Introduction

The phylum 'Verrucomicrobia' (Hedlund et al., 1997) is one of the primary lineages within the domain Bacteria. A lot of molecular phylogenetic approaches and culture-independent studies based on 16S rRNA gene sequences revealed that the members of this phylogenetic group have been detected in a very wide range of quite different habitats within the global ecosystem. At present, these lineages have been informally classified into five subdivisions numbered 1 to 5 and have also been classified into six monophyletic subdivisions numbered 1 to 6 of which three are recognized in the second edition of Bergey's Manual of Systematic Bacteriology as the families Verrucomicrobiaceae (subdivision 1), Opitutaceae (subdivision 4) and 'Xiphinematobacteriaceae' (subdivision 2). Since the six informal monophyletic subdivisions of this phylogenetic group were first proposed, the names of only a few species belonging to subdivisions 1 and 4 have been validly published. The class Opitutae, comprising two orders: Puniceicoccales containing the family Puniceicoccaceae and Opitutales containing the family Opitutaceae, was formally proposed recently for the classification of subdivision 4. However, in spite of wide ecological distribution of representatives of the phylum 'Verrucomicrobia' in nature, owing to relatively few pure cultivated and characterized species, the classification of this phylum is still ambiguous and informal. For this reason, for formal classification of the phylum 'Verrucomicrobia', it is recommended that many verrucomicrobia that thrives in a wide range of terrestrial, aquatic and marine habitats should be isolated and taxonomically investigated. In the present study, we attempted to elucidate the phylogenetic relationships of strains isolated from various marine environments using a polyphasic taxonomic approach including 16S rRNA and gyrB gene sequence analysis, together with molecular, physiological, biochemical and chemotaxonomic analyses to characterize the novel isolates.

Phylogenetic studies and analysis of the cell wall components on the strains of the class Opitutae (Subdivision 4)

Phylogenetic studies were performed using polyphasic taxonomic approaches on eleven chemoheterotrophic bacteria which were isolated from various marine environments. 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 that these isolates were clustered within the family Puniceicoccaceae of the class Opitutae (subdivision 4) (Fig.). Eleven novel strains represented three independent monophyletic lineages that were distinct from species with validly published genera with high bootstrap confidence values. Furthermore, molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (API 20E, API 50CH and API ZYM tests) and biochemical and chemotaxonomic analyses (determination of respiratory quinone system and cellular fatty acid content) were investigated on the novel isolates. In addition, increasing concentrations of the β-lactam antibiotic susceptibility tests and amino acid analysis of the cell wall hydrolysates indicated the absence of muramic acid and meso-diaminopimelic acid and the presence of relatively low molar ratios of alanine and glutamate, which suggested that these strains lack an ordinary Gram negative type of peptidoglycan in the cell wall. Based on the results of phylogenetic analysis and its polyphasic taxonomic evidences, the eleven isolates were considered to represent three novel genera and eight new species belonging to the family Puniceicoccaceae within the class Opitutae (subdivision 4), for which the names Coraliomargarita akajimensis gen. nov., sp. nov. (strain 04OKA010-24T; Yoon et al., 2007a), Pelagicoccus mobilis gen. nov., sp. nov. (strain 02PA-Ca-133T; Yoon et al., 2007b), Pelagicoccus albus sp. nov. (strain YM14-201T; Yoon et al., 2007b), Pelagicoccus litoralis sp. nov. (strains H-MN57T, H-MN48 and MN1-156; Yoon et al., 2007b), Pelagicoccus croceus sp. nov. (strain N5FB36-5T; Yoon et al., 2007d), Cerasicoccus arenae gen. nov., sp. nov. (strain YM26-026T; Yoon et al., 2007c), Cerasicoccus maritimus sp. nov. (strain YM31-114T) and Cerasicoccus frondicus sp. nov. (strains YM31-066T and YM31-067) are proposed.

Phylogenetic studies on the strains of the family Verrucomicrobiaceae and reclassification of the class Verrucomicrobiae, the order Verrucomicrobiales and the family Verrucomicrobiaceae (Subdivision 1)

Nineteen chemoheterotrophic bacterial strains were isolated from various marine environments and organisms, and were subjected to phylogenetic investigations using polyphasic taxonomic methods. Phylogenetic analyses based on the neighbour-joining (NJ) and maximum-parsimony (MP) generated comparisons of the 16S rRNA gene sequences indicated that these isolates were affiliated to the family Verrucomicrobiaceae (subdivision 1) within the phylum 'Verrucomicrobia' (Fig.). Among them, three novel strains had sequences that were related to the genus Rubritalea. Sixteen isolates studied form four different monophyletic clades that were distinct from species with validly published genera with high bootstrap confidence values. Furthermore, molecular (DNA-DNA hybridization test and determination of the DNA G+C content), physiological (API 20NE, API ZYM and Biolog tests) and biochemical and chemotaxonomic analyses (determination of respiratory quinone system and cellular fatty acid content) were investigated on the novel isolates. Some of the novel strains produced carotenoid pigments and squalene. Additionally, with these isolates, we proposed emended descriptions of the class Verrucomicrobiae, the order Verrucomicrobiales and the family Verrucomicrobiaceae for formal classification of the phylum 'Verrucomicrobia'. Analysis of the nearly complete 16S rRNA gene sequences from these strains indicated that the Rubritalea strains formed a robust clade having only limited resolution for this very tight group of species. However, the interrelationships deduced from the gyrB gene-based phylogeny showed better resolution than those based on their 16S rRNA gene sequences for the differentiation of strains at the species level. The genomic DNA-DNA relatedness values, biochemical and chemotaxonomic data (proportions of cellular fatty acids and menaquinones) also supported the results. On the basis of polyphasic evidences and phylogenetic characteristics, it was concluded that these strains should be classified as four novel genera and fourteen new species of the family Verrucomicrobiaceae within the class Verrucomicrobiae (subdivision 1), for which the names Rubritalea spongiae sp. nov. (strain YM21-132T; Yoon et al., 2007e), Rubritalea tangerina sp. nov. (strain YM27-005T; Yoon et al., 2007e), Rubritalea sabuli sp. nov. (strain YM29-052T; Yoon et al., 2007f), Persicirhabdus sediminis gen. nov., sp. nov. (strains YM20-087T and YM21-151; Yoon et al., 2007g), Roseibacillus ishigakijimensis gen. nov., sp. nov. (strain MN1-741T; Yoon et al., 2007g), Roseibacillus ponti sp. nov. (strain YM27-120T; Yoon et al., 2007g), Roseibacillus persicicus sp. nov. (strains YM26-010T, YM24-184 and YM20-122; Yoon et al., 2007g), Luteolibacter pohnpeiensis gen. nov., sp. nov. (strain A4T-83T; Yoon et al., 2007g), Luteolibacter algae sp. nov. (strains A5J-41-2T and A5J-40; Yoon et al., 2007g), Haliferula harenosa gen. nov., sp. nov. (strain YM23-227T), Haliferula pudorinus sp. nov. (strain 06SJR1-1T), Haliferula phycoensis sp. nov. (strain AK18-024T), Haliferula helveola sp. nov. (strain 05IJR53-1T) and Haliferula sargassicola sp. nov. (strains MN1-1037T and MN1-1047) are proposed.

Conclusion

We attempted to elucidate the phylogenetic relationships of the thirty novel isolates using a polyphasic taxonomic approaches including 16S rRNA and gyrB gene sequence analysis, together with molecular, physiological, biochemical and chemotaxonomic analyses to characterize the novel strains. Based on these data, it is proposed that these isolates represent seven novel genera and twenty-two new species within the phylum 'Verrucomicrobia'.

培養不能あるいは難培養微生物(VBNC)として知られているVerrucomicrobia門の細菌群は、自然界の多ぐの環境に分布しているが分離・培養などが非常に困難であるため、今までこれらの菌の分類に関する研究報告は殆んどなされていない状況である。現在Verrucomicrobia門の細菌群は主に培養に依存しないクローン解析の手法に基づいてSubdivision1-5に分けられている6そのうち3つのSubdivisionについてはBergey's Manual of Systematic Bacteriology Vo1.2に掲載されている。すなわちClass Verrucomicrobiae (subdivision 1),Class Opitutae (subdivision4)およびClass'Spartobacteriae'(subdivision2)である。最近、海洋バイオテクノロジー研究所・笠井宏朗は海中環境を模倣したin situ cultureの手法を用いて、海水からVerrucomicrobia門の細菌群を分離・培養することに成功した。本研究は海洋バイオテクノロジー研究所より上述のVerrucomicrobia門細菌株の提供を受け、それらの菌株の系統分類学的位置づけを明らかにし、これによりVerrucomicrobia門細菌の分類体系の確立をめざしたものである。

第1章では研究の背景と目的について述べた。

第2章ではVerrucomicrobia門、Opitutae綱に含まれる新属Coraliomargarita,Pelagicoccus,Cerasicoccusの提唱、およびOpitutae綱に含まれる菌種の細胞壁組成について述べた。

海洋から分離された11株について16SrRNA遺伝子に基づく系統解析、化学分類、生理生化学的性状、形態学的性状、DNA4-DNA相同性試験、などの多相分類の手法で解析を行った結果、これらの分離株はすべてOpitutae綱のPuniceicoccaceae科に含まれる新属、新種と判断されたので、それぞれCoraliomargarita akajimensis gen. nov.,sp,nov.(基準株04OKA010-24T),Pelagicoccus mobilis gen. nov.,sp nov.(基準株02PA-Ca-133T),Pelagicoccus albus sp. nov.(基準株YM14-201T),Pelagicoccus litoralis sp. nov., sp. nov(基準株H-MN57T),Pelagicoccus croceus sp. nov.(基準株N5FB36-5T),Cerasicoccus arenae gen.nov.,sp.nov.(基準株 YM26-026T),Cerasicoccus maritimus sp,nov.(基準株 YM31-114T),Cerasicoccus frondicus sp.nov.(基準株YM31-066T)とすることを提唱した。また Opitutae 綱の全菌株について細胞壁組成を調べた結果、ムラミン酸やメソ-ジァミノピメリン酸が検出されないこと、ペプチドグリカン合成阻害剤であるβ-lactam系抗生物質に高度の耐性を示すことが判明した。このことは、ほとんどの真正細菌が有する細胞壁の構成成分であるペプチドグリカンをOpitutae綱の細菌は欠如しているものと考えられた。このように、Verrucomicrobia門の Opitutae 綱の細菌はの細菌群は、通常のグラム陰性細菌よりもさらに原始的な細胞壁を有している可能性が強く示唆され、これは Plactomycetes 門に次ぐ進化学的に興味深い発見である。

第3章ではClass Verrucomicrobia の新属 Persicirhabdus, Roseibacillus, Luteolibacter, Haliferula および Rubritalea 属に見出された3新種について述べた。さらに phylum (門) Verrucomicrobia の正式提唱、および class (綱) Verrucomicrobiae, order (目)Verrucomicrobiales, family (科) Verrucomicrobiaceaeの各分類群の修正を行った。

海洋環境から分離された19株について16SrRNA遺伝子に基づく系統解析、化学分類、生理生化学的性状、形態学的性状、DNA-DNA相同性試験、などの多相分類の手法で解析を行った結果、これらの分離株はすべて'Verrucomicrobia'綱のVerrucomicrobiaceae 科に含まれる新属、新種と判断されたので、それぞれRubritalea spongiae sp.nov.(基準株YM21-132T),Rubritalea tangerina sp.nov. (基準株YM27-005T), Rubritalea sabuli sp.nov.(基準株YM29-052T),Persicirhabdus sediminis gen. nov.,sp. nov.(基準株YM20-087T), Roseibacillus ishigakijimensis gen.nov., sp. nov.(基準株 MN1-741T), Roseibacillus ponti sp. nov.(基準株 YM27-120T),Roseibacillus persicicus sp. nov.(基準株 YM26-OlOT),Luteolibacter pohnpeiensis gen. nov., sp. nov.(基準株A4T-83T),Luteolibacter algae sp. nov.(基準株A5J-41-2T),Haliferula harenosa gen. nov.,sp. nov. (基準株YM23-227T),Haliferula helveola sp. nov(基準株06SJR1-1T),Haliferula phycoensis sp. nov.(基準株AK18-024T),Haliferula helveola sp nov(基準株05IJR53-1T)およびHaliferula sargassicola sp.nov.(基準株MN1-1037T)をそれぞれ提唱した。このように、Verrucomicrobiaceae 科に4新属14新種を追加することとなった。

以上のように Verrucomicrobiaceae 科、Verrucomicrobiales 目、Verrucomicrobiae 綱に含まれる新属新種を提唱したので、それぞれの科、目、綱の分類学的定義の変更を提唱した。さらに、Verrucomicrobia 門について正式提唱を行った。

第4章は結論である。

以上、本論文は細菌分類の1大系統群であるVerrucomicrobia 門についてその分類体系を明らかにしたもので、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。