Hallam Steven J, Pagé Antoine P, Constan Lea, Song Young C, Norbeck Angela D, Brewer Heather, Pasa-Tolic Ljiljana
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada.
Methods Enzymol. 2011;494:75-90. doi: 10.1016/B978-0-12-385112-3.00004-4.
Methane production and consumption in anaerobic marine sediments is catalyzed by a series of reversible tetrahydromethanopterin (H(4)MPT)-linked C1 transfer reactions. Although many of these reactions are conserved between one-carbon compound utilizing microorganisms, two remain diagnostic for archaeal methane metabolism. These include reactions catalyzed by N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and methyl-coenzyme M reductase (MCR). The latter enzyme is central to C-H bond formation and cleavage underlying methanogenic and reverse methanogenic phenotypes. Here, we describe a set of novel tools for the detection and quantification of H4MPT-linked C1 transfer reactions mediated by uncultivated anaerobic methane-oxidizing archaea (ANME). These tools include polymerase chain reaction primers targeting ANME MCR subunit A subgroups and protein extraction methods from marine sediments compatible with high-resolution mass spectrometry for profiling community structure and functional dynamics.
厌氧海洋沉积物中的甲烷产生和消耗是由一系列可逆的四氢甲蝶呤（H(4)MPT）连接的C1转移反应催化的。尽管这些反应中的许多在利用一碳化合物的微生物之间是保守的，但仍有两个反应是古菌甲烷代谢的诊断性反应。这些反应包括由N5-甲基四氢甲蝶呤：辅酶M甲基转移酶和甲基辅酶M还原酶（MCR）催化的反应。后一种酶对于产甲烷和反产甲烷表型所基于的C-H键形成和裂解至关重要。在这里，我们描述了一套用于检测和定量由未培养的厌氧甲烷氧化古菌（ANME）介导的H4MPT连接的C1转移反应的新工具。这些工具包括针对ANME MCR亚基A亚组的聚合酶链反应引物，以及与高分辨率质谱兼容的从海洋沉积物中提取蛋白质的方法，用于分析群落结构和功能动态。
