The house that Kocuria built: A potential role for functional amyloid in the unique cube-based structure of K. varians biofilms

Biofilm is the dominant growth mode of microorganisms, providing protection, surface adhesion and synergy between co-habiting organisms. Many aspects of biofilm formation and architecture remain unknown and vary greatly among different species. Proteins that self-assemble into highly stable extracellular cross-β structures, known as amyloids, mechanically strengthen biofilm in various species, including Escherichia coli and Pseudomonas spp. Previously, we demonstrated that the microbial strain Kocuria varians G6, isolated from a food processing facility surface in a slaughterhouse, was able to engage in multi-species biofilms. Here we investigate the biofilm formation of Kocuria varians G6. We found that K. varians G6 forms a unique biofilm structure composed of unique cube-shaped clusters, which over time expand and at a specific time-point burst to release cells that then form a biofilm covering the surface. The cube surfaces bind Thioflavin T and Proteostat, suggesting the presence of amyloid as structural components. Further support for this was obtained from (a) reduced cube size when these were exposed to epigallocatechin-3-gallate (EGCG), a compound known to prevent amyloid polymerization and (b) a dense network of thin fibrils surrounding individual cells which were sensitive to both EGCG and proteinase K. We were unable to identify the potential amyloid monomer by comparative proteome analysis of whole-cell lysates with and without traditional amyloid depolymerization, indicating that the amyloids are highly stable or simply not detected by our proteome approach. This work expands our understanding of biofilm structural diversity and indicates a previously unreported role for amyloids in spatially patterned bacterial architectures.