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 σA-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 σA, 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 σA-dependent transcription whose expression itself depends on LLPS. Gp33 selectively suppresses σA-driven transcription by trapping the RNA polymerase holoenzyme in an inactive state and preventing σA-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.