CRISPRi-mediated silencing of racR reveals molecular strategies for surviving lethal stress coupled to Rac prophage excision
Cryptic prophages, abundant in bacterial genomes, often carry regulatory modules that rewire host physiology contextually. E. coli K-12 Rac prophage harbors a genetic module consisting of a RacR repressor that tightly represses the toxin genes ydaS/T. Dysregulation of racR and expression of YdaS/T lead to growth arrest, rapid loss of viability, and morphological defects. We investigated the physiological consequences of racR silencing and the host adaptive responses to lethal stress imposed by YdaS/T. We show that racR depletion establishes an inner membrane-centered stress characterized by selective hyperpolarization, increased permeability, and arrest of cytokinesis downstream of FtsZ-ring assembly, triggering copious filamentation. Transcriptomic analysis showed extensive metabolic reprogramming, revealing a coordinated, energy-conserving response marked by respiratory remodelling, repression of proton motive force-intensive pathways, and activation of global stress pathways. We show that recovery is accompanied by remarkable physiological heterogeneity with divergent filament fates, including lysis, persistent arrest, and recovery by loss of Rac. Thus, heterogeneous membrane dysfunction and metabolic states appeared to determine the balance between recovery and cell death. These findings support a model in which racR silencing activates a prophage-host stress circuit interlinking inner membrane energetics, activation of envelope stress, cell division control, and heterogeneous recovery.