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bioRxiv · 10.64898/2025.12.02.691978

Secret life of prophages: template-directed synthesis of DNA superstructures via prophage activation and rolling circle replication in bacterial biofilms

Minero, G. A. S.·Lund, L. M.·Klausen, L. H.·Ajunwa, O. M.·Dong, M.·Meyer, R. L.·Birkedal, V.·Thormann, K.
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Abstract

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 m) in S. oneidensis and wire-like structures (>50 m) 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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/691978v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@aec065org.highwire.dtl.DTLVardef@d94709org.highwire.dtl.DTLVardef@c6c228org.highwire.dtl.DTLVardef@f4f465_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Minero, G. A. S., Lund, L. M., Klausen, L. H., Ajunwa, O. M., Dong, M., Meyer, R. L., Birkedal, V., Thormann, K.. 2025-12-03. Secret life of prophages: template-directed synthesis of DNA superstructures via prophage activation and rolling circle replication in bacterial biofilms. https://doi.org/10.64898/2025.12.02.691978

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