A packaging signal-binding protein regulates the assembly checkpoint of integrative filamentous phages

Many integrative filamentous phages not only lack Ff coliphage homologues essential for assembly but also have distinct packaging signals (PS). Their encapsidation remains completely uncharacterized to date. Here we report the first evidence of a PS-dependent checkpoint for integrative filamentous phage assembly. Suppressor screening of PS-deficient phages identified an unknown protein, PSB15 (<underline>PS</underline>-<underline>b</underline>inding<underline>15</underline>kDa), crucial for encapsidation. The WAGFXF motif of the PSB15 N-terminus directly binds to PS DNA with conformational change, while suppressor mutations relieve DNA binding specificity constraints to rescue assembly arrest. PSB15 interacts with phospholipid cardiolipin via its basic helix and C-terminus, and recruits PS DNA to the inner membrane (IM). The PSB15-PS complex is released from the IM by interaction between its hydrophobic linker and thioredoxin (Trx), a host protein that is required for Ff assembly but whose mechanisms are still unclear. Live cell imaging shows that thioredoxin and DNA binding regulate the dwelling time of PSB15 at cell poles, suggesting that they both facilitate the dissociation of PSB15 from the IM. Loss of PSB15 or its PS-binding and IM-targeting/dissociation activity compromised virus egress, indicating that the PS/PSB15/Trx complex establishes a regulatory phage assembly checkpoint critical for integrative phage infection and life cycles.