Spontaneous phase separation in Vibrio cholerae bacterial biofilms

Bacterial biofilms are ubiquitous surface-associated bacterial communities embedded in a matrix. However, how the matrix components interact with each other and with cells to scaffold the biofilm structure remains poorly understood. The biofilm matrix in Vibrio cholerae (Vc), the causative agent of pandemic cholera and our model biofilm former is primarily composed of Vibrio polysaccharide (VPS), accessary matrix proteins, and in some cases extracellular DNA. Here, we couple bacterial genetics, high-resolution imaging, and biochemistry to study the self-organization of extracellular matrix in biofilms formed by Vc. We found, surprisingly, that Vc culture undergoes spontaneous phase separation (PS), both in vitro and in vivo, since Vc cells do not possess a specific attraction to their own secreted polymers. Furthermore, by coupling single-cell imaging techniques with immunostaining, we followed the evolution of VPS structures during PS. Our approach addresses the critical need to understand the formation of bacterial biofilms to posteriori use new strategies to disrupt and/or eliminate biofilms by interfering with the PS process.