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Vibrio cholerae H‐NS domain structure and function with respect to transcriptional repression of ToxR regulon genes reveals differences among H‐NS family members
Author(s) -
Nye Melinda B.,
Taylor Ronald K.
Publication year - 2003
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2003.03701.x
Subject(s) - regulon , biology , vibrio cholerae , gene , genetics , psychological repression , mutagenesis , promoter , dna binding protein , transposon mutagenesis , repressor , regulation of gene expression , gene expression , microbiology and biotechnology , mutant , transposable element , transcription factor , bacteria
Summary H‐NS is an abundant bacterial protein involved in transcriptional silencing of a variety of environmentally responsive genes during growth under non‐permissive conditions. We have previously demonstrated a direct role for H‐NS in the negative modulation of expression of several genes within the ToxR virulence regulon of Vibrio cholerae . Here we have undertaken extensive mutagenesis of the structural and functional domains of the H‐NS protein to determine the contribution of each to the regulation of gene expression. Insertions within, or truncations of, the C‐terminal conserved DNA‐binding domain prevent repression of toxT and ctx , as expected. Dominant negative experiments demonstrate that V. cholerae H‐NS represses gene expression as an oligomeric protein. Hydrophobic coiled–coil interactions have been shown to provide oligomerization capability in other H‐NS orthologues. We used site‐directed mutagenesis to construct altered V. cholerae H‐NS proteins, including an extensive internal deletion within the predicted coiled–coil domain. Remarkably, these proteins were competent to repress gene expression and to form oligomers. Chimeric H‐NS proteins, using sequences from both Escherichia coli and V. cholerae H‐NS orthologues, revealed that V. cholerae H‐NS possesses a second oligomerization domain in the N‐terminal 24 amino acids of the protein. Overall, our results suggest DNA binding and protein oligomerization, provided by either the central coiled–coil or N‐terminal domain, are required for repression of promoters responsive to H‐NS within the ToxR regulon.