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. Given their importance, it is surprising how much we still don't understand about basic bacterial cell biology. We still don't know, for instance, how bacteria generate and maintain their characteristic shapes, establish polarity, organize their genomes, segregate their chromosomes, divide, and in some cases move. In eukaryotes, all these tasks are performed by cytoskeletal filaments, but because previous imaging technologies failed to reveal analogous structures in bacteria, it was long thought that bacteria don't possess cytoskeletons. More recently, fluorescence microscopy has shown that bacteria have substantial internal order. In the first cycle of this grant, we used another young technology, electron cryotomography (ECT), to produce three- dimensional images of intact bacterial cells in a near-native state to """"""""molecular"""""""" (~4-6 nm) resolution. Using ECT we directly visualized hundreds of cytoskeletal filaments in ~20 different bacterial species, proving that the bacterial cytoskeleton is in fact both general and complex. Here we propose to develop and apply new correlated light and electron microscopy techniques to identify these filaments generally. This should allow us to resolve key discrepancies between the existing light and electron microscopical results and generate much-needed insight into the structures and functions of the bacterial cytoskeleton. We also propose work to improve EM image quality generally and expand the number of filaments being studied.

Public Health Relevance

Knowing the structure and function of the bacterial cytoskeleton will help us understand how bacteria accomplish their roles in health and disease and perhaps in the long-term future suggest new antibiotic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094800-07
Application #
8214543
Study Section
Microscopic Imaging Study Section (MI)
Program Officer
Flicker, Paula F
Project Start
2005-12-15
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
7
Fiscal Year
2012
Total Cost
$342,473
Indirect Cost
$126,753
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Oikonomou, Catherine M; Chang, Yi-Wei; Jensen, Grant J (2016) A new view into prokaryotic cell biology from electron cryotomography. Nat Rev Microbiol 14:205-20
Chang, Yi-Wei; Rettberg, Lee A; Treuner-Lange, Anke et al. (2016) Architecture of the type IVa pilus machine. Science 351:aad2001
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Chang, Yi-Wei; Chen, Songye; Tocheva, Elitza I et al. (2014) Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat Methods 11:737-9
Shikuma, Nicholas J; Pilhofer, Martin; Weiss, Gregor L et al. (2014) Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343:529-33
Pilhofer, Martin; Aistleitner, Karin; Biboy, Jacob et al. (2013) Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ. Nat Commun 4:2856
Pilhofer, Martin; Jensen, Grant J (2013) The bacterial cytoskeleton: more than twisted filaments. Curr Opin Cell Biol 25:125-33
Briegel, Ariane; Pilhofer, Martin; Mastronarde, David N et al. (2013) The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards. J Struct Biol 183:95-8
Yamaichi, Yoshiharu; Bruckner, Raphael; Ringgaard, Simon et al. (2012) A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole. Genes Dev 26:2348-60

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