【作者】 曹轶；

【导师】 吴敏；

【作者基本信息】 浙江大学 ， 植物学， 2010， 博士

【摘要】 极端微生物能够在普通微生物无法生存的极端环境中良好生长。深海和盐湖是典型的极端生境,孕育了嗜热、嗜冷、嗜压、嗜盐、嗜碱等多种极端微生物。本论文以“非培养-可培养-功能基因”为研究线索,从三个不同方面,围绕生活在深海和盐湖中的极端微生物展开研究。首先,针对采集自西南印度洋洋中脊2951米深的深海热液区沉积物样品,通过构建16SrRNA和nifH基因文库,对原核微生物的种群多样性进行分析。结果表明,古菌种系型主要分布在广古菌门的MBGE类群和Thaumarchaeota门的MGI类群。大约60%的细菌种系型属于于变形菌门,其余细菌种系型分布于放线菌门、拟杆菌门、浮霉菌门、酸杆菌门、硝化螺旋菌门、绿弯菌门、绿菌门、衣原体门、疣微菌门以及未分类类群OD1、OP11、WS3和TM6。nifH基因的发现说明西南印度洋洋中脊深海热液区存在生物固氮作用,系统发育分析表明只存在Ⅰ型和Ⅲ型微生物nifH基因,说明在此深海热液区环境中细菌发挥主要的固氮作用。其次,从波多黎各海沟6000米深处海水样品中分离到一株专性嗜压疑似新种细菌YC-1并进行多相分类学研究。结果表明,菌株YC-1能在8℃和50 MPa的条件下最适生长,在标准大气压下无法生长。主要的脂肪酸成分为C16:1、C18：1和C10：03-OH。基因组G+C mol%含量为44.6 mmol%。菌株YC-1为革兰氏阴性细菌,无鞭毛,兼性厌氧,营异养生活。16S rRNA基因的序列与深海非培养细菌Oseda_syml最为相近,序列相似性为97.2%；最近缘的可培养标准菌株属于Neptunomonas属,16SrRNA基因序列相似性介于92.7-93.2%。根据多相分类的差异,建议定义菌株YC-1为海洋螺菌科新的分类单元,命名为"Profundimonas piezophila gen. nov., sp. nov."。最后,对模式嗜盐碱古菌Natronomonas pharaonis的乙醇代谢及其功能酶进行研究。结果表明,N. pharaonis的乙醇代谢是应对环境中乙醇胁迫的一种应激机制。作为N. pharaonis乙醇代谢过程中两个关键酶,编码乙醇脱氢酶的adh基因不受乙醇的诱导表达,而编码乙醛脱氢酶的aldH2基因则受乙醇的诱导表达,说明乙醛脱氢酶对N. pharaonis的乙醇耐受性具有主要贡献,而乙醇脱氢酶可能还参与了其它代谢途径。大肠杆菌异源表达的N. pharaonis乙醇脱氢酶具有嗜盐、嗜碱和嗜热的性质,而乙醛脱氢酶则体现嗜碱、嗜热和非嗜盐的性质。更多还原

【Abstract】 Extremophiles live well in several extreme environments, where normal microorganisms cannot survive. Typical extreme habitats as Deep Ocean and Salt Lake give birth to many thermophilic, psychrophilic, piezophilic, halophilic and alkaliphilic microorganisms. Focus on the microbial life living in Deep Ocean and Salt Lake, three different studies were presented in this thesis.Firstly, a sediment sample collected from a deep-sea hydrothermal vent field located at a depth of 2,951 m on the Southwest Indian Ridge. Phylogenetic analyses were performed on the prokaryotic community and diversity based on the 16S rRNA and nifH genes libraries. Within the Archaea, the dominant clones belonged to the groups of MBGE and MGI within the phyla Euryarchaeota and Thaumarchaeota, respectively. About 60% of the bacterial clones belonged to the Proteobacteria. Additional bacterial phyla detected included the Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Nitrospirae, Chloroflexi, Chlorobi, Chlamydiae, Verrucomicrobia, and candidate divisions OD1, OP11, WS3, and TM6. The detection of nifH genes suggests that biological nitrogen fixation occurred in the hydrothermal vent field of the Southwest Indian Ridge. Phylogenetic analysis shown that only cluster I and cluster III NifH are present, indicating that Bacteria play the main role in nitrogen fixation in this hydrothermal vent environment.Secondly, a novel obligately piezophilic bacterium was isolated from seawater collected from Puerto Rico Trench at a depth of 6,000 m. The isolated strain YC-1 grew well with an optimal growth hydrostatic pressure of 50 MPa at 8℃, and no growth was observed at atmospheric pressure. The predominant cellular fatty acids are C16:1, C18:1 and C10:0 3-OH. The G+C content was 44.6 mol%. Strain YC-1 is Gram-negative, non-flagellum forming, and facultative anaerobic heterotroph. The 16S rRNA gene sequence of strain YC-1 was closely similar to the uncultured deep-sea bacterial clone Osedax_syml (97.2%), and was closely related to cultivated members of the genus Neptunomonas (92.7-93.2%). Based on taxonomic and phenotypic differences observed, the strain YC-1 appears to represent a new species within the family Oceanospirillaceae, which named Profundimonas piezophila gen. nov., sp. nov.Thirdly, we have studied the role of functional genes within ethanol metabolism of haloalkaliphilic Archaea Natronomonas pharaonis. It indicated that ethanol oxidation by N. pharaonis may be one of the mechanisms to degrade toxic compounds found in its habitat. In N. pharaonis, the adh gene and aldH2 gene encode alcohol dehydrogenase and aldehyde dehydrogenase, respectively, they are key enzymes involved in alcohol metabolism. Reverse transcription-PCR analysis showed that the aldH2 gene was inducible by ethanol, but the adh gene transcription was not affected by ethanol. It indicated that aldehyde dehydrogenase of N. pharaonis possibly contribute to the alcohol resistance, and alcohol dehydrogenase may play other physiological roles in addition to ethanol degradation. The recombinant alcohol dehydrogenase was haloalkaliphilic and thermophilic, and the recombinant aldehyde dehydrogenase was thermophilic, alkaliphilic and salt independent.更多还原