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Mol Microbiol. 1999 Jan;31(1):305-17.

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

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1
Department of Microbiology and Molecular Genetics, Shipley Institute of Harvard Medical School, Boston, MA 02115, USA.

Abstract

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using lambda repressor-ToxR chimeric proteins and a lambda repressor-controlled reporter system (OR1 PR-lacZY) established that ToxR sequences can effectively dimerize the amino-terminal domain of lambda repressor in Escherichia coli. However, in E. coli, ToxR 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 lambda-ToxR chimeras. When the OR1 PR-lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that lambda-ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of lambda-ToxR proteins to form tetramers in E. coli suggested that lambda-ToxR dimers could act co-operatively. Using a redesigned reporter system containing multiple lambda operator sites (OR1 OR2 OR3 PR-lacZY), we found that lambda-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 falled to correlate a monomer-dimer transition of the lambda-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. cholerae, deletion 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 in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity.

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