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.

This study describes the first direct functional assignment of a highly abundant extracellular protein from a key environmental and biotechnological biofilm performing an anaerobic ammonium oxidation (anammox) process. Expression levels of Brosi_A1236, belonging to a class of proteins previously suggested to be cell surface associated, were in the top one percentile of all genes in the “Candidatus Brocadia sinica”-enriched biofilm. The Brosi_A1236 structure was computationally predicted to consist of immunoglobulin-like anti-parallel β-strands, and circular dichroism conducted on the isolated surface protein indicated that β-strands are the dominant higher-order structure. The isolated protein was stained positively by the β-sheet-specific stain thioflavin T, along with cell surface- and matrix-associated regions of the biofilm. The surface protein has a large unstructured content, including two highly disordered domains at its C terminus. The disordered domains bound to the substratum and thereby facilitated the adhesion of negatively charged latex microspheres, which were used as a proxy for cells. The disordered domains and isolated whole surface protein also underwent liquid-liquid phase separation to form liquid droplets in suspension. Liquid droplets of disordered protein wet the surfaces of microspheres and bacterial cells and facilitated their coalescence. Furthermore, the surface layer protein formed gels as well as ordered crystalline structures. These observations suggest that biophysical remodeling through phase transitions promotes aggregation and biofilm formation. IMPORTANCE By employing biophysical and liquid-liquid phase separation concepts, this study revealed how a highly abundant extracellular protein enhances the key environmental and industrial bioprocess of anaerobic ammonium oxidation (anammox). Extracellular proteins of environmental biofilms are understudied and poorly annotated in public databases. Understanding the function of extracellular proteins is also increasingly important for improving bioprocesses and resource recovery. Here, protein functions were assessed based on theoretical predictions of intrinsically disordered domains, known to promote adhesion and liquid-liquid phase separation, and available surface layer protein properties. A model is thus proposed to explain how the protein promotes aggregation and biofilm formation by extracellular matrix remodeling and phase transitions. This work provides a strong foundation for functional investigations of extracellular proteins involved in biofilm development.

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a “public-good” EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.

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Recent studies on the formation, organisation and dynamics of biofilms highlight the interplay between physical forces and biological programs. Two complementary generalised pathways that explain the mechanisms driving biofilm formation have emerged. In the first pathway, where physical forces precede the biological program, the initial expansion of cells leads to cell clustering or aggregation prior to the production of extracellular polymeric substances (EPS). The second pathway describes an initial biologically prompted production of EPS, which introduces new biophysical interactions within the EPS, such as by phase separation, macromolecular crowding, excluded volume interactions and intermolecular cross-linking. In practice, which of the two pathways is adopted is ultimately determined by the specificities of the biofilm and the local microenvironment, each leading to the formation of robust, viscoelastic biofilm. Within this framework, we further highlight here recent findings on the role of higher-order structures in matrix gelation and phase separation of EPS in promoting the clustering of bacteria. We assert that examining biofilms through the combined lens of physics and biology promises new and significant methodological and conceptual advancements in our understanding of biofilms.

The secondary structure of the coiled coil peptides was regulated by altering the azido content at the hydrophobic core. These peptides were further investigated to form higher-order assemblies presumably via azido-mediated interactions.