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. Shewanella oneidensis MR-1 is a motile facultative bacterium with remarkable metabolic versatility in regards to electron acceptor utilization; it can utilize O2, nitrate, fumarate, Mn, Fe, and S0 as terminal electron acceptors during respiration. This versatility allows MR-1 to efficiently compete for resources in environments where electron acceptor type and concentration fluctuate in space and time. The ability to effectively reduce polyvalent metals and radionuclides, including solid phase Fe and Mn oxides, has generated considerable interest in the potential role of this organism in biogeochemical cycling and in the bioremediation of contaminant metals and radionuclides. In spite of considerable effort, the details of MR-1's electron transport system and the mechanisms by which it reduces metals and radionuclides remain unclear. Even less is known regarding the molecular networks in this organism that allow it to respond to compete efficiently in a changing environment. The entire genome sequence of MR-1 has, in essence, been determined and high throughput methods for measuring gene expression are now available, including mass spec-based proteome analyses developed at PNNL. Although powerful, DNA array and proteome analyses must be tightly coupled with other approaches to effectively reveal the molecular details of how MR-1 functions in, and responds to, its environment. We propose to investigate the following in a concerted fashion: gene expression (proteome and transcriptome); gene function (genetic footprinting); protein localization (transmission electron, fluorescence, and AFM-enhanced two-photon confocal microscopy); and protein-protein interactions (photon arrival time distribution analysis). This group intends to conduct EM-tomography analyses of whole Shewanella putrefaciens MR-1 cells to obtain 3-D structures as the basis for building a mesh-based cellular model. They would also like to explore the possibility of using gold particle labeling techniques to map the distribution of specific proteins on the surfaces of MR-1 cells. In addition to previously proposed work, we would like to investigate the ultrastructure of the cell envelope including the outer membrane of Shewanella oneidensis MR-1 with the emphasis on filamentous structures and exopolysaccharides. Bacterial cultures grown in conditions including flocculation or metal reduction-translocation are of particular interest in characterizing the spatial relationships between metals (oxidized and reduced), EPS,surface proteins, and potentially other structures in flocs and biofilms. The previously discussed approach of collecting images from frozen cultures using the cold stage on JEOL 4000 would be a method of choice for preserving non-collapsed morphology of EPS. Another area of interest is 3-d visualization of specific proteins, including outer membrane cytochromes and pilin or pilin-like structures, by immunogold labeling. Both pre-embedded plastic thick sections (expression of proteins associated with outer membrane), and sections obtained by ultracryomicroscopy with the follwing immunocytochemical incubation (JEOL 3100) would be prepared by our lab prior shipping them to your facility. The immunogold experiments will contribute significantly to understanding cell functions we are interested in. At last, we would like to image previously and future high-pressure frozen Shewanella . This method followed by a freeze subtitution dramatically improved inner structure of Shewanella. Previously prepared frozen cells are stored at NCMIR. We would like to use the capability of telemicroscpy for volumes collection and will need assistance with 3-d reconstruction softwa
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