Abstact

Filamentous Pf bacteriophages are widely distributed in Pseudomonas aeruginosa and profoundly influence biofilm formation and host virulence. The Pf4 prophage encodes a type II toxin-antitoxin (TA) system, PfiAT, modulating Pf4 propagation; however, its mechanistic role remains unclear. Here, through structural and biochemical analysis, we demonstrate that the PfiT toxin (ParE/RelE superfamily) has a unique C-terminal extension essential for TA complex formation. The antitoxin PfiA harbors a previously uncharacterized DNA binding domain, and its phosphorylation during biofilm formation shifts the PfiAT complex stoichiometry from a noncanonical PfiA(6)PfiT(4) to a canonical PfiA(2)PfiT(2) assembly. This phosphorylation is mediated by the prophage Pf6-encoded kinase toxin PfkA/PfkB at T5 in PfiA's DNA binding domain. This posttranslational modification eliminates the pool of free toxins through complex reorganization, thereby neutralizing PfiT toxicity and enabling rapid Pf4 propagation during P. aeruginosa biofilm development. This study uncovers the cross-talk of TA systems from two coresident prophages and the role of posttranslational modification of TA system in mediating phage-phage and phage-host dynamics.