Vibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V. cholerae infections are characterized by the co-occurrence of cells with motile and sessile behaviors, but the determinant of this phenotypic diversity remains poorly understood. We used single-cell tracking to examine the motile behaviors of two V. cholerae strains (El Tor C6706 and Classical O395) in response to direct manipulations of c-di-GMP concentration. Both motile and non-motile cells are present in a well-mixed batch culture with distributions of these phenotypes that change depending on the growth phase. We determined that the proportion of motile cells differs between El Tor and Classical strains because they maintain different levels of c-di-GMP. However, even in conditions that promote biofilm formation, V. cholerae still generates a sub-population of motile cells. C-di-GMP is known to inversely regulate assembly of mannose-sensitive haemagglutinin (MSHA) pili and flagella. We found that the most cells in clonal populations are flagellated, but MSHA piliation affords the variable opportunity to attach at single-cell level, driving behavioral switching between motile and sessile behaviors. Our results support the hypothesis that c-di-GMP regulates phenotypic diversity in V. cholerae, and it does so by simultaneous elaboration of MSHA pili and flagellum.
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