Minero, G. A. S. | Search bioRxiv

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↗