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潮间带趋磁杆菌与西沙珊瑚礁生境趋磁细菌多样性及特性研究
滕兆洁
学位类型硕士
导师肖天 研究员
2017-05-05
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词趋磁细菌 多样性 显微操作技术 珊瑚礁生境 潮间带
摘要趋磁细菌(Magnetotactic bacteria,MTB)是一类能够在胞内合成由生物膜包被的磁性纳米颗粒(磁小体),并借助鞭毛沿磁力线方向运动的细菌。趋磁细菌具有形态多样性,主要有球形、杆形、弧形、螺旋形和多细胞聚集体等形式。通常,在淡水或海水环境中,趋磁球菌占据数量优势。本实验在青岛汇泉湾潮间带发现了一株海洋趋磁杆菌QR-1,该菌丰度高,优势度达90%以上。QR-1菌体较大,平均大小为6.71 ± 1.03 μm × 1.54 ± 0.20 μm;两端各具一个多聚磷酸盐颗粒,呈长椭形,大小不一;磁小体链纵贯菌体,单个菌体内通常含有1~4条平行排列的磁小体链,磁小体呈棱柱形,主要成分为Fe3O4;QR-1为双毛菌,极生双鞭毛,鞭毛成束,每个鞭毛束均由7根鞭毛和多根纤毛构成。趋磁杆菌QR-1运动速度较快,平均速度为70 ± 28 μm/s;运动方式复杂多样,具有典型的轴向运动,具有独特的群体运动。当磁极方向调转时,菌体会响应磁场改变进行调头运动;当运动遭遇障碍时,菌体会通过暂停和绕过的方式逃离障碍物。本实验通过显微操作、全基因组扩增获得QR-1的16S rRNA基因序列,并通过FISH验证了序列的准确性,最终确定该菌为α-变形菌纲的新种。
随着MTB调查研究范围的不断扩大,本实验还在荣成月湖和法国马赛的潮间带沉积物中发现了3种形态与QR-1相似的趋磁杆菌FM-t,FM-w和LY-1。该类MTB菌体明显分为两部分,菌体两端各具内含物颗粒,中间具多个高折光颗粒,黑色磁小体链清晰可见,沿长轴纵贯菌体,均能进行轴向趋磁运动。3种菌体积巨大,FM-t,FM-w长度超过10 μm。FM-t菌体具两条平行排列的磁小体链,磁小体为棱柱形,大小为106 ± 13 nm × 90 ± 15 nm。本实验利用显微操作结合全基因组扩增技术获得3种菌的16S rRNA基因序列,分析结果显示3种趋磁杆菌为3个新种,属于3个新属,均属于α-变形菌纲。
目前,对MTB研究主要集中在水域生态系统,本论文主要针对西沙珊瑚礁生境中MTB进行调查。在众多海洋次级生态系统中,珊瑚礁生态系统中MTB鲜有报道。本实验首次对西沙珊瑚礁生境中沉积物样品进行MTB进行多样性分析,结果显示,西沙珊瑚礁生境中MTB具有极高的多样性。形态上,MTB主要以球形、杆状、弧形、螺旋状和多细胞聚集体,即MMPs等形式;磁小体形态主要包括子弹头形、棱柱形和不规则形,其中以子弹头形磁小体为主,占总磁小体数量的90%以上,部分单个菌体内具有子弹头形和不规则形两种形态的磁小体,另外,磁小体链的排列方式也具有明显的多样性;在系统发育上,本实验在珊瑚礁生态系统中共发现9个种,分属于α-变形菌纲、δ-变形菌纲和硝化螺菌门,其中包括发现了4个新种,属于2个新属。
此外,本论文通过查阅磁小体矿化作用过程相关文献,选择已完成全基因组测序且磁小体链排列相对稀疏的Magnetospira sp. QH-2作为研究对象,利用生物信息学软件,研究与磁小体成链相关的MamK蛋白的结构和系统进化。研究结果显示,mamK开放阅读框大小为1086 bp,编码361个氨基酸,MamK蛋白属于NBD_sugar-kinase_HSP70_actin超家族,为非跨膜蛋白;此外, 还利用生物信息学工具预测MamK蛋白二级和三级结构。系统进化分析显示MamK蛋白的系统进化和16S rRNA基因的系统进化有一定差异,推测磁小体岛的获得可能与菌种的进化是独立的两个过程;淡水与海水中的趋磁螺菌虽然形态相似,但系统进化地位有一定差异,可能是两者不同生境下适应性演化的结果。
其他摘要Magnetotactic bacteria (MTB) are a group of Gram-negative bacteria that synthesize magnetic crystals, enabling them to navigate in relation to magnetic field lines. Morphologies of magnetotactic bacteria include spirillum, coccoid, rod, vibrio, and multicellular morphotypes. The coccid shape is generally the most abundant morphotype among MTB magnetotactic bacteria. Here we described a species of giant rod-shaped magnetotactic bacteria (designated QR-1) collected from sediment in the low tide zone of Huiquan Bay (Yellow Sea, China). This morphotype accounted for 90% of the magnetotactic bacteria collected, and the only one taxonomic group which was detected in the sampling site. Microscopy analysis revealed that QR-1 cells averaged 6.71 ± 1.03 μm × 1.54 ± 0.20 μm in size, and contained in each cell 42~146 magnetosomes that are arranged in a bundle formed one to four chains along the long axis of the cell. The QR-1 cells displayed axial magnetotaxis with an average velocity of 70 ± 28 μm/s. Transmission electron microscopy based analysis showed that QR-1 cells had two tufts of flagella at each end. Phylogenetic analysis of the 16S rRNA genes revealed that QR-1 together with three other rod-shaped uncultivated magnetotactic bacteria is clustered into a deep branch of Alphaproteobacteria.
We found three rod-shaped MTB strains shared similar morphological characteristics with QR-1 in intertidal sediments from Lake Yuehu, Rongcheng and Marseille. They were divided into two parts, had several refractive vesicles at the middle of the cell and large inclusion particles at their poles, and were able to exhibit axial magnetotactic movement. These three strains were unusually large in size; both FM-t and FM-w were longer than 10 μm. FM-t cells had elongated prismatic magnetosome crystals and two parallel magnetsome chains arranging along the long axial of cells. In this study we used microsorting combined with single cell whole genome amplification to identify the 16S rRNA gene of QR-1 and analyze its taxonomic affiliation. The analysis results showed that the three rod-shaped MTB were three new species belonging to three new genera and affiliate with the Alphaproteobacteria.
Among all the secondary marine ecosystems, the MTB diversity in sediments of coral reef ecosystems was rarely reported. In this study, we applied ecological investigation to reveal the diversity and characteristics of MTB in sediments from Paracel Islands coral reef habitat. The results showed that the Paracel Islands coral reef habitats had a very high MTB diversity. MTB morphologies included spirillum, coccoid, rod, vibrio, and multicellular morphotypes, and morphologies of magnetosomes included bullet-shaped magnetosomes which were approximately 90% of total magnetosomes, prismatic magnetosomes, and irregular-shaped magnetosomes. Some MTB cells contained both bullet-shaped and irregular-shaped magnetosomes. Phylogenetic analysis revealed that 9 MTB species belonging to Alphaproteobacteria, Deltaproteobacteria and Nitrospirae were identified from coral reef sediments in Paracel Islands, including 4 new species belonging to the 2 new genera.
Magnetospira sp. QH-2 is a marine magnetotactic spirillum, isolated from the intertidal zone of Yellow Sea, China. It was predicted that the trait that its magnetosomes in a chain display a relatively large size distribution is related to the function of mamK-encoded protein, namely MamK. We found that mamK gene codes for 1086bp, and MamK which belonged to the NBD_sugar-kinase_HSP70_actin superfamily was composed of 361 amino acids and did not contain any trans-membrane domain. Based on the differences of the phylogenetic trees, we determined that the acquisition of magnetosome island and bacteria evolution were possibly two independent processes. Whereas the morphology feature of magnetotactic spirillum both in fresh water and marine were similar, their phylogenetic statuses were different to some extent, and that may indicated the adaptive evolutions of this magnetotactic spirillum.
学科领域微生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136541
专题海洋生态与环境科学重点实验室
作者单位中国科学院海洋研究所
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滕兆洁. 潮间带趋磁杆菌与西沙珊瑚礁生境趋磁细菌多样性及特性研究[D]. 北京. 中国科学院大学,2017.
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