深海超嗜热嗜压古菌Pyrococcus yayanosii压力适应性机制研究

发布时间：2018-05-06 15:40

本文选题：Pyrococcus + yayanosii　；参考：《中国海洋大学》2014年博士论文

【摘要】：深海是典型的高静水压环境，嗜压微生物（piezophile）是深海生态系统中的重要类群。随着深海取样技术的不断发展和深海高压微生物特殊培养装置的研发，已经从深海环境中分离到了多种嗜压微生物，其中包括不能在常压条件下生长的专性嗜压微生物。通过对分布于不同压力范围的微生物近缘种间进行比较而获得的嗜压微生物的压力适应性的认识，不能反映微生物个体水平上对静水压力变化的应激调控机制。本文通过比较严格嗜压菌株与其衍生的兼性嗜压菌株在基因组、转录组及相关生理性状上的差异，试图从微生物细胞的物质代谢与能量转化的角度分析深海嗜压微生物的压力适应性。 Pyrococcus yayanosii CH1分离自大西洋中脊4100米水深的“Ashadze”热液口，是目前已知的第一株和唯一一株严格嗜压的超嗜热古菌，其最适生长压力为52MPa，最高耐受压力超过了120MPa。本论文通过人工驯化手段，获得CH1衍生的兼性嗜压菌株A1，其最适生长压力为52MPa，但压力耐受范围变宽，可以在0.1MPa的常压条件下生长。对A1菌株进行全基因组测序，并与CH1进行比较基因组分析，结果表明两个基因组中存在23处序列差异，这些序列差异所在的基因主要与细胞周期调控、鞭毛的合成以及芳香族氨基酸的转运等相关。对A1菌株中相关基因的功能展开研究，将有助于揭示此类深海超嗜热嗜压古菌的压力适应性机制。 A1可以在常压下生长，使以该菌株为宿主建立遗传操作系统成为可能。构建了携带A1中pyrF基因侧翼同源序列和受谷氨酸脱氢酶强启动子控制的3-hydroxy-3-methylglutaryl辅酶A（HMG-CoA）还原酶超表达基因元件（SimR）的自杀质粒pLMO02。利用过表达HMG-CoA还原酶赋予A1菌株simvastatin抗性作为标记，成功的中断了A1菌株的pyrF基因（PYCH0296），获得了尿嘧啶缺陷型菌株A2。而且，又构建了SimR-pyrF基因元件，获得了无痕基因中断载体pLMO03。无痕敲除系统突破了A1菌株中可用筛选标记的限制，可以中断A1基因组中的任何非必需基因，并且筛选标记可以重复利用。单一菌株以甲酸为唯一碳源的产氢呼吸途径仅见于深海超嗜热古菌Thermococcus onnurineus NA1，是最简单的厌氧呼吸方式之一。比较基因组分析表明，Thermococcales科超嗜热古菌中只有Thermococcus onnurineus NA1、Thermococcus gammatolerans EJ3和Pyrococcus yayanosii中存在有完整的甲酸代谢基因簇（Fmr）。中断A1菌株的Fmr基因簇后，突变株中大量积累甲酸，其0.1MPa的生长也受到限制。膜结合态氢酶复合体Mbh以及胞质内氢酶复合体SHI参与了Thermococcales微生物的能量代谢，中断A1菌株的Mbh和SHI基因簇后，突变株中也积累甲酸，表明Mbh和SHI复合体也参与了A1菌株的甲酸代谢。甲酸既是代谢过程中的重要中间代谢产物，也是一些超嗜热微生物肌苷（IMP）合成途径中的一碳单元。对A1菌株在0.1MPa和52MPa压力条件下的转录组分析发现，与IMP合成途径相关的基因转录在0.1MPa压力条件下调，而与甲酸代谢及能量转换相关的氢酶的编码基因则转录上调。该结果暗示，A1中参与IMP合成代谢的甲酸被分解产能以适应低压的胁迫环境。而对于CH1在15MPa和52MPa压力条件下的转录组分析发现，，其在15MPa的边界压力下，不能有效的调节甲酸由参与IMP合成向氧化产能转换。进一步对IMP合成途径和甲酸氧化途径相关基因的启动子区域分析，发现IMP生物合成途径中的多个基因的上游区域，存在有一个保守的motif“CnTn5TGn3AAA”。以此motif为探针，通过磁珠富集的方法获得了可以与该motif结合的转录因子(PTF)。转录组和蛋白组分析结果表明，A1菌株中甲酸代谢受压力调控的可能模型为：当A1处于52MPa时，PTF高表达，并与IMP途径中相关基因的motif结合，激活IMP途径的表达。甲酸作为一碳单元，参与IMP的合成；而当A1处于0.1MPa时，PTF的表达受到抑制，进而导致IMP合成受阻，引起甲酸的累积，而甲酸的大量积累又诱导了甲酸代谢基因簇的表达。此外，在不同的温度、pH以及盐度胁迫条件下，A1菌株中的甲酸代谢基因簇也转录上调，该结果暗示在深海超嗜热嗜压古菌中通过调节甲酸代谢的流向，对其应对多重环境因子的胁迫具有重要意义。
[Abstract]:The deep-sea is a typical Gao Jing water pressure environment, and the pressure microorganism (piezophile) is an important group in the deep-sea ecosystem. With the continuous development of deep-sea sampling technology and the development of special culture device for deep-sea high pressure microorganism, a variety of eosinophils have been separated from the deep sea environment, including the inability to grow under normal pressure. Specific pressure microbes. The understanding of the pressure adaptability of the pressure microbes obtained by comparing the marginal species of microbes distributed in different pressure ranges can not reflect the stress regulation mechanism on the change of hydrostatic pressure on the microorganism's individual level. This paper compares the strict strain strain and its derived facultative strain strain. The differences in genome, transcriptional group and related physiological characters try to analyze the pressure adaptability of deep sea eosinophils from the point of view of material metabolism and energy transformation of microbial cells.
Pyrococcus yayanosii CH1 is separated from the "Ashadze" hydrothermal mouth of the 4100 meter depth of the middle ridge of the middle ridge of the Atlantic. It is the first known strain and the only strain of the strict thermophilic antithermophilic bacteria. The optimum growth pressure is 52MPa. The highest tolerance pressure exceeds the 120MPa. in this paper, and the CH1 derived facultative strain A1 is obtained by artificial domestication. The optimum growth pressure is 52MPa, but the range of stress tolerance is wide and can grow under the normal pressure of 0.1MPa. The whole genome sequencing of A1 strains and comparative genomic analysis with CH1 show that there are 23 sequences in the two genomes, which are mainly related to the regulation of cell cycle and the combination of flagellum. The study on the function of the related genes in the A1 strain will help to reveal the pressure adaptation mechanism of this kind of deep-sea hyper thermophilic bacteria.
A1 can be grown at normal pressure, making it possible to establish a genetic operating system with the strain as the host. A suicide plasmid pLMO02. that carries the pyrF gene in A1 and the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase overexpressed gene element (SimR) controlled by the strong promoter of glutamate dehydrogenase is used to express HMG. -CoA reductase endowed the A1 strain simvastatin resistance as a marker, successfully disrupted the pyrF gene of the A1 strain (PYCH0296), obtained the uracil defective strain A2. and constructed the SimR-pyrF gene element, and obtained the traceless gene interrupt carrier, pLMO03. null knockout system, which broke through the restriction of the available screening markers in the A1 strain. Cut off any non essential genes in the genome of A1, and the screening markers can be reused.
The hydrogen production pathway of the single strain with formic acid as the only carbon source is found only in the deep-sea hyper thermophilic Thermococcus onnurineus NA1, which is one of the simplest anaerobic respiration methods. Comparative genomic analysis shows that only Thermococcus onnurineus NA1, Thermococcus gammatolerans EJ3 and Pyrococcus yay are in the Thermococcales family. There is a complete formic metabolic gene cluster (Fmr) in anosii. After interrupting the Fmr gene cluster of A1 strain, a large amount of formic acid is accumulated in the mutant strain, and the growth of 0.1MPa is also restricted. The membrane bound hydrogen enzyme complex Mbh and the intracellular hydrogenase complex SHI participate in the energy metabolism of Thermococcales microbes and interrupt the Mbh and SHI gene clusters of the A1 strain. After that, formic acid was also accumulated in the mutant strain, indicating that Mbh and SHI complex were also involved in formic acid metabolism of A1 strain.
Formic acid is not only an important intermediate metabolite in metabolic process, but also a carbon unit in some hyper thermophilic inosine (IMP) synthesis pathway. The transcriptional analysis of A1 strain under the pressure of 0.1MPa and 52MPa found that the gene transcription related to the IMP synthesis pathway was downregulated in the 0.1MPa pressure condition, but with the formic acid metabolism and energy conversion phase. The results suggested that the A1 involved in IMP anabolic formic acid was decomposed to adapt to the stress environment of low pressure, and the transcriptional analysis of CH1 under the pressure of 15MPa and 52MPa found that it could not be used to regulate formic acid by participating in the IMP synthesis to oxidation capacity under the boundary pressure of 15MPa. Transformation.
Further analysis of the promoter region of the IMP synthesis pathway and the genes related to formic acid oxidation found that there was a conservative motif "CnTn5TGn3AAA" in the upstream region of the multiple genes in the IMP biosynthesis pathway. Using motif as a probe, the transcription factor (PTF), which could be combined with the motif, was obtained through the enrichment of magnetic beads. The results of recorded and proteome analysis showed that the possible model of the stress regulation of formic acid metabolism in the A1 strain was that when A1 was in 52MPa, PTF was highly expressed, and the expression of IMP pathway was activated by the binding of motif to the related genes in the IMP pathway. As a carbon unit, formic acid was involved in the synthesis of IMP; while A1 was in 0.1MPa, the expression of PTF was inhibited. The accumulation of formic acid was caused by the blocking of IMP synthesis, and the accumulation of formic acid induced the expression of formic acid metabolism gene cluster.
In addition, under the conditions of different temperatures, pH and salinity stress, the transcription of the metabolic genes of formic acid in the A1 strain is also up-regulated. The results suggest that the regulation of the flow of formic metabolism in the deep-sea hyper thermophilic bacteria is of great significance to stress the stress of multiple environmental factors.

【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：Q178.53

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