Hales B A, Edwards C, Ritchie D A, Hall G, Pickup R W, Saunders J R
Department of Genetics and Microbiology, University of Liverpool, United Kingdom.
Appl Environ Microbiol. 1996 Feb;62(2):668-75. doi: 10.1128/aem.62.2.668-675.1996.
The presence of methanogenic bacteria was assessed in peat and soil cores taken from upland moors. The sampling area was largely covered by blanket bog peat together with small areas of red-brown limestone and peaty gley. A 30-cm-deep core of each soil type was taken, and DNA was extracted from 5-cm transverse sections. Purified DNA was subjected to PCR amplification with primers IAf and 1100Ar, which specifically amplify 1.1 kb of the archaeal 16S rRNA gene, and ME1 and ME2, which were designed to amplify a 0.75-kb region of the alpha-subunit gene for methyl coenzyme M reductase (MCR). Amplification with both primer pairs was obtained only with DNA extracted from the two deepest sections of the blanket bog peat core. This is consistent with the notion that anaerobiosis is required for activity and survival of the methanogen population. PCR products from both amplifications were cloned, and the resulting transformants were screened with specific oligonucleotide probes internal to the MCR or archaeal 16S rRNA PCR product. Plasmid DNA was extracted from probe-positive clones of both types and the insert was sequenced. The DNA sequences of 8 MCR clones were identical, as were those of 16 of the 17 16S rRNA clones. One clone showed marked variation from the remainder in specific regions of the sequence. From a comparison of these two different 16S rRNA sequences, an oligonucleotide was synthesized that was 100% homologous to a sequence region of the first 16 clones but had six mismatches with the variant. This probe was used to screen primary populations of PCR clones, and all of those that were probe negative were checked for the presence of inserts, which were then sequenced. By using this strategy, further novel methanogen 16S rRNA variants were identified and analyzed. The sequences recovered from the peat formed two clusters on the end of long branches within the methanogen radiation that are distinct from each other. These cannot be placed directly with sequences from any cultured taxa for which sequence information is available.
对取自高地泥炭沼的泥炭和土壤岩芯中的产甲烷细菌进行了评估。采样区域主要覆盖着高位沼泽泥炭，还有小片红棕色石灰岩和泥炭潜育土。采集了每种土壤类型30厘米深的岩芯，并从5厘米厚的横向切片中提取DNA。纯化后的DNA用引物IAf和1100Ar进行PCR扩增，这两种引物特异性扩增1.1 kb的古菌16S rRNA基因，还用引物ME1和ME2进行扩增，这两种引物设计用于扩增甲基辅酶M还原酶（MCR）α亚基基因的0.75 kb区域。仅从高位沼泽泥炭岩芯最深的两个切片中提取的DNA获得了这两对引物的扩增产物。这与产甲烷菌群体的活性和生存需要厌氧环境的观点一致。两次扩增的PCR产物都进行了克隆，并用MCR或古菌16S rRNA PCR产物内部的特异性寡核苷酸探针筛选得到的转化体。从两种类型的探针阳性克隆中提取质粒DNA并对插入片段进行测序。8个MCR克隆的DNA序列相同，17个16S rRNA克隆中的16个序列也相同。一个克隆在序列的特定区域与其余克隆表现出明显差异。通过比较这两个不同的16S rRNA序列，合成了一种寡核苷酸，它与前16个克隆的序列区域100%同源，但与变体有6个错配。该探针用于筛选PCR克隆的原始群体，所有探针阴性的克隆都检查是否存在插入片段，然后对其进行测序。通过使用这种策略，进一步鉴定和分析了新的产甲烷菌16S rRNA变体。从泥炭中回收的序列在产甲烷菌谱系的长分支末端形成了两个彼此不同的簇。这些序列不能直接与任何有序列信息的培养分类群的序列归为一类。
