ToxR co‐operative interactions are not modulated by environmental conditions or periplasmic domain conformation

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using λ repressor–ToxR chimeric proteins and a λ repressor‐controlled reporter system (OR1 PR–lacZY ) established that ToxR sequences can effectively dimerize the amino‐termina...

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Published in:Molecular microbiology 1999-01, Vol.31 (1), p.305-317
Main Authors: Dziejman, Michelle, Kolmar, Harald, Fritz, Hans‐Joachim, Mekalanos, John J.
Format: Article
Language:English
Subjects:
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Blotting, Western
Dimerization
DNA-Binding Proteins - genetics
DNA-Binding Proteins - metabolism
Escherichia coli
Gene Expression Regulation, Bacterial
Genes, Reporter
Hydrogen-Ion Concentration
Lac Operon
Membrane Proteins - genetics
Membrane Proteins - metabolism
Osmolar Concentration
Periplasm
Protein Conformation
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Repressor Proteins
Transcription Factors - genetics
Transcription Factors - metabolism
Vibrio cholerae
Viral Proteins
Viral Regulatory and Accessory Proteins
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Summary:ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using λ repressor–ToxR chimeric proteins and a λ repressor‐controlled reporter system (OR1 PR–lacZY ) established that ToxR sequences can effectively dimerize the amino‐terminal domain of λ repressor in Escherichia coli. However, in E. coliToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of λ–ToxR chimeras. When the OR1 PR–lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that λ–ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of λ–ToxR proteins to form tetramers in E. coli suggested that λ–ToxR dimers could act co‐operatively. Using a redesigned reporter system containing multiple λ operator sites (OR1 OR2 OR3 PR–lacZY ), we found that λ–ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co‐operative interactions. However, we failed to correlate a monomer–dimer transition of the λ–ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re‐evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. choleraedeletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild‐type levels of toxin
ISSN:0950-382X
1365-2958
DOI:10.1046/j.1365-2958.1999.01173.x