Fukuba Tatsuhiro, Ogawa Mari, Fujii Teruo, Naganuma Takeshi
Institute of Industrial Science, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8508, Japan.
Mar Biotechnol (NY). 2003 Sep-Oct;5(5):458-68. doi: 10.1007/s10126-002-0091-0.
The phylogenetic diversity of dissimilatory sulfite reductase (DSR, EC 1.8.99.3) alpha-subunit genes from a deep-sea cold seep was analyzed. Bulk genomic DNA was extracted from the cold seep sediment and used for amplification by polymerase chain reaction (PCR) of DSR alpha-subunit gene. Two sizes of PCR products, 1.4 kb (expected) and 1.3 kb (unexpected), were amplified. Sixteen clones of the 1.4-kb amplicons and 16 clones of 1.3-kb amplicons, a total of 32 clones, were obtained and grouped into operational DSR units (ODUs) based on restriction fragment length polymorphism (RFLP) by digestion with HaeIII and MboI. A total of 14 ODUs, i.e., 5 ODUs from 1.4-kb amplicon clones and 9 ODUs from 1.3-kb amplicon clones, were recovered. About 400 bp of the 5' ends of all the clones was sequenced and validated the RFLP-based ODU grouping. All the 5'-end 400-bp sequences of ODUs, even from the 1.3-kb amplicons, showed the characteristic DSR amino acid sequence motifs. The ODUs from 1.4-kb amplicons were closely related to the delta-Proteobacterial lineage with the DSR genes from epsilon-Proteobacterial epibionts of the hot vent worm Alvinella pompejana. The ODUs from 1.3-kb amplicons were mostly related to the unknown but possibly archaeal lineage. The diversity of the DSR genes may indicate the diversity of sulfate reducers in the seep sediment as well as the complexity of electron donors including methane.
对来自深海冷泉的异化亚硫酸盐还原酶（DSR，EC 1.8.99.3）α亚基基因的系统发育多样性进行了分析。从冷泉沉积物中提取了大量基因组DNA，并用于通过聚合酶链反应（PCR）扩增DSR α亚基基因。扩增出了两种大小的PCR产物，1.4 kb（预期）和1.3 kb（意外）。获得了16个1.4 kb扩增子克隆和16个1.3 kb扩增子克隆，共32个克隆，并通过用HaeIII和MboI消化基于限制性片段长度多态性（RFLP）将其分组为操作DSR单元（ODU）。总共回收了14个ODU，即来自1.4 kb扩增子克隆的5个ODU和来自1.3 kb扩增子克隆的9个ODU。对所有克隆的5'端约400 bp进行了测序，并验证了基于RFLP的ODU分组。所有ODU的5'端400 bp序列，即使是来自1.3 kb扩增子的序列，都显示出特征性的DSR氨基酸序列基序。来自1.4 kb扩增子的ODU与δ-变形菌谱系密切相关，其DSR基因来自热液口蠕虫阿尔文拟盲蟹（Alvinella pompejana）的ε-变形菌外共生体。来自1.3 kb扩增子的ODU大多与未知但可能是古菌的谱系相关。DSR基因的多样性可能表明冷泉沉积物中硫酸盐还原菌的多样性以及包括甲烷在内的电子供体的复杂性。
