Stress testing reveals selective vulnerabilities in protein homeostasis.

Protein quality control (PQC) systems are essential for cellular resilience to proteotoxic stress. Despite intensive study for decades, functional redundancies in the system obscure the contributions of the collectively important individual genes. Here, we leverage transposon sequencing across bacteria strains lacking key chaperones and proteases to reveal hidden determinants of stress response in protein homeostasis. By profiling fitness under multiple proteotoxic stresses, we uncover stress-specific vulnerabilities and reveal how major players of PQC mask correlations between transcriptomic responses and gene fitness. As an illustration of unexpected connections, we identify a heat-specific synthetic lethality between the disaggregase ClpB and DNA Polymerase I (PolA) mediated by persistent aggregation of the RecA recombinase and toxic persistence of the heat shock regulon. Our findings reveal that stress-induced aggregation is not broadly toxic. Rather, it becomes lethal in specific genetic or environmental contexts due to the depletion of components only needed in those specific circumstances. This work presents a framework to reveal normally hidden fragility in stress responses using gene fitness scores adaptable to a variety of systems.