Unraveling the functions of uncharacterized transcription factors in Escherichia coli using ChIP-exo

Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, no TRN is fully characterized since the identity and activity of all the transcriptional regulators that comprise a TRN are not known. Here, we experimentally evaluate 40 uncharacterized proteins in Escherichia coli K-12 MG1655, which were computationally predicted to be transcription factors (TFs). First, we used a multiplexed ChIP-exo assay to characterize genome-wide binding sites for these candidate TFs; 34 of them were found to be DNA-binding protein. We then compared the relative location between binding sites and RNA polymerase (RNAP). We found 48% (283/588) overlap between the TFs and RNAP. Finally, we used these data to infer potential functions for 10 of the 34 TFs with validated DNA binding sites and consensus binding motifs. These TFs were found to have various roles in regulating primary cellular processes in E. coli. Taken together, this study: (1) significantly expands the number of confirmed TFs, close to the estimated total of about 280 TFs; (2) predicts the putative functions of the newly discovered TFs, and (3) confirms the functions of representative TFs through mutant phenotypes.