1 Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium that has garnered significant attention due to its entomopathogenic properties. It is widely used as a biopesticide, producing crystal proteins (Cry toxins) that are toxic to various insect orders. Despite its extensive use and study, the evolutionary mechanisms that drive the diversity and adaptability of Bt strains remain poorly understood.

Bacillus thuringiensis is a member of the Bacillus cereus group, which also includes Bacillus cereus and Bacillus anthracis. These species share a close phylogenetic relationship but differ significantly in their pathogenicity and ecological niches (Ehling-Schulz et al., 2019). Bt is distinguished by its ability to produce parasporal crystals containing Cry toxins during sporulation, which are lethal to a wide range of insect larvae upon ingestion (Deng et al., 2014). The genetic basis for this insecticidal activity lies in the presence of Cry genes, which are often located on plasmids and can be transferred between strains, facilitating rapid adaptation to new hosts (Zheng et al., 2017).

Comparative genomic analysis is a powerful tool for understanding the genetic diversity and evolutionary dynamics of bacterial species. By comparing the genomes of different Bt strains, researchers can identify core and accessory genes, elucidate the roles of mobile genetic elements, and uncover the genetic basis for host specificity and virulence (Alcaraz et al., 2010). This approach has revealed that Bt strains possess a high degree of genomic plasticity, with significant portions of their genomes dedicated to virulence factors, fitness traits, and mobile elements such as bacteriophages and transposases. Understanding these genomic features is crucial for developing more effective biopesticides and for predicting the evolutionary trajectories of Bt populations.

This study analyzes the genetic diversity and evolutionary relationships among different Bt strains, identifies key genomic features and gene clusters associated with their insecticidal properties, explores the genetic basis of host specificity and the mechanisms of host-pathogen interactions, and provides insights into the evolutionary mechanisms driving the diversification and specialization of Bt strains. This study aims to conduct in-depth comparative genomic analysis of different Bt strains to promote their development and application in biotechnology and agriculture.

2 Overview of Bacillus thuringiensis

2.1 General characteristics of Bt strains

Bt strains are characterized by their ability to form parasporal crystals during sporulation. These crystals contain insecticidal proteins that target specific insect orders, such as Lepidoptera, Coleoptera, and Diptera. The genome of Bt typically includes a circular chromosome and multiple plasmids, which harbor genes encoding for these toxins and other virulence factors. For instance, the genome of Bt strain HD521 contains a circular chromosome and six plasmids, encoding various virulence proteins such as Immune Inhibitor A, Hemolytic Enterotoxin, and Chitinase (Sun et al., 2021). Similarly, Bt strain GR007 has a circular chromosome and three megaplasmids, with multiple pesticidal protein genes (Figure 1) (Pacheco et al., 2021).

2.2 Biological functions and applications

The primary biological function of Bt is its role as a biopesticide. Bt produces a variety of insecticidal proteins, including Cry and Cyt toxins, which are effective against a wide range of insect pests. These proteins disrupt the gut cells of insects, leading to their death. Bt strains are also known for their potential in controlling plant diseases and promoting plant growth. For example, Bt strain SY49.1 exhibits significant activity against plant fungal infections and promotes plant growth through the production of bioactive compounds such as thuricin and bacillibactin (Yılmaz et al., 2022). Additionally, Bt strains have been explored for their antifungal potential, as seen in the case of Bt strain MORWBS1.1, which shows antagonistic activity against Fusarium species (Adeniji et al., 2021).

2.3 Genetic diversity among Bt strains

Bt strains exhibit considerable genetic diversity, which is reflected in their genomic composition and the variety of insecticidal proteins they produce. This diversity is often attributed to horizontal gene transfer, which allows Bt strains to acquire new genetic elements, including plasmids and transposons. For instance, the genome of Bt strain HS18-1 contains nine plasmids, encoding multiple virulence factors and insertion sequences (Sun et al., 2021). The genetic diversity of Bt strains can be assessed using techniques such as Multi-Locus Sequence Typing (MLST) and Random Amplified Polymorphic DNA (RAPD) analysis. A study on Bt strains from Kuwait revealed significant genetic variation among local isolates, highlighting the unique DNA patterns of different strains (Qasem et al., 2015). Furthermore, evolutionary genomics studies have shown that Bt strains possess a high degree of genomic plasticity, enabling them to adapt to various ecological niches and host organisms.

3 Methods for Comparative Genomic Analysis

3.1 Genomic sequencing techniques

Genomic sequencing is a fundamental step in comparative genomic analysis, providing the raw data necessary for subsequent bioinformatics and comparative studies. Various sequencing technologies have been employed to sequence the genomes of Bacillus thuringiensis (Bt) strains. For instance, next-generation sequencing (NGS) technologies, such as those used in the sequencing of Bt X022, offer high-throughput capabilities and have been instrumental in oBtaining comprehensive genomic data (Quan et al., 2016). Additionally, PacBio RS II sequencers have been utilized for complete genome sequencing, as demonstrated in the studies of Bt strain BM-Bt15426 and Bt ATCC 10792, providing long-read sequences that are crucial for assembling complex genomes and identifying plasmids (Chelliah et al., 2019).

3.2 Bioinformatics tools and software

Bioinformatics tools and software are essential for analyzing and interpreting genomic data. The RAST server is commonly used for functional annotation of sequenced genomes, as seen in the analysis of Bt X022 (Quan et al., 2016). Other tools, such as antiSMASH, are employed for in silico investigation of biosynthetic gene clusters, which can predict the production of biopesticidal metabolites (Adeniji et al., 2021). Comparative genomic studies also utilize phylogenomic analyses to clarify taxonomic relationships and evolutionary mechanisms. For example, phylogenomic trees based on core gene sequences have been used to distinguish between Bacillus cereus and Bt strains, proposing the recognition of two Bt genomovars (Baek et al., 2019). Additionally, tools for plasmid content analysis and the identification of virulence factors and antibiotic resistance genes are crucial for understanding the pathogenic potential and evolutionary adaptations of Bt strains (Bolotin et al., 2017; Liu et al., 2017).

3.3 Data integration and comparative approaches

Integrating genomic data with other omics data, such as proteomics, enhances the understanding of the functional implications of genomic variations. Comparative analyses of genomic and proteomic data have revealed discrepancies between genome annotations and protein expression profiles, highlighting the importance of multi-omics approaches. For instance, in Bt 4.0718, certain genes related to insect pathogenicity were not detected in the proteomic data, suggesting gene silencing or low expression levels (Rang et al., 2015). Comparative genomic analyses also focus on identifying evolutionary mechanisms, such as the role of mobile genetic elements, prophages, and plasmids in genome plasticity and adaptation. Studies have shown that prophages can mobilize chromosomally encoded Cry-toxins, contributing to the rapid evolution and adaptation of Bt strains. Furthermore, comparative genomic studies of Bt strains in bacterial consortia have provided insights into their cooperative mechanisms and responses to environmental factors, which are essential for optimizing their use in biotechnological applications (Jia et al., 2016).

4 Genomic Features of Bt Strains

4.1 Core and pan-genome analysis

The core and pan-genome analysis of Bacillus thuringiensis (Bt) strains reveals significant insights into their genetic diversity and evolutionary mechanisms. Comparative genomic studies have shown that Bt strains possess a highly variable genome, with a core set of genes essential for basic cellular functions and a pan-genome that includes strain-specific genes contributing to their adaptability and pathogenicity. For instance, the genome of Bt strain BM-Bt15426 contains 5 409 predicted genes, highlighting the extensive genetic repertoire of Bt strains (Liu et al., 2017). Additionally, the comparative genomic analysis of nearly 900 Bt strains has led to the proposal of two distinct genomovars, B. thuringiensis gv. thuringiensis and B. thuringiensis gv. cytolyticus, based on core gene sequences (Baek et al., 2019).

4.2 Gene content and functional categories

Bt strains are characterized by a diverse array of genes encoding for various functional categories, including virulence factors, insecticidal proteins, and metabolic enzymes. For example, the genome of Bt strain GR007 contains multiple pesticidal protein genes, including 10 Cry genes, two Vip genes, and several virulence factors (Pacheco et al., 2021). Similarly, Bt strain HD521 encodes eight types of virulence protein factors and several insertion sequences and prophage sequences, indicating a complex genomic architecture (Sun et al., 2021). The presence of genes related to antibiotic resistance and heavy metal resistance in Bt strain HM-311 further underscores the functional versatility of Bt genomes (Zuo et al., 2020).

4.3 Mobile genetic elements

Mobile genetic elements, such as plasmids, transposons, and prophages, play a crucial role in the genomic plasticity and horizontal gene transfer in Bt strains. The genome of Bt strain GR007, for instance, includes three megaplasmids that harbor multiple pesticidal protein genes and virulence factors (Pacheco et al., 2021). Bt strain HS18-1 contains nine circular plasmids, encoding various virulence factors and insertion sequences (Sun et al., 2021). The presence of numerous plasmids and transposons in Bt genomes facilitates the acquisition and dissemination of beneficial traits, such as insecticidal activity and antibiotic resistance, thereby enhancing their adaptability to diverse environments (Wang et al., 2018; Reyaz et al., 2019).

5 Evolutionary Mechanisms in Bt Strains

5.1 Horizontal gene transfer

Horizontal gene transfer (HGT) plays a significant role in the evolution of Bacillus thuringiensis (Bt) strains. The presence of mobile genetic elements such as bacteriophages, insertion sequences (IS elements), and transposases in Bt genomes suggests that these elements facilitate the transfer of genes between different strains and species. For instance, prophages have been identified as likely candidates for the mobilization of chromosomally encoded cry-toxins in Bt strains, which are crucial for their virulence and adaptability to various ecological niches (Cao et al., 2018). Additionally, the genome of Bt strain BM-Bt15426 contains multiple virulence factors and antibiotic resistance genes, indicating the acquisition of these traits through HGT (Schäfer et al., 2023).

5.2 Gene duplication and diversification

Gene duplication and subsequent diversification are key mechanisms driving the evolution of Bt strains. The presence of multiple copies of Cry-toxin genes on plasmids and chromosomes in different Bt strains highlights the importance of gene duplication in expanding the functional repertoire of these bacteria. For example, the high virulence of Bt strain MYBt18679 was associated with elevated copy numbers of plasmids containing nematicidal toxin genes, which were favored during pathogen-host coevolution. Furthermore, comparative genomic analyses have revealed the irregular distribution of pesticidal genes among Bt strains, suggesting that gene duplication and diversification contribute to the genetic diversity observed within this species (Zhou et al., 2024).

5.3 Adaptive evolution and natural selection

Adaptive evolution and natural selection are crucial in shaping the genetic landscape of Bt strains. Host-parasite coevolution experiments have demonstrated that high virulence is specifically favored during the coevolution of nematicidal Bt strains with their host organism, Caenorhabditis elegans. This adaptive process involves real-time genetic changes, including the fixation of specific cry-toxin genes in response to selective pressures. Additionally, the identification of genes involved in the biosynthesis of antimicrobial compounds, such as zwittermicin and quercetin 2,3-dioxygenase, in Bt strains underscores the role of natural selection in promoting traits that enhance the survival and competitiveness of these bacteria in diverse environments (Adeniji et al., 2021).

6 Case Studies of Specific Bt Strains

6.1 Comparison of highly virulent strains

Highly virulent strains of Bacillus thuringiensis (Bt) have been extensively studied to understand their genetic and proteomic profiles, which contribute to their insecticidal properties. For instance, the Bt strain GR007, which is toxic to Spodoptera frugiperda and Manduca sexta larvae, has been sequenced to reveal multiple pesticidal protein genes, including 10 Cry genes and two Vip genes. Proteomic analysis of the parasporal crystals of GR007 identified eight Cry proteins, with Cry1Bb and Cry1Ka showing the highest activity against S. frugiperda and M. sexta larvae, respectively (Pacheco et al., 2021). Another highly virulent strain, Bt X022, isolated from soil in China, showed strong insecticidal activity against several Lepidopteran pests. Comparative genomic and proteomic analyses revealed the presence of genes coding for Cry1Ac, Cry1Ia, Cry2Ab, and Vip3A proteins, with three insecticidal crystal proteins detected during the spore-release period (Quan et al., 2016).

6.2 Analysis of Bt strains with novel traits

Novel traits in Bt strains can provide insights into their unique insecticidal properties and potential applications. The Bt strain BLB406, for example, exhibits larvicidal activity against Aedes aegypti larvae and contains a unique combination of toxins, including five Cry genes (Cry11, Cry22, Cry2, Cry60, Cry64) and two Vip4 genes. This combination offers potential larvicidal and anti-cancer activities, making BLB406 a promising candidate for biotechnological applications (Zghal et al., 2018). Additionally, the Bt strain BM-Bt15426 has been sequenced to identify its genetic characteristics, revealing 21 virulence factors and nine antibiotic resistance genes. This strain's genome provides valuable information for further studies on its pathogenic mechanisms and phenotypes (Liu et al., 2017).

6.3 Evolutionary insights from comparative genomics

Comparative genomic analyses of Bt strains have provided significant evolutionary insights. The distribution of genomic virulence determinants in various Bt strains does not align with the established serotyping classification, indicating that serotyping may not accurately reflect the phylogenetic relationships within the species. Comparative genomic and proteomic techniques have shown that core gene sequences and accessory protein genes do not serve as distinctive bases for serovar attribution, emphasizing the need for phylogenomics approaches for accurate strain classification (Figure 2) (Shikov et al., 2021).

Shikov et al. (2021) presented a study that showcased the spore morphology, growth curves, protein electrophoresis profiles, and the distribution of functional genome classifications (COG) among different Bacillus thuringiensis (Bt) strains. Although there are similarities in morphology and growth characteristics among different Bt strains, they exhibit significant differences in protein expression and functional genome classification. Comparative genomic and proteomic techniques have not only revealed the evolutionary relationships among Bt strains but also provided profound insights into their virulence mechanisms. These findings underscore the importance of adopting systematic genomic approaches for accurate strain classification, thus better understanding the evolution and ecological adaptability of Bt strains.

The study of Bt strain 4.0718 revealed that not all products deduced from the annotated genome could be identified in the proteomic data, suggesting that some genes may be silenced or expressed at very low levels. This analysis highlighted the importance of regulatory networks in spore formation and the potential for constructing highly virulent engineered bacteria (Rang et al., 2015).

7 Functional Implications of Genomic Variability

7.1 Impact on toxin production

Genomic variability in Bacillus thuringiensis (Bt) strains significantly influences toxin production, which is crucial for their effectiveness as biopesticides. For instance, the strain GR007 contains multiple pesticidal protein genes, including 10 Cry genes and two Vip genes, which contribute to its high toxicity against specific insect larvae (Pacheco et al., 2021). Similarly, the strain BLB406 exhibits a unique combination of Cry and Vip genes, enhancing its larvicidal activity against Aedes aegypti larvae (Zghal et al., 2018). The presence of mobile genetic elements, such as transposons and plasmids, further contributes to the diversity of toxin genes, as seen in the strain H3, which harbors 11 novel Cry proteins within a highly dynamic plasmid environment (Fayad et al., 2020). This genomic plasticity allows Bt strains to adapt to different ecological niches and host organisms, thereby enhancing their biopesticidal potential (Peralta et al., 2021).

7.2 Environmental adaptations

The genomic variability of Bt strains also plays a crucial role in their environmental adaptations. Bt strains can acquire genetic material through horizontal gene transfer, which enables them to adapt to diverse ecosystems. For example, the strain MORWBS1.1 from South Africa has distinctive genomic properties that could be exploited for biopesticidal applications (Adeniji et al., 2021). Additionally, the strain YBt-1518, which is highly toxic to nematodes, contains multiple virulence factors and nematicidal crystal protein genes, allowing it to thrive in environments where nematodes are prevalent (Wang et al., 2014). The ability of Bt strains to adapt to specific ecological niches is further supported by the presence of various mobile genetic elements, such as bacteriophages and transposases, which facilitate the acquisition and dissemination of beneficial genes.

7.3 Implications for biopesticide development

The genomic variability of Bt strains has significant implications for the development of biopesticides. Understanding the genetic basis of toxin production and environmental adaptation can inform the design of more effective and sustainable biopesticides. For instance, the strain BLB406, with its unique combination of Cry and Vip genes, offers a potential solution to issues such as narrow insecticidal spectra and insect resistance (Zghal et al., 2018). Moreover, the identification of novel Cry proteins in the strain H3 highlights the potential for discovering new toxins that can be used to develop next-generation biopesticides (Fayad et al., 2020). The genomic analysis of Bt strains also provides valuable insights into their safety and potential risks, as demonstrated by the study on the biopesticidal origin of Bt in foods, which revealed a diverse virulence gene profile (Biggel et al., 2022). By leveraging the genomic variability of Bt strains, researchers can develop biopesticides that are not only effective but also environmentally friendly and safe for use in agriculture.

8 Challenges and Future Directions

8.1 Technical challenges in genomic analysis

The genomic analysis of Bacillus thuringiensis (Bt) strains presents several technical challenges. One significant issue is the difficulty in separating and purifying insecticidal active substances due to their relatively short half-life and the need to study samples at different developmental stages. Additionally, the presence of numerous plasmids and mobile genetic elements, such as bacteriophages and transposons, complicates the genomic landscape, making it challenging to achieve a comprehensive understanding of the genome (Bolotin et al., 2017). The discrepancies between genomic annotations and proteomic data further complicate the analysis, as not all predicted proteins are detectable in proteomic studies (Rang et al., 2015; Quan et al., 2016). This indicates potential gene silencing or low expression levels, which require advanced techniques to elucidate.

8.2 Gaps in current research

Despite significant advancements, there are still notable gaps in the current research on Bt. One major gap is the limited understanding of the evolutionary mechanisms and adaptive potential of Bt strains, particularly how they evolve and adapt to various ecological niches and hosts. The role of genomic plasticity and the influence of natural conditions and interaction partners, including hosts and competitors, remain underexplored. Additionally, the irregular distribution of pesticidal genes among Bt strains complicates the taxonomic classification and identification of these bacteria, suggesting a need for more robust genomic or molecular systematic features (Baek et al., 2019). Furthermore, the interaction structures between Bt and other organisms, such as fungi and plants, are not well understood, which limits the potential for developing Bt as a biocontrol agent against a broader spectrum of pests .

8.3 Future trends and research opportunities

Future research should focus on several key areas to address the current challenges and gaps. First, integrating advanced genomic and proteomic techniques, such as next-generation sequencing and mass spectrometry, can provide a more comprehensive understanding of the Bt genome and its expression profiles (Rang et al., 2015; Quan et al., 2016). This approach can help identify novel insecticidal toxins and other virulence factors that contribute to Bt's entomopathogenicity (Zhu et al., 2015). Second, exploring the evolutionary mechanisms and adaptive strategies of Bt through comparative genomics and phylogenomic analyses can shed light on the genetic basis of its ecological versatility and host specificity (Yuan et al., 2014; Elleuch et al., 2015). Third, developing more accurate and reliable taxonomic classification systems based on genomic data can improve the identification and characterization of Bt strains, facilitating their use in biocontrol applications (Baek et al., 2019). Finally, investigating the interactions between Bt and other organisms, including plants and fungi, can expand the potential applications of Bt as a biocontrol agent beyond insect pests (Adeniji et al., 2021).

9 Concluding Remarks

The comparative genomic analysis of Bacillus thuringiensis (Bt) strains has provided significant insights into the evolutionary mechanisms and adaptive strategies of this species. Key findings include the identification of various virulence factors, fitness factors, and mobile genetic elements such as bacteriophages, IS elements, and transposases, which contribute to the genomic plasticity and adaptability of Bt strains. The study also highlighted the role of plasmids in the transfer of Cry genes, which are crucial for host specialization and adaptation to different ecological niches. Additionally, the presence of multiple virulence factors on transposable elements suggests a strategy for expanding the insecticidal spectrum and overcoming host resistance. The genomic and proteomic analyses revealed that not all annotated genes are expressed, indicating potential gene silencing or low expression levels. Furthermore, the study identified two genomovars within Bt, suggesting a need for refined taxonomic classification.

Comparative genomic studies are essential for understanding the complex evolutionary processes and adaptive mechanisms in bacterial species. These studies provide a comprehensive view of the genetic diversity and functional capabilities of different strains, which is crucial for identifying key factors that contribute to pathogenicity, host specificity, and environmental adaptability. In the case of Bt, comparative genomics has elucidated the genetic basis for its ability to infect a wide range of invertebrate hosts and its potential as a biocontrol agent. Moreover, these studies facilitate the identification of novel genes and pathways that can be targeted for genetic engineering and biotechnological applications.

Future research should focus on the following areas to further advance our understanding of Bt and its applications. Conduct functional studies to validate the roles of identified genes and pathways in virulence, host adaptation, and environmental survival. This includes gene knockout and overexpression experiments to determine the phenotypic effects of specific genetic elements. Investigate the molecular mechanisms underlying host-pathogen interactions, particularly the co-evolutionary dynamics between Bt and its hosts. This can be achieved through experimental evolution studies and real-time monitoring of genetic changes during host-pathogen interactions. Further refine the taxonomic classification of Bt strains using genomic and phenotypic data. This includes the identification of additional genomovars and the development of molecular markers for accurate strain identification. Explore the biocontrol potential of Bt against a broader range of pests and pathogens, including phytopathogenic fungi. This involves screening for antifungal activities and identifying the genetic basis for such activities. Leverage the genetic diversity and metabolic capabilities of Bt for biotechnological applications, such as the production of bioinsecticides, enzymes, and other valuable bioproducts. This includes optimizing fermentation processes and genetic engineering for enhanced production. By addressing these research areas, we can enhance our understanding of Bt's evolutionary mechanisms and harness its potential for various applications in agriculture, biotechnology, and medicine.

Acknowledgments

The publisher would like to thank Dr. Fang X from the Hainan Institute of Tropical Agricultural Resources for reviewing and providing valuable feedback on the manuscript. Special thanks are also extended to the two anonymous peer reviewers for their review and valuable suggestions for improvements.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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