The TorR high-affinity binding site plays a key role in both torR autoregulation and torCAD operon expression in Escherichia coli

In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-compone nt regulatory system induces the torCAD operon, which encodes the TMAO resp iratory system of Escherichia coli, The sensor protein TorS detects TMAO an d transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD, The torR gene and the torCAD operon are divergent ly transcribed, and the short torR-torC intergenic region contains four dir ect repeats (the tot. boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers the torR transcription start site and constitute s a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites, By using torR-lacZ operon fusions in different genetic backgrounds, we showed that the torR gene is negatively autoregulat ed. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a torS mutant, In addition, this negative regulation involves only the T orR high-affinity binding site, Together, these data suggest that phosphory lated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo , thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sit es must be close to each other and located on the same side of the DNA heli x to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated Tor R to hox 3 and box 4.