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Journal of Bacteriology, December 2008, p. 8065-8074, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01121-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Different Signaling Roles of Two Conserved Residues in the Cytoplasmic Hairpin Tip of Tsr, the Escherichia coli Serine Chemoreceptor {triangledown}

Patricia Mowery,{dagger} Jeffery B. Ostler,{ddagger} and John S. Parkinson*

Biology Department, University of Utah, Salt Lake City, Utah 84112

Received 10 August 2008/ Accepted 6 October 2008

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.

* Corresponding author. Mailing address: Biology Department, University of Utah, Salt Lake City, UT 84112. Phone: (801) 581-7639. Fax: (801) 581-4668. E-mail: parkinson{at}biology.utah.edu

{triangledown} Published ahead of print on 17 October 2008.

{dagger} Present address: Department of Biology, Hobart and William Smith Colleges, Geneva, NY 14456.

{ddagger} Present address: Nelson Laboratory, 6280 S. Redwood Road, Salt Lake City, UT 84123.

Journal of Bacteriology, December 2008, p. 8065-8074, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01121-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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