Comprehensive Genomic Analysis of the pO157 Plasmid in Enterohemorrhagic Escherichia coli O157:H7 Reveals Non-synonymous Variation in Virulence Factors.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen responsible for severe human diseases, including hemolytic uremic syndrome. Its virulence plasmid, pO157, encodes toxins, proteases, and adhesins; however, the extent of genetic variability among strains remains poorly characterized. Here, we analyzed 153 complete pO157 sequences using comparative pan-genomic and structural approaches. Plasmids ranged from 88.2 to 98.1 kb, contained 84-102 coding sequences, and displayed highly conserved GC content (~ 47.85%). The pan-genome comprised 145 genes, including 60 core and 85 accessory genes, many of which encode hypothetical proteins of unknown function. Key pO157-associated genes (ehxA, katP, stcE, espP, and toxB) were present in all analyzed isolates. Subcellular localization predictions indicated that most proteins are cytoplasmic, whereas only a small subset of hypothetical proteins showed potential secretion signals. Comparative structural analyses revealed that the analyzed protein variants maintained highly conserved overall predicted architectures despite localized amino acid variability. Although some substitutions were located within predicted functional regions, the analyses revealed no major structural alterations among variants. Hemolytic activity assays performed in selected strains revealed phenotypic variability that was not exclusively associated with ehxA sequence variation, suggesting the involvement of additional regulatory or genetic factors. Overall, these findings indicate that pO157 maintains a highly conserved genomic organization while preserving limited sequence variability among virulence-associated genes. This study provides a comparative framework for future investigations aimed at understanding the functional significance of pO157-associated genetic variation in EHEC O157:H7.