A major limitation of transmission electron microscopes is the requirement for thin specimens. E. coli cells are about 1 micron across, and have traditionally been considered to be too thick for analysis by cryo electron tomography. A number of small bacterial cells are now being studied by electron tomography because they are smaller and therefore can be imaged in the transmission mode at low doses. However the biology of chemotaxis has been best studied in E. coli and B. subtilis, both of which are relatively thick cells. Steady improvements that we have made over the last four years in specimen preparation, imaging protocols and image analyis have now led to the first description of chemotaxis arrays in intact wild-type E. coli cells. We have capitalized on this advance to image cells in which we are able to manipulate the levels of different signaling components individually, and correlate structure of the chemotaxis machinery in a given cell with its physiology. Our ability to carry out such experiments is a cornerstone in the battery of structural and functional experiments now underway to dissect how the cell alters the architecture of its signaling machinery in response to changes in protein levels and external stimuli. 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. In a related development, we have addressed a long-standing mystery surrounding gliding motility of F. johnsoniae as an example of bacteria that move rapidly over surfaces in the apparent absence of macromolecular cell-surface structures such as flagella or Type IV pili. We have used cryo electron tomography to show that thin filaments are present on the surface of motile F. johnsoniae cells but not on the mutant GldF, which is a nonmotile cell. GldF is a component of an ATP-binding-cassette transporter that is required for gliding. To confirm this, we transformed mutant cells with a plasmid carrying the gene for GldF and showed that filaments were recovered. These experiments also have allowed us to demonstrate that density from GldF can be visualized in periplasmic region, and that it serves as a potential scaffold for anchoring the tufts of filaments that appear to mediate gliding motility.
| 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 |
