Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing
Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing
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DOI：
10.1046/j.1462-2920.2003.00543.x
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发表时间：
2004-02-01
影响因子：
5.1
通讯作者：
Murrell, JC
中科院分区：
文献类型：
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作者：
Hutchens, E;Radajewski, S;Murrell, JC
莫维尔洞是一个特殊的地下水生态系统，由原位化学自养生产支持。洞穴大气中含有1-2%的甲烷（CH4），尽管在气泡中发现的甲烷浓度要高得多，气泡使微生物席漂浮在水面上。由于之前对稳定碳同位素比率的分析表明甲烷氧化发生在这种环境中，我们假设在Movile洞穴中有活跃的有氧甲烷氧化细菌（甲烷氧化菌）。为了鉴定Movile洞穴水和垫料中的活性甲烷氧化菌，采用dna稳定同位素探测（DNA-SIP）实验，在10%(CH4)-C-13的顶空条件下培养了一个微环境。利用改进的离心条件，收集c -13标记的DNA片段，作为聚合酶链反应扩增的模板。对甲烷氧化菌甲烷氧化途径小亚基rRNA和关键酶的编码基因分析发现，甲基单胞菌（Methylomonas）、甲基球菌（Methylococcus）和甲基胞菌（Methylocystis/Methylosinus）能够同化(CH4)-C-13，并且这些甲烷氧化菌含有两种已知甲烷单加氧酶（MMO）的编码基因。非甲烷营养细菌和藻类的序列提供了CH4周转的证据，这可能是由于对c -13标记的代谢物或生物量的交叉取食。我们的研究结果表明，好氧甲烷氧化菌在Movile洞穴中积极地将CH4转化为复杂的有机化合物，从而有助于维持这个封闭生态系统中微生物群落的多样性。
Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%(CH4)-C-13 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a C-13-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the (CH4)-C-13, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on C-13-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.