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Bacteriophages are major drivers of Staphylococcus aureus evolution, mediating predation, lysogeny, and the horizontal transfer of virulence and antibiotic-resistance genes. However, how lifestyle shapes their genomic architecture and synonymous codon usage remains poorly understood. To address this, we performed a comparative analysis of 30 experimentally verified virulent phages, 30 temperate phages, and 30 intact prophages from diverse S. aureus strains. We assessed genome heterogeneity and patterns of synonymous codon usage across these groups. Genome- and gene-size distributions were examined, along with gene-level metrics including overall and positional GC content, Codon Adaptation Index (CAI), and the effective number of codons ( Nc ). Virulent phages showed the highest genomic heterogeneity: nearly two-thirds of pairwise genome comparisons (59.1%) displayed complete divergence with no detectable similarity; their genomes also followed a strongly bimodal size distribution (median ≈130 kb; range 16.8 kb−148.5 kb). In contrast, temperate phages had smaller, tightly constrained genomes (median ≈43 kb; IQR 1.2 kb) and formed a continuous network of recombinational connectivity without extreme divergence. Prophages displayed intermediate characteristics, with larger and more variable genome sizes. The clearest lifestyle-associated signal appeared at the synonymous level. Genes from virulent phages had significantly lower GC content at third codon positions (%GC3), higher CAI, and lower Nc than genes from temperate phages and prophages (large Cliff's δ effects, Holm-adjusted p 0.001). Importantly, these differences remained consistent across functional gene categories, including replication, structural, lysis, regulatory, and repair genes, and persisted after controlling for compositional bias. Together, these results reveal distinct evolutionary trajectories among S. aureus phages. The temperate phages appear to diversify through modular recombination within interconnected genomic networks, whereas the virulent phages show episodic divergence coupled with distinct codon usage patterns that may reflect enhanced translational selection, although partially influenced by compositional bias.
Gamkrelidze et al. (Wed,) studied this question.