Extracellular matrix and spores impart structural hierarchy and compositional heterogeneity in Bacillus subtilis biofilms

Biofilms are surface-associated soft microbial communities that may be beneficial or detrimental to their hosting environment. During biofilm development, cells protect themselves with polymeric matrix, termed the extra-cellular matrix (ECM), and organize in space into functionally distinct subgroups, in a process resembling differentiation in higher organisms. An interesting question arising is whether this division of labor in biofilms is also reflected in the molecular-level order across whole biofilms. Using combined X-ray diffraction/fluorescence, we studied the molecular order in intact Bacillus subtilis biofilms. We discovered that biofilms display a distinct spatio-temporal XRD signature that depends on highly ordered structures in spores and on cross-[beta]; sheet structures in matrix components. Furthermore, signals of spores, water molecules and metal-ions are especially enhanced along macroscopic wrinkles, known to act as water channels. Demonstrating in situ the link between molecular structures, distribution of metal-ions, and division of labor across whole biofilms in time and space, this study provides novel insight that is crucial for better understanding of biofilm development and physiology.