福州温泉嗜热菌多样性及嗜热菌Geobacillus thermoglucosidasius低温适应的机制

发布时间：2018-01-14 01:04

本文关键词：福州温泉嗜热菌多样性及嗜热菌Geobacillus thermoglucosidasius低温适应的机制　出处：《福建农林大学》2016年博士论文　论文类型：学位论文

【摘要】：温泉是地球上一类重要的热环境,其高温和贫营养环境中生存着大量的嗜热微生物。嗜热菌是一类重要的微生物,在生存机制和应用方面都吸引着越来越多关注。地球上不同地区温泉嗜热菌组成差异很大,对不同地区温泉中嗜热菌多样性研究有利于加深对该类生态系统的认识。地芽孢杆菌属(Geobacillus)是温泉可培养嗜热菌中的优势菌,也是堆肥等热环境中的常见菌种,但是随着近年来在常温和寒冷环境中不断分离到Geobacillus属菌株,这类嗜热菌适应低温生存的机制引起人们关注。本论文通过调查福州三处温泉的嗜热菌多样性,发现Geobacillus属细菌在温泉热环境中广泛分布,在此基础上进一步研究了Geobacillus属细菌的低温适应机制。本文应用Illumina MiSeq平台检测福州大汤(DaTang)、汤埕(TangCheng)和双龙(ShuangLong)三处温泉水体中的细菌16S rDNA多样性,显示三处温泉中嗜热菌多样性丰富,其中丰度较大的嗜热菌有变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、产水菌门(Aquificae),硝化螺旋菌门(Nitrospirae)、装甲菌门(Armatimonadetes),栖热菌门(Thermi), GAL15菌门,酸杆菌门(Acidobacteria),绿菌门(Chlorobi),放线菌门(Actinobacteria),绿弯菌纲(Chloroflexi), OP1菌门,OD1菌门,拟杆菌门(Bacteroidetes)和EM3菌门。不同温泉之间细菌多样性差异较大,Pearson相关性分析和RDA分析显示三处温泉微生物多样性的差异与温泉温度、Na+、NO2和Mg2+等水体理化性质有关。对福州地区三个典型温泉：大汤温泉、汤埕温泉和双龙温泉温泉底泥中的可培养嗜热菌进行了研究,结果显示大汤温泉嗜热菌的种类多于汤埕,双龙温泉嗜热菌最少。进一步分析发现三个温泉嗜热菌多样性与pH,碱解氮和有效磷有关,pH越低,碱解氮和有效磷含量越高温泉中嗜热菌越丰富。所分离的32株嗜热菌隶属于6个不同的属,其中Geobacillus属和Anoxybacillus属菌株为福州温泉中的优势嗜热菌菌种。可培养方式和非可培养方式研究对比发现温泉中可培养菌只占非常小一部分,坚壁菌门中产芽孢细菌较易培养；栖热菌,变形菌等嗜热菌没有从现有的培养方式中得到纯培养物。改善可培养方式或者应用宏基因组学手段有利于进一步挖掘温泉嗜热菌基因资源。对分离的一株嗜热菌Geobacillus sp. CHB1进行了鉴定和基因组测序。该菌生长温度范围为47℃～74℃之间,最适生长温度为60℃。16S rDNA测序发现该菌与标准菌株Geobacillus thermoglucosidasius DSM2542T同源性最近为99.26%。该菌的G+C含量为49%,与Geobacillus thermoglucosidasius DSM25421基因组杂交率为75%。但是其与标准菌株Geobacillus thermoglucosidasius DSM25421在生理生化特征和细胞膜磷脂脂肪酸组成方面又有所不同,判断该菌株为Geobacillus thermoglucosidasius种的一个亚种。对Geobacillus sp. CHB1基因组进行测序,该菌株基因组长度为3.22M,编码4122个开放阅读框。为了研究Geobacillus sp. CHB1适应低温生长的机制。以Geobacillus sp. CHB1为目标菌株,应用适应性进化技术(Experimental evolution)向低于其最低生长温度的方向进化,经过6个月的实验得到了可以在430C温度下起始生长的进化菌株。进化菌株生长速度明显高于初始菌株。进化菌株Ec细胞膜磷脂脂肪酸在低温下表现出剧烈调整的倾向,iso 15：00在45℃低温生长时相对含量显著上升,这也从另一个方面证明了进化后的菌株对低温的适应能力更好。比较蛋白组学发现进化菌株与核苷酸相关的蛋白表达量上调,而与脂肪酸代谢相关的蛋白表现下调。蛋白组学结果与进化菌株表现出较高的生长速度和较强的脂肪酸调节能力是相一致的。在前期6个月进化的基础上,以Ec为出发菌株又继续向更低生长温度进行了12个月的进化实验。温度生长实验证明,液体培养条件下三个进化群体(F1、F2和F3)都可以在38-39℃之间稳定生长传代,固体平板实验证明进化群体中挑取的菌株F3可以在39℃温度条件下生长并形成菌落。这些证明,在Ec的基础上,经过12个月的进化实验,该菌的最低生长温度又下降了至少4℃。对进化群体Ec,F2和F3进行重测序分析,发现两个群体有15个共有的突变基因,其中7个基因在两个群体中突变位置不同,说明这些基因很可能在这个阶段适应低温的过程中起到重要的作用。比较基因组学发现MFS、DNA聚合酶p亚基、AB水解酶和肽酶等是独立适应低温进化的群体共有的突变基因,认为这些基因是与低温适应有关的潜在基因突变。从F3群体中挑取两个单菌落重测序,发现个体基因组中杂合子明显少于其对应的群体F3,说明在适应低温进化过程中群体内菌株间积累了大量的不同的突变,这些突变有些还没有在群体中固定下来。在这个阶段Ec适应更低温度的进化过程中还出现了一些突变体,F3群体中出现了细胞分裂被抑制的长线形菌体,通过比较基因组结果发现该群体中FtsK基因发生了倒位突变。这些结果加深了对嗜热菌适应低温的机制的理解。过氧化氢酶在独立进化的群体EA、EB和EC均表现出比初始菌株A高出20倍以上的过氧化氢酶活性。质谱鉴定证明起作用的过氧化氢酶是一种携带血红素的单功能过氧化氢酶,针对该基因的qPCR进一步证明其在进化菌株中高表达。进化菌株的发酵液和重组过氧化氢酶都显示出该过氧化氢酶在0℃～40℃温度范围内至少有40%以上的活性,表明该酶在Geobacillus低温生长时是以活性形式存在的。有研究表明过氧化氢酶可以促进一些常温菌在低温下的生存能力。我们认为适应性进化过程中群体中过氧化氢酶高表达个体的出现是一个重要事件,该过氧化氢酶高表达个体更能适应该个体出现时的低温环境,随着该适应低温个体在群体中占据统治地位出现了整个群体对低温适应性增强的现象。总之,适应低温进化后的Geobacillus sp. CHB1群体在多个水平表现出低温适应能力的增强：如起始生长温度的明显降低了约9℃,生长速度的明显提高,细胞膜脂肪酸调节能力的改变,过氧化氢酶表达量的提高及蛋白组水平上与核酸代谢和脂肪酸代谢相关蛋白的表达变化。可能原因是进化菌株在低温条件下的生长速度高于死亡速度。通过比较基因组学找到与物质转运相关的基因MFS、与DNA复制相关的基因、DNA聚合酶p亚基以及功能未知的AB水解酶和肽酶等基因的突变。这些基因是适应低温过程的关键基因,它们的突变如何影响生长参数、脂肪酸组成和蛋白质表达需要进一步研究。
[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.

【学位授予单位】：福建农林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

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