Vibrio cholerae H-NS domain structure and function with respect to transcriptional repression of ToxR regulon genes reveals differences among H-NS family members.
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 coiledcoil 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 coiledcoil 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 coiledcoil or N-terminal domain, are required for repression of promoters responsive to H-NS within the ToxR regulon.
Nye
Melinda B
MB
Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Taylor
Ronald K
RK
D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing / Q000502:physiology; D009876:Operon; D012097:Repressor Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics* / Q000472:pathogenicity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C031908:H-NS protein, bacteria; D012097:Repressor Proteins; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria