Bt Research 2024, Vol.15, No.3, 118-130 http://microbescipublisher.com/index.php/bt 121 In a study focusing on Bt isolates from Indian soils, distinct plasmid bands of different sizes were observed, with some isolates showing plasmids above 33 500 bp. This indicates the diversity and complexity of plasmid profiles in Bt strains (Rangeshwaran et al., 2014). Another study utilized selective methods to isolate Bt from various habitats, confirming the presence of plasmids through hemolytic activity and parasporal crystal protein profiles. The extracted plasmids were comparable to reference strains, demonstrating the effectiveness of these methods in recovering plasmid DNA (Shishir et al., 2012). 4.2 Molecular characterization methods Molecular characterization of plasmids involves several techniques to understand the genetic variability and properties of Bt isolates. Polymerase Chain Reaction (PCR) is commonly used for genetic profiling and toxicity prediction. Repetitive element polymorphism (Rep-PCR) using ERIC, REP, and BOX primers helps in understanding genetic diversity. Amplified Fragment Length Polymorphism (AFLP) is another technique used to detect molecular markers and assess genetic variability (Valicente and Silva, 2017). In addition to PCR and AFLP, plasmid characterization is crucial for detecting the number and patterns of plasmids. For instance, a study on Bt isolates from Bangladesh used 16S rDNA gene sequencing for strain identification and SDS-PAGE for analyzing crystal proteins. The plasmid analysis revealed at least one 15 Kb DNA band, which was comparable to the reference strain, indicating the presence of Cry1, Cry2, and Cry9 type proteins (Shishir et al., 2012). 4.3 Bioinformatics tools for plasmid analysis Bioinformatics tools play a significant role in the comparative analysis of plasmid content, especially with the advent of whole genome sequencing (WGS). Plasmid Profiler is a pipeline designed to perform comparative plasmid content analysis without the need for de novo assembly. It rapidly identifies plasmid sequences by mapping reads to a plasmid reference sequence database and annotates predicted plasmid sequences with their incompatibility group. The results can be visualized as an interactive heat map, facilitating the analysis of plasmid genes or regions of interest (Zetner et al., 2017). PlasFlow is another tool that uses a neural network approach to identify bacterial plasmid sequences in environmental samples. It can recover plasmid sequences from assembled metagenomes with high accuracy, making it suitable for analyzing plasmidomes in diverse environments (Krawczyk et al., 2018). Similarly, Platon uses protein sequence-based replicon distribution scores to distinguish plasmid-borne from chromosome-borne contigs. It achieves high accuracy and is suitable for high-throughput taxon-independent analyses (Figure 2) (Schwengers et al., 2020). The MOB-suite is a set of modular tools for the reconstruction and typing of plasmids from draft assembly data. It provides high sensitivity and specificity in identifying plasmid contigs and offers replicon typing, relaxase typing, and prediction of conjugation potential. The MOB-suite reduces errors in plasmid reconstruction and is available as an open-source tool (Robertson and Nash, 2018). In summary, the combination of advanced extraction techniques, molecular characterization methods, and sophisticated bioinformatics tools provides a comprehensive approach to plasmid profiling in Bt isolates. These methods enable researchers to uncover the diversity and functional roles of plasmids in different habitats, contributing to our understanding of microbial adaptation and resistance mechanisms. 5 Comparative Analysis of Plasmid Profiles 5.1 Plasmid diversity in different habitats Plasmids are crucial genetic elements that facilitate the rapid adaptation and evolution of bacterial populations by transferring genes that confer selective advantages, such as antibiotic resistance and metabolic capabilities. The diversity of plasmids varies significantly across different habitats, influenced by the environmental conditions and the bacterial communities present. For instance, F-type plasmids, which are known for carrying antimicrobial resistance (AMR) genes, exhibit substantial diversity in both environmental and livestock settings. These plasmids adapt to their specific niches, with unique combinations of core and accessory genes that reflect their environmental origins (Matlock et al., 2021).
RkJQdWJsaXNoZXIy MjQ4ODYzNA==