Machine Learning-Based Characterization of Bacillus anthracis Phenotypes from pXO1 Plasmid Proteins.

The Bacillus anthracis pXO1 plasmid, encoding ~143 proteins, presents a compact model for exploring protein function and evolutionary patterns using protein language models. Due to the organism's slow evolutionary rate, its limited amino acid variation enhances detection of physiologically relevant patterns in plasmid protein composition. In this study, we applied embedding-based analyses and machine learning methods to characterize pXO1 protein modules across diverse B. anthracis lineages. We generated protein sequence embeddings, constructed phylogenies, and compared plasmid content with whole genome variation. While whole genome and plasmid-based phylogenies diverge, the composition of proteins encoded along the pXO1 plasmid revealed lineage specific structure. Association rule mining combined with decision tree classification produced plasmid-encoded targets for assessing anthrax sublineage, which yielded functionally redundant protein modules that reflected geographic and phylogenetic patterns. A conserved DNA replication module exhibited both shared and B. anthracis lineage specific features. These results show that pXO1 plasmid protein modules contain biologically meaningful and evolutionarily informative signatures, exemplifying their value in phylogeographic characterizations of bacterial pathogens. This framework can be extended to study additional virulence plasmids across Bacillus and other environmental pathogens using scalable protein language model tools.