Liquid–liquid phase separation and a phage-encoded inhibitor cooperatively drive transcriptional transition during phage SPO1 infection

Liquid–liquid phase separation (LLPS) organizes biochemical reactions in cells, yet whether this principle contributes to bacteriophage development has remained unclear. During infection, Bacillus phage SPO1 initially relies on host σ^A-dependent transcription to drive early gene expression before switching to phage-encoded σ factors for middle and late transcription. However, the mechanisms underlying these transitions, particularly how the middle σ factor Gp28 displaces σ^A, have remained elusive. Here, we show that SPO1 exploits LLPS to orchestrate its transcriptional program. We identify the phage-encoded transcription factor Gp27 as the principal driver of phase separation, forming biomolecular condensates both in vitro and in vivo. Structural and biochemical analyses reveal that Gp27 possesses a modular architecture that promotes condensate formation and concentrates the transcriptional machinery, thereby compensating for the intrinsically weak promoter-binding activity of phage-encoded σ factors. In parallel, we identify SPO1 Gp33 as an inhibitor of σ^A-dependent transcription whose expression itself depends on LLPS. Gp33 selectively suppresses σ^A-driven transcription by trapping the RNA polymerase holoenzyme in an inactive state and preventing σ^A-mediated promoter recognition. Together, these findings uncover LLPS as a previously unrecognized regulatory strategy exploited by bacteriophages, which acting in concert with a phage-encoded transcription inhibitor, as a mechanism governing phage transcriptional progression.