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J Biol Chem, Vol. 275, Issue 11, 7713-7722, March 17, 2000
From the Departments of Conjugal transfer of Agrobacterium
tumefaciens Ti plasmids is regulated by quorum sensing via the
transcriptional activator TraR and the acyl-homoserine lactone
Agrobacterium autoinducer (AAI). Unique to this system, the
activity of TraR is negatively modulated by an antiactivator called
TraM. Analyses from yeast two-hybrid studies suggest that TraM directly
interacts with the activator, but the conditions under which these
components interact and the region of TraR responsible for this
interaction are not known. Induction of traM in a strain in
which TraR was activating transcription of a reporter system led to
rapid cessation of gene expression. As assessed by a genetic assay that
measures AAI-dependent DNA binding, TraM inhibited TraR
function before and after the transcription factor had bound to its DNA
recognition site. Consistent with this observation, in gel retardation
assays, purified TraM abolished the DNA binding activity of TraR in a
concentration-dependent manner. Such inhibition occurred
independent of the order of addition of the reactants. As assessed by
far Western analyses TraM interacts with TraR by directly binding the
activator. TraM in its native form interacted with native TraR and also
with heat-treated TraR but only when SDS was included with the
denatured protein. TraM interacted with TraR on blots prepared with
total lysates of cells grown in the presence and absence of AAI. Far
Western analysis of N- and C-terminal deletion mutants localized a
domain of TraR contributing to TraM binding to the C-terminal portion
of the activator protein. Random mutagenesis by hydroxylamine treatment and error-prone polymerase chain reaction identified several residues in this region of TraR important for interacting with TraM as well as
for transcriptional activation or/and DNA binding. We conclude that
TraM inhibits TraR by binding to the activator at a domain within or
close to the helix-turn-helix motif located at the C terminus of the protein.
The Antiactivator TraM Interferes with the
Autoinducer-dependent Binding of TraR to DNA by Interacting
with the C-terminal Region of the Quorum-sensing Activator*
§,§, and¶
Crop Sciences and
¶ Microbiology, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801
*
This work was supported by Grant R01-GM52465 from the
National Institutes of Health (to S. K. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Crop
Sciences, University of Illinois at Urbana-Champaign, 240 Edward R. Madigan Laboratory, 1201 West Gregory Dr., Urbana, IL 61801. Tel.:
217-333-1524; Fax: 217-244-7830; E-mail: stephenf@uiuc.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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