Transphosphorylation of the TorR response regulator requires the three phosphorylation sites of the TorS unorthodox sensor in Escherichia coli.

Two-component regulatory systems allow cells to adapt to environmental changes. In Escherichia coli, the TorS/TorR two-component system induces the expression of the tor structural operon encoding the trimethylamine N-oxide reductase respiratory system in response to substrate availability. TorS belongs to a sensor subfamily that includes a classical transmitter domain, a receiver, and a C-terminal alternative transmitter domain. The histidine phosphorylation sites of each TorS transmitter domain and the aspartate phosphorylation site of the TorS receiver were individually changed by site-directed mutagenesis. All three phosphorylation sites proved essential for in vivo induction of the tor structural operon and for in vitro transphosphorylation of the cognate TorR response regulator. The His to Gln change in the classical transmitter domain abolished TorS autophosphorylation, whereas TorS underwent significant autophosphorylation when the phosphorylation site of its receiver or alternative transmitter was changed. Complementation between pairs of defective TorS proteins was achieved in vitro, allowing TorR transphosphorylation. This strongly suggests that TorS is a multimer in which intermolecular phosphorylation occurs. The wild-type alternative transmitter domain alone was shown to complement a TorS protein mutated in its C-terminal alternative transmitter. Interestingly, overproduction of the alternative transmitter domain led to in vivo TorR-dependent constitutive expression of the tor operon in a torS+ or torS context. Hence, the TorS alternative transmitter contains the phosphodonor site for TorR. Taken together, our results support a TorS phosphorylation cascade from the classical transmitter to the sensor receiver and the alternative transmitter phosphorylation sites.

An unorthodox sensor protein (TorS) mediates the induction of the tor structural genes in response to trimethylamine N-oxide in Escherichia coli.

We isolated and characterized three spontaneous mutations leading to trimethylamine N-oxide (TMAO)-independent expression of the tor operon encoding the TMAO-reductase anaerobic respiratory system in Escherichia coli. The mutations lie in a new for regulatory gene, the torS gene, which probably encodes a sensor protein of a two-component regulatory system. One mutation, which leads to full TMAO-constitutive expression, is a 3-amino-acid deletion within the potential N-terminal periplasmic region, suggesting that this region contains the TMAO-detector site. For the other two mutations, a further induction of the tor operon is observed when TMAO is added. Both are single substitutions and affect the linker region located between the detector and the conserved transmitter domains. Thus, as proposed for other sensors, the TorS linker region might play an essential role in propagating conformational changes between the detector and the cytoplasmic signalling regions. The TorS histidine kinase is an unorthodox sensor that contains a receiver and a C-terminal alternative transmitter domain in addition to the domains found in most sensors. Previously, we showed that TMAO induction of the for operon requires the TorR response regulator and the TorT periplasmic protein. Additional genetic data confirm that torS encodes the sensor partner of TorR and TorT. First, insertion within torS abolishes tor operon expression whatever the growth conditions. Second, overexpressed TorR bypasses the requirement for torS, whereas the torT gene product is dispensable for tor operon expression in a torS constitutive mutant. This supports a signal-transduction cascade from TorT to TorR via TorS.

The periplasmic TorT protein is required for trimethylamine N-oxide reductase gene induction in Escherichia coli.

Expression of the Escherichia coli torCAD operon, which encodes the trimethylamine N-oxide reductase system, is regulated by the presence of trimethylamine N-oxide through the action of the TorR response regulator. We have identified an additional gene, torT, located just downstream from the torR gene, which is necessary for torCAD structural operon expression. Insertion within the torT gene dramatically reduced the expression of a torA'-'lacZ fusion, while presence of the gene in trans restored the wild-type phenotype. Overproduction of TorR in a torT strain resulted in partial constitutive expression of the torA'-'lacZ fusion, suggesting that TorR acts downstream from TorT. The torT gene codes for a 35.7-kDa periplasmic protein which presents some homology with the periplasmic ribose-binding protein of E. coli. We discuss the possible role of TorT as an inducer-binding protein involved in signal transduction of the tor regulatory pathway.

Binding of the TorR regulator to cis-acting direct repeats activates tor operon expression.

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N-oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC'-lacZ fusion dramatically reduces TorR-dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel-retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the -35 promoter sequence and includes the third tor box (box3); the last is found downstream from the -35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments. We propose a model in which multiple binding sites (i.e. the tor boxes) contribute to the formation of a nucleoprotein complex, but only one particular proximal site positions TorR properly so that it interacts with RNA polymerase.

Conservation of cis-acting elements within the tor regulatory region among different Enterobacteriaceae.

The Escherichia coli (Ec) torCAD operon encoding the trimethyl amine N-oxide (TMAO) reductase system is induced by both TMAO and anaerobiosis. The tor regulatory regions from bacteria related to Ec have been amplified by the polymerase chain reaction (PCR) using degenerate oligodeoxyribonucleotide primers based on conserved sequences of the tor products. The amplified regions from Salmonella enteritidis and Sa. typhimurium (St) were the same size as that from Ec and showed 82% identity with it. Interestingly, four boxes of a 10-nucleotide motif (5'-CTGTTCATAT) were found in direct repeat at the same location in the tor regulatory region of the three species. Although the amplified fragment from Shigella sonneï (Ss) was highly homologous to the Ec corresponding segment, the first tor box was missing. In Ec, the St and Ss tor promoters were still regulated by both TMAO and anaerobiosis, but their transcriptional activities were significantly lower than that of the Ec tor promoter. Deletion of the two first boxes of the Ec tor regulatory region inactivated the tor promoter while deletion of the region just upstream from the tor boxes led to a significant decrease in tor expression. Our results strongly suggest that the tor boxes, as well as specific sequences outside the tor boxes, play an important role in the expression of the tor operon.

The torR gene of Escherichia coli encodes a response regulator protein involved in the expression of the trimethylamine N-oxide reductase genes.

Expression of the Escherichia coli torCAD operon encoding the trimethylamine N-oxide (TMAO) reductase system is induced by both TMAO and anaerobiosis. A torR insertion mutant unable to express the torA gene had previously been isolated. The torR gene was cloned and sequenced. It encodes a 25,000-Da protein which shares homology with response regulators of two-component systems and belongs to the OmpR-PhoB subclass. Overproduction of TorR mimics the presence of the inducer TMAO while the anaerobic control is unchanged, suggesting that TorR mediates only the TMAO induction. The overproduced TorR protein was purified to more than 90%. The torR gene is located just upstream of the torCAD operon, with an opposite transcription direction. The torR-torCAD intergenic region is unusual in that it contains four direct repeats of a 10-nucleotide motif. Part or all of these motifs could be involved in the binding of TorR. The gene encoding the sensor partner does not seem to be adjacent to torR, since the divergent open reading frame found immediately downstream of torR exhibits none of the features of a protein histidine kinase.