ChIP-exo analysis reveals novel uncharacterized transcription factors involved in the diverse regulatory functions of Escherichia coli

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, TRNs are not fully characterized since the identity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in the model organism Escherichia coli K-12 MG1655, which were previously predicted to be transcription factors (TFs). First, we used myc-tagging and a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs. We then compared those binding sites with those of RNA polymerase and sigma factors to increase the confidence in the measured DNA-binding events. Finally, we used these data to infer potential functional annotations for 13 candidate TFs. Our results revealed that these candidate TFs have a varied number of regulatory genes and are involved in E. coli's primary cellular processes. Taken together, thes results: (1) expand the number of confirmed TFs from 249 to 276, close to the estimated total of 280 TFs in E. coli K-12 MG1655; (2) provide a roadmap for the functional characterization of the newly discovered TFs; and (3) enable the elucidation of the first comprehensive TRN in this model organism. Overall design: Identification of genome-wide bindings for uncharacterized transcription factors in E. coli K-12 MG1655, using ChIP-exo technology