本文關(guān)鍵詞：福州溫泉嗜熱菌多樣性及嗜熱菌Geobacillus thermoglucosidasius低溫適應(yīng)的機(jī)制　出處：《福建農(nóng)林大學(xué)》2016年博士論文　論文類型：學(xué)位論文

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【摘要】：溫泉是地球上一類重要的熱環(huán)境,其高溫和貧營養(yǎng)環(huán)境中生存著大量的嗜熱微生物。嗜熱菌是一類重要的微生物,在生存機(jī)制和應(yīng)用方面都吸引著越來越多關(guān)注。地球上不同地區(qū)溫泉嗜熱菌組成差異很大,對不同地區(qū)溫泉中嗜熱菌多樣性研究有利于加深對該類生態(tài)系統(tǒng)的認(rèn)識。地芽孢桿菌屬(Geobacillus)是溫泉可培養(yǎng)嗜熱菌中的優(yōu)勢菌,也是堆肥等熱環(huán)境中的常見菌種,但是隨著近年來在常溫和寒冷環(huán)境中不斷分離到Geobacillus屬菌株,這類嗜熱菌適應(yīng)低溫生存的機(jī)制引起人們關(guān)注。本論文通過調(diào)查福州三處溫泉的嗜熱菌多樣性,發(fā)現(xiàn)Geobacillus屬細(xì)菌在溫泉熱環(huán)境中廣泛分布,在此基礎(chǔ)上進(jìn)一步研究了Geobacillus屬細(xì)菌的低溫適應(yīng)機(jī)制。本文應(yīng)用Illumina MiSeq平臺檢測福州大湯(DaTang)、湯埕(TangCheng)和雙龍(ShuangLong)三處溫泉水體中的細(xì)菌16S rDNA多樣性,顯示三處溫泉中嗜熱菌多樣性豐富,其中豐度較大的嗜熱菌有變形菌門(Proteobacteria)、厚壁菌門(Firmicutes)、產(chǎn)水菌門(Aquificae),硝化螺旋菌門(Nitrospirae)、裝甲菌門(Armatimonadetes),棲熱菌門(Thermi), GAL15菌門,酸桿菌門(Acidobacteria),綠菌門(Chlorobi),放線菌門(Actinobacteria),綠彎菌綱(Chloroflexi), OP1菌門,OD1菌門,擬桿菌門(Bacteroidetes)和EM3菌門。不同溫泉之間細(xì)菌多樣性差異較大,Pearson相關(guān)性分析和RDA分析顯示三處溫泉微生物多樣性的差異與溫泉溫度、Na+、NO2和Mg2+等水體理化性質(zhì)有關(guān)。對福州地區(qū)三個典型溫泉：大湯溫泉、湯埕溫泉和雙龍溫泉溫泉底泥中的可培養(yǎng)嗜熱菌進(jìn)行了研究,結(jié)果顯示大湯溫泉嗜熱菌的種類多于湯埕,雙龍溫泉嗜熱菌最少。進(jìn)一步分析發(fā)現(xiàn)三個溫泉嗜熱菌多樣性與pH,堿解氮和有效磷有關(guān),pH越低,堿解氮和有效磷含量越高溫泉中嗜熱菌越豐富。所分離的32株嗜熱菌隸屬于6個不同的屬,其中Geobacillus屬和Anoxybacillus屬菌株為福州溫泉中的優(yōu)勢嗜熱菌菌種。可培養(yǎng)方式和非可培養(yǎng)方式研究對比發(fā)現(xiàn)溫泉中可培養(yǎng)菌只占非常小一部分,堅(jiān)壁菌門中產(chǎn)芽孢細(xì)菌較易培養(yǎng)；棲熱菌,變形菌等嗜熱菌沒有從現(xiàn)有的培養(yǎng)方式中得到純培養(yǎng)物。改善可培養(yǎng)方式或者應(yīng)用宏基因組學(xué)手段有利于進(jìn)一步挖掘溫泉嗜熱菌基因資源。對分離的一株嗜熱菌Geobacillus sp. CHB1進(jìn)行了鑒定和基因組測序。該菌生長溫度范圍為47℃～74℃之間,最適生長溫度為60℃。16S rDNA測序發(fā)現(xiàn)該菌與標(biāo)準(zhǔn)菌株Geobacillus thermoglucosidasius DSM2542T同源性最近為99.26%。該菌的G+C含量為49%,與Geobacillus thermoglucosidasius DSM25421基因組雜交率為75%。但是其與標(biāo)準(zhǔn)菌株Geobacillus thermoglucosidasius DSM25421在生理生化特征和細(xì)胞膜磷脂脂肪酸組成方面又有所不同,判斷該菌株為Geobacillus thermoglucosidasius種的一個亞種。對Geobacillus sp. CHB1基因組進(jìn)行測序,該菌株基因組長度為3.22M,編碼4122個開放閱讀框。為了研究Geobacillus sp. CHB1適應(yīng)低溫生長的機(jī)制。以Geobacillus sp. CHB1為目標(biāo)菌株,應(yīng)用適應(yīng)性進(jìn)化技術(shù)(Experimental evolution)向低于其最低生長溫度的方向進(jìn)化,經(jīng)過6個月的實(shí)驗(yàn)得到了可以在430C溫度下起始生長的進(jìn)化菌株。進(jìn)化菌株生長速度明顯高于初始菌株。進(jìn)化菌株Ec細(xì)胞膜磷脂脂肪酸在低溫下表現(xiàn)出劇烈調(diào)整的傾向,iso 15：00在45℃低溫生長時相對含量顯著上升,這也從另一個方面證明了進(jìn)化后的菌株對低溫的適應(yīng)能力更好。比較蛋白組學(xué)發(fā)現(xiàn)進(jìn)化菌株與核苷酸相關(guān)的蛋白表達(dá)量上調(diào),而與脂肪酸代謝相關(guān)的蛋白表現(xiàn)下調(diào)。蛋白組學(xué)結(jié)果與進(jìn)化菌株表現(xiàn)出較高的生長速度和較強(qiáng)的脂肪酸調(diào)節(jié)能力是相一致的。在前期6個月進(jìn)化的基礎(chǔ)上,以Ec為出發(fā)菌株又繼續(xù)向更低生長溫度進(jìn)行了12個月的進(jìn)化實(shí)驗(yàn)。溫度生長實(shí)驗(yàn)證明,液體培養(yǎng)條件下三個進(jìn)化群體(F1、F2和F3)都可以在38-39℃之間穩(wěn)定生長傳代,固體平板實(shí)驗(yàn)證明進(jìn)化群體中挑取的菌株F3可以在39℃溫度條件下生長并形成菌落。這些證明,在Ec的基礎(chǔ)上,經(jīng)過12個月的進(jìn)化實(shí)驗(yàn),該菌的最低生長溫度又下降了至少4℃。對進(jìn)化群體Ec,F2和F3進(jìn)行重測序分析,發(fā)現(xiàn)兩個群體有15個共有的突變基因,其中7個基因在兩個群體中突變位置不同,說明這些基因很可能在這個階段適應(yīng)低溫的過程中起到重要的作用。比較基因組學(xué)發(fā)現(xiàn)MFS、DNA聚合酶p亞基、AB水解酶和肽酶等是獨(dú)立適應(yīng)低溫進(jìn)化的群體共有的突變基因,認(rèn)為這些基因是與低溫適應(yīng)有關(guān)的潛在基因突變。從F3群體中挑取兩個單菌落重測序,發(fā)現(xiàn)個體基因組中雜合子明顯少于其對應(yīng)的群體F3,說明在適應(yīng)低溫進(jìn)化過程中群體內(nèi)菌株間積累了大量的不同的突變,這些突變有些還沒有在群體中固定下來。在這個階段Ec適應(yīng)更低溫度的進(jìn)化過程中還出現(xiàn)了一些突變體,F3群體中出現(xiàn)了細(xì)胞分裂被抑制的長線形菌體,通過比較基因組結(jié)果發(fā)現(xiàn)該群體中FtsK基因發(fā)生了倒位突變。這些結(jié)果加深了對嗜熱菌適應(yīng)低溫的機(jī)制的理解。過氧化氫酶在獨(dú)立進(jìn)化的群體EA、EB和EC均表現(xiàn)出比初始菌株A高出20倍以上的過氧化氫酶活性。質(zhì)譜鑒定證明起作用的過氧化氫酶是一種攜帶血紅素的單功能過氧化氫酶,針對該基因的qPCR進(jìn)一步證明其在進(jìn)化菌株中高表達(dá)。進(jìn)化菌株的發(fā)酵液和重組過氧化氫酶都顯示出該過氧化氫酶在0℃～40℃溫度范圍內(nèi)至少有40%以上的活性,表明該酶在Geobacillus低溫生長時是以活性形式存在的。有研究表明過氧化氫酶可以促進(jìn)一些常溫菌在低溫下的生存能力。我們認(rèn)為適應(yīng)性進(jìn)化過程中群體中過氧化氫酶高表達(dá)個體的出現(xiàn)是一個重要事件,該過氧化氫酶高表達(dá)個體更能適應(yīng)該個體出現(xiàn)時的低溫環(huán)境,隨著該適應(yīng)低溫個體在群體中占據(jù)統(tǒng)治地位出現(xiàn)了整個群體對低溫適應(yīng)性增強(qiáng)的現(xiàn)象�？傊�,適應(yīng)低溫進(jìn)化后的Geobacillus sp. CHB1群體在多個水平表現(xiàn)出低溫適應(yīng)能力的增強(qiáng)：如起始生長溫度的明顯降低了約9℃,生長速度的明顯提高,細(xì)胞膜脂肪酸調(diào)節(jié)能力的改變,過氧化氫酶表達(dá)量的提高及蛋白組水平上與核酸代謝和脂肪酸代謝相關(guān)蛋白的表達(dá)變化�？赡茉蚴沁M(jìn)化菌株在低溫條件下的生長速度高于死亡速度。通過比較基因組學(xué)找到與物質(zhì)轉(zhuǎn)運(yùn)相關(guān)的基因MFS、與DNA復(fù)制相關(guān)的基因、DNA聚合酶p亞基以及功能未知的AB水解酶和肽酶等基因的突變。這些基因是適應(yīng)低溫過程的關(guān)鍵基因,它們的突變?nèi)绾斡绊懮L參數(shù)、脂肪酸組成和蛋白質(zhì)表達(dá)需要進(jìn)一步研究。
[Abstract]:Hot springs is a important thermal environment, the high temperature and oligotrophic environment exist thermophilic microorganisms large. Thermophilic bacteria is a kind of important microorganisms, are attracting more and more attention in the survival mechanism and application. The earth in different areas of thermophiles from hot springs composed of great difference, in favor of understanding of the ecological system of the thermophilic bacteria diversity in different regions in hot springs. Thermophilic Bacillus (Geobacillus) is a halophilic bacteria hot springs in bacteria and common bacteria and compost in thermal environment, but in recent years under normal temperature and cold environment continuously isolated from genus Geobacillus this kind of thermophilic bacteria strains, adaptation mechanism of low temperature survival concern. This paper through the investigation of Fuzhou three hot springs of thermophilic bacteria diversity, found Geobacillus bacteria are widely distributed in the hot environment, on the basis of Further studies of Geobacillus bacteria in low temperature adaptation mechanism. In this paper, application of Illumina MiSeq detection platform (DaTang), Fuzhou Tang Tang Cheng (TangCheng) and Ssangyong (ShuangLong) three hot water of bacteria 16S rDNA diversity, showed three hot springs in thermophilic bacteria diversity, the abundance of large L. the heat was Proteobacteria (Proteobacteria), Firmicutes (Firmicutes), aquificae (Aquificae), nitrospirae (Nitrospirae), armored door (Armatimonadetes), bacterium Thermus sp. GAL15 (Thermi), door door, acidobacteria (Acidobacteria), green bacteria (door Chlorobi), actinobacteria (Actinobacteria), green (Chloroflexi), bending fungi OP1 bacteria strain OD1 door, door, bacteriodetes (Bacteroidetes) and EM3 were different between spring. The bacterial diversity differences, Pearson correlation analysis and RDA analysis showed that three hot spring microbial diversity difference Different with the temperature of the hot spring, Na+, NO2 and Mg2+. The water physical and chemical properties of three typical hot springs in Fuzhou area: soup hot springs, hot springs and Shuanglong hot spring Tang Cheng in the sediment of culturable thermophilic bacteria were studied. The results showed that the spa thermophilic bacteria species more than Cheng soup, Ssangyong spa thermophilic bacteria. Further analysis found that at least three spring thermophilic bacteria diversity and pH, nitrogen and phosphorus, the lower the pH, nitrogen and phosphorus content is higher in the hot springs of thermophilic bacteria more abundant. The isolated 32 strains of thermophilic bacteria belonging to 6 different genera. The genus Geobacillus and Anoxybacillus strains for hot springs in Fuzhou dominant thermophilic bacteria strains. Culturable and non culturable methods comparative study found in hot springs can be accounted for only a very small portion of bacteria, bacteria Bacillus middle door hardtop is easy to culture; Thermus thermophilus no deformation bacteria etc. Get a pure culture from the culture in the existing training methods can be improved. Or the application of metagenomics means to further mining hot thermophilic bacteria gene resources. On the separation of a thermophilic bacterium Geobacillus sp. CHB1 was identified and genome sequencing. The bacterial growth temperature range is 47 to 74 degrees centigrade. The optimum growth temperature of the bacteria and the standard strain Geobacillus thermoglucosidasius DSM2542T homology 99.26%. recently the bacteria content of G+C is 49% to 60 DEG C.16S rDNA and Geobacillus thermoglucosidasius DSM25421 sequencing, genomic hybridization rate was 75%. But with the standard strain Geobacillus thermoglucosidasius DSM25421 in fatty acid composition and physiological and biochemical characteristics of membrane phospholipids and different, judge the strain as a subspecies of Geobacillus thermoglucosidasius. Geobacillus sp. CHB1 into the genome of For sequencing, the genome length of 3.22M, encoding 4122 open reading frames. In order to study the Geobacillus sp. CHB1 to adapt to the mechanism of low temperature growth. Taking Geobacillus sp. CHB1 as target strains, application of adaptive evolution technology (Experimental evolution) evolution to less than the lowest growth temperature direction, after 6 months of experimental evolution strain can grow starting at the temperature of 430C. The evolution of strain growth rate was significantly higher than the initial strain. The evolution of phospholipid fatty acid of strain Ec membrane showed a tendency to violent adjustment at low temperatures, ISO 15:00 in the low temperature of 45 DEG C growth relative content increased significantly, this is another way that the evolution of the strain on low temperature adaptability is better. Comparative proteomics found that evolution strains and related nucleotide protein expression, and protein expression associated with fatty acid metabolism under regulation. Proteomics results and evolutionary strains showed higher growth rate and higher fatty acid regulation ability is consistent. Based on the previous 6 months of evolution, with Ec as the starting strain and continue to lower growth temperature for 12 months. The evolution of growth temperature experiments prove that the liquid culture evolution under the condition of the three groups (F1, F2 and F3) can be passaged stably in 38-39 C experimental solid plate shows that the strain of F3 were in the evolution group in 39 DEG C temperature conditions to grow and form colonies. These prove that on the basis of Ec, through evolutionary experiments for 12 months, the lowest the bacteria growth temperature dropped at least 4 degrees. On the evolution of group Ec and F3, F2 sequencing analysis, two groups found 15 common mutations, including 7 genes in two groups of mutations in different positions, suggesting that these genes are likely to This stage to play an important role in the process of low temperature. Comparative genomics found MFS, DNA polymerase P subunit, AB hydrolase and peptidase were independently adapted to low temperature evolution groups shared mutations, that these genes and gene mutation related to cold acclimation potential. From the F3 group were two single colony sequencing, it was found that the groups of F3 heterozygotes was significantly less than the corresponding individual genome, in the adaptation of the group in low temperature strain evolution process between the accumulation of a large number of different mutations, these mutations have yet to be fixed. In the group also appeared in the process of evolution to lower the temperature in some mutants at this stage Ec, F3 cell line shape populations appeared to inhibit cell division, by comparing the results of FtsK genome in the group gene inversion mutation. These results deepen the understanding of L. The thermophilic bacteria adapt to the low temperature the understanding of the mechanism of the EA group. The catalase in the independent evolution, EB and EC showed that the catalase activity of more than 20 times higher than the initial strain A. Mass spectrometry that catalase effect is a monofunctional catalase heme carrying the gene of qPCR, to further prove its high expression in the evolution of strains. The fermentation broth and the recombinant catalase strains showed the evolution of catalase at 0 to 40 DEG C temperature range of at least 40% more active, indicating that the enzyme is present in the active form in Geobacillus low temperature growth. Studies have shown that catalase can promote some bacteria at room temperature the ability to survive at low temperature. We believe that the adaptive evolution of catalase expression of individual groups appeared to be an important event, the high expression of catalase The body can adapt to the low temperature environment in the presence of individual, with the individual to adapt to low temperature dominated in population in the entire group to enhance the adaptability to low temperature phenomenon. In a word, the evolution of the Geobacillus sp. adapt to the low temperature CHB1 groups showed enhanced low temperature adaptability at multiple levels such as: initial growth temperature significantly reduced about 9 DEG C, significantly increase the growth rate, the changes of cell membrane fatty acid regulation ability, catalase expression increased and protein level and nucleic acid metabolism and fatty acid metabolism related protein changes. The possible reason is the growth rate of strain evolution under the condition of low temperature is higher than the death rate by comparison. Genomics found MFS gene associated with the transfer of the substance, and the replication of DNA gene, DNA polymerase P subunit and unknown function AB hydrolase and peptidase gene mutation These genes are key genes that adapt to low temperature processes, how their mutations affect growth parameters, fatty acid composition and protein expression need to be further studied.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q93

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