Molecular cloning and transcriptional regulation of ompT, a ToxR‐repressed gene in Vibrio cholerae

Abstract

SummaryIn 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 identi®ed 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 identi®ed 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 À95 and À30 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.

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

“…We have shown previously that ToxR binds directly to the -30 to -95 region of the ompT promoter, consistent with its role as a repressor. We also observed sequencedependent activation of ompT transcription in the absence of ToxR and suggested that cAMP receptor protein (CRP) positively regulates ompT transcription (Li et al, 2000). However, how ToxR represses transcription by binding to this site and how ToxR as well as CRP mediate regulated expression of OmpT in response to environmental signals remained to be determined.…”

“…1A, in LB). Our previous observation that CRP is a potential activator of ompT gene expression implies that CRP may contribute to the upregulation of OmpT in response to certain environmental signals (Li et al, 2000). To define the optimal conditions for OmpT expression so that the contribution of CRP as an activator can be assessed, we grew V. cholerae classical strain 395 to different growth phases in rich or minimal media, varying the osmolarity and temperature.…”

“…ToxT then directly activates transcription of the genes encoding CT and TCP and autoregulates its own gene (Yu and DiRita, 1999). In the other branch, independent of TcpP and ToxT, ToxR differentially regulates the expression of two outer membrane porins, OmpU and OmpT (Chakrabarti et al, 1996), resulting in almost exclusive expression of OmpU in a toxR + strain and exclusive expression of OmpT in a toxR -strain Crawford et al, 1998;Li et al, 2000). The genes encoding CT are part of the genome of a lysogenic phage (Waldor and Mekalanos, 1996), and the genes encoding TCP are part of the Vibrio pathogenicity island, which is also apparently of phage origin (Karaolis et al, 1998;.…”

“…For ctxAB, ToxR binding to the -34 to -143 region of the promoter is sufficient to activate transcription in Escherichia coli, but not in V. cholerae Champion et al, 1997;Li et al, 2000). At the ompU promoter, DNase I footprinting revealed the protection of three distinct sites by ToxR.…”

“…It was proposed that the binding of ToxR to a region extending from -238 to -149 of the promoter is followed by co-operative binding to the downstream regions extending from -116 to -58 and from -53 to -24, where ToxR interacts directly with RNA polymerase (RNAP) and activates transcription (Crawford et al, 1998). For toxT, ToxR binding to the -108 to -65 region of the promoter recruits TcpP to bind downstream at -58 to -44, where TcpP can activate transcription directly (Krukonis et al, 2000;Li et al, 2000). ToxR was proposed as a repressor of OmpT because the expression of this protein is dramatically increased in a toxR mutant .…”