E. coli Single-Stranded DNA Binding (SSB) Protein Undergoes Dynamic Liquid-Liquid Phase Separation Controlled via Protein-Protein and Protein-DNA Interactions

Abstract

A number of eukaryotic proteins have recently been shown to form phase-separated liquid condensates (membrane-less organelles) playing key roles in normal cell physiology and stress tolerance as bioreactors, biomolecular filters, stress sensors or molecular reservoirs. However, it is still debated whether liquid-liquid phase separation (LLPS) is a fundamental process in bacteria as it is in eukaryotes. Here we report the striking observation that the E. coli SSB protein, a ubiquitous central player of practically all DNA metabolic processes, forms phase-separated condensates under physiological conditions. LLPS is generally driven by multivalent weak protein-protein or protein-nucleic acid interactions, mediated mostly by intrinsically disordered protein regions. We found that LLPS by the homotetrameric SSB protein is mediated via multifaceted inter-tetramer interactions involving all protein regions including the conserved ssDNA-binding domain, the intrinsically disordered linker (IDL) and the C-terminal peptide (CTP, a highly conserved protein-protein interaction motif binding to a variety of DNA metabolic proteins). SSB, ssDNA and SSB-interacting partners are highly concentrated within the phase-separated droplets, whereas LLPS is overall regulated by the stoichiometry of SSB and ssDNA. Our bioinformatics analysis indicates that the LLPS-forming propensity of the SSB IDL is broadly conserved across all major phylogenetic groups of Eubacteria. Together with recently observed dynamic spot-like subcellular localization patterns of SSB, our results suggest that bacterial cells store an abundant pool of SSB and SSB-interacting proteins in phase-separated condensates via a conserved mechanism. The discovered features enable rapid mobilization of SSB to ssDNA regions exposed upon DNA damage or metabolic processes to serve efficient repair, replication and recombination.