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

为了克服传统培养技术在自然环境微生物群落分析中的局限性，我们成功地利用16SrRNA基因对海洋沉积物中的微生物群落进行了非培养分析。从相模湾的两个采样站（SA和SB）和东京湾的一个采样站（TK）采集沉积物样品，提取样品DNA后，进行16SrDNA的PCR扩增和克隆。用HhaI、RsaI和HaeIII三种四碱基特异性限制性内切酶酶切后，对RFLP图谱进行分析。根据ARCHRONIC的树状图，选择37个克隆并测序，测序的克隆落入5个主要的细菌谱系：γ，δ和γ发光细菌。革兰氏阳性菌和疣微菌科。在SA中，发现了疣微菌门和变形菌门的三个亚门，绝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亚类在SB和TK中是常见的。从SB和TK收集的属于高GC革兰氏阳性菌的所有克隆都落入同一个簇中，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.