【作者】 杨靖；

【导师】 李颖； WU Long-Fei；

【作者基本信息】 中国农业大学 ， 微生物学， 2014， 博士

【摘要】 趋磁细菌在自然界广泛存在,它们能在胞内合成链状排列的Fe3O4或者Fe3S4纳米磁性颗粒(磁小体),鉴于磁小体的特性及其潜在的应用前景,提高趋磁细菌培养水平始终是人们关注的热点问题。然而由于它们对溶氧和营养条件要求苛刻,目前只有少数几种获得纯培养,且培养水平远达不到生产要求。本研究以淡水磁螺菌Magnetospirillum gryphiswaldense MSR-1和海洋磁螺菌Marine magnetic spirillum QH-2为材料,探讨了二者在不同培养条件下细胞生理变化规律及其与磁小体合成的关系,分别提出了进一步提高淡水和海洋趋磁螺菌的优化培养措施。此外,针对供试菌株共有的mamXY操纵元中唯一的尚未揭示功能的mamX基因为目标,探讨了其与整个操纵元中其它基因在控制磁小体成熟过程中的功能特点。鉴于淡水趋磁螺菌MSR-1已经经过多年的人工驯化,可在发酵罐深层培养,实验中发现,培养8-20h是磁小体大量合成的阶段,此时控制溶氧在2-20ppb有利于磁小体合成。18-20h是细胞生理代谢的一个关键时间转折点,细胞消耗碳源速度加快,需通过手动补料策略添加额外补料培养基以维持细胞的正常生长。20-26h,碳源乳酸浓度应控制在0.016-0.1g/L最有利于细胞生长和磁小体的合成。20-40h是细胞生长对数期,控制溶氧在20-40ppb可确保细胞快速生长,是磁小体组装成链和成熟过程。由此揭示了细胞生理特点变化规律,提出了进一步提高培养水平的工艺路线。QH-2菌株是唯一来自海洋的可以纯培养的趋磁螺菌,本实验成功对其细胞进行了摇床培养,并优化了原培养基配方。确定了其细胞的最适碳源为琥珀酸盐,最适氮源为氯化铵,偏好柠檬酸铁作为铁源,培养温度为25-28℃。在优化条件下显著提高了QH-2细胞的培养水平：液体摇床培养48小时OD600值大于0.6,是优化前的4倍左右；代时为6h,比优化前缩短了10个小时。为该菌株的进一步驯化奠定了基础。由此可见,趋磁螺菌来源不同,培养条件各异,分析菌株的生理特点,有针对性地提出具体培养措施是最有效的选择。为完善磁小体岛mamXY操纵元的功能解析,本研究构建了MSR-1菌株mamX基因的缺失突变株和互补菌株,表型和生理、生化检测发现,突变株可正常生长,但只能形成不规则的Fe3O4超顺磁颗粒。序列分析发现MamX蛋白含有保守的类似于细胞色素c的结合血红素的Magnetochrome结构域,经体外实验证明该蛋白可以与血红素结合,推测在磁小体成熟过程中,MamX可以参于磁小体膜内的电子传递或者铁元素的氧化/还原过程。采用qPCR的方法,探讨mamXY操纵元mamY、mamX、mamZ和ftsZ-like四个基因的转录规律,证明在mamX突变株中,mamY和ftsZ-like基因显著上调,mamZ基因则下调。细菌双杂交实验证实mamXY操纵元编码的四个蛋白之间存在明显互作关系,由此采用STRING工具描绘了以FtsZ-like为节点的蛋白互作网络图,推测这四个蛋白形成蛋白质复合体参与磁小体的成熟过程。实验结果完善了对磁小体岛上mamXY操纵元整体功能的解释,为进一步揭示趋磁螺菌的生物矿化机制提供了实验证据。更多还原

【Abstract】 Magnetotactic bacteria (MTB) are widespread prokaryotes in nature environment that synthesize a unique organelle called the magnetosome, which are usually nano-sized chain-like magnetic iron crystals, composed of Fe3O4or Fe3S4. In consideration of the characteristics and application prospects of magnetosome, researchers have been focusing on the hot topics about improving the culture level of MTB. However, few axenic culture of MTB have been acquired because of their rigorous requirements on dissolved oxygen and nutrient elements. In addition, the cultivation levels of axenic MTB cultures are far from the production demand. The freshwater Magnetospirillum gryphiswaldense MSR-1and marine magnetic spirillum QH-2are chose as target strains, and some researches are performed on the relation between the changes in cellular physiology and magnetosome formation. The optimized methods to culture freshwater and marine magneto-spirillum are suggested on the basis of research results. Besides, the target strains have magnetosome-formation-associated conserved mamXY operon, on which most genes’functions have been elucidated except for mamX gene. Studies on the function features of mamX and mamXY operon in controlling the mature process of magnetosome are conducted.The freshwater M. gryphiswaldense MSR-1has been domesticated artificially for many years, hence it can have submerged culture on the fermentor. In the process of MSR-1submerged culture, It’s found that:8-20h is the stage when a large quantity of magnetosomes are formed and the dissolved oxygen should be kept between2-20ppb.18-20h is a crucial turning point for cellular physiological metabolism while the carbon source consumption speeds up, some extra fed medium should be added by manual feeding strategy to preserve the regular cell growth. From20h to26h, the concentration of carbon source lactate should be maintained between0.016-0.1g/L, which is helpful for cell growth and magnetosome formation.20-40h is the log phase of cell growth and the phase for magnetosome chain formation and maturing, when should has a dissolved oxygen concentration as20-40ppb to ensure the rapid cell growth. Thus the changes in cellular physiological characteristics are revealed, and the process route for improving MTB culture is proposed.The QH-2strain is the only one marine magneto-spirillum that has axenic culture. In this study, the QH-2cells are cultured using shake cultivation successfully and the original medium is optimized. For QH-2cell, the optimal carbon source is tested to be succinate, optimal nitrogen source being sodium chloride, optimal iron source being ferric citrate, and optimal temperature being25-28℃. The cell culture level is evidently improved under optimized conditions:the OD600value can be over0.6after shake cultivation for48h in liquid medium, about four times higher than before; the generation time is6h,10hours less than before. These results lay foundation for QH-2’s further domestication. Evidently, when the origin of magneto-spirillum differs, the culture condition differs. So it’s the best choice to analyze the cellular physiological characteristics firstly and then find purposeful detail cultivation methods. In order to complete the function illumination of mamλY operon, a deletion mutant and a complemented strain for mamX are constructed. The mutant has normal growth, while synthesizes irregular superparamagnetic Fe3O4particles. Analysis of MamX protein sequence showing that it contains conserved heme-binding motif called Magnetochrome, similar to that of cytochrome c. The MamX protein can bind heme in vitro. Thus MamX is suspected to be involved in the electron transport inside magnetosome membrane, or in the oxidized/reduced process of iron, during magnetosome maturation. The qPCR tests show the transcription rules of the all four genes mamY, mamX, mamZ and ftsZ-like of mamXY operon:in the mamX mutant, mamY and ftsZ-like are up-regulated, while mamZ is down-regulated. The bacterial two hybrid assay shows evident interactions between the four MamXY proteins. Hence a protein association network is drew by STRING tool, indicating FtsZ-like as node. The four proteins are speculated to form protein complex and promote the magnetosome maturation. These results complement the interpretation of mamXY operon’s function, and providing supplementary evidences for further revealing the biomineralization mechanism in magnetotactic bacteria.更多还原