Biology subjects

Minero, G. A. S.

Publications and source records attributed to Minero, G. A. S..

5 recordsLinked to original sources

bioRxiv2026

Unmasking the diversity of extracellular nucleic acids in the biofilm matrix using nucleic acid-binding dyes

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven membrane-impermeant dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms. HIGHLIGHTS- Biofilms contain non-canonical structures of extracellular DNA and RNA - This study tests the ability of DNA-binding dyes to visualise such structures - Propidium iodide visualises RNA with brighter fluorescence than DNA - Red-fluorescent dyes were more versatile than green-fluorescent dyes - Combining several dyes enabled the detection of non-canonical structures

microbiology↗

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

Hemin-binding DNA structures on the surface of bacteria promote extracellular electron transfer

Recent research has shown that bacteria in anoxic layers of Pseudomonas aeruginosa biofilms can respire by transferring electrons to oxygen via extracellular DNA (eDNA) and DNA-binding redox mediators that are unique to this species1. In this study, we propose a similar but generic mechanism by which bacteria can transfer electrons via DNA in biofilms, using hemin as a redox-mediator and hemin-binding G-quadruplex (G4) DNA structures in the extracellular matrix. Using Staphylococcus epidermidis as a model organism, voltammetry showed that eDNA and hemin were needed for extracellular electron transfer (EET). Surface-associated G4-DNA formed a complex with hemin, which transferred electrons from the bacteria to an electrode under anoxic conditions. Addition of G4-DNA and hemin to growing biofilms promoted EET which was stable for days. G4-DNA/hemin is also a peroxidase-like DNAzyme, capable of transferring electrons from bacteria to H2O2. G4-DNA were only recently discovered to be abundant in the extracellular matrix of biofilms2,3. We now show that hemin turns these structures into conduits for EET. The study opens the door to new and generic mechanisms for bacterial energy conservation under oxygen-limiting conditions, and for tackling H2O2, a common host defense mechanism against bacterial infections.

microbiology↗

bioRxiv2023

Bacterial efflux pumps excrete SYTO-TM dyes from bacteria and lead to false-negative staining results

Multidrug efflux pumps excrete a range of small molecules from bacterial cells. In this study, we show that bacterial efflux pumps have affinity for a range of SYTO dyes that are commonly used to label bacteria. Efflux pump activity will there lead to false negative results from bacterial staining and SYTO dyes should be used with caution on live samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/560001v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1b8bab4org.highwire.dtl.DTLVardef@e9a84forg.highwire.dtl.DTLVardef@291058org.highwire.dtl.DTLVardef@1f0245d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Extracellular G-quadruplex and Z-DNA protect biofilms from DNase I and forms a DNAzyme with peroxidase activity

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often arises from lysed cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplex formation might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media amended with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structure in biofilms grown in vitro and in vivo using a murine implant-associated osteomyelitis model. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt, and we confirmed that G-quadruplex DNA and Z-DNA was also present in biofilms from infected implants. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/541711v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@c32012org.highwire.dtl.DTLVardef@6e8c7eorg.highwire.dtl.DTLVardef@1c9cc6corg.highwire.dtl.DTLVardef@18be51e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗