ENVIRONMENTAL MICROBIOLOGY PNNL GROUP

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 蓝细菌，以前称为蓝绿藻，是一大群光合作用的原核生物，是水环境中产氧光合作用的主要贡献者。 特别地，集胞藻属PCC 6803是一种已经广泛用于光合作用研究的蓝细菌，这是由于其完全测序的基因组、天然可转化性以及异养和自养生长的能力。 作为原核生物，蓝细菌必须在单个细胞内进行多种代谢，生物合成和组织活动。值得注意的是，蓝细菌是唯一的原核生物在拥有高度分化和区室化的膜系统。 集胞藻6803是一种革兰氏阴性细菌，其不仅具有由外膜、肽聚糖层和质膜组成的细胞被膜，而且还具有发生光合反应的类囊体膜的内部系统。 超微结构的研究已经持续了30多年，研究了蓝藻复杂的内部组织，并且薄切片透射电子显微镜清楚地显示了类囊体膜及其单独的膜囊。 然而，很少有人知道的三维亚细胞组织的类囊体膜内的细菌细胞。 重要的是，类囊体膜和质膜之间的空间关系仍然不清楚，蛋白质和其他生物分子在两个膜系统之间的运动机制仍然未知。 值得注意的是，许多研究者的研究并没有明确地证明两个膜系统之间存在任何连续性，或者存在转运囊泡。 因此，目前的知识的蓝藻细胞的内部组织仍然不完整。 到目前为止，大多数信息都是从随机薄切片或少量连续薄切片的电子显微镜中收集的。 虽然信息量大，但连续切片带来了许多技术挑战，并且在z轴方向上的分辨率受到切片厚度的限制。 该分辨率限制在~70 nm范围内，不足以解决集胞藻6803中的膜组织、相互连接和囊泡运输问题。 电子断层扫描的分辨率不受切片厚度的限制，并且可以用于生成分辨率为~3-10 nm的3D断层图像。 电子断层扫描有可能阐明在集胞藻6803中发现的复杂膜系统的结构，并有助于我们理解这种生物体如何建立和维持这样的system.This请求是进行EM-tomography分析的整个集胞藻属PCC 6803细胞，以获得3-D结构作为建立细胞模型的基础，用于调查上述膜功能。 我们还想探索使用金粒子标记技术来绘制集胞藻细胞内特定蛋白质分布的可能性。 这些研究是根据太平洋西北国家实验室环境微生物学小组的Alice Dohnalkova博士在NCMIR产生的整个细菌细胞的3D重建的建议和示例设计的。 我们的实验室正在与PNNL合作开展一项关于蓝藻膜的生物学大挑战项目，这些研究将成为华盛顿大学与PNNL合作的一部分。

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. Cyanobacteria, formerly known as blue-green algae, are a large group of photosynthetic prokaryotes that are major contributors to oxygenic photosynthesis in aqueous environments. In particular, Synechocystis sp. PCC 6803 is a cyanobacterium that has been used extensively in photosynthesis research, due its completely sequenced genome, natural transformability, and ability to grow heterotrophically as well as autotrophically. As prokaryotes, cyanobacteria must perform multiple metabolic, biosynthetic, and organizational activities within a single cell. Notably, cyanobacteria are unique among prokaryotes in possessing highly differentiated and compartmentalized membrane systems. Synechocystis 6803 is a Gram-negative bacterium that has not only a cell envelope consisting of outer membrane, peptidoglycan layer, and plasma membrane, but also an internal system of thylakoid membranes where photosynthetic reactions occur. Ultrastructural studies extending over thirty years have examined the complex internal organization of cyanobacteria, and thin-section transmission electron microscopy clearly shows the thylakoid membranes with their individual membrane sacs. However, little is known about the three-dimensional subcellular organization of the thylakoid membranes within such bacterial cells. Importantly, the spatial relationship between the thylakoid membranes and plasma membrane remains unclear, and the mechanisms of movements of proteins and other biomolecules between the two membrane systems remains unknown. It is noteworthy that examination by numerous investigators has not unequivocally demonstrated the existence of any continuity between the two membrane systems, or of the existence of transport vesicles. Thus, current knowledge of the internal organization of the cyanobacterial cell remains incomplete. The majority of information thus far has been gathered from electron microscopy of random thin sections or small numbers of serial thin sections. While informative, serial sectioning poses numerous technical challenges, and is limited in resolution in the z-axis direction by the thickness of the sections. This resolution limit, in the range of ~70 nm, is insufficient to address the questions of membrane organization, interconnectedness, and vesicle transport in Synechocystis 6803. Electron tomography is not limited in resolution by section thickness, and can be used to generated 3-D tomograms with resolution of ~3-10 nm. Electron tomography has the potential to elucidate the structure of the complex membrane systems found in Synechocystis 6803 and contribute to our understanding of how this organism builds and maintains such systems.This request is to conduct EM-tomography analyses of whole Synechocystis sp. PCC 6803 cells to obtain 3-D structures as the basis for building a cellular model for investigations of the membrane features described above. We would also like to explore the possibility of using gold particle labeling techniques to map the distribution of specific proteins within Synechocystis sp. cells. These studies were designed based on the recommendation and example of a 3-D reconstruction of a whole bacterial cell generated at the NCMIR by Dr. Alice Dohnalkova from the Environmental Microbiology Group at the Pacific Northwest National Laboratory. Our lab is partnering with PNNL on a biology Grand Challenge project on cyanobacterial membranes and these studies will be part of a collaborative effort between Washington University and PNNL.