Erin L. Mettert, Jeffrey N. Carey, Wilma Ross, Mark Goulian, Patricia J. Kiley
ABSTRACT In Escherichia coli , cell‐to‐cell variability in aerobic expression of trimethylamine N ‐oxide (TMAO) reductase serves as a bet‐hedging strategy, enabling a subset of cells to continue growing upon a shift to anaerobic conditions in the presence of TMAO. This variability has been attributed to the low abundance of TorS and TorT signaling proteins, which along with TorR, regulate transcription of torCAD encoding TMAO reductase. Here, we characterize the divergent promoters that drive expression of the bipartite sensor and kinase, TorT and TorS. In vitro transcription analysis identified two transcription start sites for torS and one start site for torT , all located in close proximity. Notably, the RNA polymerase‐σ 70 –10 element of the upstream torS promoter overlaps that of the torT promoter, defining it as a bidirectional promoter. We further demonstrate that these two promoters are subject to repression by the global regulator IscR, which binds to a site that partially overlaps the shared −10 elements. IscR binding antagonizes RNAP‐σ 70 binding, limiting transcription of both promoters and contributing to low torS/torT expression. Finally, we show that a single base substitution within the overlapping −10 elements increases torS/torT transcription by at least 4‐fold and abolishes cell‐to‐cell variability in torCAD expression, despite this promoter variant remaining sensitive to IscR‐mediated repression. Together, these findings indicate that promoter strength and transcriptional repression are critical components for regulated bet‐hedging, enabling E. coli to optimize energy metabolism in response to fluctuations in oxygen availability and TMAO levels.