Biofilm Morphology and Growth

Bacterial biofilms represent basic forms of multicellular organization that confer survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae. It is unknown how spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion. While lineage and cell trajectory maps have been made for large eukaryotic systems such as embryos, no such maps exist for bacterial biofilms. Tracing lineages and spatial trajectories of bacterial cells in biofilms is essential to understanding biofilm development and to identifying the underlying biological and physical principles governing multicellular development. A roadblock in this undertaking is that conventional confocal microscopy is insufficient to achieve the spatial and temporal resolution required to map cell paths in densely packed biofilms containing micrometer-sized bacteria.

We developed dual-view light-sheet microscopy to investigate the dynamics of biofilm development from a founder cell to a mature three-dimensional community. Tracking of individual cells revealed two distinct fates: one set of biofilm cells expanded ballistically outward, while the other became trapped at the substrate. A collective fountain-like flow transported cells to the biofilm front, bypassing members trapped at the substrate and facilitating lateral biofilm expansion. This collective flow pattern was quantitatively captured by a continuum model of biofilm growth against substrate friction. Coordinated cell movement required the matrix protein RbmA, without which cells expanded erratically. Thus, tracking cell lineages and trajectories in space and time revealed how multicellular structures form from a single founder cell. The collective cellular flow patterns and cell-matrix substrate interaction principles may be relevant in other prokaryotic and eukaryotic systems and could underpin multicellular development and organization.