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J. Biol. Chem., Vol. 278, Issue 46, 45072-45081, November 14, 2003

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SmcR and Cyclic AMP Receptor Protein Coactivate Vibrio vulnificus vvpE Encoding Elastase through the RpoS-dependent Promoter in a Synergistic Manner^*

Hye Sook Jeong, Myoung Hee Lee, Kyu-Ho Lee, Soon-Jung Park¶, and Sang Ho Choi||

From the Departments of Food Science and Technology and of Molecular Biotechnology, Biotechnology Research Institute, Chonnam National University, Kwang-Ju 500-757, the Department of Environmental Science, Hankuk University of Foreign Studies, Yongin, Kyunggi-Do 449-791, and the ^¶Department of Parasitology, Yonsei University, College of Medicine, Seoul 133-791, South Korea

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.

Received for publication, July 28, 2003 , and in revised form, August 18, 2003.

^* This work was supported by Grant 2001-015-GP0017 from Korea Research Foundation, Republic of Korea (to S. H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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. Tel.: 82-62-530-2146; Fax: 82-62-530-2149; E-mail: shchoi{at}chonnam.ac.kr.

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