氧化亚铁硫杆菌的趋磁构建及其超精细和微磁结构研究

Acidithiobacillus ferrooxidans are important strains in microbial metallurgy. They have high resistibility to many metals and submetals. The strains can adsorb the toxic heavy metals in sludge and soil culture. Some heavy metals can be enriched in the strains, and others can take part in the intracellular metabolism. Therefore, it is necessary to effectively separate the acidithiobacillus ferrooxidans from the sludge and soil culture. While, there are still no effective method for the separation. We propose to slove this problem by genetic engineering. The gene cluster of magnetosome in magnetotactic bacteria would be transferred into acidithiobacillus ferrooxidans and be expressed. Many characterized methods through the cryo field-emission transmission electron microscopy (Cryo-TEM) would be used for the detailed microstructural study of magnetosome synthetized in the acidithiobacillus ferrooxidans. The information about the shape, crystalline state, elemental composition and valent state of synthetized magnetosome would be systematic studied. The different genes of magnetosome would be expressed in acidithiobacillus ferrooxidans. The functions of the different genes of magnetosome should be maken clear through the comparative characterization of ultramicrostructure of magnetosome. Finally, the strains of acidithiobacillus ferrooxidans which can be stably express the genes of magnetosome would be obtained. In the cell of these strains, many magnetosome crystals arranged in chain would be formed. Accordingly, these strains would have strong magnetotactic characteristics and can move quickly along the externally-applied magnetic field.

氧化亚铁硫杆菌是微生物冶金领域的重要菌株，对多种金属及类金属具有极高抗性，该菌可将污泥、土壤等危险废物中毒性重金属吸附，于胞内沉淀累积或者参与胞内代谢。因此如何有效分离回收胞内富集重金属的氧化亚铁硫杆菌，是亟需解决的问题。本项目拟通过基因工程手段，将趋磁细菌体内的磁小体基因簇（38个相关基因）转入氧化亚铁硫杆菌内，综合运用冷冻场发射透射电子显微镜(cryo-TEM)的多种手段，系统研究合成磁小体颗粒的形态、晶态、元素成分、价态、结构等信息；通过超微结构对比表征不同磁小体基因在氧化亚铁硫杆菌内的表达情况，明确各个磁小体基因的功能；最终获得稳定表达磁小体基因的氧化亚铁硫杆菌株，在其体内可以合成呈链状排布的多个磁小体颗粒，使其具备强趋磁特性，在外加磁场中，实现快速定向运动。

本项目以化能自养嗜酸硫杆菌为研究对象，主要关注有经济价值的氧化亚铁嗜酸硫杆菌(Acidithiobacillus ferrooxidans)、喜温嗜酸硫杆菌(Acidithiobacillus caldus)。通过嗜酸硫杆菌基因改造途径和独特的无机硫代谢途径两种手段进行了该类菌磁性特性的构建研究及包括透射电子显微表征等在内的多手段细致表征。基于基因改造途径，我们将磁细菌内磁性基因簇克隆至团队构建的独特可穿梭于嗜酸硫杆菌的广泛宿主pMSD1质粒上，通过大肠杆菌和嗜酸硫杆菌结合转移方式，使得目的基因进入嗜酸硫杆菌。但是由于磁细菌磁性基因簇很大，远大于pMSD1质粒，整个磁性基因簇的克隆转基因操作遇到了障碍。于是我们又摸索了利用嗜酸硫杆菌独特的无机硫代谢体系，探讨通过合成纳米粒子的方式实现。嗜酸硫杆菌具有多种复杂的硫代谢途径、具有高效率的硫代谢能力、及具有超强的各类重金属抗性和代谢能力，基于此，我们首先就嗜酸硫杆菌及各类基因敲除株Δsdo1、Δsdo2、Δsdo1&sdo2进行了细致的显微结构研究，证实在嗜酸硫杆菌胞内积累的黑色球状物质为多聚磷酸盐，首次发现胞外亦大量积累多聚磷酸盐球，且被富硫物质包裹。基于嗜酸硫杆菌胞外多聚磷酸盐球产物、胞内有机硫代谢酶O-succinylhomoserine sulfhydrylase、无机硫代谢酶-硫醌氧化还原酶(Sulfide:quinone oxidoreductase)等多途径，我们探究了，以镉、铜、铅、铁、锌、锰等金属在嗜酸硫杆菌生物体作用下合成各类纳米硫化物及其独特的荧光、磁性性能进行了详尽的研究，取得了系列突破性成果。成功获得了具备明显荧光特征的硫化镉量子点、硫化铜量子点、硫化铅量子点等；探究了锰、铁掺杂的硫化锌量子点的荧光特性、磁性性能；并就嗜酸硫杆菌生物体合成的各类硫化物纳米粒子等进行了详尽性能和显微结构表征。

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