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J. Biol. Chem., Vol. 283, Issue 35, 23610-23618, August 29, 2008

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A Consensus Sequence for Binding of SmcR, a Vibrio vulnificus LuxR Homologue, and Genome-wide Identification of the SmcR Regulon^*

Dong Hwan Lee¹, Hye Sook Jeong¹², Hee Gon Jeong, Kyung Mo Kim, Heebal Kim, and Sang Ho Choi³

From the National Research Laboratory of Molecular Microbiology and Toxicology, Center for Agricultural Biomaterials and the Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. In the present study, DNA targets of SmcR, a Vibrio vulnificus LuxR homologue, were selected from a random pool of DNA fragments by using a cycle selection procedure consisting of in vitro DNA-SmcR interaction, purification of SmcR-DNA complexes, and PCR amplification of SmcR-bound DNA. The amplified DNA fragments were cloned and analyzed separately by electrophoretic mobility shift assay to verify the specific binding of SmcR to the DNA. The DNA sequences bound by SmcR were determined by DNase I footprinting, and alignment of the resulting 29 sequences revealed a 22-bp consensus SmcR-binding sequence, 5'-TTATTGATWWRWTWNTNAATAA-3' (where W represents A or T, R is G or A, and N is any nucleotide), with an 8-bp (TTATTGAT) inverted repeat. The consensus sequence revealed greater efficiency for the binding of SmcR than the SmcR-binding sequence previously identified within P_vvpE. Mutational analysis demonstrated that the 9th and 10th bases from the center are the most essential for SmcR binding. A genome-wide search using the consensus sequence predicted that at least 121 genes are under the control of SmcR, and 10 of these newly identified SmcR regulon members were verified as being regulated by SmcR in V. vulnificus as well as in vitro. The consensus sequence and newly identified genes should be of use for elucidating the regulatory mechanism of SmcR and provide further insight into the role of the quorum sensing in V. vulnificus pathogenesis.

Received for publication, February 25, 2008 , and in revised form, May 27, 2008.

^* This study was supported by grants from the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science, and Technology, the MarineBio 21 Project, Ministry of Maritime Affairs and Fisheries, and National Research Laboratory Grant, Korea Science and Engineering Foundation, R0A-2007-000-20039-0, 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Tables 1 and 2.

¹ Both authors contributed equally to this work.

² Present address: Division of Enteric and Hepatitis Viruses, Center for Infectious Diseases, National Institute of Health, Seoul 122-701, South Korea.

³ To whom correspondence should be addressed: Dept. of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea. Tel.: 82-2-880-4857; Fax: 82-2-873-5095; E-mail: choish{at}snu.ac.kr.

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