Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae.
In pathogenic Vibrio cholerae, at least 17 genes are co-ordinately regulated by ToxR. Most of these genes, including those that encode cholera toxin (CT), toxin co-regulated pilus (TCP), accessory colonization factor (ACF) and OmpU, are positively regulated. OmpT is the only identified protein under negative regulation of ToxR. To understand the molecular mechanism by which ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR. Sequence analysis revealed that ompT encodes a predicted 35.8 kDa outer membrane porin of V. cholerae. Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon. Both primer extension analysis and promoter fusion studies showed that ToxR represses OmpT expression at the transcriptional level. Promoter fusion studies also suggest that cyclic AMP receptor protein (CRP) is involved in ompT activation. Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediates repression of ompT transcription by directly binding to an A/T-rich region between -95 and -30 of the ompT promoter. To further understand how the interaction of ToxR with different promoters results in its function as an activator or repressor, we have also mapped the regions on the ctxAB and toxT promoters to which ToxR binds. The regions protected by ToxR on each of these promoters are all A/T rich and large in size, although they are positioned differently relative to each transcriptional start site.
Li
C C
CC
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, School of Medicine, Baltimore, MD 21201, USA.
Crawford
J A
JA
DiRita
V J
VJ
Kaper
J B
JB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D018272:Porins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014734:Vibrio cholerae / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D018272:Porins; D014157:Transcription Factors; C405564:ompT protein, bacteria; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; C075626:tcpN protein, Vibrio cholerae