Extracellular polysaccharides are crucial components for biofilm development. Although Bacillus subtilis is one of the most characterized Gram-positive biofilm model system, the structure-function of its exopolysaccharide, EpsA-O, remains to be elucidated. By combining chemical analysis, NMR spectroscopy, rheology, and molecular modeling, high-resolution data of EpsA-O structure from atom to supramolecular scale was obtained. The repeating unit is composed of the trisaccharide backbone [-3)-α-D-GlcpNAc-(1-3)-β-D-QuipNAc4NAc-(1-3)-β-D-GalpNAc-(1-]n, and the side chain β-D-Galp(3,4-S-Pyr)-(1-6)-β-D-Galp(3,4-S-Pyr)-(1-6)-α-D-Galp-(1- linked to C4 of GalNAc. Close agreement between the primary structure and rheological behavior allowed us to model EpsA-O macromolecular and supramolecular solution structure, which can span the intercellular space forming a gel that leads to a complex 3D biofilm network as corroborated by a mutant strain with impaired ability to produce EpsA-O. This is a comprehensive structure-function investigation of the essential biofilm adhesive exopolysaccharide that will serve as a useful guide for future studies in biofilm architecture formation.
