Sa3ms, a lysogenic bacteriophage encoding the staphylococcal enterotoxins SEA, SEG, and SEK and the fibrinolytic enzyme staphylokinase (Sak), was identified in the unannotated genome sequence of the hypervirulent community-acquired Staphylococcus aureus strain 476. We found that mitomycin C induction of Sa3ms led to increased transcription of all four virulence factors. The increase in sea and sak transcription was a result of read-through transcription from upstream latent phage promoters and an increase in phage copy number. The majority of the seg2 and sek2 transcripts were shown to initiate from the upstream phage cI promoter and hence were regulated by factors influencing cI transcription. The lysogeny module of Sa3ms was shown to have some -like features with divergent cI and cro genes. Band shift assays were used to identify binding sites for both CI and Cro within the region between these genes, suggesting a mechanism of control for the Sa3ms lytic-lysogenic switch. Our findings suggest that the production of phage-encoded virulence factors in S. aureus may be regulated by processes that govern lysogeny.Staphylococcus aureus is a leading cause of nosocomial and community-acquired infections worldwide (42). S. aureus causes a wide range of diseases that vary in severity from mild skin infections, such as boils and furuncles, to life-threatening diseases, like toxic shock syndrome and endocarditis. The ability of S. aureus to cause such a wide variety of diseases is thought to be due in part to its elaboration of a large number of secreted and cell-surface associated virulence factors (1,5,21,36,48).Much of the variation between S. aureus strains appears to be attributable to mobile genetic elements, such as plasmids, bacteriophages, pathogenicity islands, transposons, insertion sequences, and the staphylococcal chromosomal cassette (2, 27). The bacteriophages and pathogenicity islands of S. aureus encode many virulence factors (35). The known phage-encoded virulence factors include Panton-Valentine leukocidins (24, 33), exfoliative toxin type A (47), and staphylococcal enterotoxins (SEs) (3). The SEs constitute a family of related proteins whose activities are associated with staphylococcal food poisoning, toxic shock syndrome, and possibly several autoimmune disorders (17, 41). SEs are powerful superantigens that activate subsets of T lymphocytes to liberate various cytokines, including gamma interferon and tumor necrosis factor (41). In addition to these characterized phage-encoded virulence factors, a number of putative S. aureus virulence factors, such as staphylokinase (Sak), are also encoded in phage genomes (25). Staphylokinase, a potent plasminogen activator, has been hypothesized to aid in the dissemination of S. aureus from fibrinous clots and abscesses (1).The genome sequences of two S. aureus isolates derived from life-threatening community-acquired infections have been determined (2; unpublished data). The methicillin-resistant strain MW2 (sequenced at Juntendo University, Tokyo, Japan) was i...