Genomic diversification and modular recombination of Stx2 prophages associated with chromosomal integration sites...

Shiga toxin 2 (Stx2) production in Shiga toxin-producing Escherichia coli (STEC) is mediated by lambdoid Stx2-converting bacteriophages, which play a central role in the evolutionary and pathogenic potential of STEC lineages. However, the mechanisms through which Stx2 phages diversify and evolve during natural outbreaks remain unclear. Here, we analyzed a collection of 20 related Stx2 phage and prophage genomes recovered from STEC O111 and O157 isolates associated with a foodborne disease outbreak in Japan. These included 2 infectious, Stx2-converting phages isolated directly from bloody stool samples, 17 Stx2 prophages, and 1 stx-negative prophage. Genome-wide single-nucleotide polymorphism analysis identified 282 polymorphic sites, resolving two predominant lineages associated with distinct chromosomal integration sites (argW or wrbA), corresponding largely to the STEC O111 and O157 backgrounds, respectively. Despite this lineage structure, the genomes exhibited extensive mosaicism, including lineage-incongruent haplotype blocks and multiple recombination junctions, indicating frequent modular exchanges among co-circulating phages. The preferential association between specific haplotypes and chromosomal integration sites further suggests that the host genomic context contributes to prophage genomic variation. Additionally, the Stx2-converting phages and argW-integrated prophages encoded functionally active restriction-modification systems, indicating that prophage-encoded defense mechanisms may contribute to genome stability and diversification. Collectively, these findings highlight the genetic plasticity of Stx2 phages and prophages during a single outbreak and offer genome-based insights into the evolutionary dynamics and epidemiological significance of toxin-converting bacteriophages in STEC populations.IMPORTANCEShiga toxin-converting bacteriophages are key drivers of virulence and genomic evolution in Shiga toxin-producing Escherichia coli (STEC). However, the diversity of these phages during natural disease outbreaks remains unclear. By analyzing a set of closely related Stx2 phages and prophages recovered from a single foodborne disease outbreak, this study revealed that Stx2 prophage populations are structured by their chromosomal integration sites, primarily at the argW or wrbA loci. Despite this lineage structure, phage genomes exhibit extensive mosaicism generated by modular recombination. We further showed that prophage-encoded restriction-modification systems are functionally active, suggesting that prophage defense mechanisms may influence genome stability and diversification. Collectively, these findings elucidate how chromosomal context, recombination, and prophage-encoded functions drive the evolution of toxin-converting bacteriophages. These dynamics have important implications for STEC population structure, outbreak dynamics, and the emergence of new pathogenic strains.