海洋趋磁细菌多样性和特性研究及其分离纯化培养

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	海洋趋磁细菌多样性和特性研究及其分离纯化培养
其他题名	Diversity, Characterization and Culture of Marine Magnetotactic Bacteria
	潘红苗
学位类型	博士
	2008-05-25
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	海洋趋磁细菌趋磁球菌磁小体系统发育分析荧光原位杂交变形菌纲培养
摘要	趋磁细菌是一类革兰氏阴性的原核生物，广泛分布于淡水和海水环境中的有氧-无氧过渡区。本文研究了青岛汇泉湾沿岸一个海水养殖池塘中两个不同亚区内趋磁细菌的多样性。一个是常年水深在0.5 ~ 3 m，类似潮下带区域；另一个是在落大潮的时候沉积物能暴露在空气中，类似潮间带区域。在该池塘类似潮下带区域的沉积物中发现了大量海洋趋磁细菌，最大丰度可达105 cells/cm3。透射电镜观察发现该菌菌体形态多样，有球形或卵球形、长短杆状、弧状和螺旋状，其中球形或卵球形趋磁细菌占绝对优势。电镜观察还发现该菌磁小体的排列方式呈多样化，大多数呈链状排列，有单链、双链及多链，还有的呈环状或者成簇排列。磁小体的形态也多种多样，有正方体、棱柱体、立方八面体、子弹头状、片状和齿状。用RFLP方法分析了70个克隆，测序得到10条不同序列。经16S rDNA系统发育分析，发现9个属于α-变形菌亚纲，1个属于γ-变形菌亚纲，共有8个不同的属，优势种属于未培养的海洋趋磁球菌。所有克隆与最接近的海洋趋磁球菌的相似性并不高（76.4% ~ 89.4%），表明该区域的趋磁细菌为新发现的微生物资源。而在该池塘类似潮间带区域的沉积物中发现了单一种群结构的趋磁细菌。透射电镜观察显示菌体形态为球形至卵球形，大小为1.8 ~ 2.3 × 2.0 ~ 2.8 μm，细胞侧生两簇鞭毛，极性趋磁运动，每个细胞内都含有两条磁小体链。统计结果显示，两条磁小体链上的磁小体数目60%都相差1个。单个菌体中磁小体数目从7到31个不等，平均为18个，其中包含19个磁小体的菌体占多数。磁小体的形状多为长方体，平均长度和宽度分别为101 + 24 nm和83 + 21 nm，形态因子约为0.83 + 0.09，为单磁畴晶体。能谱显示磁小体的成分为Fe3O4。磁滞回线的测量得到单个磁小体的磁距为2.6 × 10-13 emu。用RFLP方法分析了98个克隆，测序得到3条不同序列，而三条序列之间的相似性都在98%以上，可认为是同一个种，FISH也证实了该处趋磁细菌为单一种群。经16S rDNA系统发育分析显示，该处趋磁细菌隶属于α-变形菌亚纲中的一个新属。初步结果显示了趋磁细菌对潮间带环境的适应性。本文还采用半固体培养基培养了一株海洋趋磁螺菌，电镜下观察，菌体大小约为3 × 0.8 μm，体内包含一条磁小体链，磁小体形态不规则，大小分布不均。目前，纯化工作正在进行。
其他摘要	Magnetotactic bacteria are Gram-negative and represent a diverse group of microorganisms with respect to morphology, physiology and phylogeny. They have a cosmopolitan distribution in water columns or sediments with horizontal chemical stratification. The highest numbers of magnetotactic bacteria are generally found at the oxic-anoxic transition zone or redoxcline. Here, we studied the diversity of magnetotactic bacteria in a seawater pond within an intertidal zone at Huiquan Bay in the China Sea. The pond is composed of a permanently submerged part and a low tide subregion. The magnetotactic bacteria collected from the permanently submerged part display diversity in morphology and taxonomy. Their abundance reached up to 105 cells/cm3. Their diverse morphotypes, including cocci, ovoids, rods, vibrios and spirilla were observed, and the coccoid shape was most abundant. The magnetosomes of magnetotactic bacteria arranged in one, two or multiple chains. Irregular arrangement of magnetosomes was also evident. Morphology of magnetosome crystals was homogenous in a given cell, but displayed various shapes, including square, cubo-octahedron, parallelepiped, elongated hexagonal prismatic, bullet-shaped and tooth-shaped in different cells. The morphological diversity of marine magnetotactic bacteria was confirmed by restriction fragment length polymorphism analysis (RFLP) and sequence analysis of 16S rDNA. At least 10 different taxonomic groups were identified. Among them, nine belonged to α-Proteobacteria and one exception was γ-Proteobacteria. Compared to known axenic or uncultivated magnetotactic bacteria, the 16S rDNA sequences of most magnetotactic bacteria collected from the permanently submerged part exhibited low sequence identities ranging from 76.4% to 89.4%. These results suggest a specific geo-distribution of the magnetotactic bacteria collected from the China Sea. In contrast, we found a virtually homogenous population of ovoid-coccoid magnetotactic bacteria in the low tide subregion of the pond. The dimensions of the cells ranged from 1.8 to 2.3 mm in width and 2.0 to 2.8 mm in length. They were bilophotrichously flagellated and exhibited polar magnetotactic behaviour. Almost all cells contained two chains of magnetosomes. The results of statistic analysis in TEM show that sixty per cent of cells had a difference of one magnetosome between the two chains. The number of magnetosomes per cell ranged from 7 to 31 with average of 18 and the occurrence of 19 magnetosomes per cell was the highest. Most of the magnetosome crystals in cells had a rectangular projected shape with a size of 101 + 24 nm (mean with standard deviation) in length and 83 + 21 nm in width. That gives a value of shape factor (width/length) about 0.83 + 0.09. They are likely to be single-domain magnetite. Analysis by energy dispersive X-ray spectroscopy (EDXS) unambiguously identified the ‘iron-oxide’ nature of the magnetosome crystals. In addition, selected area electron diffraction analysis of individual crystals confirmed the magnetite nature of the magnetosomes in cells. The result of the magnetic hysteresis loop measurement indicated the magnetic moment of a single magnetosome was 2.6 × 10-13 emu. The morphological diversity of marine magnetotactic bacteria was confirmed by RFLP and sequence analysis of 16S rDNA. At least 3 different taxonomic groups were identified. Pair-wise comparison revealed that 16S rDNA sequence identity showed >98% sequence identity each of them. The combination of restriction RFLP and sequencing of cloned 16S rDNA genes from the low tide subregion samples as well as fluorescence in situ hybridization (FISH) revealed the presence of a homogenous population. Moreover, phylogenetic analysis indicated that the Qingdao Huiquan low tide magnetotactic bacteria belong to a new genus affiliated with the a-subclass of Proteobacteria. This finding suggests the adaptation of the magnetotactic bacterial population to the marine tide. In this study, a novel marine magnetospirillum magneticum was cultured in semi-solid medium. Examined by TEM, the dimensions of the cells ranged from 3 mm in length and 0.8 mm in width. The cells contained one chain of magnetosomes. These magnetosomes were irregular in shape and the distribution of magnetosomes was asymmetric. But the pure strain was not obtained at present. This work is been doing now.
页数	80
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/535
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	潘红苗. 海洋趋磁细菌多样性和特性研究及其分离纯化培养[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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