1 Introduction

Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium renowned for its insecticidal properties due to the production of crystal (Cry) proteins. These proteins are highly specific to various insect orders, including Lepidoptera, Diptera, and Coleoptera, making Bt a valuable tool in integrated pest management (IPM) and agricultural biotechnology (Hằng et al., 2021). The insecticidal activity of Bt results from the formation of pores in the midgut epithelial cells of susceptible insects, leading to cell lysis and death. Bt has been extensively utilized in the development of biopesticides and genetically modified crops, such as Bt cotton and Bt corn, which express Cry proteins to protect against insect pests (Hằng et al., 2021; Lazarte et al., 2021).

Comparative genomics, which involves the analysis and comparison of genetic material from different organisms, helps to understand their evolutionary relationships, functional genomics, and genetic diversity. In the study of Bt and related Bacillus species, comparative genomics is crucial for several reasons. It aids in identifying new insecticidal genes and understanding their mechanisms of action, thereby developing more effective biopesticides (Hằng et al., 2021). It provides insights into the genetic basis of insect resistance to Bt toxins, enabling the design of strategies to mitigate resistance development (Chen et al., 2021; Lazarte et al., 2021). Furthermore, comparative genomics can reveal the evolutionary adaptations and ecological niches of different Bacillus strains, contributing to a broader understanding of their roles in natural and agricultural ecosystems (Reyaz et al., 2019; Crickmore et al., 2020).

This study provides a comprehensive analysis of the comparative genomics of Bt and related Bacillus species, summarizing current knowledge on the genetic diversity and evolutionary relationships among Bt strains and related Bacillus species. It identifies and characterizes new insecticidal genes and their potential applications in pest management, discusses the mechanisms of insect resistance to Bt toxins and potential strategies to overcome resistance, and explores the ecological and functional roles of Bt and related Bacillus species in various environments. By synthesizing findings from multiple research studies, this study aims to advance our understanding of Bt genomics and its applications in sustainable agriculture and pest management.

2 Overview of Bacillus Genomics

2.1 General characteristics of Bacillus genomes

Bacillus species are known for their diverse metabolic capabilities and adaptability to various environments. The genomes of Bacillus species typically range in size from approximately 5 to 6.5 million base pairs (bp) and have a G+C content around 35%-36% (Liu et al., 2017). For instance, the genome of Bacillus thuringiensis strain BM-BT15426 is 5 246 329 bp long with a G+C content of 35.40% (Liu et al., 2017), while Bacillus thuringiensis HM-311 has a genome size of 6,019,481 bp and a G+C content of 35.85%. These genomes often contain numerous plasmids, which carry genes responsible for various functions, including virulence factors and antibiotic resistance (Reyaz et al., 2019; Zuo et al., 2020).

2.2 Sequencing technologies and methods

The sequencing of Bacillus genomes has evolved significantly with advancements in sequencing technologies. Early sequencing efforts involved labor-intensive methods, but modern techniques such as PacBio RS II and Illumina HiSeq 4000 platforms have streamlined the process, allowing for high-throughput and accurate sequencing (Liu et al., 2017; Zuo et al., 2020). For example, the genome of Bacillus thuringiensis strain BM-BT15426 was sequenced using the PacBio RS II sequencer, which provides long-read sequencing capabilities, and assembled de novo using the HGAP pipeline (Liu et al., 2017). Similarly, Bacillus thuringiensis HM-311 was sequenced using both PacBio RS II and Illumina HiSeq 4000 platforms, combining long-read and short-read sequencing to achieve a comprehensive genome assembly (Zuo et al., 2020).

2.3. Genome annotation and analysis

Genome annotation is a critical step in understanding the functional capabilities of Bacillus species. Automated annotation tools such as the RAST server and NCBI's annotation pipeline are commonly used to predict coding sequences, identify genes, and assign functions based on homology (Quan et al., 2016; Reyaz et al., 2019). For instance, the genome of Bacillus thuringiensis X022 was annotated using the RAST server, which identified genes coding for insecticidal proteins and metabolic pathways (Quan et al., 2016). Similarly, the genome of Bacillus thuringiensis isolate T414 was annotated to reveal the presence of parasporal crystal protein genes and various virulence factors (Reyaz et al., 2019).

Comparative genomics further enhances our understanding by identifying core and pan-genomes, which distinguish conserved genes from those unique to specific strains. This approach has revealed significant functional differences among Bacillus species, such as variations in carbohydrate utilization and signal transduction pathways (Alcaraz et al., 2010). Additionally, comparative analysis of genomic and proteomic data can provide insights into gene expression and regulation, as demonstrated in studies of Bacillus thuringiensis strains (Rang et al., 2015; Quan et al., 2016).

3 Genomic Features of Bt

3.1 Genome structure and organization

Bacillus thuringiensis (Bt) exhibits a complex genome structure characterized by a circular chromosome and multiple plasmids. For instance, the Bt GR007 strain has a circular chromosome and three megaplasmids, with the two largest megaplasmids containing multiple pesticidal protein genes (Pacheco et al., 2021). Similarly, the Bt isolate T414 contains a chromosome and 15 different plasmids, which harbor various insecticidal genes (Reyaz et al., 2019). The genome of Bt X022 consists of one circular chromosomal DNA and seven plasmids, which include genes coding for several Cry proteins and a vegetative insecticidal protein (Quan et al., 2016).

3.2 Toxin gene clusters

3.2.1 Types of toxins

Bt produces a wide variety of insecticidal proteins, including Cry, Cyt, and Vip toxins. The GR007 strain, for example, contains 10 cry genes, two vip genes, and two binary toxin genes (Pacheco et al., 2021). The T414 strain harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab, and cyt1) and a vegetative insecticidal protein gene (vip3Aa) (Reyaz et al., 2019). Additionally, the X022 strain contains genes coding for Cry1Ac, Cry1Ia, Cry2Ab, and Vip3A proteins (Quan et al., 2016).

3.2.2 Gene expression and regulation

The expression of Bt toxin genes is tightly regulated and can be influenced by various factors. For instance, proteomic analysis of the parasporal crystals of the GR007 strain revealed the presence of eight Cry proteins, with differential toxicity against different insect larvae (Pacheco et al., 2021). In the case of Bt X022, the presence of Cu2+ was found to increase the production of insecticidal crystal proteins, indicating a regulatory mechanism linked to environmental factors (Quan et al., 2016).

3.2.3 Evolution of toxin genes

The evolution of Bt toxin genes is marked by horizontal gene transfer and recombination events. For example, the coexistence of cry9A and vip3A genes in the same plasmid in certain Bt strains suggests a recombination mechanism responsible for their association (Wang et al., 2020). Phylogenetic analysis of toxin genes in the mosquitocidal Bt strain S2160-1 also indicates evolutionary relationships among various Cry and toxin proteins (Liu et al., 2018).

3.3 Plasmids and mobile genetic elements

Plasmids play a crucial role in the genetic diversity and adaptability of Bt. In the Bt GR007 strain, plasmids pGR340 and pGR157 contain multiple insecticidal protein genes, including cry and vip genes. These findings help understand the evolutionary mechanisms of Bt and its adaptability in different environments (Figure 1) (Pacheco et al., 2021). Similarly, the plasmid in the T414 strain carries various insecticidal genes and additional toxic factors, such as chitinase and protease (Reyaz et al., 2019). Mobile genetic elements, including insertion sequences and prophages, significantly contribute to the plasticity of the Bt genome. For example, the presence of IS3 and bcr1 elements in B. cytotoxicus highlights the role of mobile genetic elements in genomic diversity and horizontal gene transfer (Fayad et al., 2021).

Figure 1 Genome of B. thuringiensis strain GR007. (A) Circular maps of chromosome and plasmids. The distribution of circles from outside to inside indicate CDS forward, CDS reverse, COG functional assignation, GC skew, GC content. The pathogenic islands (PAI) identified are indicated in the plasmids pGR340 and pGR157. (B) Representation of PAIs. Pesticidal proteins are represented in green arrows, transposases in blue arrows and additional ORF into the PAIs with purple arrows (Adopted from Pacheco et al., 2021)

Figure 1 shows the genomic structure of the Bt GR007 strain, including one circular chromosome and three plasmids (pGR340, pGR157, and pGR55). These plasmids carry various insecticidal protein gene clusters, particularly the toxin gene clusters on plasmids pGR340 and pGR157, demonstrating the crucial role of Bt in genetic diversity and environmental adaptation. The pGR340 plasmid contains cry and vip gene clusters, which are exchanged and recombined among different Bt strains through horizontal gene transfer, thereby enhancing Bt's adaptability and insecticidal efficacy. Figure 1 provides a detailed depiction of these plasmids and gene clusters, offering important references for studying the genetic structure and function of Bt insecticidal protein genes.

4 Comparative Genomics with Related Bacillus Species

4.1 Phylogenetic relationships

The phylogenetic relationships within the Bacillus genus, particularly between Bacillus thuringiensis (Bt) and other Bacillus species, have been extensively studied using whole-genome sequencing and phylogenomic analyses. A study involving nearly 900 whole genome sequences of Bacillus cereus and Bt strains revealed that these species form a well-supported monophyletic clade, distinct from other Bacillus species (Baek et al., 2019). This clade is further divided into two genomovars: B. thuringiensis gv. thuringiensis and B. thuringiensis gv. cytolyticus, indicating significant genomic diversity within Bt itself (Baek et al., 2019). Additionally, the Genome BLAST Distance Phylogeny (GBDP) approach has been used to classify Bacillus cereus group strains into distinct clusters, further elucidating their phylogenetic relationships (Liu et al., 2015).

4.2 Conserved and unique genomic regions

Comparative genomic analyses have identified both conserved and unique genomic regions within Bacillus species. A large-scale bioinformatics analysis of 1 566 Bacillus genomes uncovered that the majority of specialized metabolites produced by Bacillus species are highly conserved, with significant roles in bacterial physiology and development (Grubbs et al., 2017). However, unique biosynthetic gene clusters (BGCs) were also identified, which are scattered across the genus and may encode unknown natural products (Grubbs et al., 2017). In Bt, specific pesticidal protein genes, such as cry and vip genes, are often located on megaplasmids, which are not uniformly distributed across all strains, indicating a level of genomic uniqueness (Pacheco et al., 2021). Furthermore, taxonomically restricted genes (TRGs) in Bacillus species, which are unique to specific lineages, have been identified and are thought to contribute to lineage-specific biological properties (Karłowski et al., 2023).

4.3 Horizontal gene transfer

Horizontal gene transfer (HGT) plays a crucial role in the genomic evolution of Bacillus species, including Bt. HGT events have been identified as a significant source of genomic novelty, contributing to the acquisition of virulence factors and antibiotic resistance (Sevillya et al., 2020). In Bt, the presence of multiple pesticidal protein genes on megaplasmids suggests that these genes may have been acquired through HGT. Additionally, a study on the CRISPR-Cas system in Mycobacterium tuberculosis highlighted the role of HGT in the evolution of this system, providing a parallel to similar mechanisms that may occur in Bacillus species (Singh et al., 2021). The distribution of virulence genes in Bt does not always correlate with phylogenetic positions, further supporting the role of HGT in shaping the genomic landscape of these bacteria (Shikov et al., 2021).

In summary, the comparative genomics of Bt and related Bacillus species reveal a complex interplay of conserved and unique genomic regions, shaped significantly by phylogenetic relationships and horizontal gene transfer. These findings underscore the dynamic nature of bacterial genomes and the evolutionary processes that drive their diversity and adaptation.

5 Functional Genomics

5.1 Gene expression profiling

Gene expression profiling in Bacillus thuringiensis (Bt) and related Bacillus species has been extensively studied to understand the regulation of insecticidal protein production and other metabolic processes. For instance, the comparative analysis of the Bt X022 strain revealed that the presence of Cu²⁺ significantly influences the metabolic regulation of carbon flux, leading to increased production of insecticidal crystal proteins (ICPs) during the spore-release period (Quan et al., 2016). Similarly, in Bt strain 4.0718, gene expression profiling indicated that certain genes, such as cry2Ab and cry1Ia, were either silenced or expressed at very low levels due to the lack of functional promoters or the presence of transposons (Rang et al., 2015). These findings underscore the importance of gene expression profiling in identifying the regulatory mechanisms that control the production of key proteins in Bacillus species.

5.2 Proteomics and metabolomics

Proteomics and metabolomics provide a comprehensive understanding of the protein expression and metabolic pathways in Bacillus species. In Bt X022, proteomic analysis during the spore-release period identified several ICPs, including Cry1Ca, Cry1Ac, and Cry1Da, which were not predicted by genomic data alone (Quan et al., 2016). This highlights the complementary nature of proteomics in validating genomic predictions. Additionally, the proteomic analysis of Bt strain 4.0718 revealed the presence of sporulation-related proteins such as Spo0A~P, SigF, SigE, SigK, and SigG, which play crucial roles in the spore formation regulatory network (Rang et al., 2015). Metabolomic studies have also been instrumental in elucidating the metabolic pathways involved in the production of secondary metabolites. For example, the comparative genomic and functional analyses of Bacillus cereus strains identified conserved genes related to plant-growth-promoting traits, which are crucial for their ecological adaptation (Zeng et al., 2018). These studies demonstrate the power of proteomics and metabolomics in uncovering the functional aspects of Bacillus species.

5.3 Functional studies of specific genes

Functional studies of specific genes in Bacillus species have provided insights into their roles in various biological processes. In the Bacillus cereus group, genes under positive selection were found to be involved in antibiotic resistance, DNA repair, nutrient uptake, metabolism, cell wall assembly, and spore structure, indicating their importance in ecological adaptation (Rasigade et al., 2018). Additionally, the large-scale bioinformatics analysis of Bacillus genomes uncovered conserved roles of natural products in bacterial physiology, with certain specialized metabolites acting as developmental signals that inhibit sporulation (Grubbs et al., 2017). These functional studies are crucial for understanding the genetic basis of the diverse phenotypic traits observed in Bacillus species and for developing strategies for their genetic modification and application in various fields.

Functional genomics approaches, including gene expression profiling, proteomics, metabolomics, and functional studies of specific genes, have significantly advanced our understanding of the complex regulatory networks and metabolic pathways in Bt and related Bacillus species. These insights are essential for harnessing the full potential of these bacteria in biotechnological and agricultural applications.

6. Ecological and Evolutionary Insights

6.1. Adaptation to environmental niches

Bacillus species exhibit remarkable adaptability to diverse environmental niches, driven by genomic variations and selective pressures. For instance, Bacillus pumilus group strains show distinct genomic features that facilitate their survival in marine and terrestrial environments. Marine strains are enriched with genes related to transcription, phage defense, DNA recombination, and repair, while terrestrial strains possess genes aiding survival in land-specific niches (Fu et al., 2021). Similarly, Bacillus mycoides, a member of the Bacillus cereus group, demonstrates significant genomic diversity, with adaptive genes enabling it to thrive in various ecological niches, particularly in cold climates (Fiedoruk et al., 2021). These adaptations are often facilitated by horizontal gene transfer and the presence of mobile genetic elements, which enhance the genomic plasticity of these bacteria (Du et al., 2023).

6.2 Co-evolution with hosts

The co-evolution of Bacillus species with their hosts is a significant driver of their evolutionary trajectory. Bacillus thuringiensis, for example, has evolved high virulence through the selective advantage of its Cry toxin genes during co-evolution with nematode hosts. This co-evolutionary process is distinct from unidirectional selection and involves the accumulation of multiple virulence factors, which enhance the pathogen's ability to infect and adapt to various hosts (Masri et al., 2015). Additionally, Bacillus thuringiensis has specialized to exploit multiple invertebrate hosts, with host switching occurring at both major clade and subclade levels. The transfer of plasmids carrying cry genes plays a crucial role in this adaptation, allowing the bacteria to target specific insect orders effectively (Zheng et al., 2017).

6.3 Implications for biocontrol and biotechnology

Insights into the ecology and evolution of Bacillus species are of great significance for biocontrol and biotechnology. It is known that Bacillus amyloliquefaciens strains with biocontrol properties possess a set of core genes involved in the production of secondary metabolites, volatile compounds, and biofilm formation, which are crucial for their beneficial interactions with plants. These genetic elements not only enhance plant growth but also induce host defense responses, making these strains valuable in agricultural applications (Magno-Pérez-Bryan et al., 2015). Moreover, the metabolic characterization of Bacillus species reveals their nutritional preferences and metabolic capabilities (Figure 2), information that can be used to develop targeted biocontrol strategies and enhance the efficacy of these bacteria under various environmental conditions (Chang et al., 2020). Additionally, the genomic analysis of Bt provides a theoretical basis for developing more effective biopesticides and highlights the potential of genomics in addressing insect resistance.

Figure 2 Metabolic rates correspond to certain chemical properties of nutrients (Adopted from Chang et al., 2020) Image caption: (a–d) Four chemical properties of nutrients examined with the structure of an example from each category (i): (a) carbohydrates (shown: D-glucose), (b) amino acids (shown: L-alanine), (c) lipids (shown: caproic acid), and (d) hydrophilicity as represented by partition coefficient (shown: tyramine and L-arginine). (ii) Heat maps of maximum metabolic rates for nutrients with nutrients in the category for chemical property under question (+ or lesser) or did not (− or greater). Nutrients are hierarchically clustered by their chemical structural similarities using atom-pair distances. Ba: B. anthracis, Bc: B. cereus, Bs: B. subtilis, Sa: S. aureus. (iii) Average maximum metabolic rates for nutrients by chemical property (blue: carbohydrates, red: amino acids, green: lipids, yellow: hydrophilicity/partition coefficient). Bars represent averages of all nutrients categorized by chemical property. Error bars represent standard error of the mean. Maximum metabolic rate for each nutrient is an average from three independent experiments (n = 3, *: p < 0.05, unpaired Student’s t-test). Figure created with R v3.5.3 (https://www.R-project.org) and GraphPad Prism 5 (https://www.graphpad.com) (Adopted from Chang et al., 2020)

Chang et al. (2020) demonstrated the nutritional preferences in carbon utilization, highlighting the metabolic characteristics of Bacillus species. Through the analysis of utilization rates of nutrients with different chemical properties, the study revealed significant differences in the metabolic capacities of Bacillus species. The study showed that different strains have distinct utilization rates for carbohydrates, amino acids, and lipids. This information is crucial for developing targeted biocontrol strategies. Especially for Bt and related Bacillus species, analyzing their metabolic preferences and capacities under specific environmental conditions can enhance the efficacy of these bacteria in various environments, providing a scientific basis for biocontrol.

7 Applications and Implications

Bt's applications in agriculture, medicine, and industry highlight its versatility and potential for promoting sustainable practices. However, careful regulatory oversight is essential to mitigate any potential risks associated with its use.

7.1 Agricultural applications of Bt

Bacillus thuringiensis (Bt) has revolutionized agricultural practices through its use as a biopesticide and in genetically modified crops. Bt produces insecticidal proteins, particularly Cry proteins, which are highly effective against a variety of insect pests. These proteins have been incorporated into crops such as maize, cotton, and brinjal, providing them with inherent resistance to pests and reducing the need for chemical pesticides (Gutiérrez et al., 2019). The use of Bt in agriculture not only enhances crop yields but also promotes environmentally friendly farming practices by reducing the reliance on synthetic chemicals (Jouzani et al., 2017; Dame et al., 2021). Additionally, Bt-based biopesticides are considered safe for humans and non-target organisms, making them a preferred choice for integrated pest management (Koch et al., 2015; Gutiérrez et al., 2019).

7.2 Medical and industrial uses

Beyond agriculture, Bt has shown potential in various medical and industrial applications. Recent studies have highlighted the use of Bt in bioremediation, where it helps in the detoxification of heavy metals and other pollutants (Jouzani et al., 2017). Bt has also been explored for its ability to produce polyhydroxyalkanoate biopolymers, which are valuable in the production of biodegradable plastics. In the medical field, Bt's parasporins have demonstrated anticancer properties, offering a novel approach to cancer treatment (Melo et al., 2016; Jouzani et al., 2017). Furthermore, Bt's chitinase enzyme, which degrades chitin, has industrial applications due to its abundance in nature and its role in enhancing the efficacy of Bt insecticides (Melo et al., 2016).

7.3 Regulatory and safety considerations

The widespread use of Bt in agriculture and other fields necessitates rigorous regulatory and safety evaluations. Bt proteins, particularly those expressed in transgenic crops, must undergo comprehensive biosafety assessments to ensure they do not pose ecological risks or harm non-target organisms (Li et al., 2022). These evaluations include studies on the environmental fate of Bt proteins, their impact on soil microbial diversity, and potential unintended effects on ecosystems. Additionally, the potential for gene flow from Bt crops to wild relatives is assessed to prevent ecological imbalances (Koch et al., 2015). Regulatory frameworks are in place to monitor and manage the use of Bt products, ensuring their safe and sustainable application in various industries (Koch et al., 2015; Li et al., 2022).

8 Concluding Remarks

Comparative genomics studies have revealed significant insights into the genetic diversity, insecticidal properties, and potential applications of Bacillus thuringiensis (Bt) and related Bacillus species. Bt strains exhibit a wide range of insecticidal proteins, such as Cry and Vip proteins, which are crucial for their effectiveness as biopesticides. For instance, Bt X022 contains genes for Cry1Ac, Cry1Ia, Cry2Ab, and Vip3A, while Bt GR007 harbors multiple cry and vip genes, demonstrating their genetic variability and potential for targeted pest control.

Proteomic analyses complement genomic data by identifying the expression of specific insecticidal proteins during different growth stages. For example, proteomic analysis of Bt X022 during the spore-release period detected Cry1Ca, Cry1Ac, and Cry1Da proteins, which could not be predicted solely by genomic data. Studies also identified various virulence factors and resistance genes in Bt strains, such as antibiotic and heavy metal resistance genes in Bt HM-311, which may contribute to their survival in contaminated environments.

Comparative genomic and proteomic techniques provide more accurate phylogenetic relationships among Bt strains than traditional serotyping methods, offering important insights into Bt classification and evolutionary history. Future Bt genomics research should further annotate Bt genomes functionally, particularly identifying and characterizing new insecticidal proteins and virulence factors, and exploring the regulatory mechanisms governing their expression under different environmental conditions.

In practical applications, utilizing genomic and proteomic data to develop Bt strains with specific insecticidal profiles tailored to particular pests can enhance biopesticide efficacy and reduce environmental impact. Advanced genomic tools should be used for routine monitoring of Bt biopesticide residues in food products to ensure food safety and regulatory compliance. Through these measures, future research and practical applications can maximize the benefits of Bt and related Bacillus species in sustainable agriculture and pest management.

Acknowledgments

The MicroSci Publisher is grateful to the two anonymous peer reviewers for their insightful feedback and suggestions on this manuscript.

Conflict of Interest Disclosure

The authors affirm 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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