Vibrio cholerae biofilm growth program and architecture revealed by single cell live imaging
Significance Biofilms are surface associated bacterial communities embedded in an extracellular matrix. Connections between biofilm architectural, material, and mechanical features have never been systematically studied at the individual cell level due to inadequate optical resolution. Here, we develop imaging, experimental, and modeling tools to analyze living, growing bacterial biofilms at single cell resolution. We discover that Vibrio cholerae biofilms undergo a 2D to 3D transition as a consequence of directional cell division and anisotropic pressure caused by cell to surface adhesion. Moreover, deletion of a single gene responsible for cell to cell adhesion changes the biofilm growth mode from directional cell growth to expansion caused by the extracellular matrix. The technology reported here enables future studies of single cell gene expression in bacterial communities. Biofilms are surface associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state of the art microscopy techniques to enable live single cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA. Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell to cell heterogeneity in response to external stimuli.
Proceedings of the National Academy of Sciences