Affiliation:
1. Department of Biologic and Materials Sciences, University of Michigan School of Dentistry
2. Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan
Abstract
ABSTRACT
ToxR-dependent recruitment of TcpP to the
toxT
promoter facilitates
toxT
transcription in
Vibrio cholerae
, initiating a regulatory cascade that culminates in cholera toxin expression and secretion. Although TcpP usually requires ToxR to activate the
toxT
promoter, TcpP overexpression can circumvent the requirement for ToxR in this process. To define nucleotides critical for TcpP-dependent promoter recognition and activation, a series of
toxT
promoter derivatives with single-base-pair transversions spanning the TcpP-binding site were generated and used as plasmid-borne
toxT-lacZ
fusions, as DNA mobility shift targets, and as allelic replacements of the chromosomal
toxT
promoter. When present in Δ
toxR V. cholerae
overexpressing TcpP, several transversions affecting nucleotides within two direct repeats present in the TcpP-binding region (TGTAA-N
6
-TGTAA) caused defects in TcpP-dependent
toxT-lacZ
fusion activation and toxin production. Electrophoretic mobility shift assays demonstrated that these same transversions reduced the affinity of the
toxT
promoter for TcpP. The presence of ToxR suppressed transcription activation defects associated with most, but not all, transversions. Particularly, the central thymine nucleotide of both pentameric repeats was essential for efficient
toxT
activation, even in the presence of ToxR. These results suggest that the
toxT
promoter recognition function provided by ToxR can facilitate the interaction of TcpP with the
toxT
promoter but is insufficient for promoter activation when the TcpP-binding site has been severely compromised by mutation. Thus, the interaction of TcpP with nucleotides of the direct repeat sequences appears to be a prerequisite for
toxT
promoter activation.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
31 articles.
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