Quantitative Imaging and Analysis of Bacterial Biofilms from the Single Cell to the Collective

PROJECT ABSTRACTIt is now understood that in their natural environments, bacteria primarily exist in multicellular, surface-boundcommunities called biofilms. Biofilms cause major problems in medicine as they are inherently resistant toantibiotics and cause chronic infections; in industry, biofilms foul surfaces and clog filtration devices. Cells inbiofilms display striking differences from planktonic cells, such as extracellular matrix production, a 1,000-foldincrease in tolerance to antibiotics, and unique gene expression patterns that are specific to particular locationswithin the biofilm. Because biofilms are three dimensional, heterogeneous, and rearrange over time,investigations have been limited to optical studies of biofilm formation when only few cells are present or to grosscharacterization of the entire structure. We recently made a research breakthrough: We resolved the individualcells in living, growing biofilms up to a depth of 30 microns, using customized spinning-disk confocal microscopy,fluorescent reporters, and automated cell-segmentation software. This is the first time anyone has peered 'into'a biofilm, to watch it develop, cell by cell, in the presence of flow, under conditions that model environmental,medical, and industrial systems. Thus, we are in a position to use three-dimensional imaging, combined with keytechnological advancements they propose to make in photo-activation and optogenetics, to characterize biofilmsfrom the gene to the genome and from the cell to the collective. Central questions to be addressed for the firsttime include how do quorum sensing and genome-wide expression profiles vary in space and time within growingbiofilms? Experimental design and interpretation of measurements will be guided by biophysical modeling. Wewill launch the studies with the human pathogen Vibrio cholerae, known for rapid but transient biofilm formation.Specifically, we will pioneer a comprehensive examination of biofilm formation, development, and signaltransduction from the single-cell to multi-cell levels and in realistic environments that mimic the spatial, temporal,and physical constraints found in nature. The interdisciplinary work will lead to understanding of gene regulation,cell-to-cell communication, and the spatial and temporal organization of biofilms, which in turn, dictate the large-scale features and ecological fitness of these multicellular systems. The proposal is unusually interdisciplinary:it teams Bassler, a microbiologist who is a leader in quorum sensing and biofilms, with Stone, an engineer whosefocus is imaging, fluid dynamics, and the modeling of transport processes, and Wingreen, a theoreticalbiophysicist who models bacterial signaling circuits and biofilm development. The approach of direct imaging,beyond connecting genetics to biophysics, promises new insights relevant to understanding and manipulatingbiofilms with the goal of improving human health.