Studies on development of a molecular method for analyzing microbial community structure of chemolithotrophic ammonia oxidizers by quantifying abundance of phylogenetic groups

本研究的目的是开发一种新的分子方法来分析微生物群落结构在环境中，针对好氧化学无机营养型氨氧化细菌（AOB），通过量化每个系统发育组组成的社会。氨氧化细菌在全球氮循环中起着关键作用，其单系性使其特别适合于分子方法的应用。由于氨氧化细菌生长缓慢，因此对其群落结构进行了分子生物学分析。本研究采用真实的时间PCR方法对环境样品中的AOB细胞进行计数，并考察该方法是否能准确地检测AOB。本研究采用了一对针对不同AOB特异性基因的引物，以及从几个具有相对不同系统发育位置的AOB菌株和分离物中提取的模板DNA。引物组和菌株/分离物的每种组合提供了用于定量的标准曲线，并且其相关性非常好。然而，不同菌株/分离株的标准曲线并不重叠，这将导致在从混合群体中扩增DNA时，根据所选择的用于创建标准曲线的染料，错误地估计准确的靶基因数目。根据结果，我们指定了正确估计所需的引物组和模板DNA的类型的组合。在此基础上，讨论了利用现有技术表达群落中AOB各系统发育类群丰度的可行策略。

Aim of the present study is to develop a novel molecular method for analyzing microbial community structure in environments, targeting aerobic chemolithotrophic ammonia-oxidizing bacteria(AOB), by quantifying each phylogenetic group comprising a community. AOB play a pivotal role in the global nitrogen cycle, and their monophyletic nature makes them especially suitable for the application of molecular methods. Since their poor growth, community structure of AOB has been analyzed by molecular methods. Recently, PCR has been introduced for quantifying AOB in environmental samples and its potential and reliability has been examined.In this study, real time PCR method, one of quantitative PCR methods which potentially allow to precisely estimating number of target cells in a sample, was applied to enumerate AOB cells, and was examined if the method can properly estimate AOB. A couple of primer sets which target different genes specific for AOB, and template DNA preparations extracted from several AOB strains and isolates which have relatively diverse phylogenetic positions were employed. Every combination of primer set and strain/isolate provided a standard curve for quantification, and its correlation was very good. However, standard curves obtained from different strains/isolates did not overlap, which would result in incorrect estimations of the exact target numbers when amplifying DNA from mixed communities, depending on the stain selected for creating a standard curve.We examined the factors affect the shape of standard curve, and elucidated the mechanism of incorrect estimation of the target genes. Based on the results, we specified the combination of types of primer set and template DNA necessary for correct estimation. Based on the results, realistic strategies for expressing abundance of each phylogenetic group of AOB in a community using available techniques were also discussed.