Biophysical mechanisms of phenotypic segregation in bacterial biofilms*

Biofilms are ubiquitous surface-adhered communities of bacteria, encased within extracellular matrices of biopolymers, that play many significant roles in health, nature, and industry. Recent breakthroughs in experimental techniques have enabled the detailed characterization of the fundamental mechanical interactions that shape biofilm development, but how phenotypic heterogeneity—which is known to be widespread in microbial communities—influences biofilm development remains largely unknown. Here, we discuss our use of agent-based models (ABMs) to identify the biophysical mechanisms through which spatiotemporal heterogeneity in the signaling of a key intracellular second-messenger, cyclic di-GMP (c-di-GMP), can affect the organization of Vibrio cholerae biofilms. Cyclic di-GMP controls the planktonic-to-biofilm transition in many bacteria, by upregulating biofilm-related phenotypes (e.g., matrix production) while downregulating planktonic phenotypes (e.g., flagella assembly). Recent work from our laboratory has revealed that, contrary to the classical assumption that biofilm formation is a coordinated process, cells within growing V. cholerae biofilms exhibit significant spatiotemporal heterogeneity in c-di-GMP levels, and that this phenotypic heterogeneity gives rise to phenotypic segregation: high-c-di-GMP cells are enriched within the biofilm core, whereas low-c-di-GMP cells occupy the periphery. We have used ABMs to identify the biophysical mechanisms that can (and cannot) give rise to this phenotypic segregation. Our findings demonstrate how molecular signaling and mechanical interactions can come together to shape biofilm development, in a manner echoing the development of more complex multicellular collectives such as eukaryotic tissues.