硝酸盐还原酶Nap影响磁小体生物矿化的机制研究

Magnetic nanoparticles (MNPs) are important for various applications of biotechnology and biomedicine due to its proven biocompatibility. However, it is remained difficult to produce particles with a narrow size distribution and to control the morphology of the particles in chemical synthesis routes. Magnetotactic bacteria (MTB) are capable of producing unique intracellular membrane-enveloped organelles, the magnetosomes, which provides new strategies to obtain and utilize magnetic nanoparticles. However, MTB are recalcitrant to cultivate in the laboratory, fastidious to grow and/or cumbersome to analyze by genetic methods, and moreover, magnetosomes are only synthesized under anaerobic or microaerobic conditions, which altogether hinder the potential applications of magnetosome. Our previous work showed that periplasmic nitrate reductase Nap is involved in poising redox conditions for magnetite biomineralization. Deletion of nap genes not only abolished anaerobic growth and delayed aerobic growth, but also severely affected magnetite synthesis and led to the formation of fewer, smaller and irregular magnetosomes during denitrification and microaerobic oxygen respiration. However, it is unclear how Nap plays such an important role in magnetite biomineralization. Therefore, we plan to further investigate the function of the whole nap operon (napFDAGHBC) in detail. By deleting each of the nap genes, we will test the activity of nitrate reduction and magnetosome biosynthesis in different nap mutants. The nap gene(s) which are shown to be involved in magnetite biomineralization will be further characterized by different methods, including TEM, fluorescence microscopy, intracellular redox measurement and Western Blot, thereby to elucidate the role of Nap in magnetosome formation. Therefore, our studies will demonstrate (1) which Nap protein(s) are required for nitrate reducing and thus to uncover the function of Nap in nitrate reduction; (2) which nap gene(s) are essential for wild type-like magnetosome biosynthesis and further elucidate how Nap protein(s) affect biomineralization. Furthermore, our work will also try to improve the efficiency of nitrate reduction by genetic engineering, and consequently increase magnetosome production. Altogether, our project will not only expand the knowledge on nitrate reductase Nap, but also provide a new insight into genetic engineering of magnetotactic bacteria by improving some ubiquitous metabolism pathways, such as denitrification.

磁性颗粒因其优质的生物学性能而广泛应用于各个领域，但人工合成纳米级形状规则的磁性颗粒非常困难，趋磁细菌则能够在胞内合成规则的磁性颗粒——磁小体，这种独特的矿化过程为磁性颗粒的开发开辟了新途径。但苛刻的生物矿化条件极大地限制了磁小体的利用，因此，揭示磁小体合成机制，阐明其关键限速步骤，对于合理改造趋磁细菌，提高磁小体产量具有重要意义。前期研究发现，趋磁细菌硝酸盐还原酶Nap的缺失会严重损害磁小体的合成，但作用机制尚不明确。据此，项目以趋磁细菌napFDAGHBC操纵子作为研究对象，通过基因敲除构建不同突变株，酶活测定确定硝酸盐还原的必需nap基因，阐述其催化机制；通过分子生物学、生物化学和显微镜技术等，探索Nap参与生物矿化的作用机理，优化硝酸盐还原反应，提高磁小体产量。本研究将从硝酸盐还原酶Nap这个新视点探讨磁小体合成的机制，不但能够拓展对Nap功能的认知，还为提高磁小体产量提供新思路。

趋磁细菌是一类可以沿磁场运动的革兰氏阴性细菌，广泛存在于海洋和湖泊中，其独特之处在于胞内能够合成由生物膜包被的纳米级磁性颗粒——磁小体。趋磁细菌仅在微好氧和厌氧条件才能够合成磁小体，而好氧环境会完全抑制磁小体的合成。前期研究发现，厌氧呼吸反硝化作用在趋磁细菌MSR-1生物矿化过程发挥着重要的作用，其中以反硝化作用的第一个还原酶——硝酸盐还原酶Nap的影响最为显著。当编码硝酸盐还原酶的napFDAGHBC基因簇缺失后，趋磁细菌反硝化途径完全被阻断，磁小体的合成量明显降低且形态不规则，这暗示着在趋磁细菌中Nap（NapFDAGHBC）不单单只是一个硝酸盐还原酶，它可能存在其它新的未知的功能，进而参与到磁小体的生物合成过程中，但各个Nap蛋白的催化机制以及其在磁小体生物矿化中的作用机理仍然未知。据此，项目开展了如下研究内容：1）同源重组逐个敲除nap基因簇各个基因，并检测其对细菌生长，尤其是在反硝化过程发挥的作用；2）检测各nap基因在生物矿化过程的作用；3）通过在nap基因簇前加入强启动子来优化反硝化作用，以期提高MSR-1的生长和磁小体的产量。研究发现，NapA、NapB、NapC和NapD是趋磁细菌MSR-1反硝化过程的必需蛋白，任何一个基因的缺失都会导致趋磁细菌MSR-1丧失反硝化能力。在生物矿化过程中，只有ΔnapD突变株磁小体合成受到影响，合成的磁小体纳米颗粒变小且形状不规则；进一步研究发现，NapD可能在磁小体合成过程发挥氧化还原作用。另一方面，前期研究表明，Nap催化的硝酸盐还原为亚硝酸盐反应是整个反硝化过程的限速步骤，且启动子转录水平极低，因此，为提高趋磁细菌MSR-1的产量，通过导入强启动子的方式提高Nap的表达量，目前研究仍在进行中。综上所述，项目通过对生物矿化关键代谢蛋白Nap的深入解析，确定其参与生物矿化的关键蛋白，为后期合成生物学改造反硝化途径，提高细菌产量和磁小体生成量提供了重要理论基础。