ELECTRON TOMOGRAPHY OF THE PHOTOSYNTHETIC APPARATUS OF CHLOROTHRIX HAL

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Chlorothrix halophila is a newly discovered anoxygenic photosynthetic bacterium with a unique photosynthetic apparatus. The in situ structure of this photosynthetic apparatus is being investigated to help us to understand how this organism undertakes photosynthesis and how there is transfer of energy from the chlorosome antenna complex to harvest the sun¿s energy. The question of how bacteriochlorophyll is packed into the chlorosomes for efficient energy transfer would also be addressed by investigating the structure of isolated chlorosomes by cryo electron tomography. BackgroundPhotosynthesis is the process used by living organisms to extract energy from the sun. Energy derived from the process of photosynthesis is vital for almost all life on earth and is harvested by people in many forms ranging from food to fossil fuels. An understanding of the biophysics, biochemistry, and ultrastructure of the various types of photosynthesis will aid in our understanding of this important process. The focus of our research group is to investigate the evolution of photosynthetic organisms and the different types of photosynthetic apparatus. The organism of interest for this proposal to the NCMIR research facility is Chlorothrix halophila, a filamentous anoxygenic phototroph tolerant to hypersaline conditions. This organism is interesting as initial results indicate that Cx. halophila, while being most closely related to the filamentous anoxygenic phototrophs (green non-sulfur bacteria) phylogenetically, has a photosynthetic antenna system that is most similar to the green sulfur bacteria (Klappenbach and Pierson, 2004). Surprisingly also, there has been no biochemical or spectroscopic evidence of a reaction center complex, making this organism an enigma. This unique organism opens avenues to investigate novel mechanisms of photosynthesis and adaptation to an extreme environment. The objective of this proposal is to characterize structurally this organism¿s photosynthetic apparatus both in situ within the cell and as isolated components. Whilst this research is not directly biomedically related as is the focus of the NCMIR, the facilities at your institute would be ideal for our research purposes as is the proximal location to Arizona. We therefore hope that you consider our proposal to undertake research at your facility.ProposalOur proposed research is to investigate the in situ structure and overall placement of the photosynthetic apparatus in the Cx. halophila cell by resin-embedded electron tomography and, at a higher resolution, the structure of the isolated photosynthetic components by cryo electron tomography. The chlorosome antenna complex (200 nm x 50 nm x 50 nm), which initially captures photons for photosynthesis in this organism, is clearly distinguished in standard transmission electron micrographs. In other species of photosynthetic bacteria that contain chlorosomes, these structures are associated directly with reaction centers embedded within a membrane. In Cx. halophila, the chlorosomes, whilst often associated with the cytoplasmic membrane, are also found within the central cytoplasmic region without apparent connection to any membranous material. As all reaction centers to date have been found associated with membranes, this species suggests the intriguing possibility of an alternate mechanism of photosynthesis. Unfortunately, the exact distribution and associations of the chlorosomes throughout the cell has not been determined because of the inherent limitations of resolution in the Z-axis of thin section transmission electron microscopy. Electron tomography is the ideal technique required to determine this distribution.We are able to successfully prepare Cx. halophila cells for resin-embedded electron tomography at Arizona State University using cryofixation by high pressure freezing followed by freeze substitution with a standard mix of glutaraldehyde and osmium tetroxide, and embedment in Spurr¿s resin. Selective staining of hydrophilic, bacteriochlorophyll-containing chlorosomes has also been successful with a mix of potassium permanganate and osmium tetroxide (Hohmann-Marriott et al., 2005). Potassium permanganate also enhances membrane contrast. With these two fixation methods along with electron tomography we hope to be able to obtain a high-resolution map of the Cx. halophila cell, the association of the chlorosomes and membrane system, and the in situ macromolecular associations of the photosynthetic apparatus. These results will help to understand the biophysical profile that we see from spectroscopic analysis. The small diameter (1 ¿m) of Cx. halophila makes it an ideal candidate for electron tomography with few serial sections required to obtain a complete cell.To further investigate these associations, we hope to use cryo electron tomography on isolated photosynthetic complexes. Chlorosomes can be isolated from Cx. halophila and cryo electron tomography is the best technique by which the chlorosome structure can be investigated without artifact. Single particle reconstruction is not an option as these are heterogenous structures varying in both size and shape. If cryo electron tomography of the isolated complexes cannot be done at the NCMIR, whole cells could be brought over, high pressure frozen and cryo sectioned and a tilt series obtained of the photosynthetic apparatus at high magnification.Experiments to be done/Facilities to be usedResin-embedded electron tomography Preparation of the samples for electron tomography will be undertaken at Arizona State University. The use of either the JEM-3200EF IVEM (300 kV) or the JEM-4000EX IVEM (400 kV) would be required to obtain the tilt series for electron tomography of serial sections of whole cells and also some tilt series at higher magnification of the photosynthetic apparatus within the cell. Cryo electron tomography The samples (isolated chlorosomes) will be brought to the NCMIR research facility. Freezing of the samples for cryo electron tomography will need to be undertaken at the NCMIR. The JEOL-2000EX (200 kV) CryoElectron Microscope would be used and help with reconstruction of these tomograms and image analysis would be required. Alternately, whole cells could be brought to the NCMIR, frozen at the facility and cryosectioned before further capturing of tomograms.Software Any training in software required to undertake the reconstruction of tomograms and relevant image analysis software that could be transferred to Arizona State University would be greatly appreciated.ReferencesHohmann-Marriott, M.F., Blankenship, R.E., and Roberson R.W. (2005) The ultrastructure of Chlorobium tepidum chlorosomes revealed by electron microscopy. Photosynth. Res. (2005) 86: 145¿154.Klappenbach, JA and Pierson, BK (2004) Phylogenetic and physiological characterization of a filamentous anoxygenic photoautotrophic bacterium ¿Candidatus Chlorothrix halophila¿ gen nov., sp. Nov., recovered from hypersaline microbial mats. Arch. Microbiol. 181: 17-25.