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

Abstract

In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces the torCAD operon, which encodes the TMAO respiratory system of Escherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torR gene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers the torR transcription start site and constitutes 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 autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in a torS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated 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 sites must be close to each other and located on the same side of the DNA helix 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 TorR to box 3 and box 4.

FIG. 1

Primer extension analysis of torR and torCAD. The labeled primers were annealed to RNA from MC4100 cells grown anaerobically in the absence (− lanes) or presence (+ lanes) of 10 mM TMAO and extended with reverse transcriptase. Lanes A, C, G, and T are a sequencing ladder of the torC DNA region made with the same primer as that used in the primer extension reaction of torC.

FIG. 2

Activity of the torR promoter mutated or not mutated on tor boxes in different genetic backgrounds. (Left) The tor boxes are overlined, and the transcription start site of torR is indicated by a +1 arrow. Except for pPR5, the 5′ part of the cloned tor sequence is not shown (as indicated by −//−). The 5′ end of the cloned tor fragment corresponds to position −124, relative to the torR transcription start site for pPR1 to pPR4 and to position −53 for pPR5; the 3′ extremity of the cloned fragment for all pPR plasmids corresponds to position +15. For plasmids pPR2, pPR3, and pPR4, only the point mutations are indicated. (Right) The LCB506 (tor wild-type [wt]), LCB507 (torR), and LCB434 (torS) strains containing the pPR plasmids were grown anaerobically in the presence (+) or absence (−) of TMAO. β-Galactosidase activity of the plasmid-borne torR-lacZ fusions is expressed in Miller units.

FIG. 3

Activity of the tor operon promoter with base pair deletions or insertions between box 2 and box 3. (Left) The tor boxes are overlined, the sequence insertions are in italics, and the transcription start site of torC is indicated by a +1 arrow. The 3′ part of the cloned tor sequence is not shown (as indicated by −//−). The 3′ end of the cloned tor fragment corresponds to position +275. (Right) The LCB506 (tor wild-type [wt]) and LCB507 (torR) strains containing the pPTor plasmids were grown anaerobically in the presence (+) or absence (−) of TMAO. β-Galactosidase activity of the plasmid-borne torC-lacZ fusions is expressed in Miller units. bp, the number of base pairs inserted (+) or deleted (−) between box 2 and box 3; ND, not determined.