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J. Biol. Chem., Vol. 281, Issue 26, 17727-17735, June 30, 2006

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Differential Recognition of Citrate and a Metal-Citrate Complex by the Bacterial Chemoreceptor Tcp^*

Tomonori Iwama¹, Yasuaki Ito, Hisaaki Aoki, Hiroshi Sakamoto, Shuzo Yamagata, Keiichi Kawai, and Ikuro Kawagishi^¶

From the Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu 501-1193 and the Division of Biological Science, Graduate School of Science, and the ^¶Institute for Advanced Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the 1-2 and 3-4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn⁶⁷, which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn⁶⁷ and allosteric switching.

Received for publication, February 2, 2006 , and in revised form, April 18, 2006.

^* This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science (to I. K.). 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.: 81-58-293-2932; Fax: 81-58-293-2932; E-mail: iwamatom{at}cc.gifu-u.ac.jp.

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