(795c) Super-Resolution Imaging of the Bacterial Chemotaxis System in Bacillus Subtilis | AIChE

(795c) Super-Resolution Imaging of the Bacterial Chemotaxis System in Bacillus Subtilis


Agrawal, U. - Presenter, University of Illinois at Urbana-Champaign
Schroeder, C. M., University of Illinois at Urbana-Champaign
Rao, C. V., University of Illinois, Urbana Champaign

In this work, we apply fluorescence nanoscopy to elucidate a near molecular scale view of key proteins involved in bacterial motility. The ability to probe cellular structures and events using fluorescence microscopy is of paramount importance for understanding dynamic biological processes. However, optical diffraction limits the spatial resolution of conventional fluorescence microscopy to ≈ 250 nm, which precludes spatially resolved analysis of nanometer-scale cellular protein assemblies that mediate key biological processes, such as microbial motility. Recent advances in super-resolution imaging have enabled imaging at 20-25 nm spatial resolution1.

Chemotaxis refers to the movement of microorganisms under the influence of chemical gradients used by microbes to search for nutrients for survival. Chemotaxis is exploited industrially for designing microbial biosensors for detection and degradation of a broad spectrum of chemicals including toxins, environmental pollutants, as well as bio-warfare agents. In general, bacteria perform chemotaxis via run-and-tumble strategy using an intricate internal signaling network. Chemotaxis is mediated by protein clusters, which display dynamic changes in spatial organization and architecture in response to chemical cues. These chemotactic receptor clusters play a key role in response to stimulants, eventually eliciting behavioral response post signal transduction via the chemotactic network. As chemotactic clusters are several fold smaller than the diffraction limit of light (200 nm), the application of fluorescence nanoscopy is imperative to develop an accurate model for chemotaxis.

We are employing single molecule localization based super-resolution imaging to directly visualize dynamic changes in nano architectures of key chemotactic proteins in response to chemical stimulation in B. subtilis to elucidate near molecular scale view on signal amplification and signal transduction. Recent studies on B. subtilis have revealed that receptor lattices are not static and undergo significant structural rearrangement when exposed to ligands2. However, the understanding of mechanism of this rearrangement, activity modulation by receptors and eventual signal transduction remains unclear. Our work has revealed marked differences in the subcellular localization and clustering patterns of receptor proteins upon chemical stimulation in individual bacterial cells. In first-of-its-kind super-resolution visualization in B. subtilis, we observed that receptor rearrangement is characterized by a largely polar localization in unstimulated cells to a more diffused polar-lateral configuration in cells that have been exposed to ligand. Our results conform to the previous diffraction limited studies2. We achieved a spatial resolution of ≈ 25 nm using immunofluorescence based stochastic optical reconstruction microscopy (STORM). Our super-resolution data also provides crucial information on changes in structure and composition of the polar and lateral receptor clusters in B. subtilis during chemotaxis towards asparagine. This work provides a novel platform to study how chemotaxis clusters form, reorganize and localize during chemotaxis. Our data also embodies individual receptor molecules or small cluster sizes, which were previously inaccessible using conventional fluorescence microscopy. Overall, this work presents tremendous potential to provide complete molecular-scale understanding of chemotactic response using receptor dynamics by tapping information on cluster densities, cluster sizes, cluster shapes and number of receptors forming a cluster. Our work will provide novel insights to help discern long-standing mystery of mechanistic details of receptor organization and signal transduction during chemotaxis in B. subtilis.


1. Agrawal, U., Reilly, D. T. & Schroeder, C. M. Current opinion in biotechnology 1–8 (2013).

2. Wu, K., Walukiewicz, H. E., Glekas, G. D., Ordal, G. W. & Rao, C. V. The Journal of biological chemistry 286, 2587–95 (2011).