Bacteria are nearly ubiquitous, play vital roles in industry and the environment, and are important actors in both health and disease for humans and other organisms. They are also small, easily-manipulable model cells that can be used to study basic cell biological studies. Motile bacteria, including many important pathogens, constantly monitor their environment in order to swim towards nutrients and away from toxins, a process called chemotaxis. Attractants and repellents bind to chemoreceptors, which are typically found at the poles of cells grouped together in highly cooperative, ordered arrays. Activated chemoreceptor arrays phosphorylate a protein messenger which in turn binds to flagellar motors, governing the rate of motor reversals and, ultimately, whether the cell continues to move forward or changes direction. While powerful methods like X-ray crystallography and NMR spectrometry have revealed the structures of individual domains of certain chemoreceptors at near-atomic resolution, they have not revealed how the chemoreceptors are arranged inside living cells or the structural basis of array cooperativity. Instead, we have begun to address these issues with an emerging technology, electron cryotomography, which can produce 3-D reconstructions of intact bacterial cells at "macromolecular" (1-5 nm) resolution, which is sufficient to visualize individual receptor dimers. Briefly, bacterial cultures are plunge-frozen in thin films across EM grids and then imaged from a range of angles as the sample is tilted incrementally around one or two axes. 3-D reconstructions are then calculated from the images, and sub-regions with common features can be averaged to increase the signal-to-noise ratio. Following recent work in which we showed that bacterial chemoreceptor arrays are universally arranged in a conserved, 12-nm hexameric lattice of trimers-of-receptor-dimers, here we propose to extend that work in resolution and by imaging fully-activated and -deactivated states. This should reveal how the proteins are arranged within the array as well as the structural basis of activation and array cooperativity. This information will in turn help us understand how bacteria accomplish their roles in health and disease and perhaps suggest new antibiotic targets or strategies.

Public Health Relevance

Many bacteria can swim towards nutrients and away from toxins by sensing how the concentrations of these molecules change in their environment as they move forward. We propose to image the protein machines responsible for this ability with an emerging technology, electron cryotomography. Understanding the molecular mechanisms may suggest new targets or strategies to combat bacterial infections, and will reveal basic principles of how cells sense and respond to their environments, ultimately helping us understand and combat other diseases such as chronic inflammation, cancer, and diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM101425-01A1
Application #
8507076
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Deatherage, James F
Project Start
2013-04-01
Project End
2017-02-28
Budget Start
2013-04-01
Budget End
2014-02-28
Support Year
1
Fiscal Year
2013
Total Cost
$276,344
Indirect Cost
$106,344
Name
California Institute of Technology
Department
None
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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
Briegel, Ariane; Ames, Peter; Gumbart, James C et al. (2013) The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state. Mol Microbiol 89:831-41