Robust Modulation of a Bacterial Kinase by Protein Phase Separation

Abstract

The diversity of molecular function and material properties in cytoplasmic biomolecular condensates contributes to the robustness of enzymatic interactions in a crowded cellular environment. However, molecular principles that organize and control chemical reactions in these condensates are poorly understood. We utilized a multi-scale approach combining biochemical reconstitution and single molecule imaging to gain insights into a kinase signaling reaction localized in a phase separated polar microdomain in the Gram-negative bacterium Caulobacter crescentus. Caulobacter divides asymmetrically to yield a sessile stalked cell and a flagellated swarmer cell. Much of this asymmetric division is regulated through different signaling pathways that reside in microdomains at the two cell poles prior to cell division. Specifically, at the stalked pole, two intrinsically disordered proteins, PopZ and SpmX, form a stable complex and sequester the histidine kinase DivJ whose autophosphorylation contributes to the regulation of cell cycle progression. Here, I will discuss the biophysical basis for phase separation of SpmX and PopZ and the implications of this phenomenon in the context of the Caulobacter cell cycle. Using biochemistry and state of the art 3-D single molecule imaging, we have identified molecular features that regulate DivJ diffusion and autophosphorylation in the polar microdomain. We further establish the role of protein topology, crowding, and the extracellular environment on DivJ regulation via phase separation. Our results underscore the relationship between enzyme activity and unstructured biomolecular condensates that govern the physical environment in signaling hubs for robust modulation of biochemical reactions a crowded cytoplasm.