硫还原地杆菌与含铁矿物之间的胞外电子传导机理研究

This proposed research focuses on a crucial scientific question of ‘how Geobacter sulfurreducens transfers electrons from the cytoplasmic membranes, through the periplasm, across the outer membrane and to the surfaces of Fe(III)-bearing minerals’. By using the biochemical methods, cytochromes CbcL and PpcA as well as Pcc protein complexes will be purified from the cytoplasmic membrane, the periplasm and the outer membrane of G. sulfurreducens, respectively. The purified CbcL is then characterized for its ability to oxidize quinol in the cytoplasmic membrane, substrate specificity and electron transfer mechanisms. Similarly, the electron transfer rates from CbcL to PpcA and from PpcA to Pcc complexes will be determined, respectively. Furthermore, the protein-protein interactions between CbcL and PpcA as well as between PpcA and Pcc protein complexes in the cells of G. sulfurreducens will be investigated with in vivo chemical cross-linking in conjunction with immunochemical detection. Finally, the electron transfer rates from Pcc complexes to the Fe(III)-bearing minerals are measured by using the proteoliposome-based and the modern mineralogical methods. Systematic understanding of electron transfer processes of CbcL→PpcA→Pcc protein complexes→Fe(III)-containing minerals will not only reveal new insights into the molecular electron transfer mechanisms by which G. sulfurreducens reduces extracellular Fe(III)-bearing minerals, but also lay the scientific foundations for biotechnological applications of G. sulfurreducens in bioremediation of contaminants in subsurface environment and in other areas.

围绕‘硫还原地杆菌如何将电子经细胞质膜、周质空间、外膜传导到含铁矿物表面’这一关键科学问题，首先运用生物化学手段，分别纯化硫还原地杆菌细胞质膜上的细胞色素CbcL、周质空间的细胞色素PpcA和外膜中的Pcc蛋白质复合体。然后表征CbcL氧化质膜中氢醌的能力、对底物的专一性和电子传导机理，测定CbcL→PpcA和PpcA→Pcc复合体的电子传导速率。利用活细胞化学固定和免疫化学检测技术，验证细菌体内CbcL-PpcA及PpcA-Pcc复合体的相互作用。采用蛋白质脂质体和现代矿物学方法，检验Pcc复合体→含铁矿物的电子传导速率。通过系统刻画CbcL→PpcA→Pcc复合体→含铁矿物的电子传导过程，从分子水平阐述硫还原地杆菌在还原含铁矿物过程中胞外电子传导机理，为有效利用开发硫还原地杆菌在修复地下污染物和在其它生物技术中的应用提供科学依据。

微生物的胞外电子传递将微生物细胞内的代谢反应与细胞外的氧化还原反应有效地耦合在一起，直接参与到碳、氮、硫、铁和碘元素的生物地球化学循环。本项目以硫还原地杆菌为模式菌株，采用微生物学、分子生物学及生物化学相结合的方法，系统地用本项目制备的OmaB、OmbB、OmcB、CbcL和PpcA多肽抗体，分析比对用甲醛、DSS、DST和BS3化学交联的、在还原三价铁条件下培养的硫还原地杆菌野生菌株及及ΔppcA和ΔcbcL基因敲除株。结果不仅首次检测到OmaB、OmbB和OmcB之间，并且发现三个PpcA和一个CbcL之间在硫还原地杆菌内的蛋白质-蛋白质相互作用。说明硫还原地杆菌细胞质膜上的CbcL可将质膜内的电子传递给细胞周质空间的PpcA,多个PpcA分子然后共同地将电子传递通过周质空间至细胞外膜上的OmaB-OmbB-OmcB复合体，OmaB-OmbB-OmcB复合体将电子由周质空间、穿过细胞外膜传递至细胞表面的含铁矿物。揭示了硫还原地杆菌还原铁矿物的胞外电子传递分子机理。本项目的研究成果不仅提升了从分子水平上理解微生物胞外电子传递过程，而且可用于合成生物学设计和改造微生物燃料电池及碳中和地合成高附加值的生化产品。