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A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein–protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation

A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein–protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation

Abstract

Abstract The Vibrio cholerae transcriptional regulator ToxR is anchored in the cytoplasmic membrane by a single transmembrane segment, its C-terminal domain facing the periplasm. Most of its N-terminal cytoplasmic domain shares sequence similarity with the winged helix–turn–helix (wHTH) motif of OmpR-like transcriptional regulators. In the heterologous host Escherichia coli ToxR activates transcription at the V.cholerae ctx promoter in a dimerization-dependent manner, which has led to its employment as a genetic indicator for protein–protein interactions. However, although offering a broader potential application range than other prokaryotic two-hybrid systems described to date, ToxR has so far only been used to study interactions between heterologous transmembrane segments or to monitor homodimerization of C-terminal fusion partners in the periplasm and the cytoplasm of E.coli . Here we show that the ToxR-system also allows the detection of heterodimerization in both cellular compartments of E.coli . In addition, to better understand ToxR's mode of action at ctx in E.coli , we have investigated the minimal requirements for its function as a transcriptional activator. We show that the wHTH motif of ToxR's N-terminal domain constitutes the minimal structural element required to activate transcription at ctx in E.coli when fused to a dimerizing protein module.
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