We are carrying out a sustained and systematic approach to address the challenge of predicting bacterial chemotaxis behavior. In studies reported over the course of the last two years, we have demonstrated that it is possible to directly visualize and determine structures of molecular components of the chemotaxis machinery and cytoskletal components in intact gram-negative bacteria. Our work on direct visualization and spatial organization of chemoreceptor arrays in intact E. coli cells using cryo-electron tomography shows that in wild-type cells, ternary complexes are arranged as an extended lattice, with significant variations in the size and specific location among cells in the same population. In the absence of CheA and CheW, chemoreceptors do not form observable clusters and are diffusely localized to the cell pole. Our results firmly establish that chemotaxis in E. coli requires the presence of chemoreceptor arrays, and that the formation of these arrays requires the scaffolding interactions of the signaling molecules CheA and CheW. In an extension of these studies to C. crescentus, we demonstrated that chemoreceptors in this Gram-negative bacterium are organized as trimers of receptor dimers, forming partially ordered, hexagonally-packed arrays of signaling complexes in the cytoplasmic membrane. This novel receptor organization at the order/disorder interface suggests how receptors and effectors can be packed in signaling assemblies to respond dynamically in the activation and adaptation steps of bacterial chemotaxis. In efforts to define the molecular structures of the individual receptors in the native environment of whole bacterial cells, we used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that were engineered to overproduce the serine chemoreceptor Tsr. We demonstrated that chemoreceptors are organized as trimers of receptor dimers and display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer. Ligand binding and methylation alter the distribution of chemoreceptors between the two conformations, with serine binding favoring the expanded conformation, and chemoreceptor methylation favoring the compact conformation. The distinct positions of chemoreceptor HAMP domains within the context of a trimeric unit are thus likely to represent important aspects of chemoreceptor structural changes relevant to chemotaxis signaling. Based on these results, we propose that the compact and expanded conformations represent the kinase on and kinase off states of chemoreceptor trimers, respectively. The fact that we can determine structures of molecular complexes in intact cells revolutionizes our approach to carrying out meaningful calculations of the dynamic changes in the chemotaxis apparatus and cytoskeletal architecture, and to compare how changing the physiology of the cells by genetic alterations can be used to probe and understand the behavior of the underlying complex machinery. In more recent studies, we have further extended the analysis to describe and compare the spatial distribution, localization and architecture of chemoreceptor arrays in three different Gram-negative bacteria using cryo-electron tomography. We show that although each organism shares a seemingly common arrayed architecture, E. coli arrays are disperse and extended, in contrast to the compact arrays observed in Caulobacter and Bdellovibrio cells. Chemoreceptor arrays are also more consistent in size and are closely associated with the single polar flagella in Caulobacter and Bdellovibrio cells, while they vary greatly in size and demonstrate no discernable spatial correlation to the multiple flagella present in E. coli cells. Tomographic averaging results demonstrate that with the application of hexagonal symmetry, all three organisms have a similar unit spacing and trimer-of-dimer distance. However, analysis of receptor distribution in individual arrays reveals substantial variations in nearest neighbor proximities from one species to another, and in response to changes in growth medium. We are now using the information derived from the structural analyses to obtain statistically meaningful receptor distribution plots and to use them to generate computational models aimed at predicted the chemotactic behavior of Gram-negative bacteria.
| Earl, Lesley A; Falconieri, Veronica; Subramaniam, Sriram (2018) Microbiology catches the cryo-EM bug. Curr Opin Microbiol 43:199-207 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature 558:553-558 |
| Kang, Yanyong; Kuybeda, Oleg; de Waal, Parker W et al. (2018) Publisher Correction: Cryo-EM structure of human rhodopsin bound to an inhibitory G protein. Nature : |
| Earl, Lesley A; Falconieri, Veronica; Milne, Jacqueline Ls et al. (2017) Cryo-EM: beyond the microscope. Curr Opin Struct Biol 46:71-78 |
| Wohlbold, Teddy John; Podolsky, Kira A; Chromikova, Veronika et al. (2017) Broadly protective murine monoclonal antibodies against influenza B virus target highly conserved neuraminidase epitopes. Nat Microbiol 2:1415-1424 |
| 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 |
| 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 |
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