNAs, 51 tRNAs, 3 rRNA genes comprising three operons (5S, 16S, 23S), 5 non-coding RNAs (ncRNAs), and 120 pseudogenes. We have identified four CRISPR repeats in strains AS_3 and AS_5 and three Cas clusters in strain AS_3 (Tables S1 and S2). The genomic features of strains AS_3 and AS_5 are summarized in Table 1. The data from this Whole Genome Shotgun project for strains AS_3 and AS_5 have been deposited in DDBJ/ENA/ GenBank under the accession numbers JAPETG000000000 and JAPEVU000000000, respectively.

3.2. Functional Annotation

Phylogenomic classification and identification of strains AS_3 and AS_5 were determined using the Type Strain Genome Server (TYGS) (https://tygs.dsmz.de, accessed on 7 November 2024) platform. Additionally, the ANI value of strains AS_3 and AS_5 with other closely related Bacillus species was calculated using the OrthoANI tool. The phylogenetic analysis of the whole genome showed that strains AS_3 and AS_5 were closely related to Bacillus cereus type strain ATCC 14579T and Bacillus thuringiensis type strain ATCC 10792T (Tables S3 and S4). Strain AS_3 was closely related to B. cereus type strain ATCC 14579T with a DNA–DNA hybridization (dDDH) value of 85.8% and a 0.11% difference in G+C content; and B. thuringiensis type strain ATCC 10792T with a dDDH value of 71.5% and a 0.35% difference in G+C content (Table S3). Similarly, strain AS_5 was closely related to B. cereus type strain ATCC 14579T with a dDDH value of 86% and a 0.11% difference in G+C content, and to B. thuringiensis type strain ATCC 10792T with a dDDH value of 71.5% and a 0.35% difference in G+C content (Table S4). The dDDH values of both Bacillus species exceeded a recognized threshold of dDDH value > 70% as the cutoff point of species delineation [41,42]. The ANI analysis revealed that AS_3 was the closest to AS_5 with an ANI value of 100%, exceeding the species boundary threshold ANI > 95–96%, as shown in Figure 1 [43,44], which indicates that they could be the same species. Moreover, strain AS_3 had an ANI value of 98% with Bacillus cereus type strain ATCC 14579T and 96.52% with Bacillus thuringiensis type strain ATCC 10792T, whereas strain AS_5 had an ANI value of 98.01% with Bacillus cereus type strain ATCC 14579T and 96.53% with Bacillus thuringiensis type strain ATCC 10792T, which were above the species threshold values. In contrast, strains AS_3 and AS_5 had an ANI value of 91.44% and 91.45% with B. fungorum 17-SMS-01, respectively.
The RAST annotation server was utilized for the annotation and classification of genes in strains AS_3 and AS_5 (Figure 2). Subsystem coverage indicates genes proportion assigned to known functions (23%) against hypothetical genes (77%). Subsystem category distribution presents gene assignments visual pie chart across high-level functional categories, and subsystem feature counts list the exact number of annotated features for each category. A wide range of putative housekeeping genes is required to support an endophytic lifestyle for bacterial reproduction and growth by facilitating the transport and uptake of essential nutrients. The RAST annotation analysis of the AS_3 and AS_5 genomes identified putative genes involved in carbohydrate and amino acid metabolism, cofactors, lipids, fatty acids, and vitamins, which are essential for promoting plant growth and sustaining an endophytic lifestyle. Genes encoding carbohydrate metabolism play significant roles in the uptake and movement of nutrients, including nitrogen, iron, and phosphate, and are essential for the symbiotic interaction between plants and bacteria [45]. The identified putative and functional genes in strains AS_3 and AS_5, based on genome annotation and similarity to previously characterized gene clusters, suggest potential applications such as genetic engineering and bioremediation.
Most of the genes identified within the genomes of strains AS_3 and AS_5 that promote the endophytic lifestyle include those involved in siderophore production, other iron acquisition mechanisms, potassium metabolism genes, and nitrogen metabolism genes [29]. Furthermore, genes responsible for phytohormone production, stress tolerance, and virulence, as well as disease and defense mechanisms, play a crucial role in promoting and facilitating host plant growth and the symbiotic interaction between plant and bacterial endophytes [46,47].
A Venn diagram was used to determine the number of unique and shared genes in the whole genomes of Bacillus cereus AS_3, Bacillus cereus AS_5, and selected related species (Figure 3). A total of 4067 genes were shared among AS_3, AS_5, and all selected comparison species. Among these, strains AS_3 and AS_5 shared a total of 446 unique genes when compared to selected species. Moreover, strain AS_3 had three unique genes while strain AS_5 had four unique genes that are responsible for coding transport and transcriptional regulators, which are important in bacterial endophytes and have been previously found in other bacterial endophyte species [48,49]. The ANI and dDDH results suggest that strains AS_3 and AS_5 belong to the same species, sharing an ANI value of 100%. In contrast, the Venn diagram results suggest that AS_3 and AS_5 might exhibit genetic variations with unique genes between each other. Horizontal gene transfer (HGT) is the primary mechanism contributing to genetic variation and, consequently, evolutionary differentiation among closely related species [50]. Furthermore, the adaptive traits that facilitate survival in new environments are often acquired through HGT, especially genes involved in antibiotic resistance and metabolism [51,52,53,54]. Strains AS_3 and AS_5 were found to contain multiple sets of gene clusters obtained through horizontal gene transfer. These genomic islands (GIs) found in bacterial endophytes play a crucial role by encoding diverse accessory genes that support environmental adaptability, symbiosis, and the diversification of a bacterium within its host [55].
The GI viewer platform was utilized to screen and detect genomic islands within strains AS_3 and AS_5, revealing multiple clusters of genomic regions that displayed evidence of horizontal gene transfer. As such, seven GIs (Figure 4) were identified in both AS_3 and AS_5 genomes when compared against Bacillus cereus ATCC 10987 as the reference genome. Moreover, several genes have been identified in the seven GIs that encode glycosyltransferase, cupin, penicillin-binding transpeptidase, ornithine cyclodeaminase family protein, putative quinol monooxygenase, and phage major capsid. Genes encoding cupin have been identified among multiple endophytes and significantly contribute to plant cell wall polysaccharide modification [49].

3.2.1. In Silico Identification and Characterization of Biosynthetic Gene Clusters

Bacterial secondary metabolites, commonly referred to as natural products, are an essential source of biologically active compounds for agricultural and healthcare applications [56]. The key enzymes responsible for the biosynthesis of secondary metabolites catalyze the formation of structurally diverse, low-molecular-weight compounds, including non-ribosomal peptides (NRPs), polyketide synthases (PKSs), terpenoids, saccharides, ribosomally synthesized and post-translationally modified peptides (RiPPs), and a plethora of hybrids [57]. The two main clusters responsible for the biosynthesis of secondary metabolites in bacteria are polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) [58]. AntiSMASH 5.0 and PRISH online databases were utilized to analyze the genomes of strains AS_3 and AS_5 to explore and screen the biosynthetic gene clusters of secondary metabolites. A total of 10 biosynthetic gene clusters were predicted for each genome of strains AS_3 and AS_5, including one terpene-type molybdenum cofactor, one NI-siderophore-type petrobactin, two RiPPs, four NRPSs, one NRP metallophore, one NRPS-type bacillibactin, one linear azole-containing peptide (LAP), and one betalactone-type fengycin (Table 2 and Table 3). When comparing the predicted gene clusters, the petrobactin-encoded gene cluster demonstrated a percentage similarity of 100% (to B. anthracis str. Ames) for both strains, while the bacillibactin-encoded gene cluster had a percentage similarity of 100% (to B. subtilis subsp. subtilis str.) for AS_3 and 71% for AS_5. Moreover, the molybdenum cofactor-encoded gene cluster showed a percentage similarity of 17% (to S. carnosus) for both strains, whereas the fengycin-encoded gene cluster exhibited a percentage similarity of 40% (to B. velezensis FZB 42) for strains AS_3 and AS_5. However, among the biosynthetic gene clusters including RiPPs, NRPSs, and LAPs, no similarity was detected, suggesting that some secondary metabolites may be specific to strains AS_3 and AS_5. The identified biosynthetic gene clusters are involved in the synthesis of natural products with a variety of biological activities, such as antiviral, antimicrobial, anticancer, iron acquisition, and insecticidal activities. The biosynthetic gene cluster NRPS-type bacillibactin is a class of siderophore antibiotics secreted by the genus Bacillus and is reported to have antibacterial properties [59]. Although the presence of secondary metabolite gene clusters is common among Bacillus species, to the best of our knowledge, this study is the first to report such traits in strains isolated from A. sessiliflora, an unexplored host. Moreover, certain gene clusters with no homology to known databases suggest unexplored metabolic capabilities. While the study presents genomic evidence for secondary metabolite biosynthetic genes such as non-ribosomal peptides (NRPs), polyketide synthases (PKSs), and siderophores, it remains a primarily descriptive analysis. Further studies are required to investigate the expression of these gene clusters under specific conditions and to establish their correlation with actual phenotypic traits.

3.2.2. Genes Involved in Endophytic Lifestyle

This study identified several putative genes involved in the bacterial endophytic lifestyle and behavior to further understand the factors influencing Bacillus cereus AS_3 and Bacillus cereus AS_5’s lifestyle and colonization (Table S5). Genes putatively associated with transcriptional regulators, stress protection, detoxification, motility, transport, adhesion, substrate utilization, plant cell wall modification, and secretion and delivery systems were identified in AS_3 and AS_5. Moreover, several genes related to chemotaxis and motility, including methyl-accepting chemotaxis proteins, chemotaxis protein CheA, CheC, CheD, and flagellar motor switch protein FliG, were predicted to be essential for bacterial colonization of plant hosts [63]. These genes have been previously linked to endophytic colonization and plant growth promotion [2,64]. Methyl-accepting chemotaxis proteins (MCPs) play a crucial role in the initial colonization stage, where free-living bacteria attach to the rhizoplane [45]. Additionally, these transmembrane sensors enable the detection of surrounding molecules, directing bacteria either away from repellents or toward attractants [65]. Genes encoding sensor histidine were identified in the genomes of strains AS_3 and AS_5. These genes are involved in the early detection of phytopathogens, thereby contributing to the host’s protection mechanism against pathogen invasion [66].
Transcriptional regulators are critical as they allow bacterial endophytes to rapidly and accurately respond to environmental changes and colonization of their host plants [45,67,68]. Regulators of adhesion and biofilm formation are vital, as they are often controlled through quorum sensing at the bacterial population level [45]. Furthermore, the quorum-sensing molecule diffusible signal factor (DSF) in bacterial endophyte Stenotrophomonas maltophilia R551-3 has been reported to regulate cell motility, chemotaxis, multidrug efflux pumps, and biofilm formation. The TetR/AcrR family transcriptional regulators are involved in several fundamental roles, including morphogenesis and antibiotic production, multidrug resistance, catabolic pathways, osmotic stress, virulence of pathogenic bacteria, and the modification and clearance of toxic compounds [69]. The GntR family transcription regulators identified in the genomes of strains AS_3 and AS_5 control multiple cellular processes, including cell motility, bacterial resistance, glucose metabolism, pathogenesis, and virulence [69]. Similarly, LysR family proteins regulate gene expression related to metabolism, motility, and quorum [70]. The PadR transcription regulators, primarily found in Bacillus endophytic species, were identified in the genomes of AS_3 and AS_5. They are recognized for their roles as environmental sensors and play crucial roles in detoxifying harmful phenolic compounds [71]. Moreover, PadR-regulated pathways enable bacteria to tolerate and detoxify aromatic pollutants and lignin-derived compounds, supporting their potential applications in biofuel and bioremediation production from plant materials [72].
Several genes encoding antibiotic and virulence factors were predicted in the genomes of strains AS_3 and AS_5, including the YihY/virulence factor BrkB family protein, class A beta-lactamase Bla1, cell wall-active antibiotic response protein LiaF, antibiotic biosynthesis monooxygenase, conserved virulence factor C family protein, and beta-lactamase family protein. The YihY/virulence factor BrkB family protein is a novel virulence factor first discovered in the pathogenic bacteria Bordetella pertussis that demonstrates significant resistance to complement-dependent death by normal human serum [73]. Class A beta-lactamase Bla1 is a group of antimicrobial agents that hydrolyze penicillin [74], and antibiotic biosynthesis monooxygenase plays a key role in the biosynthetic pathway for polyketide antibiotics, including alnumycin, daunomycin, and tetracenomycin [75,76,77].
The KEGG-based pathways of phytohormones in AS_3 and AS_5 have been predicted and found to contain a wide range of essential plant hormones, including abscisic acid, auxin, ethylene, jasmonic acid, cytokinin, salicylic acid, and gibberellins (Figure S1). The predicted phytohormone pathways contribute significantly to plant growth and development through biological mechanisms including cell elongation, stress tolerance, defence against phytopathogen division and differentiation, and access to nutrients [48,78]. Similarly, the jasmonic acid plant hormone identified in the AS_3 and AS_5 genomes plays a crucial role in plant growth and development, defence, and regulated response to environmental stress [78]. The biosynthetic gene for the alkaline phosphatase synthesis transcriptional regulatory protein (phoP) was identified in the genomes of strains AS_3 and AS_5 (Table 4), which contribute significantly to solubilizing phosphate. Singh and Arona [79] reported that the application of phosphate-solubilizing endophytic Pseudomonas sp., as a bioinoculant, substantially promotes the growth and productivity of the medicinal plant Withania somnifera under a nutrient-limited saline environment. Furthermore, the detection of alkaline phosphatase activity in the soil was also observed following bio-inoculation.

3.2.3. Genes Responsible for Bioremediation

Several species within the genera Mycobacteria, Burkholderia, and Pseudomonas [80] have been commonly utilized in bioremediation processes due to their ability to tolerate and detoxify toxic compounds, including heavy metals, and survive in toxic environments [81,82]. Moreover, bacteria use biosorption as the main mechanism for metal bioremediation [82,83]. As such, bacterial endophytes have been identified and used as potential candidates for bioremediation because of their symbiotic relationships with the host plant, which can grow and survive under unfavourable conditions [84,85]. Bacterial endophyte Bacillus spp. has been reported to have the ability to tolerate and survive in the presence of toxic heavy metals such as Zinc (Zn), Cadmium (Cd), and Palladium (Pd) [86,87]. The gene for arsenate reductase (arsC) and the ACR3 family arsenite efflux transporter (arsB), which provide resistance to antimony (Sb) and arsenic (As), were identified in the genomes of AS_3 and AS_5. Additionally, the molybdate ion transporter protein modA, which transports tungsten (W), was identified in the genomes.
The genomes of Bacillus cereus AS_3 and AS_5 specifically indicate the presence of several genes associated with heavy metal resistance, including those responsible for copper resistance, such as the CopC/CopD family protein, which encodes a heavy metal translocating P-type ATPase, and a magnesium transporter. Copper-resistant gene clusters and operons have been identified in other bacterial species, including Pseudomonas, Xanthomonas, and Sphingobium yanoikuye [88,89,90]. These copper resistance genes are induced at different copper (Cu) ion levels and contribute to the copper ion transport mechanism across bacterial cells as well as the oxidation of Cu+ to less toxic Cu2+ ions [88,90]. We have identified the ssuD gene in AS_3 and AS_5, which plays a key role in the biodegradation of organosulphonate compounds such as n-hexadecane. The degradation of organosulphonates is essential for bacterial survival under oxidative stress in sulfur-deprived conditions [91]. Moreover, organosulphonates are commonly present in pharmaceuticals, detergents, pesticides, and other industrial waste. Bacteria species with the ssuD gene possess the biodegradative capacity to degrade sulfonated xenobiotics, making them essential for the bioremediation of contaminated soils and wastewater [92]. Strains AS_3 and AS_5 encode the flavin reductase (dszD) gene responsible for reductase activities that cleave carbon-sulfur bonds [93]. The presence of these genes emphasizes their essential contribution to the bio-desulfurization of petroleum oil [94]. The recG gene, which encodes an ATP-dependent DNA helicase, was found in the genomes of AS_3 and AS_5 and is associated in the literature with resistance to metals like chromium, selenium (Se), and tellurium (Te). Moreover, several genes present in AS_3 and AS_5, including arsC, corA, modA, recG, and DsbA, were predicted in silico to be involved in heavy metal resistance and remediation, suggesting potential application in the bioremediation of contaminants, pending experimental validation.

3.3. Phenotypic Characterisation

Bacillus cereus AS_3 and AS_5 were Gram-stain positive, rod-shaped, spore-forming, and motile bacteria with a growth temperature range of 15–45 °C and an optimum temperature of 30 °C. Strain AS_3 and AS_5 cells were able to grow in various concentrations of NaCl ranging from 0 to 7% (w/v), with the optimum concentration between 0–3% (w/v), demonstrating that the strains are halotolerant bacteria [95]. Moreover, the strain cells could grow in different pH ranges from 5 to 10, with the optimum pH between 5 and 8. The phenotypic characteristics of strains AS_3 and AS_5 were determined and compared to closely related type strain species, Bacillus dicomae MHSD28, B. parenthracis Mn5, B. albus N35-10-2, and Bacillus cereus type strain ATCC 14579T (Table 5). Strains AS_3 and AS_5 were unable to ferment glucose, mannitol, and inositol in API20E. However, they tested positive for oxidase, catalase, and hydrolysis of skim milk and casein. Strains AS_3 and AS_5 tested positive for D-ribose, glycerol, D-glucose, arbutin, and N-acetylglucosamine in API 50CH, similar to other reference species. Detailed differential phenotypic characteristics of AS_3 and AS_5, together with those of the reference type strains, are presented in Table 5.
The phenotypic characteristic results confirm that strains AS_3 and AS_5 belong to the same species, as they share similar properties. The ANI similarity, dDDH value, and phenotypic characteristics provided sufficient resolution to enable the categorization of AS_3 and AS_5 as the same species. These results were supported by a study conducted by [96], which showed that three Methylobacteriaceae strains isolated from the International Space Station (ISS) exhibited 100% ANI similarity and dDDH values among each other and possessed similar phenotypic properties, indicating that the strains belong to the same species [44,97,98,99]. Moreover, the dDDH method provides a high-resolution tool for taxonomy based on genomes, enabling precise species differentiation in the genomic era and allowing clear differentiation between closely related species that may appear identical based on other phenotypic traits [41,100]. The dDDH value and ANI similarity of 100% between strains AS_3 and AS_5 suggest that they belong to the same species. This conclusion is further supported by the Genome-to-Genome Distance Calculator (GGDC) [42], which shows a 98.29% similarity between the two strains (Table S6), corroborating that they are the same species. The ANI similarity, dDDH value, and phenotypic properties confirm that strains AS_3 and AS_5 are also closely related to Bacillus cereus and Bacillus thuringiensis. B. thuringiensis and B. cereus are the most developed and commercialized entomopathogens used against agricultural pests and hold medical importance due to their insecticidal activity [101]. Although B. thuringiensis is a well-known entomopathogen, B. cereus is more commonly reported as a biocontrol agent against phytopathogenic fungi and as a foodborne pathogen due to its ability to produce toxins. They have been isolated from various sources, including water, dust, soil, phylloplane, and dead insects; however, few species have been recently found within plant tissue [102,103]. Endophytic strains within the genus B. thuringiensis have been widely isolated from maize (Zea mays), kudzu (Pueraria thunbergiana), cotton (Gossypium hirsutum), and soybean (Glycine max) [102,103,104,105,106]. Recent studies have shown the relationship between B. thuringiensis and plants, indicating its potential role as a plant-growth-promoting bacterium, as well as its ability to produce plant hormones and colonize roots [107].

4. Conclusions

This study presents a genome and phenotypic-based characterization of two endophytic Bacillus cereus strains (AS_3 and AS_5) from Alectra sessiliflora, a medicinal plant with limited genomic and ecological information. The whole genomic sequencing and in silico analysis revealed that strains AS_3 and AS_5 possess putative genes with potential biotechnological applications in bioremediation, biofertilization, and biocontrol. Putative genes involved in endophytic lifestyle, bioremediation, and biosynthetic gene clusters were identified. Several biosynthetic gene clusters, compounds such as petrobactin, bacillibactin, siderophore, and other protein-coding genes were predicted and are crucial for antimicrobial compounds production, plant growth-promoting traits, biocontrol activities, and plant-microbial interaction and resistance to abiotic stresses such as heavy metals. Comparative genomics and orthologous gene clustering with related Bacillus species provided additional insights into specific strain genetic diversity and functional specialization. These findings highlight the potential biotechnological applications of the strains, particularly in agriculture, pharmaceutical, and environmental applications; however, the study is entirely computational. Therefore, the biological functions of predicted genes must be experimentally validated through metabolomic, transcriptomic, and phenotypic assays under defined conditions. The genomic and phenotypic analysis demonstrated that strains AS_3 and AS_5 share very similar genomic and phenotypic characteristics, and are closely related to the Bacillus cereus type strain ATCC 14579T, supporting their classification within the Bacillus cereus species. The ANI similarity, dDDH value, Genome-to-Genome Distance Calculator (GGDC), and phenotypic properties were sufficient to categorize AS_3 and AS_5 as the same species. The genome study established a foundational framework for future research aimed at exploring the biotechnological applications of Bacillus cereus endophytes and their potential role in the host plant.

Supplementary Materials

The following supporting information can be downloaded at:https://www.mdpi.com/article/10.3390/microbiolres16090198/s1, Table S1: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequence present within Bacillus cereus AS_3; Table S2: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequence present within Bacillus cereus AS_5; Table S3: Pairwise comparisons of Bacillus cereus AS_3 vs. type strain genomes; Table S4: Pairwise comparisons of Bacillus cereus AS_5 vs. type strain genomes; Table S5: Genome to Genome Distance calculator of Bacillus cereus AS_3 aligned with Bacillus cereus AS_5 as reference; Table S6: Genes responsible for endophytic Lifestyle in strain AS_3 and AS_5; Figure S1: KEGG mapping of the pathway predictions involved in the Biosynthesis of plant hormones in Bacillus cereus AS_3 (A) and Bacillus cereus AS_5 (B). Orange arrows represent the Mevalonic acid pathway, and blue arrows represent the MEP/DOXP pathway.

Author Contributions

K.T. contributed the experimental work and data analysis. data analysis. M.H.S.-D. Conceptualized the study, acquired funding, project administration, and provided the materials used for the characterisation studies, and was the supervisor of the project. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Research Foundation of South Africa Grant. Number: CSUR240404212233. The APC was funded by University of Johannesburg, Faculty of Science.

Conflicts of Interest

The authors declare no conflict of interest.

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Microbiolres 16 00198 g001
Figure 1. Heatmap showing the OrthoANI values of Bacillus cereus AS_3 and AS_5 with other closely related Bacillus species.
Figure 1. Heatmap showing the OrthoANI values of Bacillus cereus AS_3 and AS_5 with other closely related Bacillus species.
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Microbiolres 16 00198 g002
Figure 2. Functional categorization of coding sequences in the genomes of (A) Bacillus cereus AS_3 and (B) Bacillus cereus AS_5 based on SEED subsystem classification. Color schemes are automatically assigned by the SEED Viewer and may vary between strains.
Figure 2. Functional categorization of coding sequences in the genomes of (A) Bacillus cereus AS_3 and (B) Bacillus cereus AS_5 based on SEED subsystem classification. Color schemes are automatically assigned by the SEED Viewer and may vary between strains.
Microbiolres 16 00198 g002
Microbiolres 16 00198 g003
Figure 3. Venn diagram of the unique and shared genes of Bacillus cereus AS_3, Bacillus cereus AS_5, and selected related comparison species. Bacillus cereus type strain ATCC 14579T (1); Bacillus dicomae strain MHSD28 (2); Bacillus cereus AS_3 (3); Bacillus cereus AS_5 (4); Bacillus thuringiensis serovar berliner type strain ATCC 10792T(5).
Figure 3. Venn diagram of the unique and shared genes of Bacillus cereus AS_3, Bacillus cereus AS_5, and selected related comparison species. Bacillus cereus type strain ATCC 14579T (1); Bacillus dicomae strain MHSD28 (2); Bacillus cereus AS_3 (3); Bacillus cereus AS_5 (4); Bacillus thuringiensis serovar berliner type strain ATCC 10792T(5).
Microbiolres 16 00198 g003
Microbiolres 16 00198 g004
Figure 4. Genomic islands in the (A) Bacillus cereus AS_3 and (B) Bacillus cereus AS_5 aligned against the reference genome Bacillus cereus type strain ATCC 10987T, complete genome. A total of seven genomic islands were predicted on both genomes using IslandViewer. The green outer circle represents the scale line of the genome in Mbp, and the obtained genomic islands (GIs) are represented by the following colors: IslandPath-DIMOB (blue) and integrated detection (red).
Figure 4. Genomic islands in the (A) Bacillus cereus AS_3 and (B) Bacillus cereus AS_5 aligned against the reference genome Bacillus cereus type strain ATCC 10987T, complete genome. A total of seven genomic islands were predicted on both genomes using IslandViewer. The green outer circle represents the scale line of the genome in Mbp, and the obtained genomic islands (GIs) are represented by the following colors: IslandPath-DIMOB (blue) and integrated detection (red).
Microbiolres 16 00198 g004
Table 1. Genomic features of Bacillus cereus AS_3 and Bacillus cereus AS_5.
Table 1. Genomic features of Bacillus cereus AS_3 and Bacillus cereus AS_5.
Genome AttributeValue of AS_3Value of AS_5
Genome size (bp)5,503,5425,510,121
G+C content35.235.2
Number of contigs5938
Total number of genes 56795688
Protein-coding genes 54955508
Number of RNAs *6260
Number of tRNAs5151
Number of rRNAs 2, 1, 3 (5S, 16S, 23S)2, 1, 1 (5S, 16S, 23S)
Number of ncRNAs 55
Number of pseudogenes **122120
CRISPR repeats regions44
Cas cluster 30
* The number of RNA genes includes rRNAs, tRNAs, and ncRNAs. rRNA: ribosomal RNA; tRNA: transfer RNA; ncRNA: non-coding RNA gene; CRISPR: clustered regularly interspaced short palindromic repeats. ** Pseudogenes were identified using the NCBI-PGAP annotation pipeline.
Table 2. Biosynthetic gene clusters responsible for the production of secondary metabolites in Bacillus cereus AS_3.
Table 2. Biosynthetic gene clusters responsible for the production of secondary metabolites in Bacillus cereus AS_3.
From (bp)To (bp)TypeProductionSimilarity (%)Reference
122,014143,867TerpeneMolybdenum cofactor17Staphylococcus carnosus [60].
145,009176,716NI-siderophorePetrobactin 100Bacillus anthracis str. Ames [61].
36,839 47,105RiPP-like--
62,547 109,563NRPS--
74,891 118,472NRPS-like--
1 43,017NRP metallophore, NRPSBacillibactin85Bacillus subtilis subsp. subtilis str. 168 [62].
24,528 48,034LAP--
51,086 116,994 NRPS--
2295 12,591RiPP-like--
65,464 90,702BetalactoneFengycin 40
Table 3. Biosynthetic gene clusters responsible for the production of secondary metabolites in Bacillus cereus AS_5.
Table 3. Biosynthetic gene clusters responsible for the production of secondary metabolites in Bacillus cereus AS_5.
From (bp)To (bp)TypeProductionSimilarity (%)Reference
461,957485,463LAP--
164,310196,017NI-siderophorePetrobactin100Bacillus anthracis str. Ames [61].
473,136524,884NRP metallophore, NRPSBacillibactin71Bacillus subtilis subsp. subtilis str. 168 [62].
137,812159,665TerpeneMolybdenum cofactor17Staphylococcus carnosus [60].
101,600111,866RiPP-like--
127,308174,324NRPS--
74,891118,472NRPS-like--
10,67476,582NRPS--
229512,591RiPP-like--
65,464 90,702BetalactoneFengycin40Bacillus velezensis FZB42 [56]
Table 4. A summary of genes with biotechnological potential and responsible for bioremediation identified in Bacillus cereus AS_5 and Bacillus cereus AS_3.
Table 4. A summary of genes with biotechnological potential and responsible for bioremediation identified in Bacillus cereus AS_5 and Bacillus cereus AS_3.
GenesGene Product Role
CopC/CopDCopper resistance/copper homeostasis membrane protein CopAIncreased sensitivity to copper and increased copper uptake
corAmagnesium/cobalt transporter CorATransport of magnesium and cobalt
cutCCopper homeostasis protein CutCContribute to the cellular processes of copper uptake, storage, delivery, and efflux
copZCopper chaperone CopZCopper ion binding
McoMulticopper oxidase family proteinPossess high oxidase activity toward diverse substrates and are essential for iron transport in eukaryotes and prokaryotes
ptsPPhosphoenolpyruvate-protein phosphotransferase Contribute to regulating nitrogen metabolismPhosphoenolpyruvate-protein phosphotransferase activity
HcpHydroxylamine reductaseExhibits oxidoreductase activity, utilizing nitrogenous compounds as electron donors and iron–sulfur cluster binding
hisHImidazole glycerol phosphate synthase subunit hisHThe hisH subunit contributes to the catalysis of the glutamine hydrolysis to glutamate and ammonia during the biosynthesis of AICAR and IGP, with the subsequent transfer of the released ammonia molecule to the active site of hisH.
pdaAPolysaccharide deacetylase family proteinCatalyze the removal of either N-linked acetyl group from N-acetylglucosamine residues through hydrolysis
trxBThioredoxin-disulfide reductaseHioredoxin-disulfide reductase activity
trxAThioredoxinDisulfide oxidoreductase activity
msrAPeptide-methionine (S)-S-oxide reductase MsrAReducing the S-stereoisomer of methionine sulfoxide (MetSO) to methionine
BcpThioredoxin-dependent thiol peroxidaseAntioxidant activity and oxidoreductase activity
arsCarsenate reductase (thioredoxin)Arsenate reductase
arsBACR3 family arsenite efflux transporterArsenite secondary active transmembrane transporter activity
modAMolybdate ABC transporter substrate-bindingMolybdate ion binding, and transport; ABC-type molybdate transporter activity
tusASulfurtransferase TusAContribute to sulfurtransferase-mediated tRNA modification and biosynthesis of molybdenum cofactor
dhbA2,3-dihydro-2,3-dihydroxybenzoate dehydrogenaseCatalyzes the NAD(+)-dependent oxidation of the dihydroaromatic substrate 2,3-dihydro-2,3-dihydroxybenzoate (2,3-diDHB) to the aromatic catecholic
dhbCIsochorismate synthase DhbCBiosynthesis of the siderophore bacillibactin
IucA/IucCIucA/IucC family siderophore biosynthesis proteinBiosynthesis of siderophores, including desferrioxamine, achromobactin, and petrobactin
sodCSuperoxide dismutase [Cu-Zn]Breaks down superoxide radicals
sodASuperoxide dismutase [Mn]Inactivate harmful superoxide radicals
phoPTranscriptional regulatory protein PhoPInvolved in adaptation to low Mg(2+) restricted environments and in the transcriptional regulation of acid resistance genes
PpkPolyphosphate kinaseReversible synthesis of polyphosphate
kynA
kynB
kynU		Tryptophan 2,3-dioxygenase/arylformamidaseTryptophan catabolic process to kynurenine
ssuDFMNH2-dependent alkanesulfonate monooxygenaseAcquisition of sulfur from alkanesulfonates
recGATP-dependent DNA helicase RecGATP binding
dszDFavin reductase family proteinSulfur-specific reductase
Activities
Table 5. Differential phenotypic characteristics between Bacillus cereus AS_3 (1), Bacillus cereus AS_5 (2), and phylogenetically close strains: B. dicomae MHSD28 (3), B. parenthracis Mn5 (4), B. albus N35-10-2 (5), Bacillus cereus type strain ATCC 14579T (6).
Table 5. Differential phenotypic characteristics between Bacillus cereus AS_3 (1), Bacillus cereus AS_5 (2), and phylogenetically close strains: B. dicomae MHSD28 (3), B. parenthracis Mn5 (4), B. albus N35-10-2 (5), Bacillus cereus type strain ATCC 14579T (6).
Characteristic123456
Temperature Range
Optimum temperature 		15–45
30		15–45
30		16–45
30		15–45
30		15–45
30		10–48
30
pH range
Optimum pH		5–10
6–8		5–10
5–8		5–11
6–7		5–10
7–8		5–10
7		5.5–9
7
NaCl (%w/v) tolerance range
Optimum NaCl (%w/v) 		0–9
0–3		0–9
0.5–3		1–6
1–2		0–9
1–2		0–9
0.5–1.0		0–7
0.5–1.0
Oxidase+++++-
API 20E test:
ON PG
Arginine dihydrolase
Citrate utilization
Voges-Proskauer
gelatinase hydrolysis
Fermentation of mannitol
Fermentation of glucose
Fermentation of sorbitol
Fermentation of inositol
-
+
-
-
+
-
-
-
-
-
+
-
-
+
-
-
-
-
-
-
-
-
-
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
API 50 tests:
Glycerol
D-Ribose
D-Glucose
D-Mannose
N-Acetylglucosamine
Amygdalin
Arbutin
Salicin
Gluconate
+
+
+
-
+
-
+
-
-
+
+
+
-
+
-
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
-
-
-
-
-
+
-
+
+
+
+
_
+
+
+
+
+
+
+
+
+
Hydrolysis of
Starch
Casein
+
+
+
+
+
+
+
+
+
+
+
+
DNA genomic G+C content (mol%)35.2035.2035.2335.2035.0035.30
Note: + positive, - negative.
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Tshishonga, K.; Serepa-Dlamini, M.H. Genome Sequence and Characterization of Bacillus cereus Endophytes Isolated from the Alectra sessiliflora and Their Biotechnological Potential. Microbiol. Res. 2025, 16, 198. https://doi.org/10.3390/microbiolres16090198

AMA Style

Tshishonga K, Serepa-Dlamini MH. Genome Sequence and Characterization of Bacillus cereus Endophytes Isolated from the Alectra sessiliflora and Their Biotechnological Potential. Microbiology Research. 2025; 16(9):198. https://doi.org/10.3390/microbiolres16090198

Chicago/Turabian Style

Tshishonga, Khuthadzo, and Mahloro Hope Serepa-Dlamini. 2025. "Genome Sequence and Characterization of Bacillus cereus Endophytes Isolated from the Alectra sessiliflora and Their Biotechnological Potential" Microbiology Research 16, no. 9: 198. https://doi.org/10.3390/microbiolres16090198

APA Style

Tshishonga, K., & Serepa-Dlamini, M. H. (2025). Genome Sequence and Characterization of Bacillus cereus Endophytes Isolated from the Alectra sessiliflora and Their Biotechnological Potential. Microbiology Research, 16(9), 198. https://doi.org/10.3390/microbiolres16090198

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