Abstract:

The poles of rod-shaped bacteria emerge as regulatory hubs. We have shown that enzyme I (EI), the major bacterial sugar metabolism regulator, is sequestered when not needed in TmaR phase-separated condensates in Escherichia coli cell poles. Here, we combined genetic and automated microscopy screens to identify residues in EI and TmaR that are important for their interaction and colocalization. Mutating these residues affects EI-TmaR interaction in bacteria and impairs co-phase separation in yeast. The results were used to generate an EI-TmaR interaction model, which agrees with coevolution data and is supported by conservation of the interacting residues and EI-TmaR colocalization in other species. Mutating residues predicted to interact electrostatically further supports our model. The model explains how TmaR controls EI activity and its interaction with the phosphoprotein HPr and, hence, sugar uptake. Our study highlights the importance of sugar metabolism spatial regulation during evolution and presents a way to unravel protein-protein interactions.