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 year, 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, which may or may not be ordered, 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. At disproportionately high receptor levels, membrane invaginations containing non-functional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be re-established by increasing cellular levels of CheA and CheW. These results demonstrate 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. (see Zhang, Khursigara et al (2007) and Zhang, Weis et al (2007) for more details). In related studies we have used cryo-electron tomography to determine the architecture of polar chemoreceptor arrays in wild-type Caulobacter crescentus cells. 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. (see Khursigara, Wu and Subramaniam (2008) for more details). In continuing efforts to define the molecular structures of the individual receptors in the native environment of whole bacterial cells, we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We have 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. (see Khursigara, Wu, Zhang et al (2008) for more details).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 year, 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, which may or may not be ordered, 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. At disproportionately high receptor levels, membrane invaginations containing non-functional, axially interacting receptor assemblies are formed. However, functional chemoreceptor arrays can be re-established by increasing cellular levels of CheA and CheW. These results demonstrate 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. (see Zhang, Khursigara et al (2007) and Zhang, Weis et al (2007) for more details). In related studies we have used cryo-electron tomography to determine the architecture of polar chemoreceptor arrays in wild-type Caulobacter crescentus cells. 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. (see Khursigara, Wu and Subramaniam (2008) for more details). In continuing efforts to define the molecular structures of the individual receptors in the native environment of whole bacterial cells, we have used cryo-electron tomography combined with 3D averaging to determine the in situ structure of chemoreceptor assemblies in Escherichia coli cells that have been engineered to overproduce the serine chemoreceptor Tsr. We have 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 [summary truncated at 7800 characters]
| Hirai, Teruhisa; Subramaniam, Sriram (2009) Protein conformational changes in the bacteriorhodopsin photocycle: comparison of findings from electron and X-ray crystallographic analyses. PLoS One 4:e5769 |
| Milne, Jacqueline L S; Subramaniam, Sriram (2009) Cryo-electron tomography of bacteria: progress, challenges and future prospects. Nat Rev Microbiol 7:666-75 |
| Zhang, Peijun; Weis, Robert M; Peters, Peter J et al. (2007) Electron tomography of bacterial chemotaxis receptor assemblies. Methods Cell Biol 79:373-84 |
| Zhang, Peijun; Khursigara, Cezar M; Hartnell, Lisa M et al. (2007) Direct visualization of Escherichia coli chemotaxis receptor arrays using cryo-electron microscopy. Proc Natl Acad Sci U S A 104:3777-81 |
| Liu, Jun; McBride, Mark J; Subramaniam, Sriram (2007) Cell surface filaments of the gliding bacterium Flavobacterium johnsoniae revealed by cryo-electron tomography. J Bacteriol 189:7503-6 |
