Extracellular DNA (eDNA) plays crucial roles in biofilm formation and function, yet the role of bacteriophages (phages) in controlling eDNA synthesis, structure and activity remains obscure. Here, we demonstrate that phages harbored by environmental bacteria can be exploited for programmable synthesis of functional eDNA superstructures. We designed a 112-nucleotide circular template (T1) and used it to direct rolling circle replication (RCR) of G-quadruplex (GQ) motifs in Shewanella oneidensis and Bacillus subtilis . Under nutrient-limiting conditions, prophage activation triggered cell lysis and subsequent extracellular DNA synthesis, producing multimeric GQ concatemers that self-assembled into distinct morphologies: spherical structures (≤10 μm) in S. oneidensis and wire-like structures (>50 μm) in B. subtilis . Real-time monitoring using fluorescent reporter strains revealed that DNA synthesis occurred predominantly after bacterial lysis, coinciding with prophage replication. The resulting DNA superstructures exhibited peroxidase activity through GQ-hemin DNAzyme formation and enhanced the electrochemical properties of S. oneidensis biofilms, showing a 3-fold increase in current density. This work unveils a previously unknown mechanism by which prophages contribute to biofilm architecture and establishes a biotechnological platform for engineering functional DNA materials in living bacterial communities, with potential applications in biotechnology and synthetic biology. ![Figure][1]</img> ### Competing Interest Statement The authors have declared no competing interest. Villum Fonden, https://ror.org/05nqkay65, 00050284 Danmarks Frie Forskningsfond, https://ror.org/05svhj534, 2032-00294B, 9040-00323B Carlsberg Foundation, https://ror.org/01kpjmx04, CF20-0364 Novo Nordisk Foundation, https://ror.org/04txyc737, NNF20OC0061417 [1]: pending:yes

Extracellular DNA (eDNA) plays crucial roles in biofilm formation and function, yet the role of bacteriophages (phages) in controlling eDNA synthesis, structure and activity remains obscure. Here, we ...

Extracellular DNA (eDNA) plays crucial roles in biofilm formation and function, yet the role of bacteriophages (phages) in controlling eDNA synthesis, structure and activity remains obscure. Here, we ...

Published in Plasmid

Given that prophages are particularly overlooked and merit increased attention due to their vital implications for microbiomes and their hosts, we created Prophage-DB to advance our understanding of prophages in microbiomes through a comprehensive characterization of prophages in publicly available …

Gastrointestinal health has a significant impact on quality of life, and increasing profiling of the gut microbiome is identifying key players involved in disease states. However, evidence of the asso...

Gastrointestinal health has a significant impact on quality of life, and increasing profiling of the gut microbiome is identifying key players involved in disease states. However, evidence of the asso...

Abstract. Transformation occurs when bacteria import exogenous DNA via the competence machinery and integrate it into their genome through homologous recom

Despite bifidobacteria playing essential roles in human and infant health, large-scale studies of their interactions with phages are scarce. Docherty et al. uncover a diverse repertoire of antiviral defenses, including phage-specific CRISPR-Cas and prophage counter-defenses. The observed coevolutionary dynamics shape genomic diversity and may have potential biomedical and commercial relevance.