Spontaneous phenotypic heterogeneity in c-di-GMP signaling within developing Vibrio cholerae biofilms generates a spatially sorted pattern through differential drag mechanisms, wherein high-c-di-GMP cells experience greater matrix-mediated friction and remain in the biofilm core while low-c-di-GMP cells are preferentially transported to the periphery and continuously shed as planktonic cells, thereby enabling bet-hedging that enhances fitness in fluctuating environments.

How phenotypic heterogeneity shapes biofilm architecture and development remains poorly understood. Motivated by this, we developed imaging tools to track intracellular levels of c-di-GMP, a key second messenger that controls the motile-to-sessile transition, in developing Vibrio cholerae biofilms at single-cell resolution. We show that c-di-GMP levels spontaneously bifurcate into a bimodal distribution that forms a spatially sorted pattern: High-c-di-GMP cells dominate the biofilm core while low-c-di-GMP cells localize to the periphery. Combining single-lineage tracing, mutant analysis, and agent-based modeling, we reveal that this pattern arises from differential viscosity and surface friction mediated by matrix-dependent interactions between cells and their microenvironments. We demonstrate that this heterogeneity and phenotypic sorting enable continuous emergence and shedding of planktonic cells, enhancing fitness in fluctuating environments. Our findings uncover a differential drag mechanism for pattern formation in multicellular systems, and expand the classical picture of biofilm lifecycle by highlighting the functional significance of phenotypic heterogeneity.