Exploring the function of bacterial condensates in adaptation and evolution

Project Summary Physical organization of biomolecules within the cell is critical for controlling key life processes in health and disease. It has recently been realized that in addition to membrane bound compartments such as the nucleus or mitochondria, membraneless compartments may play a pivotal role in organizing biomolecules in the cell. Called biomolecular condensates, these assemblies form by weak multivalent interactions and are known for their spherical shapes and viscoelastic behavior. A biomolecular condensate can reversibly localize or delocalize biomolecules in response to environmental cues. They can also interact with numerous biomolecules in a non- stoichiometric manner. There is growing evidence suggesting that certain proteins in bacteria may function as condensates. While they provide a means of compartmentalization for cells lacking traditional organelles, their most impactful role could be as rapid response sensors. However, studying condensates in bacteria is particularly challenging. Most evidence comes from laboratory-based reconstitution experiments that fail to accurately replicate the true cellular environment, including context-dependent interactions. Due to the lack of suitable tools, understanding the effects of condensates on phenotypes and inhibiting bacterial growth through this novel mechanism have proven difficult. Current methods to assess condensate formation rely on fluorescence microscopy, which is limited by the diffraction limit of light and cannot resolve structures smaller than approximately 250 nm. Alternatively, proximity ligation-based assays followed by mass spectrometry are often biased towards capturing strong interactions, thus neglecting dynamic interactions. In this proposal, we aim to develop a novel approach that combines modular fluorophores and correlative super-resolution microscopy to investigate the structure, dynamics, and interactome of condensates using genetically encoded tags. Our goal is to apply this opto-proteomic toolbox to understand the role of biomolecular condensates in various aspects of the bacterial cell cycle, surface colonization, and evolution within both free and host environments. This approach will be readily applicable for the study of condensates in other bacteria and beyond, while shedding light on the nanoscale mechanisms that impact macroscopic phenotypes.