Superresolution Imaging of Intact Microbial Communities Reveals Molecular Architecture of Biofilm Development and Bacterial Organization

Most bacteria live as a biofilm community in their natural habitat. This surface-attached social life form is commonly found in antibiotic-resistant infections and chronic diseases. For example, bacterial biofilms are a leading cause of lung infection and death among cystic fibrosis patients. Biofilms are also crucial for bioenergy research since cellulose degrading bacteria in the gut of termites are organized as heterogenic biofilms and believed to communicate throughout these tissue-like structures. To gain structural and molecular insight on biofilm formation, we imaged intact bacterial biofilms at different developmental stages without using any fixing agents by STORM microscope. These three-dimensional superresolution images revealed ten to twenty biofilm-promoting exopolysaccharide-rich regions that are sparsely distributed on the cell surface. A few hours after surface attachment of bacteria, these small globular structures expanded to ∼100 nm in size and protruded from the cell surface. During the initial stage of biofilm formation, we observed extensive interactions among neighboring cells through these globular exopolysaccharides. Moreover, we identified straight cable-like cell-to-cell and cell-to-substrate connections, up to 5 microns in length, originating from globular structures. These physical interactions may explain how bacteria form initial microcluster on the surface, first stage of commitment to biofilm formation. Microcluster formation depends on bacterial twitching motility on the surface and exopolysaccharide synthesis.These results are shifting the paradigm in the biofilm field which states that bacteria are randomly embedded in an extracellular matrix in biofilms. Our data suggests that bacteria actively build their house similar to a spider web by synthesizing sticky globular polysachharides on the cell surface, which are then extended to cable-like structures by twitching motility on the surface.