【作者】 谢伟；

【导师】 何光源； 徐安龙；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2010， 博士

【摘要】 深海热液口烟囱是通过来自地壳的熔浆与海水交互作用而形成的特殊的地质结构,该环境被认为与地球早期环境相似,在热液口烟囱的喷口中,不断有气体、金属离子喷出,且形成了热量和化学物质浓度梯度。这些热量与化学物质浓度梯度为微生物的生存提供了保证,大量的化能自养微生物通过氧化这些无机物获取能量,将该环境中的无机碳转化为有机碳,从而为该环境丰富的生物群落提供大量初级营养物质。尽管对这些化能自养型微生物的多样性研究较为深入,但是对它们的代谢特征的研究才刚刚起步。我们对胡安得富卡洋中脊硫化物样品4143-1宏基因组文库的2880个Fosmid克隆子进行454高通量测序,一共获得308,034条序列,平均长度为227bp。对这些序列进行拼接后得到15.3 MB序列,包括31,405条拼接序列和22,968不能拼接的序列,其中21,836条序列能够与NCBI非冗余蛋白数据库在期望值为10-5下有较好的注释。所有注释序列中有70%能够分布在COG的分类项目中。首先,我们利用双向聚类的方法对包括4143-1样品和Lost City热液口烟囱样品的18个环境宏基因组数据做比较分析发现,两个热液口烟囱能够聚为一支,且参与DNA错配修复和同源重组的基因序列丰度均显著高于其他环境,表明该环境微生物具备丰富的DNA修复系统以应对该环境下有毒物质对基因组的损伤。另外,两个热液口烟囱均含有丰度较高的化学趋化性和鞭毛运动相关基因,反应出这些微生物对于烟囱壁动态变化环境的一种适应性。其次,通过对4143-1宏基因组数据分析发现,该环境微生物群落代谢方面具有以下特征:通过硫氧化物复合体依赖性途径或者腺苷-5’-磷酸硫酸酐依赖途径氧化硫化物提供电子,偶联硝酸盐还原提供能量,以卡尔文循环为主要途径进行二氧化碳固定。通过对代谢关键酶基因簇的分析发现,兼性厌氧和专性厌氧微生物均对该环境初级营养物质的产生发挥作用。最后,通过高通量测序获取的基因片段信息,设计特异引物筛选对应的Fosmid克隆子,可获取对应基因或基因簇的全长信息,我们从宏基因文库中筛选到两个分别含聚酮合成酶基因簇和聚酮聚肽杂合型基因簇的克隆子,虽然两个克隆子对三种标准菌均未表现出抑菌活性,这些模块仍表现出一些与目前已知的PKS基因簇不同的特征,为组合生物合成研究增加了新的成员。总而言之,我们的研究不仅在物质能量水平揭示了胡安得富卡洋中脊热液口烟囱壁微生物群落对该极端环境的适应性机制,也为进一步开发利用该环境的基因资源打下基础。更多还原

【Abstract】 Deep-sea hydrothermal vent chimneys that form by interactions between hot fluids and cold seawater are regarded as primordial environments reminiscent of early Earth conditions, with reactive gases, dissolved elements, and thermal and chemical gradients operating over spatial scales of millimeters and centimeters up to meters. These chemical and thermal gradients along and inside the sulfide chimneys provide a wide range of microhabitats for chemolithoautotrophic microorganisms that fix inorganic carbon using chemical energy obtained through the oxidation of reduced inorganic compounds contained in the hydrothermal fluids, converting the geothermally derived energy into microbial biomass. Although the phylogenetic diversity of these microorganisms has been described previously, the adaptation and metabolic potential of the microbial communities is only beginning to be revealed. A pyrosequencing approach was utilized to directly obtain sequences from a fosmid library constructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at the Juan de Fuca Ridge. A total of 308,034 reads with an average sequence length of 227 bp were generated. The sequenced microbiome of the hydrothermal vent sample consisted of 31,405 contigs and 22,968 singletons, a total of 15.3 MB. A total of 21,836 open reading frames (ORFs) were predicted from the data set, more than 70% of which could be classified into function sets in the COG category.Firstly, comparative genomic analyses of metagenomes from a variety of environments by two-way clustering of samples and functional gene categories demonstrated that the 4143-1 metagenome clustered most closely with that from a carbonate chimney from Lost City; both are highly enriched in genes for mismatch repair and homologous recombination, suggesting that the microbial communities have evolved extensive DNA repair systems to cope with the extreme conditions that have potential deleterious effects on the genomes. As previously reported for the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion of transposases, implying that horizontal gene transfer may be a common occurrence in the deep-sea vent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highly enriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highly dynamic conditions present within the chimney walls.Secondly, we reconstruct the metabolic pathways at the metegenome level. The result indicated that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfur oxidation through sulfur-compound oxidation (sox) dependent and adenosine-5’-phosphosulfate (APS) dependent pathways, putatively coupled to nitrate reduction to perform inorganic carbon fixation through the Calvin-Benson-Bassham cycle (CBB cycle). Based on the genomic organization of the key genes of the carbon fixation and sulfur oxidation pathways contained in the large genomic fragments, both obligate and facultative autotrophs appear to be present and contributing to biomass production.Then, we screened the library with specific primers and got two fomsid containing PKS/NPRS gene fragments. Although neither of them had antimicrobial functions towards Escherichia coli, Bacillus subtilis or Saccharomyces cerevisiae. There are some characters of the two PKS/NPRS clusters, which are different from knows.In summary, in addition to the understanding of novel mechanism for the formation of such unique environment driven by this special community of microbiota, the genetic resources from the deep-sea vent environments will be available for further functional analyses as gene treasure house through this novel approach.更多还原