The biofilm matrix scaffold of Pseudomonas aeruginosa contains G-quadruplex extracellular DNA structures

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.


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Pseudomonas aeruginosa wild type and RSCV.  Fig 5B).

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Oscillating rheograms ( Figure 1C, Supp Figure 1) are now presented in terms of tan (δ) versus frequency, in order to simplify their interpretation, where tan (δ) is defined as the ratio of viscous to elastic response (in response to point 4). tan (δ) values less than 1 indicate that elasticity dominates the viscoelastic properties of the material, which is consistent with a gel. tan (δ) is presented as mean +/-standard deviation for three biological replicates. Normal force measurements were interpreted more quantitatively, to demonstrate that eDNA was solubilized and that eDNA dominated the elastic response of the dissolved biofilm. This was achieved by fitting the data to a polymer model used to describe flexible/semi-flexible polymers in solution, and calculating the power law dependency of normal force difference on shear rate (which also indicated that the dominant polymer in solution was semi-flexible like DNA). Results from triplicate analyses were therefore required to fit the model parameters (i.e. n = 3 The antibody used was the anti-DNA G-quadruplex (G4) antibody, clone 1H6 (Sigma-Aldrich), which binds to tetramolecular and unimolecular DNA G-quadruplex structures without sequence specificity and does not bind to either non-G4 ssDNA or dsDNA (Henderson et al. Detection of G-quadruplex DNA in mammalian cells).

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