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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Anaerobic ammonium oxidation (anammox) performing bacteria self-assemble into compact biofilms by expressing extracellular polymeric substances (EPS). Anammox EPS are poorly characterized, largely due to their low solubility in typical aqueous solvents. Pronase digestion achieved 19.5 ± 0.9 and 41.4 ± 1.4% (w/w) more solubilization of Candidatus Brocadia sinicaenriched anammox granules than DNase and amylase respectively. Nuclear magnetic resonance profiling of the granules confirmed that proteins were dominant. We applied ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and N,N-dimethylacetamide (EMIM-Ac/DMAc) mixture to extract the major structural proteins. Further treatment by anion exchange chromatography isolated homologous S/T-rich proteins BROSI_A1236 and UZ01_01563, which were major components of the extracted proteins and sequentially highly similar to putative anammox surface-layer (S-layer) protein KUSTD1514. EMIM-Ac/DMAc extraction enriched for these putative S-layer proteins against all other major proteins, along with six monosaccharides (i.e. arabinose, xylose, rhamnose, fucose, galactose and mannose). The sugars, however, contributed <0.5% (w/w) of total granular biomass, and were likely coenriched as glycoprotein appendages. This study demonstrates that S-layer proteins are major constituents of anammox biofilms and can be isolated from the matrix using an ionic liquidbased solvent.