Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments
Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments
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DOI：
10.1128/aem.65.11.5066-5074.1999
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发表时间：
1999-11
影响因子：
4.4
通讯作者：
A. Costello;M. Lidstrom
中科院分区：
文献类型：
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作者：
A. Costello;M. Lidstrom
从华盛顿湖沉积物中提取甲烷氧化高峰期沉积物的总DNA，用聚合酶链式反应(PCR)扩增甲烷氧化细菌(甲烷氧化菌)自然种群的16S rRNA和pmoA基因。为每个基因构建克隆文库，利用限制性片段长度多态性(RFLP)和四聚体限制性内切酶MspI、HaeIII和HhaI对每个文库中的约200个克隆进行分析。根据其RFLP图谱对扩增产物进行分组，并对每一组的代表性进行测序和分析。对获得的16S rRNA数据的研究表明，当这些数据与从相同环境中获得的纯培养数据相比较时，现有的引物不能揭示存在的总甲烷营养多样性。针对甲基单胞菌属、甲基单胞菌属和甲基胞囊菌属的甲烷氧化菌，开发了新的特异性引物，构建了更完整的克隆文库。此外，还针对甲烷氧化菌颗粒甲烷单加氧酶基因之一pmoA设计了一条新的引物。16S rRNA和pmoA基因序列的系统发育分析表明，新的引物可以在甲烷氧化菌的已知多样性上检测到这些基因。除了这些发现外，本研究中获得的16S rRNA数据与先前描述的系统发育数据相结合，以确定可用于在属水平上鉴定甲烷氧化菌的可操作分类单位。
ABSTRACT The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymesMspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the generaMethylomonas, Methylosinus, andMethylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.