北臺灣酸性熱泉中微生物群集的總體基因體分析

Abstract

極端環境微生物生態系的探查，數十年來是為地球歷史、生物演化、天體生物學領域發展的基礎研究之一。溫泉是臺灣常見的極端環境生態系統，早期在北部大屯山區對溫泉微生物生態系的調查偏重使用聚合酶連鎖反應(polymerase chain reaction, PCR) 偵測原核生物16 S rRNA 基因，再加以轉殖與定序，對於微生物族群結構與功能的探究深度及廣度較為不足。近五年來，次世代定序的技術逐漸成熟與普及，使序列資料的獲得更有效率，也能得知更接近真實的自然界樣貌。在這篇研究中，選定於大屯山區四磺坪地區溫泉進行採樣，利用總體基因體學結合IlluminaTM 定序平臺分析大屯火山群的四磺坪酸性熱泉中的微生物群集，希望了解其主要的群集結構與功能。 經過調查，四磺坪區域的溫泉，泉溫平均為73°C，pH 平均為2.8，估計溫泉水中浮游原核生物密度約為7.3×105/mL，低於一般陸地淡水常溫水體與鹽湖大約十倍。浮游微生物總體基因隨後建構為一包含九千餘株樣本的fosmid library，自library中取一千餘株樣本與另一批直接自四磺坪泉水萃取的總體基因同時進行次世代定序。經由生物資訊分析定許結果，發現細菌為此溫泉生態系的強勢種，估計與古細菌的比例約為7:3 到8:2 之間。細菌主要以化學自營性細菌phylum Aquificae成員為主，其次為 Proteobacteria 和Firmicutes；古細菌則以phylum Crenarchaeota佔主要位置。在本次調查中，部分分析顯示Aquificae 之下的 Hydrogenobaculum屬佔有相當高的相對豐度。在比對模式種基因體序列後，發現無法完整重建本區 Hydrogenobaculum 類似物種的基因體，可能暗示著此二者間存有某種程度上的序列差異性。藉由Protein COG 的分佈模式分析，並與其他相似案例比較，推測此熱泉微生物群集的整體基因體組成偏重於代謝相關功能。

Researches of ecosystems in extreme environments have been critical to the accumulation of fundamental knowledge in fields of earth history, evolution biology and astrobiology. Novel enzymes, molecules possessed by microorganisms in extreme environments also provide sources for potent molecular biological and biotechnical applications. Extreme environments such as hot springs are commonly seen in Taiwan. Previous studies relevant to hot spring microbial ecosystems in the Tatun Volcanic Group area (TVG, northern Taiwan) relied mainly on SSU rRNA gene sequence analyses, which were unilateral and could be possibly biased. To obtain a more realistic view of prokaryotic diversity by acquiring sufficient sequence information, next-generation sequencing (NGS) was applied to this study with the aid of bioinformatics. Through the investigations on metagenomes from microbial community in a hot spring in SHP area, this study was aimed to obtain an overview on prokaryotic diversity, community structure and function in the hydrothermal spring system. According to the results, the average pH of the hot spring in SHP area was 2.8, and average temperature was 73°C. Cell density of planktonic prokaryotes was estimated to be 7.3×105/mL, which was about 10 times lower than that of terrestrial fresh water and salt lakes. Metagenomic DNAs extracted from SHP hot spring were used for construction of a fosmid library containing 9481 clones. DNAs extracted from hot spring water and DNAs of 1485 clones from fosmid library were sequenced using IlluminaTM platform. Analyses of community composition revealed dominance of bacteria over archaea by 7:3 to 8:2. Members of bacterial phylum Aquificae were abundant, followed by Proteobacteria and Firmicutes; members of phylum Crenarchaeota were most abundant among archaea. Sequences from predominant members of genus Hydrogenobaculum, affiliated with Aquificae, were mapped to the reference genome and certain level of sequence difference was found in the comparison. By analysis of the protein COG distribution pattern and comparing results from the other research, the hot spring prokaryotic community in SHP displayed a higher proportion of metabolism-related functions encoded in their genomes.