Bt Research 2024, Vol.15, No.2, 76-86 http://microbescipublisher.com/index.php/bt 81 harboring parasporal crystal protein genes such as cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab, and cyt1, as well as vegetative insecticidal protein gene vip3Aa (Reyaz et al., 2019). Similarly, the sequencing of the pBtoxis plasmid in Bt subsp. israelensis identified 125 potential coding sequences, many of which are involved in toxin production and gene regulation (Stein et al., 2006). Another study on a novel Bt strain H3 identified 11 novel Cry proteins through whole-genome sequencing, highlighting the dynamic nature of toxin-carrying plasmids (Fayad et al., 2020). 6.2 Proteomic analysis Proteomic analysis complements genomic sequencing by identifying and characterizing the proteins expressed by the toxin genes. Techniques such as SDS-PAGE and MALDI-TOF/TOF are used to analyze the protein composition of Bt strains. For example, SDS-PAGE analysis of the spore-crystal mixture of Bt isolate T414 showed the presence of two major protein bands, 130 and 65 kDa, corresponding to the Cry and Cyt toxins (Reyaz et al., 2019). In another study, MALDI-TOF/TOF was used to confirm the identity of Cry8 proteins in the parasporal body of Bt strain FCC 7, which was toxic against various lepidopterans and coleopterans (Lazarte et al., 2021). These proteomic techniques are crucial for verifying the expression and functionality of the identified toxin genes. 6.3 Functional assays Functional assays are essential for determining the bioactivity of the identified toxins. These assays involve testing the toxicity of the Bt strains or their purified toxins against target insect species. For instance, the novel Bt strain H3 was tested for its mosquitocidal activity, showing a unique killing profile with higher toxicity against Aedes albopictus and Anopheles gambiae compared to Culex pipiens (Fayad et al., 2020). Similarly, the toxicity of Bt strain FCC 7 was evaluated against the cotton boll weevil, Anthonomus grandis, confirming its insecticidal properties (Lazarte et al., 2021). These assays provide practical insights into the effectiveness of the toxins and their potential applications in pest control. 6.4 Bioinformatics approaches Bioinformatics approaches are employed to analyze and predict the functions of the genes and proteins identified through genomic and proteomic methods. Tools such as NCBI BLAST, RAST, and BtToxin_scanner are used to annotate genomes and identify toxin genes. For example, the genome of Bt strain BLB406 was analyzed using BtToxin_scanner, revealing a unique combination of cry and vip genes that contribute to its larvicidal activity against Aedes aegypti (Zghal et al., 2018). Additionally, bioinformatics analysis of the pGI1 plasmid in Bt H1.1 identified a toxin-antitoxin system, tasA-tasB, which is widely distributed among various microorganisms (Fico and Mahillon, 2006). These computational tools are invaluable for understanding the genetic and functional diversity of Bt toxins. 7 Ecological and Evolutionary Implications 7.1 Role in Bt evolution The evolution of Bacillus thuringiensis (Bt) is significantly influenced by the presence of plasmid-encoded toxins. These toxins, particularly the Cry and Cyt proteins, play a crucial role in the pathogen's adaptation and specialization to various hosts. The coevolution between Bt and its hosts, such as nematodes and insects, drives the selection of virulent strains with high toxin gene copy numbers, as demonstrated by the fixation of the BT-679 genotype in coevolutionary experiments (Argôlo-Filho and Loguercio, 2013; Masri et al., 2015). Additionally, the acquisition and maintenance of plasmids carrying these toxin genes are essential for Bt's pathogenicity and host specificity, facilitating rapid adaptation to new ecological niches (Figure 3) (Masri et al., 2015; Zheng et al., 2017).
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