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During infection, Bacillus phage SPO1 initially relies on host \u03c3A-dependent transcription to drive early gene expression before switching to phage-encoded \u03c3 factors for middle and late transcription. However, the mechanisms underlying these transitions, particularly how the middle \u03c3 factor Gp28 displaces \u03c3A, 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 \u03c3 factors. In parallel, we identify SPO1 Gp33 as an inhibitor of \u03c3A-dependent transcription whose expression itself depends on LLPS. Gp33 selectively suppresses \u03c3A-driven transcription by trapping the RNA polymerase holoenzyme in an inactive state and preventing \u03c3A-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.<\/jats:p>","DOI":"10.1093\/nar\/gkag742","type":"journal-article","created":{"date-parts":[[2026,7,15]],"date-time":"2026-07-15T11:49:57Z","timestamp":1784116197000},"source":"Crossref","is-referenced-by-count":0,"title":["Liquid\u2013liquid phase separation and a phage-encoded inhibitor cooperatively drive transcriptional transition during phage SPO1 infection"],"prefix":"10.1093","volume":"54","author":[{"given":"Zonglan","family":"Yu","sequence":"first","affiliation":[{"name":"Department of Infectious Diseases, the First Affiliated Hospital of Xi\u2019an Jiaotong University , Shaanxi 710061 ,","place":["China"]},{"name":"Key Laboratory of Surgical Critical Care and Life Support, Biobank, Centre for Biobank and Advanced Medical Research of 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