In Bacteria, transcription factors (TFs) consist of sensory ligand-binding and DNA-binding helix-turn-helix (HTH) domains to respond to the environmental and internal stimuli. Although Escherichia coli is a well-studied model bacterium, yet half of its TFs’ characteristics remain unclear, including 72 genes of unknown function. Using E. coli synthetic genetic array, a quantitative approach to scoring the fitness of single and double mutant genes, I was able to measure pairwise genetic interactions (GIs) among all TFs in rich and minimal media (RM and MM) to create a differential (DF) GI network. DF network analysis demonstrated GIs altered in RM or MM growth conditions. Both static and DF 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 similar GI profiles are co-conserved in bacterial evolution. Moreover, the E. coli TF GI network provided deep insights on conserved genetic association across bacterial phyla that can be valuable to build TF machinery organization, even in pathogens.