Biophysical traits of surface (S-) layer protein involved in biofilm matrix assembly and function

Abstract

Extracellular proteins play crucial roles in bacterial biofilm formation, serving as cellular adhesives, structural scaffolds, and matrix stabilizers. However, many of these proteins remain uncharacterized due to a lack of reference protein databases, challenges in extracellular polymer extraction, and disagreement on the best biophysical methods for resolving functions. Direct functional characterization of isolated exoproteins remains the only strategy available for understanding their role in biofilm matrix formation in different microbial systems. In this study, the ionic liquid (IL), 1-ethyl-3-methylimidazolium acetate, was employed to extract the major extracellular protein from anaerobic ammonium oxidation (anammox) bacteria biofilms. The extract contained a glycoprotein which was homologous to a putative surface (S-) layer protein identified from Candidatus Kuenenia stuttgartiensis, KUSTD 1514. Structural characterization demonstrated that the exoprotein adopted an anti-parallel β-sheet secondary structure with an unstructured C-terminus containing two highly disordered domains. The recombinantly produced disordered domains and biofilm exoprotein isolate underwent liquid-liquid phase separation (LLPS) to form liquid condensates in suspension. Additionally, the disordered domains bound to and facilitated the aggregation of negatively charged latex microspheres and bacteria, indicating their molecular adhesive property. Immunofluorescence and fluorescence in situ hybridization (FISH) staining methods demonstrated that the extracellular glycoproteins form envelopes around the anammox cells, supporting their identity as a S-layer protein. The S-layer protein also coated a member of filamentous non-S-layer protein producing Chloroflexi bacteria, surrounding the anammox bacteria in the biofilm granules. Therefore, the S-layer protein is likely transported through the matrix as an extracellular polymeric substance (EPS) to serve as an anammox biofilm structural scaffold stabilizer. Collectively, the anammox exoprotein isolates formed gels and ordered crystalline structures, and the observation of various forms of S-layer protein on anammox cells suggests that the exoprotein can exist in multiple phases for biofilm formation.