Bt Research 2024, Vol.15, No.3, 118-130 http://microbescipublisher.com/index.php/bt 120 In conclusion, the study of Bt isolates from different habitats provides valuable insights into the diversity and distribution of this bacterium. Environmental factors such as soil composition, climate, and host insects significantly influence Bt distribution. The use of various collection and isolation techniques, including selective culturing, molecular characterization, and advanced electrophoresis methods, ensures the recovery of diverse Bt isolates with potential applications in biopesticide development and insect pest contro (Nair et al., 2018). 3 Plasmid Composition and Structure in Bt 3.1 Types of plasmids in Bt Bacillus thuringiensis (Bt) is known for its diverse plasmid content, which plays a crucial role in its adaptability and pathogenicity. Plasmids in Bt can be categorized based on their incompatibility groups and the functions they encode. For instance, IncF plasmids are prevalent and often carry multiple resistance genes, as seen in various studies (Doumith et al., 2012; Ajayi et al., 2021; Douarre et al., 2020). These plasmids are not only limited to antibiotic resistance but also include genes that confer resistance to heavy metals and other environmental stressors (Falgenhauer et al., 2017; Dolejská et al., 2018). Additionally, plasmids from different environments, such as wastewater treatment plants and livestock farms, show a wide range of resistance and virulence genes, indicating their adaptability to diverse habitats (Falgenhauer et al., 2017; Ajayi et al., 2021). 3.2 Plasmid structure and genetic elements The structure of Bt plasmids is highly complex, often comprising multiple replicons and a variety of genetic elements such as transposons, integrons, and insertion sequences. For example, the IncF/MOBF12 plasmid pFEMG (209 357 bp) isolated from wastewater treatment plants harbors a cluster of resistance genes interspersed with transposons and insertion sequences, which facilitate horizontal gene transfer (Ajayi et al., 2021). Similarly, the IncHI2 plasmids found in livestock farms carry a mosaic of resistance genes and heavy metal resistance determinants, indicating a high level of genetic recombination and evolution (Falgenhauer et al., 2017). The presence of multiple addiction systems, such as toxin-antitoxin modules, ensures the stable maintenance of these plasmids within their bacterial hosts (Doumith et al., 2012). 3.3 Functions of plasmid-encoded genes Plasmid-encoded genes in Bt serve a variety of functions that enhance the bacterium's survival and pathogenicity. These functions can be broadly categorized into antibiotic resistance, heavy metal resistance, and virulence factors. For instance, plasmids carrying the blaCMY-42, blaTEM-1β, and blaNDM-5 genes confer resistance to beta-lactam antibiotics, while genes like mphA-mrx-mphR provide resistance to macrolides (Ajayi et al., 2021). Heavy metal resistance genes such as ter, mer, and sil are also commonly found on Bt plasmids, enabling the bacteria to thrive in contaminated environments (Falgenhauer et al., 2017; Dolejská et al., 2018). Additionally, plasmids often carry virulence genes that contribute to the pathogenicity of Bt, such as those encoding for toxins and other virulence factors (García et al., 2018). In summary, the plasmid composition and structure in Bt are highly diverse and complex, reflecting the bacterium's ability to adapt to various environmental conditions. The presence of multiple resistance and virulence genes on these plasmids underscores their importance in the survival and pathogenicity of Bt. Further research into the genetic elements and functions of these plasmids will provide deeper insights into the mechanisms of horizontal gene transfer and the evolution of antibiotic resistance and virulence in Bt. 4 Methods for Plasmid Profiling 4.1 Extraction and purification techniques The extraction and purification of plasmids fromBacillus thuringiensis (Bt) isolates are critical steps in plasmid profiling. Traditional methods often struggle with separating plasmid DNAs with molecular masses greater than 25 Kb. Pulsed Field Gel Electrophoresis (PFGE) has emerged as an ideal method for separating and identifying plasmid profiles, especially for large plasmids. PFGE leverages regular changes in the direction and size of the electric field to enable the separation of high molecular weight DNAs, making it suitable for Bt strains that commonly contain multiple plasmids ranging from 10 Kb to over 600 Kb (Zhou et al., 2014).
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