ENVIRONMENT SENSING, METAB RESPONSE & REGULATORY NETWORK IN RESPIRATORY PATHWAY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心，不一定是研究者所在机构。希瓦氏菌（Shewanella oneidensis）MR-1是一种在电子受体利用方面具有显著代谢多样性的能动兼性细菌;它可以在呼吸过程中利用O2、硝酸盐、富马酸盐、Mn、Fe和SO作为末端电子受体。这种多功能性使MR-1能够在电子受体类型和浓度在空间和时间上波动的环境中有效地竞争资源。有效地减少多价金属和放射性核素，包括固相铁和锰氧化物的能力，产生了相当大的兴趣，这种生物体的潜在作用，在地球化学循环和污染金属和放射性核素的生物修复。尽管付出了相当大的努力，MR-1的电子传输系统的细节以及它还原金属和放射性核素的机制仍然不清楚。关于这种生物体中的分子网络，使其能够在不断变化的环境中有效地竞争，我们所知甚少。MR-1的整个基因组序列基本上已经确定，现在可以使用高通量方法测量基因表达，包括PNNL开发的基于质谱的蛋白质组分析。虽然功能强大，但DNA阵列和蛋白质组分析必须与其他方法紧密结合，以有效揭示MR-1如何在其环境中发挥作用并对其做出反应的分子细节。我们建议以协调一致的方式研究以下内容：基因表达（蛋白质组和转录组）;基因功能（遗传足迹）;蛋白质定位（透射电子，荧光和AFM增强的双光子共聚焦显微镜）;和蛋白质-蛋白质相互作用（光子到达时间分布分析）。 该小组打算对整个腐败希瓦氏菌MR-1细胞进行电磁断层扫描分析，以获得三维结构，作为建立基于网格的细胞模型的基础。他们还想探索使用金颗粒标记技术来绘制MR-1细胞表面特定蛋白质分布的可能性。除了以前提出的工作，我们想调查的超微结构的细胞包膜，包括外膜的希瓦氏菌oneidensis MR-1的丝状结构和胞外多糖的重点。在包括絮凝或金属还原易位的条件下生长的细菌培养物在表征金属（氧化和还原）、EPS、表面蛋白以及絮凝物和生物膜中的潜在其他结构之间的空间关系方面特别感兴趣。先前讨论的使用JEOL 4000上的冷台从冷冻培养物收集图像的方法将是用于保存EPS的非塌陷形态的选择方法。 另一个感兴趣的领域是通过免疫金标记对特定蛋白质的三维可视化，包括外膜细胞色素和菌毛蛋白或菌毛蛋白样结构。预包埋的塑料厚切片（表达与外膜相关的蛋白质）和通过以下免疫细胞化学孵育（JEOL 3100）通过超低温显微镜获得的切片将由我们的实验室制备，然后将其运送到您的机构。免疫金实验将大大有助于理解我们感兴趣的细胞功能。 最后对高压冷冻希瓦氏菌进行了展望。这种方法后，冷冻冷藏显着改善希瓦氏菌的内部结构。将先前制备的冷冻细胞储存在NCMIR。 我们想利用远程显微镜的能力进行体积收集，并需要三维重建软件的帮助。

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