Lund, L. M. | Search bioRxiv

bioRxiv2025

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

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

microbiology↗

bioRxiv2024

Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we identify a mechanistic link between ECM stiffness and pHi dynamics in driving vasculogenic mimicry (VM), an aggressive cancer phenotype associated with poor prognosis. We performed single-cell imaging of pHi in lung and breast metastatic cell lines cultured on tunable-stiffness hydrogel systems. We used two tunable-stiffness hydrogel systems to independently model stiffness induced by increased protein secretion (Matrigel) and increased protein crosslinking (Hyaluronic acid gels). We show that increased ECM stiffness lowers single-cell pHi in both lung and breast metastatic cell lines. We also observed that stiff ECM promotes a distinct morphological phenotype called vasculogenic mimicry (VM). Importantly, we show that low pHi is a necessary mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes. We also find that lowering pHi on soft ECM was sufficient to induce VM in the absence of extracellular stiffening. We characterized {beta}-catenin as a pH-dependent molecular mediator of VM, where stiffness-driven increases in {beta}-catenin abundance can be overridden by high pHi, which destabilizes {beta}-catenin to reduce VM on stiff ECM. In contrast, the transcription factor FOXC2 is activated by ECM stiffness but is insensitive to pHi, and its activity alone is insufficient to maintain VM at high pHi when {beta}-catenin is lost. We uncover a novel mechanotransduction axis in which ECM stiffness regulates intracellular pH to drive {beta}-catenin-induced VM. We also show pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment. Thus, our work positions pHi as an integrator of mechanotransduction in cancer, suggesting a new framework for therapeutically targeting pHi in cancer and perhaps in other diseases driven by ECM remodeling.

cell biology↗