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.
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.
Hennecke
Frank
F
Abteilung Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Müller
Arne
A
Meister
Roland
R
Strelow
Astrid
A
Behrens
Susanne
S
D001426:Bacterial Proteins / Q000737:chemistry*; D002772:Cholera Toxin / Q000096:biosynthesis / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; D002772:Cholera Toxin