基于原位超分辨技术的微生物胞外电子传递终端协作机制研究

Extracellular electron transfer (EET) is the key feature of the microbial extracellular respiration, a very unique and new energy metabolism style (totally different from conventional intracellular respiration with soluble electron acceptors). EET played crucial roles on the biogeochemical elements cycling and pollutants transformation. It also showed great potential in bioenergy production and wastewater treatment. Outer membrane cytochrome C (OMC) proteins are the main players for EET at the outer cell membrane (the key final step for EET). Most of the bacteria with extracellular respiration function have multiple OMCs. However, due to no suitable in-situ investigation approach, the knowledge about the coexisted OMCs is still very limited, which makes it hard to understand the biological significance for the coexistence of the OMCs. .By using Shewanella oneidensis MR-1 as the model microorganism, we found that there is interesting synergetic effect between these OMCs during EET. More interestingly, we reported that graphene oxide (GO) can serve as the extracellular electron acceptor for S. oneidensis MR-1 and the bacteria can reduce the GO to reductive GO (rGO), resulting in a remarkable color change and fluorescence spectrum shift. Based on these preliminary results, we proposed to develop a new nanoscale, in-situ, dynamic method to monitor the EET of the OMCs in living cell based on super-resolution microscopy and GO nanoparticles. By employing this new method in combination with comparison between different mutants, we will explore the distribution of each OMC at nanoscale and analysis the relationship among these OMCs. With the rational control of EET flow (eg., active or inactive; up or down regulation), we will further characterize the EET function of each OMC (final electron exportation OMC, electron relay OMC or the assistant OMC without EET capability). Furthermore, by using genetic approaches and dynamic EET monitoring method developed in this study, we will elucidate the EET network among the OMCs and the electron flux distribution among each pathway. Finally, a model for EET with OMCs (emphasis on their synergetic effect) will be proposed and validated with different methods (such as pure protein analysis or other super-resolution techniques). .This project will bring the breakthrough on the method for EET in-situ investigation, provide new insight on the mechanism for the electron transfer with OMCs at nanoscale. These new knowledge and methods to be delivered will help to understand the biological significance for the coexistence of multiple OMC proteins and add new dimension to EET research to unveil the mystery of the microbial extracellular respiration.

胞外电子传递(EET)是独特的微生物能量代谢过程，是地球元素循环和污染物迁移转化的重要驱动力。外膜细胞色素C(OMC)蛋白是EET终端关键功能蛋白，胞外呼吸菌有多种OMC蛋白共存，但其原位工作模式及共存的生物学意义尚属未知。我们前期研究预示OMC蛋白间存在协同作用，且报道了OMC蛋白对氧化石墨烯(GO)的EET致变色效应。据此，本项目将基于GO的生物电致变色规律、结合超分辨显微技术，建立OMC蛋白EET的活细胞、原位、纳米尺度研究新方法；在此基础上，揭示多种OMC蛋白的空间分布规律及关系，原位解析OMC蛋白的功能及协作关系；结合突变株和EET原位动态分析，阐明OMC蛋白间的电子传递网络及流量分布规律；提出并多角度验证EET终端协作机制模型。预期将实现EET原位研究方法纳米尺度的突破、揭示OMC蛋白的电子传递协作机制，为深入理解OMC蛋白共存的生物学意义及EET过程机制提供新理论、新方法。

以胞外电子传递(Extracellular Electron Transfer, EET)为特征的胞外呼吸，完全区别于传统认知的“胞内呼吸”(含好氧和厌氧)，是一种特殊的、全新的微生物能量代谢模式。胞外呼吸赋予了微生物更灵活的能量代谢方式，扩展了微生物生存范围，也是地球元素循环、环境污染物(如重金属、放射性元素)迁移转化的重要驱动力。因此，本项目聚焦模式胞外呼吸菌Shewanella oneidensis MR-1，成功搭建了用于微生物活细胞蛋白成像分析的STED超分辨显微系统；设计并建立了微生物活细胞蛋白超分辨显微成像标记方法；建立了单细胞电子传递实时监测技术；揭示了氧化石墨烯生物还原规律及机理；发现了希瓦氏菌细胞周质电子阱；设计、创建并阐明了希瓦氏菌杂合跨膜电子传递链；阐明了希瓦氏菌胞外电子传递关键终端蛋白MtrC与OmcA，OmcA与MtrF的协作关联机理，建立了希瓦氏菌胞外电子传递终端蛋白的协作机理模型；创建了生物活性石墨烯水凝胶、单细胞电子捕集器等希瓦氏菌胞外电子传递强化调控新策略，突破了单细胞电子传递效率极限；在深入认识希瓦氏菌高效胞外电子传递性能及机制的基础上，成功开发了基于希瓦氏菌胞外电子传递能力的全细胞生物电化学传感器、CO2高效生物电化学转化系统和石墨烯基杂合生物产氢体系。以上研究成果，为微生物胞外呼吸研究提供了新方法和新技术，阐明了微生物胞外电子传递末端协作机理，建立了胞外电子传递系列新策略并突破了单细胞胞外电子传递极限，为进一步深入认识微生物胞外呼吸提供了新的理论依据，为推动微生物胞外呼吸的应用提供了新的基础和支撑。基于以上研究成果，项目实施期间共发表SCI论文17篇，含Nature Communications杂志论文1篇，申请发明专利6项（授权2项），培养博士毕业生2名，硕士毕业生2名，圆满完成了项目任务。

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