Uncovering the Molecular Composition and Architecture of the Bacillus subtilis Biofilm via Solid-State NMR Spectroscopy.

The complex and dynamic compositions of biofilms, along with their sophisticated structural assembly mechanisms, endow them with exceptional capabilities to thrive in diverse conditions that are typically unfavorable for individual cells. Characterizing biofilms in their native state is significantly challenging due to their intrinsic complexities and the limited availability of noninvasive techniques. Here, we utilized solid-state nuclear magnetic resonance (NMR) spectroscopy to analyze Bacillus subtilis biofilms in-depth. Our data uncover a dynamically distinct organization within the biofilm: a dominant, hydrophilic, and mobile framework interspersed with minor, rigid cores of limited water accessibility. In these heterogeneous rigid cores, the major components are largely self-assembled. TasA fibers, the most robust elements, further provide a degree of mechanical support for the cell aggregates and some lipid vesicles. Notably, rigid cell aggregates can persist even without the major extracellular polymeric substance (EPS) polymers, although this leads to slight variations in their rigidity and water accessibility. Exopolysaccharides are exclusively present in the mobile domain, playing a pivotal role in its water retention property. Specifically, all water molecules are tightly bound within the biofilm matrix. These findings reveal a dual-layered defensive strategy within the biofilm: a diffusion barrier through limited water mobility in the mobile phase and a physical barrier posed by limited water accessibility in the rigid phase. Complementing these discoveries, our comprehensive, in situ compositional analysis is not only essential for delineating the sophisticated biofilm architecture but also reveals the presence of alternative genetic mechanisms for synthesizing exopolysaccharides beyond the known pathway.