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J. Biol. Chem., Vol. 281, Issue 34, 24398-24404, August 25, 2006

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AI-1 Influences the Kinase Activity but Not the Phosphatase Activity of LuxN of Vibrio harveyi^*

Melanie Timmen, Bonnie L. Bassler, and Kirsten Jung¹

From the Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Strasse 1a, D-80638 München, Germany and the Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

The Gram-negative bacterium Vibrio harveyi produces and responds to three autoinducers, AI-1, AI-2, and CAI-1 to regulate cell density dependent gene expression by a process referred to as quorum sensing. The concentration of the autoinducers is sensed by three cognate hybrid sensor kinases, and information is channeled via the HPt protein LuxU to the response regulator LuxO. Here, a detailed biochemical study on the enzymatic activities of the membrane-integrated hybrid sensor kinase LuxN, the sensor for N-(D-3-hydroxybutanoyl)homoserine lactone (AI-1), is provided. LuxN was heterologously overproduced as the full-length protein in Escherichia coli. LuxN activities were characterized in vitro and are an autophosphorylation activity with an unusually high ATP turnover rate, stable LuxU phosphorylation, and a slow phosphatase activity with LuxUP as substrate. The presence of AI-1 affected the kinase but not the phosphatase activity of LuxN. The influence of AI-1 on the LuxN LuxU signaling step was monitored, and in the presence of AI-1, the kinase activity of LuxN, and hence the amount of LuxUP produced, were significantly reduced. Half-maximal inhibition of kinase activity by AI-1 occurred at 20 µM. Together, these results indicate that AI-1 directly interacts with LuxN to down-regulate its autokinase activity and suggest that the key regulatory step of the AI-1 quorum sensing system of Vibrio harveyi is AI-1-mediated repression of the LuxN kinase activity.

Received for publication, May 1, 2006 , and in revised form, June 27, 2006.

^* This work was financially supported by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg 612) and the German Academic Exchange Service (DAAD; Project-based Personnel Exchange Programme with the United States), the Howard Hughes Medical Institute, and National Institutes of Health Grant R01 GM 065859 (to B. L. B.). 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.

¹ To whom correspondence should be addressed. Tel.: 49-89-2180-6120; Fax: 49-89-2180-6122; E-mail: kirsten.jung{at}lrz.uni-muenchen.de.

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