Prophage Activation Triggers Extracellular Synthesis of DNA in Bacterial Biofilms via Rolling Circle Replication.

Extracellular DNA (eDNA) plays crucial roles in biofilm formation and function, yet the role of bacteriophages, which can exist in a dormant state as prophages, in controlling eDNA synthesis, structure, and activity remains obscure. Here, we demonstrate that prophages harbored by environmental bacteria can be exploited for synthesis of programmable eDNA superstructures with a distinct function. We designed a custom circular template and used it to direct rolling circle replication of G-quadruplex (GQ) motifs in Shewanella oneidensis and Bacillus subtilis. Under nutrient-limiting conditions, prophage activation and proliferation triggered cell lysis and subsequent eDNA synthesis, producing multimeric GQ concatemers that self-assembled into spherical (≤10 µm) and wire-like (>50 µm) superstructures in S. oneidensis and B. subtilis, respectively. Real-time monitoring using fluorescent reporter strains revealed that DNA synthesis occurred predominantly after bacterial lysis, coinciding with prophage replication. The resulting eDNA superstructures enhanced the electrochemical properties of S. oneidensis and exhibited peroxidase activity through GQ-hemin DNAzyme formation in S. oneidensis and B. subtilis cocultured biofilm. This work unveils a previously unknown mechanism by which prophages contribute to biofilm architecture and establishes a novel biotechnological platform for engineering functional DNA materials in living bacterial communities with potential applications in synthetic biology.