本文選題：Pyrococcus + yayanosii　； 參考：《中國海洋大學(xué)》2014年博士論文

【摘要】：深海是典型的高靜水壓環(huán)境，嗜壓微生物（piezophile）是深海生態(tài)系統(tǒng)中的重要類群。隨著深海取樣技術(shù)的不斷發(fā)展和深海高壓微生物特殊培養(yǎng)裝置的研發(fā)，已經(jīng)從深海環(huán)境中分離到了多種嗜壓微生物，其中包括不能在常壓條件下生長的專性嗜壓微生物。通過對分布于不同壓力范圍的微生物近緣種間進行比較而獲得的嗜壓微生物的壓力適應(yīng)性的認識，不能反映微生物個體水平上對靜水壓力變化的應(yīng)激調(diào)控機制。本文通過比較嚴格嗜壓菌株與其衍生的兼性嗜壓菌株在基因組、轉(zhuǎn)錄組及相關(guān)生理性狀上的差異，試圖從微生物細胞的物質(zhì)代謝與能量轉(zhuǎn)化的角度分析深海嗜壓微生物的壓力適應(yīng)性。 Pyrococcus yayanosii CH1分離自大西洋中脊4100米水深的“Ashadze”熱液口，是目前已知的第一株和唯一一株嚴格嗜壓的超嗜熱古菌，其最適生長壓力為52MPa，最高耐受壓力超過了120MPa。本論文通過人工馴化手段，獲得CH1衍生的兼性嗜壓菌株A1，其最適生長壓力為52MPa，但壓力耐受范圍變寬，可以在0.1MPa的常壓條件下生長。對A1菌株進行全基因組測序，并與CH1進行比較基因組分析，結(jié)果表明兩個基因組中存在23處序列差異，這些序列差異所在的基因主要與細胞周期調(diào)控、鞭毛的合成以及芳香族氨基酸的轉(zhuǎn)運等相關(guān)。對A1菌株中相關(guān)基因的功能展開研究，將有助于揭示此類深海超嗜熱嗜壓古菌的壓力適應(yīng)性機制。 A1可以在常壓下生長，使以該菌株為宿主建立遺傳操作系統(tǒng)成為可能。構(gòu)建了攜帶A1中pyrF基因側(cè)翼同源序列和受谷氨酸脫氫酶強啟動子控制的3-hydroxy-3-methylglutaryl輔酶A（HMG-CoA）還原酶超表達基因元件（SimR）的自殺質(zhì)粒pLMO02。利用過表達HMG-CoA還原酶賦予A1菌株simvastatin抗性作為標記，成功的中斷了A1菌株的pyrF基因（PYCH0296），獲得了尿嘧啶缺陷型菌株A2。而且，又構(gòu)建了SimR-pyrF基因元件，獲得了無痕基因中斷載體pLMO03。無痕敲除系統(tǒng)突破了A1菌株中可用篩選標記的限制，可以中斷A1基因組中的任何非必需基因，并且篩選標記可以重復(fù)利用。 單一菌株以甲酸為唯一碳源的產(chǎn)氫呼吸途徑僅見于深海超嗜熱古菌Thermococcus onnurineus NA1，是最簡單的厭氧呼吸方式之一。比較基因組分析表明，Thermococcales科超嗜熱古菌中只有Thermococcus onnurineus NA1、Thermococcus gammatolerans EJ3和Pyrococcus yayanosii中存在有完整的甲酸代謝基因簇（Fmr）。中斷A1菌株的Fmr基因簇后，突變株中大量積累甲酸，其0.1MPa的生長也受到限制。膜結(jié)合態(tài)氫酶復(fù)合體Mbh以及胞質(zhì)內(nèi)氫酶復(fù)合體SHI參與了Thermococcales微生物的能量代謝，中斷A1菌株的Mbh和SHI基因簇后，突變株中也積累甲酸，表明Mbh和SHI復(fù)合體也參與了A1菌株的甲酸代謝。 甲酸既是代謝過程中的重要中間代謝產(chǎn)物，也是一些超嗜熱微生物肌苷（IMP）合成途徑中的一碳單元。對A1菌株在0.1MPa和52MPa壓力條件下的轉(zhuǎn)錄組分析發(fā)現(xiàn)，與IMP合成途徑相關(guān)的基因轉(zhuǎn)錄在0.1MPa壓力條件下調(diào)，而與甲酸代謝及能量轉(zhuǎn)換相關(guān)的氫酶的編碼基因則轉(zhuǎn)錄上調(diào)。該結(jié)果暗示，A1中參與IMP合成代謝的甲酸被分解產(chǎn)能以適應(yīng)低壓的脅迫環(huán)境。而對于CH1在15MPa和52MPa壓力條件下的轉(zhuǎn)錄組分析發(fā)現(xiàn)，，其在15MPa的邊界壓力下，不能有效的調(diào)節(jié)甲酸由參與IMP合成向氧化產(chǎn)能轉(zhuǎn)換。 進一步對IMP合成途徑和甲酸氧化途徑相關(guān)基因的啟動子區(qū)域分析，發(fā)現(xiàn)IMP生物合成途徑中的多個基因的上游區(qū)域，存在有一個保守的motif“CnTn5TGn3AAA”。以此motif為探針，通過磁珠富集的方法獲得了可以與該motif結(jié)合的轉(zhuǎn)錄因子(PTF)。轉(zhuǎn)錄組和蛋白組分析結(jié)果表明，A1菌株中甲酸代謝受壓力調(diào)控的可能模型為：當A1處于52MPa時，PTF高表達，并與IMP途徑中相關(guān)基因的motif結(jié)合，激活I(lǐng)MP途徑的表達。甲酸作為一碳單元，參與IMP的合成；而當A1處于0.1MPa時，PTF的表達受到抑制，進而導(dǎo)致IMP合成受阻，引起甲酸的累積，而甲酸的大量積累又誘導(dǎo)了甲酸代謝基因簇的表達。 此外，在不同的溫度、pH以及鹽度脅迫條件下，A1菌株中的甲酸代謝基因簇也轉(zhuǎn)錄上調(diào)，該結(jié)果暗示在深海超嗜熱嗜壓古菌中通過調(diào)節(jié)甲酸代謝的流向，對其應(yīng)對多重環(huán)境因子的脅迫具有重要意義。
[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.

【學(xué)位授予單位】：中國海洋大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：Q178.53

【參考文獻】

相關(guān)期刊論文 前1條

1 李學(xué)恭;徐俊;肖湘;;深海微生物高壓適應(yīng)與生物地球化學(xué)循環(huán)[J];微生物學(xué)通報;2013年01期

本文編號：1852889