ENVIRONMENTAL MICROBIOLOGY PNNL GROUP

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。蓝藻，以前被称为蓝绿藻，是一大类光合作用原核生物，是水环境中氧光合作用的主要贡献者。其中，Synechocystis sp. PCC 6803是一种广泛用于光合作用研究的蓝藻，由于其基因组完全测序，自然转化能力，以及异养和自养生长的能力。作为原核生物，蓝藻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，蓝藻在原核生物中具有高度分化和区隔化的膜系统是独一无二的。Synechocystis 6803是一种革兰氏阴性细菌，它不仅具有由外膜、肽聚糖层和质膜组成的细胞包膜，而且具有发生光合反应的类囊体膜内部系统。三十多年来的超微结构研究已经检查了蓝藻复杂的内部组织，薄层透射电子显微镜清楚地显示了类囊体膜及其单个膜囊。然而，对这些细菌细胞中类囊体膜的三维亚细胞组织知之甚少。重要的是，类囊体膜和质膜之间的空间关系尚不清楚，蛋白质和其他生物分子在两种膜系统之间的运动机制尚不清楚。值得注意的是，许多研究者的检查并没有明确地证明两个膜系统之间存在任何连续性，也没有证明存在运输囊泡。因此，目前的蓝藻细胞的内部组织的知识仍然不完整。迄今为止，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。虽然信息丰富，但连续切片带来了许多技术挑战，并且受截面厚度的限制，在z轴方向上的分辨率有限。这个分辨率限制在~70 nm的范围内，不足以解决Synechocystis 6803的膜组织、互联性和囊泡运输问题。电子层析成像不受切片厚度分辨率的限制，可以生成分辨率为~3-10 nm的三维层析成像。电子断层扫描有可能阐明在Synechocystis 6803中发现的复杂膜系统的结构，并有助于我们了解这种生物如何建立和维持这种系统。本请求是对整个Synechocystis sp. PCC 6803细胞进行em断层扫描分析，以获得三维结构，作为建立细胞模型以研究上述膜特征的基础。我们还想探索使用金颗粒标记技术来绘制聚囊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组的Alice Dohnalkova博士在NCMIR生成的整个细菌细胞的三维重建的建议和实例设计的。我们的实验室正在与PNNL就蓝藻膜的生物学大挑战项目进行合作，这些研究将成为华盛顿大学和PNNL之间合作努力的一部分。

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.