NONCULTURE ANALYSES OF MARINE MICROBIAL COMMUNITY IN THE SEDIMENTS USING 16S rRNA GENE

为了避免传统培养技术在自然环境中评估微生物群落的局限性，我们成功地开发了利用16S rRNA基因对沉积物中海洋微生物群落进行非培养分析的方法。沉积物取样地点分别为SA和SB的相模湾2个采样站和TK的东京湾1个采样站。提取样品DNA后，对样品进行16S rDNA的PCR扩增和克隆。然后用3种四碱基特异性限制性内切酶（HhaI、RsaI和HaeIII）酶切后检测RFLP模式。根据ARDRA的树突图选择了37个无性系进行测序。测序后的克隆可分为5个主要的细菌谱系：伽马光细菌、德尔塔光细菌和ε光细菌。革兰氏阳性菌和疣菌。在SA，发现了Verrucomicrobia和变形菌门的3个亚纲。大多数克隆序列属于γ变形菌门。另一方面，高GC革兰氏阳性菌和变形菌门的γ亚纲在SB和TK中普遍存在。从SB和TK收集的高gc革兰氏阳性菌的所有克隆都属于同一簇，并被认为是未知放线菌群的成员。

To avoid the limitation of traditional culturing techniques for assessing the microbial communities in natural environments, we have successfully developed nonculture analyses of marine microbial communities in the sediments using 16S rRNA gene. Sediment samples were taken from two sampling stations in Sagami Bay, SA and SB, and one station in Tokyo Bay, TK.After DNA extraction from the samples, PCR amplification and cloning of 16S rDNA for the samples was conducted. Then the RFLP patterns were examined after digested with three four-base-specific restriction enzymes (HhaI, RsaI and HaeIII). Thirty-seven clones were selected and sequenced according to dendrograms derived from ARDRA.Sequenced clones fell into five major lineages of the domain Bacteria : the gamma, delta and epsilon Photobacteria. Gram-positive bacteria and the division Verrucomicrobia. At SA, the Verrucomicrobia and the three subclass of the Proteobacteria were found. Most clone sequences belonged to the gamma Proteobacteria. On the other hand, the high GC Gram-positive bacteria and the gamma subslass of the Proteobacteria were common at SB and TK.All clones belonging to the high-GC Gram-positive bacteria collected from SB and TK fell into the same cluster and are regarded as members of an unknown actinomycetes group.