Journal of Bacteriology, April 2006, p. 2502-2511, Vol. 188, No. 7
0021-9193/06/$08.00+0 doi:10.1128/JB.188.7.2502-2511.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Organization of FliN Subunits in the Flagellar Motor of Escherichia coli
Koushik Paul and
David F. Blair*
Department of Biology, University of Utah, Salt Lake City, Utah 84112
Received 21 December 2005/
Accepted 10 January 2006
FliN is a major constituent of the C ring in the flagellar basal body of many bacteria. It is present in >100 copies per flagellum and together with FliM and FliG forms the switch complex that functions in flagellar assembly, rotation, and clockwise-counterclockwise switching. FliN is essential for flagellar assembly and switching, but its precise functions are unknown. The C-terminal part of the protein is best conserved and most important for function; a crystal structure of this C-terminal domain of FliN from Thermotoga maritima revealed a saddle-shaped dimer formed mainly from ß strands (P. N. Brown, M. A. A. Mathews, L. A. Joss, C. P. Hill, and D. F. Blair, J. Bacteriol. 187:2890-2902, 2005). Equilibrium sedimentation studies showed that FliN can form stable tetramers and that a FliM1FliN4 complex is also stable. Here, we have examined the organization of FliN subunits by using targeted cross-linking. Cys residues were introduced at various positions in FliN, singly or in pairs, and disulfide cross-linking was induced by oxidation. Efficient cross-linking was observed for certain positions near the ends of the dimer and for some positions in the structurally uncharacterized N-terminal domain. Certain combinations of two Cys replacements gave a high yield of cross-linked tetramer. The results support a model in which FliN is organized in doughnut-shaped tetramers, stabilized in part by contacts involving the N-terminal domain. Electron microscopic reconstructions show a bulge at the bottom of the C-ring whose size and shape are a close match for the hypothesized FliN tetramer.
* Corresponding author. Mailing address: Department of Biology, University of Utah, Salt Lake City, UT 84112. Phone: (801) 585-3709. Fax: (801) 581-4668. E-mail: blair{at}bioscience.utah.edu.
Supplemental material for this article may be found at http://jb.asm.org/.
Journal of Bacteriology, April 2006, p. 2502-2511, Vol. 188, No. 7
0021-9193/06/$08.00+0 doi:10.1128/JB.188.7.2502-2511.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Copyright © 2006 by the American Society for Microbiology. All rights reserved.