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.
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