Bacterial chemotaxis is among the best-studied signal transduction pathways, where decades of analysis of structural, biochemical, genetic and physiological components of signaling have contributed to a broad understanding of the overall signaling process. We have taken significant steps over the last year both in structural imaging of intact bacterial cells and in the development of predictive computational models that capture cellular responses to changes in their environment, thus contributing to a central goal of modern cellular biology. Bacteria sense many of the changes in their local chemical environment by the binding of ligands to a family of chemotaxis receptors, which in turn trigger the activation of a signaling pathway that ultimately regulates the rotation of the flagellar motor. A detailed understanding of the spatial and temporal architecture of the bacterial apparatus for chemotaxis is a problem of fundamental interest because it will provide a framework to integrate the extensive genetic, physiological, biochemical and structural analyses of this process. With the advent of advanced imaging methods that allow spatial localization of specific protein complexes within the cell, the prospect of developing an integrated structural understanding of whole bacterial cells at the molecular level is potentially within range. Chemotaxis is a particularly tractable signaling pathway given the small number of components involved and the knowledge of the spatial localization of the front end of the signal transduction cascade. Over the last few years, we have taken systematic steps to bridge the gap from structure to physiology as it relates to deriving a quantitative model for chemotaxis that takes into account the spatial and molecular organization of the protein components in the signaling pathway. We began with first establishing the feasibility of localizing the receptors in the bacterial cell, and moved on to establishing the possibility of obtaining 3D structures of receptor proteins when they are still in an intact bacterium. This in turn, led to the discovery of the partially ordered hexagonal arrangement of receptors in the plane of the membrane. We have now extended these foundations to translating the information on the structure of the receptors, their spatial distribution, and richness of the growth medium into a testable, predictive computational model for chemotaxis signaling. Highlights of progress over the last year include (i) discovery of the partially ordered arrangement of chemoreceptor arrays in multiple gram-negative bacteria;(ii) definitive evidence of the trimer-of-dimer organization in isolated receptor assemblies;(iii) extension of cryo-electron tomographic studies to the nucleoid to establish the connection between nucleoid structure and receptor organization;(iv) development of computational models that correctly predict the changes in receptor arrangement and density with changing nutrient conditions in the medium and (v) progress towards applying combined EM/SIMS imaging to intact bacterial cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010824-06
Application #
8552846
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2012
Total Cost
$905,109
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Earl, Lesley A; Falconieri, Veronica; Milne, Jacqueline Ls et al. (2017) Cryo-EM: beyond the microscope. Curr Opin Struct Biol 46:71-78
Meyerson, Joel R; Chittori, Sagar; Merk, Alan et al. (2016) Structural basis of kainate subtype glutamate receptor desensitization. Nature 537:567-571
Subramaniam, Sriram; Earl, Lesley A; Falconieri, Veronica et al. (2016) Resolution advances in cryo-EM enable application to drug discovery. Curr Opin Struct Biol 41:194-202
Stuart, David I; Subramaniam, Sriram; Abrescia, Nicola G A (2016) The democratization of cryo-EM. Nat Methods 13:607-8
Matthies, Doreen; Dalmas, Olivier; Borgnia, Mario J et al. (2016) Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating. Cell 164:747-56
Subramaniam, Sriram; Kühlbrandt, Werner; Henderson, Richard (2016) CryoEM at IUCrJ: a new era. IUCrJ 3:3-7
Frank, Gabriel A; Shukla, Suneet; Rao, Prashant et al. (2016) Cryo-EM Analysis of the Conformational Landscape of Human P-glycoprotein (ABCB1) During its Catalytic Cycle. Mol Pharmacol 90:35-41
Bartesaghi, Alberto; Merk, Alan; Banerjee, Soojay et al. (2015) 2.2 Å resolution cryo-EM structure of ?-galactosidase in complex with a cell-permeant inhibitor. Science 348:1147-51
Patwardhan, Ardan; Ashton, Alun; Brandt, Robert et al. (2014) A 3D cellular context for the macromolecular world. Nat Struct Mol Biol 21:841-5
Meyerson, Joel R; Rao, Prashant; Kumar, Janesh et al. (2014) Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Sci Rep 4:7084

Showing the most recent 10 out of 26 publications