Genomic Characterization of Bacillus subtilis for Species-Level Discrimination Among Closely Related Bacillus Species

Bacillus subtilis is well-known for its various applications in agriculture, industry, and biotechnology. Even so, the significant genetic similarity with the rest of the B. subtilis group frequently complicates precise identification, leading to potential misclassification and inappropriate species utilization. The primary objective of this study is to overcome these obstacles by identifying potential genetic markers that enable precise taxonomic resolution and delineating the genetic diversity of B. subtilis strains, subspecies, and closely related species. An analysis of the pan-genome, comprising 87 Bacillus genomes, was conducted using the Roary pipeline, resulting in the identification of 906 conserved core genes shared by all genomes. Phylogenetic analyses utilizing these core genes elucidated evolutionary relationships within the group, confirming that B. subtilis can be distinctly differentiated from its close relatives. Furthermore, the core genes were analyzed to pinpoint nucleotide variant positions that are specific to B. subtilis, resulting in the identification of 601 variants distributed across 379 core genes. These variants were evaluated for diagnostic specificity by comparing them against identical nucleotide positions in other Bacillus species available in NCBI database through nucleotide BLAST, aiming to validate their potential as diagnostic molecular markers for the accurate differentiation of B. subtilis from its closely related Bacillus species. Together, our findings reveal key aspects of genetic diversity within the B. subtilis group and highlight potential molecular markers that can enhance B. subtilis detection protocols. These improvements will strengthen species-level identification and reduce the risk of species misapplication, supporting the safe and effective use of B. subtilis across diverse domain of real-world applications.