Shen Wenli, Wang Danrui, Li Jiangtao, Liu Yue, Wang Yinzhao, Yang Xingsheng, Peng Xi, Xie Bingliang, Su Lei, Wei Ziyan, He Qing, Wang Zhiyi, Feng Kai, Du Wenbin, Deng Ye
CAS Key Laboratory of Environmental Biotechnology, Research Center for Eco-Environmental Sciences Chinese Academy of Sciences Beijing China.
Institute for Marine Science and Technology Shandong University Qingdao China.
mLife. 2025 Feb 20;4(1):70-82. doi: 10.1002/mlf2.12159. eCollection 2025 Feb.
Anaerobic methanotrophic (ANME) microbes play a crucial role in the bioprocess of anaerobic oxidation of methane (AOM). However, due to their unculturable status, their diversity is poorly understood. In this study, we established a microfluidics-based epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) to fuse the 16S rRNA gene and gene to reveal the diversity of ANME microbes ( gene hosts) in three sampling push-cores from the marine cold seep. A total of 3725 16S amplicon sequence variants (ASVs) of the gene hosts were detected, and classified into 78 genera across 23 phyla. Across all samples, the dominant phyla with high relative abundance (>10%) were the well-known , and some bacterial phyla such as , , and ; however, the specificity of these associations was not verified. In addition, the compositions of the gene hosts were significantly different in different layers, where the archaeal hosts increased with the depths of sediments, indicating the carriers of AOM were divergent in depth. Furthermore, the consensus phylogenetic trees of the gene and the 16S rRNA gene showed congruence in archaea not in bacteria, suggesting the horizontal transfer of the gene may occur among host members. Finally, some bacterial metagenomes were found to contain the gene as well as other genes that encode enzymes in the AOM pathway, which prospectively propose the existence of ANME bacteria. This study describes improvements for a potential method for studying the diversity of uncultured functional microbes and broadens our understanding of the diversity of ANMEs.
厌氧甲烷氧化（ANME）微生物在甲烷厌氧氧化（AOM）的生物过程中起着至关重要的作用。然而，由于它们无法培养，对其多样性了解甚少。在本研究中，我们建立了一种基于微流控的epicPCR（乳液、配对分离和串联PCR）方法，将16S rRNA基因与[具体基因名称缺失]基因融合，以揭示来自海洋冷泉的三个采样推芯中ANME微生物（[具体基因名称缺失]基因宿主）的多样性。共检测到[具体基因名称缺失]基因宿主的3725个16S扩增子序列变体（ASVs），并将其分为23个门的78个属。在所有样本中，相对丰度较高（>10%）的优势门是众所周知的[具体门名称缺失]，以及一些细菌门，如[具体细菌门名称缺失]、[具体细菌门名称缺失]和[具体细菌门名称缺失]；然而，这些关联的特异性尚未得到验证。此外，[具体基因名称缺失]基因宿主的组成在不同层中存在显著差异，其中古菌宿主随着沉积物深度的增加而增加，这表明AOM的载体在深度上存在差异。此外，[具体基因名称缺失]基因和16S rRNA基因的一致系统发育树在古菌中显示出一致性，而在细菌中则不然，这表明[具体基因名称缺失]基因可能在宿主成员之间发生水平转移。最后，发现一些细菌宏基因组包含[具体基因名称缺失]基因以及AOM途径中其他编码酶的基因，这前瞻性地提出了ANME细菌的存在。本研究描述了一种用于研究未培养功能微生物多样性的潜在方法的改进，并拓宽了我们对ANME多样性的理解。
