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J. Biol. Chem., Vol. 281, Issue 41, 30310-30314, October 13, 2006
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1
2
From the
Department of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071 and the
Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm SE-171 77, Sweden
The ubiquitous bacterial second messenger c-di-GMP controls exopolysaccharide synthesis, flagella- and pili-based motility, gene expression, and interactions of bacteria with eukaryotic hosts. With the exception of bacterial cellulose synthases, the identities of c-di-GMP receptors and end targets have remained unknown. Recently, Amikam and Galperin (Amikam, D., and Galperin, M. (2006) Bioinformatics 22, 36) hypothesized that the PilZ domains present in the BcsA subunits of bacterial cellulose synthases function in c-di-GMP binding. This hypothesis has been tested here using the Escherichia coli PilZ domain protein YcgR, its individual PilZ domain and the PilZ domain from Gluconacetobacter xylinus BcsA. YcgR was purified and found to bind c-di-GMP tightly and specifically, Kd 0.84 µM. Individual PilZ domains from YcgR and BcsA also bound c-di-GMP, albeit with lesser affinity, indicating that PilZ is sufficient for binding. The site-directed mutagenesis performed on YcgR implicated the most conserved residues in the PilZ domain directly in c-di-GMP binding. It is suggested that c-di-GMP binding to PilZ brings about conformational changes in the protein that stabilize the bound ligand and initiate the downstream signal transduction cascade. While the identity of the downstream partner(s) of YcgR remains unknown, it is shown that YcgR regulates flagellum-based motility in a c-di-GMP-dependent manner. The inactivation of ycgR improves swimming and swarming motility of the poorly motile yhjH mutants of Salmonella enterica serovar Typhimurium UMR1. Therefore, biochemical and genetic evidence presented here establishes PilZ as a long sought after c-di-GMP-binding domain and YcgR as a c-di-GMP receptor affecting motility in enterobacteria.
Received for publication, July 10, 2006 , and in revised form, August 16, 2006.
* This work was supported in part by National Science Foundation Grant MCB-0316270 (to M. G.), the Karolinska Institutet (Elitforskartjänst to U. R.), and Vetenskapsrådet (621-2004-3979 (to U. R)). 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 Experimental Procedures and Tables S1 and S2.
1 To whom correspondence may be addressed: Karolinska Inst., MTC, Box 280, SE-171 77 Stockholm, Sweden. Tel.: 46-8-524-87319; Fax: 46-8-330744; E-mail: Ute.Romling{at}ki.se. 2 To whom correspondence may be addressed: Dept. of Molecular Biology, University of Wyoming, 1000 E. University Ave., Dept. 3944, Laramie, WY 82071. Tel.: 307-766-3522; Fax: 307-766-3875; E-mail: Gomelsky{at}uwyo.edu.
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