SRP341328 PRJNA771186 Auxotrophic and Prototrophic Conditional Genetic Networks Reveal the Functional Rewiring of Transcription Factors in Escherichia coli Bacterial transcription factor (TF) is the widely studied machinery in Escherichia coli, where regulatory decision made in response to environmental stimuli and genetic perturbations modulate the expression of many genes. Yet, it remains unclear how individual genes in the underlying pathways of TF machinery operate together during environmental challenge. Here, we address this by applying an unbiased, quantitative synthetic genetic interaction (GI) approach to measure pairwise GIs, among all TF genes in E. coli under auxotrophic (rich medium) and prototrophic (minimal medium) static growth conditions. The resulting differential epistasis map revealed condition-dependent GIs, widespread changes among TF genes in metabolism, and many new gene functions undetected in static conditions. Both static and differential GI networks were also effective in detecting TF pathways, highlighting new roles for uncharacterized TFs (YjdC, YneJ, YdiP) as regulators of cell division, putrescine pathway and efflux pump, and cold shock adaptation, respectively. Pan-bacterial conservation suggests that TF genes with GIs are co-conserved in evolution. Together, our results provide deep insight on the global organization of TF machinery, condition-specific rewiring of TF processes, and remodeling of genetic backup systems for TF regulation under environmental change, which will be vital for controlling bacterial pathogenesis.