Affiliation:
1. Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755
Abstract
ABSTRACT
ToxR and ToxS are integral membrane proteins that activate the transcription of virulence genes in
Vibrio cholerae
. ToxR can be separated into three different domains: an N-terminal cytoplasmic DNA binding domain, a central transmembrane domain, and a C-terminal periplasmic domain. ToxS is thought to enhance ToxR-mediated transcriptional activation through a periplasmic interaction. By P22 challenge phage selection for DNA binding, in combination with a screen for cholera toxin gene transcription, 12
toxR
and
toxS
positive control mutants producing variant ToxR proteins from the
toxRS
operon that bind to the cholera toxin promoter but that fail to activate transcription were isolated. One mutation in
toxR
specifies an E82K change in the predicted helix-loop-helix DNA binding domain and destroys ToxR-mediated activation. Seven
toxR
mutations included frameshifts and stop codons introduced into the periplasmic domain, and six of these mutations appeared to produce proteolytically processed shorter forms of ToxR, suggesting that even short periplasmic deletions alter the folding of ToxR in the periplasm. Deletion of
toxS
did not alter the steady-state level of ToxR, and ToxR was found to be capable of binding to DNA in the absence of ToxS even though it did not activate transcription. However, the ToxS L33S variant rendered ToxR susceptible to proteolysis, suggesting that the natural function of ToxS is to complex with ToxR. Therefore, certain alterations that map to the ToxR cytoplasmic DNA binding domain, to the periplasmic domain, or to ToxS separate DNA binding activity from activator function. These data support a model where proper assembly or stability of the periplasmic domain of ToxR is enhanced by ToxS. This chaperone-like activity of ToxS may be required for the formation of the transcriptional activation complex but not the ToxR-DNA complex.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
39 articles.
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