Bt Research 2024, Vol.15, No.2, 76-86 http://microbescipublisher.com/index.php/bt 80 4.3 Mechanisms of horizontal gene transfer Horizontal gene transfer (HGT) plays a crucial role in the dissemination of toxin genes among Bacillus thuringiensis and related species. The plasmid pTAND672-2 is a prime example, as it can transfer horizontally fromB. thuringiensis serovar israelensis to Lysinibacillus sphaericus, where it integrates into the chromosome via site-specific recombination (Geng et al., 2023). This plasmid and others like it within the Bacillus cereus group share a similar genetic backbone and exhibit conjugative capabilities, facilitating the spread of toxin genes across different bacterial species (Geng et al., 2023). The presence of mobile genetic elements, such as insertion sequences and transposable elements, in plasmids like pH3-180 further underscores the importance of HGT in the evolution and adaptation of Bt strains (Fayad et al., 2020). 5 Functional Analysis of Toxins 5.1 Insecticidal activity Bacillus thuringiensis (Bt) produces a variety of Cry and Cyt toxins that exhibit potent insecticidal activity against a broad range of insect pests. The insecticidal activity of these toxins is primarily determined by their ability to bind to specific receptors on the midgut epithelial cells of target insects, leading to cell lysis and death. For instance, the Cry1Ca toxin has been shown to be highly effective against Spodoptera exigua larvae, with specific mutations in domains II and III significantly reducing its toxicity, thereby highlighting the importance of these domains in determining insect specificity (Herrero et al., 2004). Additionally, the Cry4Ba toxin exploits specific residues in its C-terminal domain to interact with target receptors, such as the Aedes aegypti membrane-bound alkaline phosphatase, which is crucial for its insecticidal activity (Thammasittirong et al., 2021). 5.2 Specificity to target insects The specificity of Bt toxins to target insects is largely influenced by the presence of high-affinity binding sites on the brush border membrane of the insect midgut. For example, the Bt2-toxin and Bt4412-toxin exhibit high-affinity binding to the midgut vesicles of Manduca sexta and Pieris brassicae, respectively, correlating with their toxicity to these species. Similarly, the Cry8Ea1 toxin is specifically toxic to the underground larvae of Holotrichia parallela, with its mode of action involving the formation of transmembrane pores upon binding to midgut receptors (Guo et al., 2009). The Cry1Ca toxin's specificity towards S. exigua is also determined by its ability to form oligomeric structures upon activation, a process that is disrupted in mutants with altered domain II and III, thereby reducing their binding affinity and toxicity (Herrero et al., 2004). 5.3 Mechanisms of toxicity The mechanisms of toxicity of Bt toxins involve a series of interactions with midgut proteins that facilitate the formation of oligomeric structures and their insertion into the membrane, leading to pore formation and cell lysis. The Cry toxins, for instance, undergo proteolytic activation to form active toxins that bind to specific receptors on the midgut epithelial cells, initiating the formation of lethal transmembrane pores (Pardo-López et al., 2013). The Cry4Ba toxin's interaction with the Aedes aegypti membrane-bound alkaline phosphatase via its C-terminal domain is a critical step in mediating larval toxicity (Thammasittirong et al., 2021). Additionally, the Cry1Ca toxin's ability to form oligomeric structures upon activation is essential for its insecticidal activity, with mutations in domain II and III affecting this process and thereby altering its specificity and toxicity (Herrero et al., 2004). The coexistence of Cry9 and Vip3a genes in the same plasmid also suggests a synergistic mechanism of toxicity, enhancing the overall insecticidal efficacy and delaying resistance development in target pests (Wang et al., 2020). 6 Methods for Characterizing Plasmid-encoded Toxins 6.1 Genomic sequencing Genomic sequencing is a fundamental method for characterizing plasmid-encoded toxins in Bacillus thuringiensis (Bt). Whole-genome sequencing allows for the identification and annotation of genes responsible for toxin production. For instance, the genome sequencing of Bt isolate T414 revealed the presence of multiple plasmids
RkJQdWJsaXNoZXIy MjQ4ODYzNA==