Phase separation of a bacterial disordered chaperone mediated by electrostatic interactions

Abstract Liquid–liquid phase separation (LLPS) is emerging as a key mechanism for organizing cellular components and regulating stress responses. Although LLPS has been extensively studied in intrinsically disordered proteins, whether highly charged and intrinsically disordered molecular chaperones undergo LLPS remains poorly understood. Here, we demonstrate that the Escherichia coli acid shock protein Asr, a highly charged and intrinsically disordered chaperone, undergoes LLPS driven by electrostatic interactions and forms dynamic liquid condensates with polyanions such as DNA, RNA, heparin, and acidic proteins. Asr phase separation critically depends on positively charged clusters, polyanion length, ionic strength, and pH. Guided by Asr's physicochemical features, we identify three additional molecular chaperones, Anhydrin, Hero7, and HCVncd, that also exhibit LLPS behavior in vitro but display distinct condensate properties and pH responsiveness consistent with their individual charge compositions and distributions. In vivo, Asr–EGFP forms non–canonical compartments in 37% of E. coli cells at pH 7.5, increasing to 80% under acidic conditions (pH 4.5). These compartments disassemble under high–salt conditions after cell lysis, suggesting electrostatic mediation. In cell imaging and FRAP analyses further reveal that charge–enhanced Asr mutants and homologs form canonical condensates in vivo , predominantly co–localizing with acidic proteins. Notably, Asr*3 fusion drives condensate formation of the aggregation–prone client thereby reducing stress–induced aggregation, indicating that Asr functions as an LLPS–promoting module to mitigate protein aggregation. These findings advance our understanding of LLPS in highly charged, intrinsically disordered molecular chaperones and lay the foundation for exploring their roles in cellular homeostasis and potential applications in engineering synthetic biomolecular condensates.