TRANSPHOSPHORYLATION OF THE TORR RESPONSE REGULATOR REQUIRES THE 3 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 indu ces the expression of the tor structural operon encoding the trimethyl amine N-oxide reductase respiratory system in response to substrate av ailability. TorS belongs to a sensor subfamily that includes a classic al transmitter domain, a receiver, and a C-terminal alternative transm itter domain. The histidine phosphorylation sites of each TorS transmi tter domain and the aspartate phosphorylation site of the TorS receive r were individually changed by site-directed mutagenesis. All three ph osphorylation 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 Gin change in the classical trans mitter domain abolished TorS autophosphorylation, whereas TorS underwe nt significant autophosphorylation when the phosphorylation site of it s receiver or alternative transmitter was changed. Complementation bet ween pairs of defective TorS proteins was achieved in vitro, allowing TorR transphosphorylation. This strongly suggests that TorS is a multi mer in which intermolecular phosphorylation occurs. The wild-type alte rnative transmitter domain alone was shown to complement a TorS protei n mutated in its C-terminal alternative transmitter. Interestingly, ov erproduction of the alternative transmitter domain led to in vivo TorR -dependent constitutive expression of the tor operon in a torS(+) or t orS context. Hence, the TorS alternative transmitter contains the phos phodonor site for TorR. Taken together, our results support a TorS pho sphorylation cascade from the classical transmitter to the sensor rece iver and the alternative transmitter phosphorylation sites. (C) 1997 A cademic Press Limited.