ENVIRONMENT SENSING, METAB RESPONSE & REGULATORY NETWORK IN RESPIRATORY PATHWAY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。白氏希瓦氏菌MR-1是一种能移动的兼性细菌，在电子受体利用方面具有显著的代谢多样性；它能在呼吸过程中利用O2、硝酸盐、富马酸、锰、铁和S0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。能够有效地还原多价金属和放射性核素，包括固相铁和锰氧化物，引起了人们对这种生物在生物地球化学循环和污染金属和放射性核素的生物修复中的潜在作用的极大兴趣。尽管做出了相当大的努力，但MR-1的S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不清楚。关于这种有机体中的分子网络，使其能够在不断变化的环境中做出反应并有效竞争，人们所知的更少。从本质上讲，MR-1的整个基因组序列已经确定，现在已经有了测量基因表达的高通量方法，包括PNNL开发的基于质谱学的蛋白质组分析。尽管功能强大，DNA阵列和蛋白质组分析必须与其他方法紧密结合，才能有效地揭示MR-1如何在其环境中发挥作用和对其做出反应的分子细节。我们建议以一致的方式研究以下问题：基因表达(蛋白质组和转录组)；基因功能(基因足迹)；蛋白质定位(透射电子、荧光和原子力显微镜增强的双光子共聚焦显微镜)；以及蛋白质-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烂希瓦氏杆菌MR-1细胞进行EM-CT分析，以获得3D结构，作为建立基于网格的细胞模型的基础。他们还希望探索使用金颗粒标记技术来绘制特定蛋白质在MR-1细胞表面的分布图的可能性。除了先前提出的工作外，我们还想研究包括圆锥希瓦氏菌MR-1外膜在内的细胞膜的超微结构，重点是丝状结构和胞外多糖。在絮凝或金属还原-移位等条件下生长的细菌培养物对表征絮体和生物膜中金属(氧化和还原)、EPS、表面蛋白以及潜在的其他结构之间的空间关系特别感兴趣。前面讨论的使用JEOL 4000冷冻台从冷冻培养物中收集图像的方法将是保存EPS未塌陷形态的一种选择方法。另一个令人感兴趣的领域是通过免疫金标记法对特定蛋白质的三维可视化，包括外膜细胞色素和菌毛素或类似菌毛素的结构。无论是预包埋的塑料厚切片(与外膜相关的蛋白质的表达)，还是通过超低温显微镜和随后的免疫细胞化学孵育(JEOL 3100)获得的切片，都将在运往您的设施之前由我们的实验室准备好。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，我们要想象一下以前和未来的高压冷冻希瓦内拉。这种方法加上冷冻置换，极大地改善了希瓦纳菌的内部结构。之前准备的冷冻细胞储存在NCMIR。我们希望使用远程显微扫描功能进行卷收集，并需要3-D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