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.