【作者】 廖丽；

【导师】 吴敏； Colleen Cavanaugh； Peter Girguis；

【作者基本信息】 浙江大学 ， 微生物学， 2012， 博士

【摘要】 海洋占据地球70%以上的表面积,是全球最大的生物栖息地,拥有高度多样的生境和巨大的生物多样性。海洋跟全球的生物地球化学元素循环、气候变化以及人类的健康生存密切相关。海洋中,在数量上和功能上占据主要地位的是“看不见的主导者”,即包括细菌和古菌在内的微生物。然而,栖息在各种海洋环境中的海洋微生物并没有得到很好的认识和研究,深海微生物尤其如此。因此,为了增加对海洋环境中微生物的了解,本论文针对多个深海环境中的微生物群体,从系统发育分类、功能、代谢能力以及与无脊椎动物的共生生活方式等多个角度展开了不同层次的研究,旨在探讨海洋微生物的多样性,了解海洋微生物资源并为开发和利用海洋微生物提供第一手资料。深海沉积物是地球上仅次于海水的第二大生物栖息系统,拥有巨大的微生物资源和丰富的多样性,其中绝大多数不为人知。本论文的第一部分集中探讨来自气水合物区(第二章)和富钻结壳区(第三章)的深海沉积物中细菌和古菌的多样性,以及群体组成。通过分子生态学的手段,利用非培养技术构建16S rRNA基因的序列文库。结果表明,在上述两个研究中均发现了较高的细菌多样性和极低的古菌多样性。不同的环境因素,例如气水合物区异常的甲烷气浓度或者富钴结壳区偏高的多金属含量,可能影响其微生物的种类和结构,而微生物群体又在一定程度上反应了对环境因素的适应性和喜好,例如在富钴结壳区发现了较多的微生物跟金属代谢微生物具有高度相似的16S序列,推测可能参与金属元素的循环。通过分析微生物群体组成,该部分的研究为将来进行功能基因、转录组学和原位分析提供了参考资料。微生物的多样性除了体现在系统发育类群上,也体现在功能方面。其中,酶是微生物功能多样性的承载者。琼胶降解细菌能分泌琼胶酶,水解海藻细胞壁中的主要成份琼脂糖,生成琼胶寡糖并最终转化为半乳糖供微生物利用。论文的第四章阐述了一个来自海洋细菌Vibrio sp. CN41的新琼胶酶基因的克隆和表达以及酶活性分析。该酶为GH50家族的琼胶酶,序列新颖,并具有商业应用的潜能。本论文最后部分从自由生存的海洋微生物过渡到共生细菌,集中研究了深海热液环境中的管虫和化能自养细菌之间的共生关系。互利共生是极为普遍的现象,在深海热液环境中尤为突出。关于共生最为主要的两个方面是共生菌的传递和营养物质的供应。共生菌的传递机制主要有两种：水平传递(即从环境中获取共生菌)和垂直传递(即共生菌在生殖细胞中由上一代传给后代)。已知管虫共生菌为水平传递,但是共生菌在热液环境中的分布丰度,以及在宿主体内和在环境中的多样性并没有报道,因此论文第五章主要集中解决这两个问题。通过在离管虫堆从近到远的四个不同位置放置玄武岩条块,定量PCR测定条块上总DNA提取物中共生菌单拷贝的16S和ITS(内转录间隔区)拷贝数,推测共生菌的丰度。结果发现只有在管虫聚集的地方共生菌丰度最高,剩下三个离其不同距离的位置仅有极低的共生菌基因拷贝数。这表明可能宿主通过某种方式重新接种共生菌到周围环境中,或者共生菌在管虫聚集区能大量增殖。焦磷酸测序宿主体内和海水中的共生菌ITS序列表明共生菌具有极低的菌株多样性,仅有一个主要的ITS类型和另外三个少数类型。针对该部分结果提出了有新意的假设,值得将来进一步研究和验证。关于共生的第二个主要方面是共生菌对营养物质的利用以及如何跟宿主分工合作。管虫无口无消化器官,因此依靠共生菌为其提供营养物质,包括碳、硫、氮和磷等。氮源(氨和或硝酸盐)及其利用(合成代谢和或呼吸作用)比较复杂,并且尚未弄清。因此,利用管虫Ridgeia piscesae作为模式系统,第六章探讨了两种Ridgeia表型,目Plong skinny (LS)和short fat (SF)表型,在具有不同氨氮浓度的热液环境中对氮源的利用情况。通过逆转录实时定量PCR检测氮源利用途径中相关基因的表达,结果表明在不同外界氮源浓度和不同的宿主表型中,氨可能是主要的氮源,而且主要由共生菌合成氨为有机氮。氮利用基因的表达在不同氮源条件和不同的宿主表型下有差异,表明氮的利用可能受到环境中氮源可获得性的影响。总之,本论文探讨了从自由生存的海洋微生物到海底热液管虫-化能合成共生菌系统,展现了一个宽广的视野。研究的结果补充和发展了现有的知识体系。论文中提出了多个值得进一步研究和验证的假设,能发展成为新的研究课题。更多还原

【Abstract】 The ocean is the largest habitat on Earth, harboring diverse niches and tremendous biodiversity, and is closely related to global biogeochemical cycling, climate change, as well as human well-being. However, the unseen majority, i.e., bacteria and archaea, in marine environments is mostly unexplored. To understand diverse microbial communities in the deep sea, this thesis investigated marine microbial diversity in multiple habitats, from phylogenetic, functional and metabolic aspects, as well as in symbiotic association with invertebrates.Deep-sea sediments, the second largest habitat on Earth after the seawater column, harbor diverse microorganisms that are mostly unknown. The first part of this thesis (chapters2and3) focused on investigation of phylogenetic diversity of microorganisms in sediments from gas hydrates and cobalt-rich crust regions. Relatively high bacterial diversity, but low archaeal diversity was found in both studies. Special habitats may play a role in structuring microbial communities,﹕pecially their abnormal amount of chemicals, e.g., high methane concentrations in gas hydrate regions and enriched metals in cobalt-rich crust region. Interesting discoveries were found by examining the microbial communities, which have laid the foundation for future research into topics such as functional genes, transcriptomes and ecological associations studied through in situ hybridization.Microbial diversity can also be investigated through their functional differences, which are carried out by enzymes. To utilize agar, the cell wall component of algae, agarolytic microorganisms produce agarase to digest the neutral chain (agarose) and yield oligosaccharides, which is finally converted to galactose for utilization. The fourth chapter described cloning, expression and characterization of a new agarase from Vibrio strain, CN41. It is a GH50agarase, and has special characteristics that are promising for commercial applications.The last part of this thesis moved from free-living marine microorganisms to symbiotic microorganisms within tubeworms at deep-sea hydrothermal vents. It has been shown that tubeworm symbionts are acquired from the environment (horizontal transmission), rather than inherited from their parents (vertical transmission). However, it is unknown how abundant and diverse the symbionts are in the environment, critical for the acquisition and selection of symbionts from the environment. Hence, the fifth chapter aimed to answer these questions. To retrieve microorganisms in the vent environment, basaltic blocks were deployed for one year at four locations, i.e., among, adjacent and away from tubeworm aggregations, as well as off axis. Quantitative PCR was used to determine the copies of symbiont16S rRNA and ITS, which occur as single copy per genome. Results showed that the abundance of free-living symbionts was highest among tubeworm aggregation, but decreased dramatically with increasing distance. This indicated either replenishment of symbionts through hosts or proliferation of free-living symbionts. The symbiont diversity was investigated through pyrosequencing of ITS region from trophosome tissue and seawater filter. Surprisingly, results suggested tubeworm symbiont population was quite homogeneous within and outside tubeworms at vents, with only one dominant phylotype and three others were found.Another important question is how the symbionts utilize chemicals and produce nutrients for themselves and their hosts. Since these tubeworms are gutless and mouthless, they depend on their chemosynthetic symbionts for nutrients including carbon, nitrogen and sulfur etc. The source of nitrogen is of particular interest, since it is indispensible and various, but underemphasized. Tubeworm species, Ridgeia piscesae, was used as a model to examine nitrogen metabolism at various environmental nitrogen concentrations and of different Ridgeia phenotypes, i.e., long skinny (LS) and short fat (SF) Ridgeia. Gene expression analyses suggested that ammonium was assimilated by symbionts of both phenotypes at sites with different ammonium concentrations, but minor amounts of nitrate respiration and no nitrate assimilation were detected. Differences were found between phenotypes and vent sites, suggesting nitrogen utilization was probably influenced by environmental nitrogen availability and may be different between phenotypes.To summarize, this thesis was developed with a focus on microbial diversity from phylogenetic, metabolic, and functional perspectives. From free-living microorganisms to symbionts of vent invertebrates, this thesis demonstrated a broad view of marine microorganisms. Results promoted our understanding of marine microorganisms and updated the current knowledge. Interesting hypotheses proposed here can be developed into future projects.更多还原