The oceans occupy 71% of earth's surface, with a volume of 1.46×109 Km3 and average depth of 4,000 m, and maximal depth of approximately 11,000 m.. The main problem in studying species distribution of on marine bacteria lies in the methods used to obtain viable cultures. Whether the isolates obtained by current culture methods represent indigenous population is unknown. Determining the representative physiological studies that can be performed on the vast numbers of marine bacteria that have yet to be cultured is still a matter of guesswork (Schute et al.,1993). Marine sponges are the most ancient multicellular animals, dating back to the Precambrian period, more than 635 million years ago. Their natural product diversity is among the highest found in nature. There is growing evidence that bacterial symbionts play a crucial role as producers of sponge-derived metabolites. In many sponge species, termed "high microbial abundance" (HMA) sponges, up to half the biomass can consist of microbial symbionts, whereas "low microbial abundance" (LMA) species, which live in the same habitats, contain much fewer symbionts. Often, distantly related HMA sponges from different oceans share remarkably similar microbial communities.

In this study we attempted to elucidate the taxonomic position of nine novel marine bacterial strains that were isolated from marine seawater and marine sponges within the phylum Proteobacteria and Bacteroidetes by using polyphasic taxonomic approach.

The phylogenetic trees based on the neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) generated comparisons of the 16S rRNA gene sequences revealed individual taxonomic positions. Also molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (API 20E, API 20NE, API 50CH and API ZYM tests) and biochemical and chemotaxonomic analyses (determination of respiratory quinine system and cellular fatty acid content) were investigated on the novel isolates.

As a results, six of them were determined to be four new species of three novel genera (Oceanicoccus, Psychrosphaera, Halicoccus) in the Gammaproteobacteria. Individualy, it was concluded that strains SA4-31, SA4-46 and SA4-48T should be classified as representing a new genus and species of the family Pseudoalteromonadaceae, for which the name Psychrosphaera saromensis gen. nov., sp. nov. is proposed. Strain PZ-5T represents a novel genus and species, for which the name Oceanicoccus sagamiensis gen. nov., sp. nov., is proposed. Two strains S1-36 T and S1- 72T present a novel genus and two species as Halicoccus marinus and H. pacifica were proposed. The genus Oceanicoccus and the genus Halicoccus formed a cluster with genera Spongiibacter, Melitea, Dasania, Haliea and Congregibacter at a family level, for which the name Dasaniaceae the Class Gammaproteobacteria, is proposed (Fig. 1). Two strains SG-29T and S1-66T were determined to be two species of two novel genera (Rubricoccus, Aureimonas) belonging to the phylum Bacteroidetes. Also phylogenetic position based on 16S rRNA gene sequence, molecular, physiological and biochemical and chemotaxonomic analyses and cellular fatty acid content) were investigated. Strain SG-29T was classified as a novel genus and species, for which the name Rubricoccus marinus gen. nov., sp. nov., within family 'Rhodothermaceae', is proposed. Strain S1-66T represents a species of novel genus as Aureimonas marinu was proposed.

The other hands, about 300 colonies were isolated from about 20 species of marine sponge were investigated to find novel bacterial strains using medium P and SN. On the medium P screen results (107 colonies), 27 genera in the 5 classes were isolated from marine sponge. On the medium SN screen results (132 colonies), 29 genera in the 5 classes were obtained. Interestingly we didn't find any strain belong to the phylum Bacteroidetes and the other class that previously reported using culture independent methods. But we obtained some 6 candidates showed≦96% partial 16s rRNA gene sequences sequence similarity were re-sequenced of full 16S rRNA gene sequences and compared to data base information (Genbank) also respectively phylogenentic tree of six candidates were constructed. Finally strain MS-31T isolated from marine sponge Hymenicacidon flavia was classified as Sphingomonas Jejuensis sp. nov., in the Alphaproteobacteria.

We attempted to elucidate the phylogenetic position of nine novel isolates using a polyphasic taxonomic approaches 16s rRNA gene sequence, physiological, biochemical and chemotaxonomic analyses to characterize the novel strains. Based on the data it is present that these isolates represent 7 species of the 5 genera in the phylum Proteobacteria and Bacteroidetes

Figure 1. Neighbour-joining(NJ) phylogenetic tree based on the 16S rRNA gene sequences showing the position of strain S1-36T among the currently known and related species of the class Gammaproteobacteria. Numbers at nodes are bootstrap percentages derived from 1000 replications (NJ/ML). Sequences of Simiduia agarivorans SA1T (EF617350) was used as an outgroup. Bar, 0.01 substitutions per nucleotide position. " * " indicates recovered nodes with <50% bootstrap values in the Maximum-likehood(ML) tree.

海洋は地球表面の71%を占め、その容積は 1.46 x 109 立法キロメートルで、平均深度 4,000 m、最深 11,000 mである。海洋環境中には未だ培養化されていない重要な系統群が多数存在していることが知られているが、海洋細菌の種の分布の研究では多くの場合培養株として得ることが困難である、ということが最も問題となっている。本研究では海綿および海水から新規系統群に属する細菌を分離・培養し、系統分類学的解析を行った結果について述べた。

研究の背景と意義について述べた緒論に続き、第1章では海綿からの海洋細菌の分離を行った。韓国済州島で採集した20種の海綿からP培地とSN培地を用いて細菌株300株を分離した。これらの株の16S rRNA 遺伝子配列を調べ、新規性を調べた。P培地で分離した107株は5つの綱の27の既知属に位置し、SN培地で分離した132株は5つの綱の29の既知属に位置した。16S rRNA遺伝子の部分塩基配列(約800塩基)の比較で96%以下を示した6株については全塩基配列を求め、データベースと比較した。その結果、海綿 Hymenicacidon flaviaから得た菌株MS-31株が新規性の高い株であり、分類学的性質を調べた結果、この菌株を新種Sphingomonas jejuensis sp. nov.とする事を提唱した。

第3章では日本近海の海水から分離されたGammaproteobacteria 綱に属する海洋細菌分離株6株(SA-4-31, SA4-46, SA4-48T, PZ-5T, S1-36 T, S1-72 T株)について調べた結果を述べた。16S rRNA 遺伝子塩基配列、細胞形態、生理・生化学的性状、化学分類学的性状に基づき、SA-4-31, SA4-46, SA4-48T株は Pseudoalteromonadaceae 科の新属新種と結論されたので、これらの菌株を

Psychrophaerae saromensis gen. nov., sp. nov.とする事を提唱した。また、PZ-5T 株も新属新種と結論されたので、本菌株をOceanicoccus sagamiensis gen. nov., sp. nov.とする事を提唱した。S1-36 T 株と S1-72 T株は新属 Halicoccusの別々の種と同定されたので、それぞれ Halicoccus marinus gen. nov., sp. nov.とHalicoccus pacifica sp. nov.とすることを提唱した。このOceanicoccus 属とHalicoccus属はSpongibacter, Melitea, Dasania, Haliea, Congregibacter 属と共に1つの新しい科にまとまることから、これをDasaniaceae fam. nov.とすることを提唱した。

第4章では日本近海太平洋の海水から分離されたBacteroidetes門に属する分離株SG-29T 株とS1-66 T株について分類学的性質を調べた。その結果、SG-29T 株は "Rhodothermaceae" 科の新属新種 Rubricoccus marinus gen. nov., sp. nov., またS1-66 T株は Flavobacteriaceae 科の新属新種 Aureimonas marina gen. nov., sp. nov.とすることを提唱した。

第5章は総括である。本研究では海洋細菌9分離株について16S rRNA 遺伝子解析、生理・生化学的解析、化学分類学的解析に基づいた多相分類の手法で分類学的位置を解析した結果、これらはProteobacteria 門および Bacteroidetes 門の5新属、7新種に同定した。

以上、本研究は新規系統群に属する海洋細菌について系統解析を行ったもので、学術上、応用上貢献するところが少なくない。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。