ELECTRON TOMOGRAPHY OF THE PHOTOSYNTHETIC APPARATUS OF CHLOROTHRIX HAL

绿丝菌 HAL 光合装置的电子断层扫描

• 批准号: 7601059
• 负责人: ROBERT BLANKENSHIP
• 金额: $ 0.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7601059
• 关键词: Address; Arizona; Bacteria; Bacteriochlorophylls; Biochemical; Biochemistry; Biophysics; Caliber; Cell membrane; Cells; Chlorobi; Chlorobium; Chloroflexi; Complex; Computer Retrieval of Information on Scientific Projects Database; Computer software; Condition; Cryopreservation; Cryoultramicrotomy; Electron Microscopy; Electrons; Energy Transfer; Environment; Evolution; Food; Fossil Fuels; Freeze Substitution; Freezing; Funding; Glutaral; Grant; Harvest; Image Analysis; In Situ; Institutes; Institution; Life; Location; Maps; Membrane; Methods; Microscope; Microtomy; Morphologic artifacts; Organism; Osmium Tetroxide; Photons; Photosynthesis; Photosynthetic Complexes; Phylogenetic Analysis; Physiological; Placement; Plant Resins; Potassium Permanganate; Preparation; Process; Purpose; Range; Reaction; Research; Research Personnel; Resolution; Resources; Sampling; Series; Shapes; Source; Staining method; Stains; Standards of Weights and Measures; Structure; System; Techniques; Tomogram; Training; Transmission Electron Microscopy; United States National Institutes of Health; Universities; electron tomography; interest; microbial; novel; particle; photosynthetic bacteria; pressure; reconstruction; research facility; sample fixation; size; transmission process

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 嗜盐氯丝菌是一种新发现的产氧光合细菌，具有独特的光合器官。 正在研究这种光合作用装置的原位结构，以帮助我们了解这种生物体如何进行光合作用以及如何从叶绿体天线复合体转移能量以收集太阳能。 通过冷冻电子断层扫描研究分离的叶绿体的结构，也可以解决细菌叶绿素如何填充到叶绿体中以实现有效能量转移的问题。 背景光合作用是生物体从太阳中提取能量的过程。 光合作用过程中产生的能量对于地球上几乎所有生命都至关重要，人们以多种形式获取能量，从食物到化石燃料。 了解各种类型光合作用的生物物理学、生物化学和超微结构将有助于我们理解这一重要过程。 我们研究小组的重点是研究光合生物的进化和不同类型的光合器官。 NCMIR 研究机构的这项提案感兴趣的生物体是嗜盐氯丝菌，它是一种丝状无氧光养生物，能耐受高盐条件。 这种生物体很有趣，因为初步结果表明 Cx。嗜盐菌虽然在系统发育上与丝状缺氧光养生物（绿色非硫细菌）关系最密切，但其光合天线系统与绿色硫细菌最相似（Klappenbach 和 Pierson，2004）。 同样令人惊讶的是，没有任何生化或光谱证据表明存在反应中心复合体，这使得这种生物体成为一个谜。这种独特的生物体为研究光合作用和适应极端环境的新机制开辟了途径。 该提案的目的是从结构上表征该生物体的光合作用装置，无论是在细胞内原位还是作为分离的组件。 虽然这项研究与 NCMIR 的重点不直接与生物医学相关，但您所在研究所的设施非常适合我们的研究目的，因为您所在的研究所距离亚利桑那州最近。 因此，我们希望您考虑我们在您的设施中进行研究的建议。建议我们提出的研究是调查 Cx 中光合装置的原位结构和整体位置。通过树脂嵌入电子断层扫描技术观察嗜盐菌细胞，并通过冷冻电子断层扫描技术以更高分辨率观察分离的光合成分的结构。 叶绿体天线复合体（200 nm x 50 nm x 50 nm）最初捕获用于该生物体中光合作用的光子，在标准透射电子显微照片中清晰可见。 在含有叶绿体的其他光合细菌物种中，这些结构与嵌入膜内的反应中心直接相关。 在CX。在嗜盐菌中，叶绿体虽然通常与细胞质膜相关，但也存在于中央细胞质区域内，与任何膜材料没有明显的联系。 由于迄今为止发现的所有反应中心都与膜有关，因此该物种表明了光合作用替代机制的有趣可能性。 不幸的是，由于薄片透射电子显微镜 Z 轴分辨率的固有限制，整个细胞中叶绿体的确切分布和关联尚未确定。 电子断层扫描是确定这种分布所需的理想技术。我们能够成功制备 Cx。亚利桑那州立大学使用高压冷冻冷冻固定，然后用戊二醛和四氧化锇的标准混合物冷冻替代，并包埋在 Spurr 树脂中，用于树脂包埋电子断层扫描的嗜盐杆菌细胞。 使用高锰酸钾和四氧化锇的混合物也可以成功地对亲水性、含细菌叶绿素的叶绿体进行选择性染色（Hohmann-Marriott 等，2005）。 高锰酸钾还可以增强膜对比度。 通过这两种固定方法以及电子断层扫描，我们希望能够获得 Cx 的高分辨率图。嗜盐细胞，叶绿体和膜系统的结合，以及光合装置的原位大分子结合。 这些结果将有助于理解我们从光谱分析中看到的生物物理特征。 Cx 的小直径 (1 µm)。 halophila 使其成为电子断层扫描的理想候选者，获得完整细胞所需的连续切片很少。为了进一步研究这些关联，我们希望对分离的光合复合物使用冷冻电子断层扫描。 染色体可以从 Cx 中分离出来。 halophila 和冷冻电子断层扫描是可以在无伪影的情况下研究叶绿体结构的最佳技术。 单粒子重建不是一种选择，因为它们是大小和形状各异的异质结构。 如果无法在 NCMIR 上对分离的复合物进行冷冻电子断层扫描，则可以将整个细胞带过来，进行高压冷冻和冷冻切片，并在高放大倍数下获得光合装置的倾斜系列。要进行的实验/要使用的设施树脂嵌入式电子断层扫描用于电子断层扫描的样品的制备将在亚利桑那州立大学进行。 需要使用 JEM-3200EF IVEM (300 kV) 或 JEM-4000EX IVEM (400 kV) 来获得整个细胞连续切片的电子断层扫描倾斜系列，以及细胞内光合装置更高放大倍数下的一些倾斜系列。 冷冻电子断层扫描 样品（分离的叶绿体）将被带到 NCMIR 研究设施。 冷冻电子断层扫描样品的冷冻需要在 NCMIR 进行。 将使用 JEOL-2000EX (200 kV) 冷冻电子显微镜来帮助重建这些断层图像，并需要进行图像分析。 或者，可以将整个细胞带到 NCMIR，在设施中冷冻并冷冻切片，然后进一步捕获断层图。软件 任何进行断层图重建所需的软件培训以及可以转移到亚利桑那州立大学的相关图像分析软件将不胜感激。参考文献Hohmann-Marriott, M.F.、Blankenship, R.E. 和 Roberson R.W. (2005) 超微结构 电子显微镜显示的温热绿菌叶绿体。光合作用。资源。 (2005) 86: 145¿154.Klappenbach, JA 和 Pierson, BK (2004) 丝状缺氧光合自养细菌 ¿Candidatus Chlorothrix halophila gen nov., sp. 的系统发育和生理学特征。十一月，从高盐微生物垫中回收。拱。微生物。 181：17-25。