Dam-dependent epigenetic memory regulates prophage reintegration in Salmonella

Bacterial epigenetics has emerged as a critical mechanism for regulating gene expression in response to environmental cues, yet whether such modifications persist beyond initial stress remains unresolved. Here, we uncover an epigenetic memory system in Salmonella enterica that facilitates prophage reintegration during infection. Using an in vitro model mimicking stages of the Salmonella infection cycle, including the Salmonella-containing vacuole (SCV), we found that DNA adenine methyltransferase (Dam) plays stage-specific roles in adaptation and survival of Salmonella cells. Early during SCV-like stress, oxidative stress contributes to excision of the cryptic prophage ST64B. However, at later infection stages, levels of excised phage DNA decline as global methylation increases, notably at the promoter of intA, a prophage integrase encoded by Salmonella. This methylation persists after stress removal and maintains active intA transcription, establishing a form of epigenetic memory. Functional assays revealed that intA expression is required for efficient ST64B reintegration and that this process depends on methylation at a critical GATC site within the promoter. Mechanistically, we show that SCV stress disrupts binding of the integration host factor, a repressor of intA, thereby enabling Dam-mediated methylation. Sustained intA activation stabilizes prophage reintegration, highlighting stress-responsive epigenetic control important for adaptation during Salmonella pathogenesis.