High-resolution visualization of biofilm matrix development in space and time using fluorescent stains for cellulose

The establishment of microbial communities enmeshed in self-produced extracellular matrices (ECM), i.e. biofilms, raises increasing concerns for health and antimicrobial management. Understanding biofilm formation is crucial for developing effective control and eradication strategies. In response to environmental cues, planktonic bacterial cells switch to a sessile lifestyle coordinating growth with spatiotemporal matrix production. We follow here the production of the cellulose biofilm matrix of Pseudomonas sp. IsoF in real-time using single-step fluorescent stains. Live-tracking of polysaccharide production provides facile insights into biofilm ECM progression, revealing the dynamic matrix arrangements that shape the final biofilm structure. We show that cellulose determines substratum adherence, cell-to-cell contacts, and colony patterning in IsoF. We found the biofilms developed by IsoF in flow-cells and at the air-liquid interface remarkably similar in composition, progression, and architecture. Artificially raising intracellular c-di-GMP levels led to cellulose-dependent biofilm structures differing greatly from the wild type, enabling the production of a secondary exopolysaccharide. Our fluorescent probes allow real-time matrix visualization for dissecting biofilm architecture and regulation in routine laboratory settings.