The chemotactic signal transduction pathway of Escherichia coli is representative of a large family of signal transduction pathways that are distributed throughout the Bacteria and Archaea. These pathways are already known to mediate chemotaxis and phototaxis in free-swimming and surface-associated bacteria, but are being recognized increasingly to regulate multicellular aggregation, pilus expression, and biofilm formation - medically relevant phenomena that are characteristic of pathogenic microbes. The relative simplicity of the E. coli system, combined with the well-developed and sophisticated tools for its study, provides a strong rationale to determine in detail the sensory physiology, biochemistry and structural biology of the E. coli pathway. Transmembrane signaling in the E. coli system occurs in the context of a heterogeneous cluster, or array, of receptor proteins that are associated with the cytoplasmic adaptor protein, CheW, and the central signaling kinase, CheA. Attractant binding to the receptors causes subtle conformational changes in the receptor that are somehow propagated across the membrane to produce two effects (i) inhibition of CheA, and (ii) stimulation of receptor methylation. Kinase inhibition exhibits a positive cooperativity indicative of regulation by a cluster of receptors. The key to understanding the detailed mechanism lies in understanding the manner in which these clusters of receptor complexes are assembled and remodeled during function. In this project we will investigate the interactions among receptors and with the signaling proteins in samples of successively greater complexity (in vitro to in vivo), to determine what structures and interactions are critical to the control of kinase and methylation activity. The simplest system, active complexes of the cytoplasmic domain, CheA, and CheW assembled on vesicle surfaces, will be studied in the greatest detail, with a combination of biochemical and biophysical tools (activity assays, disulfide crosslinking, fluorescence, and solid-state NMR). Studies of assemblies of complexes of the intact receptor in vesicles will determine how the interactions in the array are modulated by the other receptor domains and by ligand. Finally, in vivo fluorescence studies will investigate remodeling of receptor complexes and arrays during function. The approaches developed and insights gained will be applicable to other systems in which changes in both conformation and subunit association play a role in the mechanism of transmembrane signaling.
This project seeks to determine the function of signaling proteins in the chemotaxis pathway of E. coli through the use of isolated protein components reassembled into functioning signaling complexes and living bacterial cells. These samples will be tested with a combination of biochemical and biophysical techniques to correlate structure with activity. This E. coli system is representative of a large family of signaling pathways in microbes, including disease-causing bacteria, and therefore, the information obtained through the course of this project will be relevant to a great number of pathways, which may benefit the development of novel antimicrobial agents.
|Harris, Michael J; Struppe, Jochem O; Wylie, Benjamin J et al. (2016) Multidimensional Solid-State Nuclear Magnetic Resonance of a Functional Multiprotein Chemoreceptor Array. Biochemistry 55:3616-24|
|PrÃ¼Î², Birgit M; Liu, Jun; Higgs, Penelope I et al. (2015) Lessons in Fundamental Mechanisms and Diverse Adaptations from the 2015 Bacterial Locomotion and Signal Transduction Meeting. J Bacteriol 197:3028-40|
|Briegel, Ariane; Wong, Margaret L; Hodges, Heather L et al. (2014) New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography. Biochemistry 53:1575-85|
|Koshy, Seena S; Li, Xuni; Eyles, Stephen J et al. (2014) Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains. Biochemistry 53:7755-64|
|Briegel, Ariane; Ladinsky, Mark S; Oikonomou, Catherine et al. (2014) Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling. Elife 3:e02151|
|Mukherjee, Supratim; Thompson, Lynmarie K; Godin, Stephen et al. (2014) Population level analysis of evolved mutations underlying improvements in plant hemicellulose and cellulose fermentation by Clostridium phytofermentans. PLoS One 9:e86731|
|Mudiyanselage, Aruni P K K Karunanayake; Yang, Meili; Accomando, Lee A-R et al. (2013) Membrane association of a protein increases the rate, extent, and specificity of chemical cross-linking. Biochemistry 52:6127-36|
|Koshy, Seena S; Eyles, Stephen J; Weis, Robert M et al. (2013) Hydrogen exchange mass spectrometry of functional membrane-bound chemotaxis receptor complexes. Biochemistry 52:8833-42|
|Fowler, Daniel J; Harris, Michael J; Thompson, Lynmarie K (2012) Heat management strategies for solid-state NMR of functional proteins. J Magn Reson 222:112-8|
|Sferdean, Fe C; Weis, Robert M; Thompson, Lynmarie K (2012) Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms. Biochemistry 51:6920-31|
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