Phase transition drives bacterial single-stranded DNA binding (SSB) protein mobilization during stress response and metabolic adaptation

Single-stranded DNA-binding proteins (SSBs) are ubiquitous factors of genome metabolism, recently recognized for their ability to undergo liquid–liquid phase separation (LLPS). While Escherichia coli SSB (EcSSB) has emerged as a model for bacterial LLPS in vitro , its phase behavior and functional dynamics in vivo have remained unexplored. Here, we define the subcellular organization of EcSSB under diverse physiological and stress conditions using super-resolution microscopy coupled with newly developed image analysis tools. We demonstrate that EcSSB forms dynamic condensates during exponential growth, which disassemble in response to DNA damage, oxidative stress, antibiotic exposure, and metabolic adaptation. These phase transitions are attenuated in stationary-phase cells, indicative of reduced responsiveness. Moreover, we show that EcSSB is spatially segregated from nucleoid regions under stress-free conditions, but relocalizes to DNA-rich regions upon stress induction, supporting a regulated condensate-to-function transition. Our findings provide direct in vivo evidence for stress- and growth-phase-dependent modulation of EcSSB phase behavior, corroborated by biophysical characterization of EcSSB condensates bridging in vitro observations with physiological relevance. Our work establishes a quantitative framework for dissecting LLPS dynamics in bacterial cells and sets the stage for future strategies to target condensate-regulated pathways in antimicrobial development.