Abstract
The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoS-dependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.
Highlights
The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE
The binding sites for cyclic AMP receptor protein (CRP) and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of promoter S (PS), respectively. These results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoSdependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase
We showed that transcription of the vvpE gene of V. vulnificus is initiated by two different types of promoter, promoter L (PL)1 and promoter S (PS), in a growth phase-dependent manner [9]
Summary
Which is under the positive control of both CRP and RpoS This differential utilization of two promoters can permit precise levels of elastase in response to modifications of the environment and growth stage. It has been reported that SmcR, the Vibrio harveyi LuxR homolog, is involved in the positive control of vvpE expression [10]. In an effort to elucidate the regulatory mechanism of vvpE expression at a molecular level, this study examined the influence of the mutation of smcR on the activity of vvpE promoters. The relationship between RpoS, CRP, and SmcR was examined by determining the cellular level of the proteins in an rpoS, crp, and smcR background. The binding of CRP and SmcR directly to the upstream portion of vvpE was demonstrated, and the binding sites for CRP and SmcR were determined
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