Single-Cell Microscopy Reveals That Levels of Cyclic di-GMP Vary among Bacillus subtilis Subpopulations

Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.