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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.

Here we provide a comprehensive review of the knowledge base of soft tribology, the study of friction, lubrication, and wear on deformable surfaces, with consideration for its application toward oral tribology and food lubrication. Studies on “soft‐tribology” have emerged to provide knowledge and tools to predict oral behavior and assess the performance of foods and beverages. We have shown that there is a comprehensive set of fundamental literature, mainly based on soft contacts in the Mini‐traction machine with rolling ball on disk configuration, which provides a baseline for interpreting tribological data from complex food systems. Tribology‐sensory relationships do currently exist. However, they are restricted to the specific formulations and tribological configuration utilized, and cannot usually be applied more broadly. With a careful and rigorous formulation/experimental design, we envisage tribological tools to provide insights into the sensory perception of foods in combination with other in vitro technique such as rheology, particle sizing or characterization of surface interactions. This can only occur with the use of well characterized tribopairs and equipment; a careful characterization of simpler model foods before considering complex food products; the incorporation of saliva in tribological studies; the removal of confounding factors from the sensory study and a global approach that considers all regimes of lubrication.

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Soft tribology is used to probe the lubrication behaviour of molten chocolate between soft contacts, analogous to in-mouth interactions between the tongue and palate. Molten chocolate is a concentrated suspension...

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17While the array of emergent properties assigned to biofilms is extensive (e.g. antimicrobial 18 tolerance), the mechanisms that underpin these are largely unknown. In particular, the 19 extracellular matrix, a defining feature of biofilms, remains poorly understood in terms of its 20 composition and contribution to biofilm structure and function. Here we demonstrate that 21 extracellular DNA exists in a complex with RNA that forms the main cross-linking exopolymer 22 of Pseudomonas biofilms, and explains biofilm elasticity. The RNA has a high purine content 23 and our solid-state NMR data indicate the formation of Hoogsteen guanine base pairs. This may 24 suggest the presence of G-quadruplexes, which is also corroborated by the enhancement of 25 biofilm formation in the presence of potassium. The finding that non-canonical interactions 26 mediate networking of matrix-forming extracellular nucleic acids addresses how eDNA is 27 organized and contributes to matrix biophysical properties. This understanding will allow for the 28 development of more effective biofilm control strategies. 29 30 42 traits in P. aeruginosa biofilms, including three exopolysaccharides (Colvin et al., 2012), four 43 proteins (Allesen-Holm et al., 2006; Borlee et al., 2010; Seviour et al., 2015a) and extracellular 44 DNA (eDNA) (Okshevsky and Meyer, 2015). Each putative exopolysaccharide (Colvin et al., 45 2012) has been identified as a primary structural agent, suggesting the existence of functional 46 redundancy. Other exopolymers have multiple roles (Irie et al., 2012) and a wide range of 47 2014). We undertook to identify the foundation polymer/s in P. aeruginosa biofilms, which are 54 defined here as those that either dominate biofilm elasticity or constitute the primary structural 55 agent/s. 56 Results 57 eDNA dominates the elastic response of Pseudomonas aeruginosa. 58 To characterize the foundation polymer of P. aeruginosa biofilms we exploited the reported 59 ability of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate (EMIM-Ac) to dissolve a range 60 of recalcitrant biopolymers, including DNA(Zhao, 2015) and cellulose (Vitz et al., 2009), which 61 led us to demonstrate this also for P. aeruginosa biofilm exopolymers (Seviour et al., 2015b). 62Here, when P. aeruginosa biofilms were dissolved in EMIM-Ac, the subsequent fluid was highly 63 viscoelastic. We measured non-linear elasticity as a shear-rate dependent high normal stress 64 difference (N 1 -N 2 ), where N 1 and N 2 are primary and secondary normal stress differences 65 respectively. Elasticity dominated the viscous flow properties for the wild type biofilm in 66 EMIM-Ac, with (N 1 -N 2 ) an order of magnitude greater than shear stress ( Figure 1A; Wild type). 67 The solvent (EMIM-Ac) alone exhibited no elasticity, indicating that the elastic properties are 68 transferred to the EMIM-Ac from the biofilm matrix. Viscosity was slightly shear-thinning 69 (Supplementary Figure 1A and Supplementary Table 1), which would be expected from dilute 70 polymer soluti...