W. Seth Childers, Dylan T. Tomares, Samuel W. Duvall, Kimberly A. Kowallis, Sara Whitlock, Michael J Collins, Wei Zhao, Chao Zha,  University of Pittsburgh, Department of Chemistry

One defining difference between bacteria and eukaryotes is the absence of membrane-bound organelles in bacteria. Recently, phase separation of proteins as biomolecular condensates has been recognized as a fundamental way to organize subcellular space as a “membraneless organelle.”Here, we will describe our discoveries of how biomolecular condensates organize and regulate mRNA decay and signal transduction processes in bacteria. Overall, our discoveries further suggest a new understanding of the bacterial cytoplasm as a crowded "bag of biomolecular condensates".

One significant challenge in cell biology is understanding if these membraneless organelles have any functional significance? To consider the function of biomolecular condensates in vivo, the Childers lab engineered a fluorescence biosensor imaging strategy to visualize changes in histidine kinase structure with subcellular resolution. Application of this engineered FRET biosensor visualized that cell pole localized membraneless organelles alter the structure of a critical cell-fate determining protein. Thus, this FRET biosensor strategy provides a strategy to demonstrate the functional significance of biomolecular condensates. It may also be utilized to map signals perceived by two-component systems.

Given our new understanding of the molecular organization of the bacterial cytoplasm, we considered the potential of biomolecular condensate in bacterial synthetic biology. Towards this goal, we revisited the phase properties of a family of ABC triblock peptide nanomaterials that forms hydrogels for drug delivery at high weight percent. We found ABC triblock proteins assemble as gel-like biomolecular condensates at low micromolar concentrations in vitro. Moreover, expression of the coiled-coil triblock peptides in bacteria leads to cell pole accumulation that could sequester clients at the cell pole with the potential to serve as synthetic membraneless organelles in bacteria. In summary, our studies suggest that phase separation allows bacteria to form membraneless organelles that alter our view of bacterial cell biology and present new opportunities in synthetic biology and new antibiotic targets.