Phase Transitions by an Abundant Protein in the Anammox Extracellular Matrix Mediate Cell-to-Cell Aggregation and Biofilm Formation

Abstract

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.

Microbial biofilms are shaped by the constant dialogue between biological and physical forces in the extracellular matrix

“…This protein also contains IDRs, and undergoes LLPS to produce liquid droplets under crowded conditions. These liquid droplets could wet and fuse cells, supporting the aforementioned mechanism for LLPS in promoting initial cell–cell adhesion and microcolony formation (Seviour et al, 2020) (Figure 3). In addition, the S‐layer protein also displays a predominately ß‐sheet secondary structure.…”

Surface-layer protein is a public-good matrix exopolymer for microbial community organisation in environmental anammox biofilms

“…Several mechanisms exist by which the S-layer protein could achieve this. The anammox biofilm S-layer protein possesses intrinsically disordered repeat domains (IDRs) at the C-terminus, which could facilitate phase transitions and the passaging of the S-layer protein through the matrix [45,46]. Alternatively, the glycosylated S-layer protein could adopt different proteoforms throughout the biofilm (e.g., glycosylated or unglycosylated forms) to facilitate the transition of S-layer protein to cells in the biofilm and its transformation into EPS [47][48][49].…”

Protein conformation and biomolecular condensates

“…Extracellular deposits of proteins are ubiquitous. These include but are not limited to collagen, blood clot forming fibrin, insect elastic matrix protein resilin, hinge ligament of bivalve molluscs abductin, spider and insect silks, matrix proteins of squid suckers, attachment fibers and adhesives of mussels, and bacterial biofilms ( Muiznieks et al., 2018 ; Urosev et al., 2020 ; Seviour et al., 2020 ). Only two representative extracellular protein condensates, elastin and silk, will be briefly described.…”