Phosphorylation of PfiA modulates Pf4 phage production through PfiA/PfiT stoichiometric reconfiguration in <i>Pseudomonas aeruginosa</i>.

Abstract

Filamentous Pf bacteriophages are widely distributed in <i>Pseudomonas aeruginosa</i> 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 PfiA₆PfiT₄ to a canonical PfiA₂PfiT₂ 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 <i>P. aeruginosa</i> 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.