The genome of the highly virulent group A Streptococcus (GAS) serotype M3 strain MGAS315 has six prophages that encode six proven or putative virulence factors. We examined prophage induction and expression of prophage-encoded virulence factors by this strain under in vitro conditions inferred to approximate in vivo conditions. Coculture of strain MGAS315 with Detroit 562 (D562) human epithelial pharyngeal cells induced the prophage encoding streptococcal pyrogenic exotoxin K (SpeK) and extracellular phospholipase A 2 (Sla) and the prophage encoding streptodornase (Sdn). Increased gene copy numbers after induction correlated with increased speK, sla, and sdn transcript levels. Although speK and sla are located contiguously in prophage ⌽315.4, these genes were transcribed independently. Whereas production of immunoreactive SpeK was either absent or minimal during coculture of GAS with D562 cells, production of immunoreactive Sla increased substantially. In contrast, despite a lack of induction of the prophage encoding speA during coculture of GAS with D562 cells, the speA transcript level and production of immunoreactive streptococcal pyrogenic exotoxin A (SpeA) increased. Exposure of strain MGAS315 to hydrogen peroxide, an oxidative stressor, induced the prophage encoding mitogenic factor 4 (MF4), and there was a concomitant increase in the mf4 transcript. All prophages of strain MGAS315 that encode virulence factors were induced during culture with mitomycin C, a DNA-damaging agent. However, the virulence factor gene transcript levels and production of the encoded proteins decreased after mitomycin C treatment. Taken together, the results indicate that a complex relationship exists among environmental culture conditions, prophage induction, and production of prophage-encoded virulence factors.Group A Streptococcus (GAS) has a wide range of disease presentation and tissue tropism, and strains of relatively few M protein serotypes account for the majority of particular clinical syndromes. For example, serotype M1 and M3 strains often cause deep tissue infections, such as necrotizing fasciitis, whereas serotype M18 strains have been repeatedly linked to contemporary cases of acute rheumatic fever in the United States (23,25,30). The genomes of one strain each of serotype M1, M3, and M18 GAS have been sequenced (3,10,30). Each strain is polylysogenic and has a unique array of prophageencoded virulence factors that may increase bacterial fitness during host colonization, infection, and evasion of the immune system. Although prophages contribute substantially to the diversity in gene content in GAS and other pathogens (1, 27), the environmental signals and regulatory mechanisms that influence expression of prophage-encoded virulence factors are poorly understood for most bacterial pathogens.Recent evidence from study of a GAS serotype M1 strain indicates that prophage induction is sensitive to environmental signals. For example, during coculture of GAS with a human pharyngeal epithelial cell line, increased gene copy...