Crosstalk between inovirus core gene and accessory toxin-antitoxin system mediates polylysogeny

Polylysogeny, the harboring of multiple prophages within a single bacterial genome, is common among bacterial pathogens and enhances virulence and genome plasticity. Inoviruses (filamentous phages) are often present in multiple copies in major pathogens, leading to polylysogeny. Two highly similar filamentous phages (Pf4 and Pf6) are integrated into the widely distributed model Pseudomonas aeruginosa strain, and both prophages are activated during biofilm formation. It remains unclear whether the two prophages function competitively or cooperatively. Here, we show a crosstalk between Pf4’s core region protein RepG4 (PA0717) and Pf6’s accessory KKP (kinase-kinase-phosphatase) toxin-antitoxin module that coordinates their propagation. RepG4, involved in Pf4 phage replication, triggers kinase-mediated toxicity of KKP in a dose-dependent manner by degrading the phosphatase antitoxin. This crosstalk serves as a molecular brake, preventing excessive Pf4 production and coordinating the release of both Pf4 and Pf6 phages during biofilm maturation. Our findings provide valuable insights into the significance of the tight regulation between phage core genes and accessory genes in establishing a mutualistic interaction between co-resident prophages.