Significance While the transcriptional regulatory network (TRN) of Escherichia coli has expanded considerably in recent years through new chromatin immunoprecipitation (ChIP) data, an open question remains: Does the global TRN, reconstructed by combining ChIP data for individual transcriptions factors, consistently explain observed differential gene expression? We have reconstructed a high-confidence TRN, determined its consistency with transcriptomics and predictive capabilities across multiple conditions, extracted 10 functional regulatory modules, and characterized this network at the sequence and structural levels. Our multiomics algorithmic pipeline is expected to facilitate rigorous validation and prioritization of experiments to elucidate TRNs in other bacteria.