JP2004159599A - New microorganism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for effectively decomposing a environmental pollutant, particularly petroleum or a polycyclic aromatic hydrocarbon in the petroleum and making the substance harmless to clean the polluted environment and provide a means for simply and rapidly carrying out monitoring, evaluation, etc., of the polluted environment. <P>SOLUTION: A new microorganism belonging to the genus Sphingomonas is isolated and the environmental pollutant is decomposed and cleaned by using the microorganism. Detection and determination of bacteria having the environmental pollutant-decomposing ability are simply and rapidly carried out and monitoring and evaluation of the polluted environment is carried out by using a probe prepared from 16S rDNA/RNA of the new microorganism. <P>COPYRIGHT: (C)2004,JPO

Description

これらの菌学的性質に基づき、Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ， Ｖｏｌｕｍｅ １（１９８４） （ Ｋｒｉｅｇ， Ｎ． Ｒ．， ａｎｄ Ｈｏｌｔ， Ｊ． Ｇ．： Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ Ｖｏｌ． １． Ｗｉｌｌｉａｍｓ ＆ Ｗｉｌｋｉｎｓ， Ｍａｒｙｌａｎｄ ， １９８４） およびＢｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ， Ｎｉｎｔｈ Ｅｄｉｔｉｏｎ （１９９４） （Ｈｏｌｔ， Ｇ．， Ｋｒｉｅｇ， Ｎ．Ｒ．， Ｓｎｅａｔｈ， Ｐ．Ｈ．Ａ．， Ｓｔａｌｅｙ， Ｊ．Ｔ．， ａｎｄ Ｗｉｌｌｉａｍｓ， Ｓ．Ｔ． （ｅｄｓ．）： Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｄｅｔｅｒｍｉｎａｔｉｖｅ Ｂａｃｔｅｒｉｏｌｏｇｙ （９ｔｈ ｅｄ．）． Ｗｉｌｌｉａｍｓ ａｎｄ Ｗｉｌｋｉｎｓ， Ｍａｒｙｌａｎｄ， １９９４）
を参考にして分類・同定を行った結果、ＡＮＩ７Ａ菌株はＳｐｈｉｎｇｍｏｎａｓ属に属する細菌と同定された。
【０００８】
次ぎに、ＡＮＩ７Ａ菌株とＡＮＩ７Ｐ菌株について述べる。
Ａタイプコロニー（実施例１参照）から得られた純培養株、ＡＮＩ７Ａ菌株のコロニー形態（ＮＳＷ＋ＡＮ二層培地（表２）、２０℃、２６日培養）は、コロニー形状：円形（ｃｉｒｃｕｌａｒ）、大きさ：２．０ｍｍ、表面：平滑（ｓｍｏｏｔｈ）、隆起状態：半レンズ状（ｃｏｎｖｅｘ）、周縁：全縁（ｅｎｔｉｒｅ）、色調：ｌｉｇｈｔ ｂｒｏｗｎ（コロニー中心部の色調が縁部より濃い）であった。一方、Ｐタイプコロニーからの純培養株、ＡＮＩ７Ｐ菌株の上記培養条件下でのコロニー形態は、コロニー形状：円形（ｃｉｒｃｕｌａｒ）、大きさ：１．８ ｍｍ、表面：平滑（ｓｍｏｏｔｈ）、隆起状態：半レンズ状（ｃｏｎｖｅｘ）、周縁：全縁（ｅｎｔｉｒｅ）、色調：ｌｉｇｈｔ ｂｒｏｗｎ（コロニー色調は全体に均質で光沢がある）であった。このようにＡＮＩ７Ａ菌株とＡＮＩ７Ｐ菌株のコロニー形態は類似しているが、コロニーの色調、光沢が異なり、また培地の種類や培養期間によっては両菌株のコロニーの隆起状態が異なっていた。
【０００９】
上記のようにＡＮＩ７Ａ菌株とＡＮＩ７Ｐ 菌株のコロニー形態は若干違っていた。しかし、両菌株の１６Ｓ ｒＤＮＡの塩基配列に基づく分子系統解析を行った結果、両菌株の１６Ｓ ｒＤＮＡの塩基配列の相同性は９９．７％であつたことから、ＡＮＩ７Ａ菌株とＡＮＩ７Ｐ 菌株は同一種と推定された。ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ 菌株の１６ＳｒＤＮＡの塩基配列を配列表の配列番号１と２に示した。また、図１にＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ 菌株の分子系統樹を示した。
【００１０】
なお、表現形質に基づく分類・同定は、本発明者らが開示している「重油分解方法」（特開２００１−３７４６６公報（公開日２００１．２．１３）およびＲ．Ｍ．Ｓｍｉｂｅｒｔ ａｎｄ Ｎ．Ｒ．Ｋｒｅｉｇ： Ｐｈｅｎｏｔｙｐｉｃ Ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ． Ｍｅｔｈｏｄｓ ｆｏｒ Ｇｅｎｅｒａｌ ａｎｄ Ｍｏｌｅｃｕｌａｒ Ｂａｃｔｅｒｉｏｌｏｇｙ （Ｐ．Ｇｅｒｈａｒｄｔ， Ｒ．Ｇ．Ｅ．Ｍｕｒｒａｙ， Ｗ．Ａ．Ｗｏｏｄ ａｎｄ Ｎ．Ｒ．Ｋｒｉｅｇ），ｐ．６０７−６５４， Ａｍｅｒｉｃａｎ Ｓｏｃｉｅｔｙ ｆｏｒ Ｍｉｃｒｏｂｉｏｌｏｇｙ， Ｗａｓｈｉｎｇｔｏｎ，Ｄ．Ｃ．（１９９４）に述べている方法に準じて行った。また、１６Ｓ ｒＤＮＡの配列決定は本発明者らが開示している「新規低温細菌を検出するためのＤＮＡプローブ」（特開２０００−３３３６８０公報（公開日２０００．１２．５）に述べている方法に準じて行った。さらにＤＮＡデーターベースより入手したＳｐｈｉｎｇｏｍｏｎａ属および代表的な微生物種の塩基配列を並列させてアライメント処理を行い、比較不能なギャップを取り除いた後、ＮＪ法により分子系統解析を実施した。得られた系統樹の各分岐の確度は、１００回のブーストラップ解析により算出した（Ｍａｒｕｙａｍａ， Ａ．， Ｄ． Ｈｏｎｄａ， Ｈ． Ｙａｍａｍｏｔｏ， Ｋ． Ｋｉｔａｍｕｒａ ａｎｄ Ｔ． Ｈｉｇａｓｈｉｈａｒａ ： Ｐｈｙｌｏｇｅｎｅｔｉｃ ａｎａｌｙｓｉｓ ｏｆ ｐｓｙｃｈｒｏｐｈｉｌｉｃ ｂａｃｔｅｒｉａ ｉｓｏｌａｔｅｄ ｆｒｏｍ ｔｈｅ Ｊａｐａｎ Ｔｒｅｎｃｈ， ｉｎｃｌｕｄｉｎｇ ａ ｄｅｓｃｒｉｐｔｉｏｎ ｏｆ ｔｈｅ ｄｅｅｐ−ｓｅａ ｓｐｅｃｉｅｓ Ｐｓｙｃｈｒｏｂａｃｔｅｒ ｐａｃｉｆｉｃｅｎｓｉｓ ｓｐ． ｎｏｖ． Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｊｏｕｒｎａｌ ｏｆ Ｓｙｓｔｅｍａｔｉｃ ａｎｄ Ｅｖｏｌｕｔｉｏｎａｒｙ Ｍｉｃｒｏｂｉｏｌｏｇｙ． ５０， ８３５−８４６， ２０００）。
【００１１】
ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ 菌株の分子系統樹の位置は表現形質による分類位置と同様に、Ｓｐｈｉｎｇｏｍｏｎａｓに属することを示した。さらにＡＮＩ７Ａ菌株と最も分子系統的に近縁なＳｐｈｉｎｇｏｍｏｎａｓ ｓｕｂａｒｃｔｉａと本菌株の１６Ｓ ｒＤＮＡの相同性は９５．８％であつた。
一方、「細菌の種は系統的にほぼ７０％またはそれ以上のＤＮＡ−ＤＮＡ相同性を示す菌株である」と定義されている（国際細菌分類命名委員会特別委員会報告、Ｌ． Ｇ． Ｗａｙｎｅ， Ｄ． Ｊ． Ｂｒｅｎｎｅｒ， Ｒ． Ｒ． Ｃｏｌｗｅｌｌ， Ｐ． Ａ． Ｄ． Ｇｒｉｍｏｎｔ， Ｏ． Ｋａｎｄｌｅｒ， Ｍ． Ｉ． Ｋｒｉｃｈｅｖｓｋｙ， Ｌ． Ｈ． Ｍｏｏｒｅ， Ｗ． Ｅ． Ｃ． Ｍｏｏｒ， Ｒ． Ｇ． Ｅ． Ｍｕｒｒａｙ， Ｅ． Ｓｔａｃｋｅｂｒａｎｄｔ， Ｍ． Ｐ． Ｓｔａｒｒ ａｎｄ Ｈ． Ｇ． Ｔｒｕｐｅｒ： Ｒｅｐｏｒｔ ｏｆ ｔｈｅ ａｄ ｈｏｃ ｃｏｍｍｉｔｔｅｅ ｏｎ ｒｅｃｏｎｃｉｌｉａｔｉｏｎ ｏｆ ａｐｐｒｏａｃｈｅｓ ｔｏ ｂａｃｔｅｒｉａｌ ｓｙｓｔｅｍａｔｉｃｓ． Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｓｙｓｔｅｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ， ３７， ４６３−４６４， １９８７）が、Ｓｔａｃｋｅｒｂｒａｎｄｔらは、上記定義におけるＤＮＡ−ＤＮＡ相同性と１６ＳｒＤＮＡの相同性との関係について、ＤＮＡ−ＤＮＡ相同性と１６ＳｒＤＮＡの相同性の比較からＤＮＡ−ＤＮＡ相同性７０％以上のものと１６ＳｒＤＮＡの相同性９７％以上のものは対応するとし、１６Ｓ ｒＤＮＡの相同性９７％以上のものを同一の種とみなされると述べている（Ｓｔａｃｋｅｂｒａｎｄｔ， Ｅ． ａｎｄ Ｇｏｅｂｅｌ， Ｂ． Ｍ．： Ｔａｘｏｎｏｍｉｃ ｎｏｔｅ： ａ ｐｌａｃｅ ｆｏｒ ＤＮＡ−ＤＮＡ ｒｅａｓｓｏｃｉａｔｉｏｎ ａｎｄ １６ＳｒＲＮＡ ｓｅｑｕｅｎｃｅ ａｎａｌｙｓｉｓ ｉｎ ｔｈｅ ｐｒｅｓｅｎｔ ｓｐｅｃｉｅｓ ｄｅｆｉｎｉｔｉｏｎ ｉｎ ｂａｃｔｅｒｉｏｌｏｇｙ． Ｉｎｔ． Ｊ． Ｓｙｓｔ． Ｂａｃｔｅｒｉｏｌ．， ４４， ８４６−８４９， １９９４）。
【００１２】
以上のことから、ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ 菌株はＳｐｈｉｎｇｏｍｏｎａｓ 属の新種として、Ｓｐｈｉｎｇｏｍｏｎａｓ 属ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ菌株と命名した。
これらの菌株は独立行政法人産業技術総合研究所特許生物寄託センターに、受託番号ＦＥＲＭＰ−１９０９５（Ｓｐｈｉｎｇｏｍｏｎａｓ 属ＡＮＩ７Ａ菌株）および受託番号ＦＥＲＭＰ−１９０９６（Ｓｐｈｉｎｇｏｍｏｎａｓ 属ＡＮＩ７Ｐ菌株）として寄託されている。
また、上記の１６Ｓ ｒＤＮＡの相同性と種の関係からみれば、ＡＮＩ７Ａ菌株または ＡＮＩ７Ｐ菌株と１６ＳｒＤＮＡの相同性において９７％以上の菌株であれば、これら菌株と同一種とみなされ、本発明に包含される。
【００１３】
〔Ｓｐｈｉｎｇｍｏｎａｓ属微生物の機能〕
Ｓｐｈｉｎｇｍｏｎａｓ属はＹａｂｕｕｃｈｉらによつて１９９０年に提唱された新属で、細胞膜にスフィンゴ糖脂質を有し、キノン系はユビキノンＱ１０で、黄色のカロチノイド色素（Ｎｏｓｔｏｘａｎｔｈｉｎ）を生成すことが特徴としてあげられている（Ｙａｂｕｕｃｈｉ， Ｅ．， Ｙａｎｏ，Ｉ．， Ｏｙａｉｚｕ， Ｈ．， Ｈａｓｈｉｍｏｔｏ， Ｙ．， Ｅｚａｋｉ， Ｔ．， ａｎｄ Ｙａｍａｍｏｔｏ， Ｈ．： Ｐｒｏｐｏｓａｌｓ ｏｆ Ｓｐｈｉｎｇｏｍｏｎａｓ ｐａｕｃｉｍｏｂｉｌｉｓ ｇｅｎ． ｎｏｖ． ａｎｄ ｃｏｍｂ． ｎｏｖ．， Ｓｐｈｉｎｇｏｍｏｎａｓ ｐａｒａｐａｕｃｉｍｏｂｉｌｉｓ ｓｐ．ｎｏｖ．， Ｓｐｈｉｎｇｏｍｏｎａｓ ｙａｎｏｉｋｕｙａｅ ｓｐ． ｎｏｖ．， Ｓｐｈｉｎｇｏｍｏｎａｓ ａｄｈａｅｓｉｖａ ｓｐ． ｎｏｖ． ， Ｓｐｈｉｎｇｏｍｏｎａｓ ｃａｐｓｕｌａｔａ ｃｏｍｂ． ｎｏｖ．， ａｎｄ ｔｗｏ ｇｅｎｏｓｐｅｃｉｅｓ ｏｆ ｔｈｅ ｇｅｎｕｓ Ｓｐｈｉｎｇｏｍｏｎａｓ． Ｍｉｃｒｏｂｉｏｌ． Ｉｍｍｕｎｏｌ．， ３４， ９９−１１９， １９９０）。
最近、Ｓｐｈｉｎｇｍｏｎａｓ属細菌は、ビフェニール（ＢＰ）等環境ホルモン、クロロフェノール、ヘキサクロロシクロヘキサン等の有機塩素化合物、キシレン、ナフタレン、フェナントレン等の芳香族炭化水素、除草剤等の農薬、ポリエチレングリコール等の合成高分子化合物など非常に広範な種々の環境汚染物質に対する強力な分解能を有することが明らかにされるとともに、さらに新たな環境汚染物質分解能の発見が期待されることなどから世界的に注目されている（Ｓｐｈｉｎｇｍｏｎａｓ属細菌の最新の知見をまとめた特集号、「Ｔｈｅ ｇｅｎｕｓ Ｓｐｈｉｎｇｏｍｏｎａｓ」、Ｊｏｕｒｎａｌ ｏｆ Ｉｎｄｕｓｔｒｉａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ ＆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ２３（Ｎｏ．４／５）， ２３１−４４５， １９９９）。我が国においても、Ｓｐｈｉｎｇｍｏｎａｓ属細菌は、きわめて強力な有害環境汚染物質の分解能を有し、バイオレメディエーションの切り札として注目されている細菌群であることから、環境浄化にＳｐｈｉｎｇｍｏｎａｓ属細菌の能力を利用する研究を進展させるため、この細菌の生態（構造と機能）、分解能（酵素と遺伝子、分子育種）、使用法（汚染源への細菌導入または栄養源供給）、工学的側面（増殖制御、環境制御、複合微生物系制御）等の総合的研究（文部科学省、科学研究費補助金、特定領域研究）の提案に向けての調査研究が行われている（シンポジウム 科学研究費補助金基盤研究（Ｃ）（企画調査）「スフィンゴモナス属細菌を用いる環境浄化システム構築基盤−スフィンゴモナス属細菌とバイオレメディーエション」講演要旨集、２００１）。また、このシンポジウムを端緒に「環境微生物研究会（スフィンゴモナス研究会）」が設立された。
【００１４】
海洋環境から単離されたＳｐｈｉｎｇｏｍｏｎａｓ 属のＰＡＨ分解細菌としては、ＰＨＥ分解細菌（Ｋｏｍｕｋａｉ−Ｎａｋａｍｕｒａ， Ｓ．， Ｓｕｇｉｕｒａ， Ｋ．， Ｙａｍａｕｃｈｉ−Ｉｎｏｍａｔａ， Ｙ．， Ｔｏｋｉ， Ｈ．， Ｖｅｎｋａｔｅｓｗａｒａｎ， Ｋ． ， Ｙａｍａｍｏｔｏ， Ｓ．， Ｔａｎａｋａ， Ｈ．， ａｎｄ Ｈａｒａｙａｍａ， Ｓ．： Ｃｏｎｓｔｒｕｃｔｉｏｎ ｏｆ ｂａｃｔｅｒｉａｌ ｃｏｎｓｏｒｔｉａ ｔｈａｔ ｄｅｇｒａｄｅ Ａｒａｂｉａｎ ｌｉｇｈｔ ｃｒｕｄｅ ｏｉｌ． Ｊ． Ｆｅｒ．Ｂｉｏｅｎｇ．， ８２， ５７０−５７４ ， １９９６、 Ｂｅｒａｒｄｅｓｃｏ， Ｇ．， Ｄｙｈｒｍａｎ， Ｓ．， Ｇａｌｌａｇｈｅｒ， Ｅ． ａｎｄ Ｓｈｉａｒｉｓ， Ｍ．Ｐ．： Ｓｐａｔｉａｌ ａｎｄ ｔｅｍｐｏｒａｌ ｖａｒｉａｔｉｏｎ ｏｆ ｐｈｅｎａｎｔｈｒｅｎｅ−ｄｅｇａｒｄｉｎｇ ｂａｃｔｅｒｉａ ｉｎ ｉｎｔｅｒｔｉｄａｌ ｓｅｄｉｍｅｎｔｓ． Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ６４， ２５６０−２５６５， １９９８）や２−ｍｅｔｈｙｐｈｅｎａｔｈｒｅｎｅ分解細菌が知られている（Ｇｉｌｅｗｉｃｚ， Ｍ．， Ｎｉｍａｔｕｚａｈｒｏｈ， Ｎａｄａｌｉｇ， Ｔ．， Ｂｕｄｚｉｎｓｋｉ， Ｈ．， Ｄｏｕｍｅｎｑ， Ｐ．， Ｍｉｃｈｏｔｅｙ， Ｖ．， ａｎｄ Ｂｅｒｔｒａｎｄ， Ｊ． Ｃ．： Ｉｓｏｌａｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｍａｒｉｎｅ ｂａｃｔｅｒｉｕｍ ｃａｐａｂｌｅ ｏｆ ｕｔｉｌｉｚｉｎｇ ２−ｍｅｔｙｌｐｈｅａｎａｔｈｒｅｎｅ． Ａｐｐｌ． Ｍｉｃｒｏｂｉｏｌ． Ｂｉｏｔｅｃｈｎｏｌ．， ４８， ５２８−５３３ １９９７）。また、海洋環境から得られたＳｐｈｉｎｇｏｍｏｎａｓ 属細菌の中にはＰＡＨ以外にトルエン、キシレン等芳香族炭化水素など各種の炭化水素を分解する細菌が報告されている（Ｒ． Ｃａｚｉｃｃｈｉｏｌ， Ｆ． Ｆｅｇａｔｅｌｌａ， Ｍ． Ｏｓｔｒｏｗｓｋｉ， Ｍ． Ｅｇｕｃｈｉ， Ｊ． Ｇｏｔｔｓｃｈａｌ： Ｓｐｈｉｎｇｏｍｏｎａｄｓ ｆｒｏｍ ｍａｒｉｎｅ ｅｎｖｉｒｏｎｍｅｎｔｓ， Ｊｏｕｒｎａｌ ｏｆＩｎｄｕｓｔｒｉａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ ＆ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ， ２３， ２６８−２７２， １９９９）。さらに、Ｓｐｈｉｎｇｏｍｏｎａｓ ａｒｏｍａｔｉｃｉｂｏｒａｎｓ Ｆ１９９菌株はトルエン、ｏ， ｍ， ｐ−キシレン、ｐ−クレゾール、ナフタレン、ビフェニール（ＢＰ）、ジベンゾチオフェン、フルオレン、サリチル酸、安息香酸などを分解し、一種一菌株で多様な有害物質分解能を有している（Ｊ．Ｋ． Ｆｒｅｄｒｉｃｋｓｏｎ， Ｆ． Ｊ． Ｂｒｏｃｋｍａｎ， Ｄ． Ｊ． Ｗｏｒｋｍａｎ， Ｓ． Ｗ． Ｌｉ ａｎｄ Ｔ． Ｏ． Ｓｔｅｖｅｎｓ： Ｉｓｏｌａｔｉｏｎ ａｎｄ ｃｈａｒａｃｔｅｒｉｚａｔｉｏｎ ｏｆ ａ ｓｕｂｓｕｒｆａｃｅ ｂａｃｔｅｒｉｕｍ ｃａｐａｂｌｅ ｏｆ ｇｒｏｗｔｈ ｏｎ ｔｏｌｕｅｎｅ， ｎａｐｈｔｈａｌｅｎｅ， ａｎｄ ｏｔｈｅｒ ａｒｏｍａｔｉｃ ｃｏｍｐｏｕｎｄｓ， Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ５７， ７９６−８０３， １９９１； Ｊ．Ｋ． Ｆｒｅｄｒｉｃｋｓｏｎ， Ｄ． Ｌ． Ｂａｌｋｗｉｌｌ， Ｇ． Ｒ． Ｄｒａｋｅ， Ｍ． Ｆ． Ｒｏｍｉｎｅ， Ｄ． Ｂ． Ｒｉｎｇｅｌｂｅｒｇ ａｎｄ Ｄ． Ｃ． Ｗｈｉｔｅ：Ａｒｏｍａｔｉｃ−ｄｅｇｒａｄｉｎｇ Ｓｐｈｉｎｇｏｍｏｎａｓ ｉｓｏｌａｔｅｓ ｆｒｏｍ ｔｈｅ ｄｅｅｐ ｓｕｂｓｕｒｆａｃｅ， Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ６１， １９１７−１９２２， １９９５； Ｄ． Ｌ． Ｂａｌｋｗｉｌｌ， Ｇ． Ｒ． Ｄｒａｋｅ， Ｒ． Ｈ． Ｒｅｅｖｅｓ， Ｊ．Ｋ． Ｆｒｅｄｒｉｃｋｓｏｎ， Ｄ． Ｃ． Ｗｈｉｔｅ， Ｄ． Ｃ． Ｒｉｎｇｅｌｂｅｒｇ， Ｄ． Ｐ． Ｃｈａｎｄｌｅｒ， Ｍ． Ｆ． Ｒｏｍｉｎｅ， Ｄ． Ｗ． Ｋｅｎｎｅｄｙ ａｎｄ Ｃ． Ｍ． Ｓｐａｄｏｎｉ， Ｔａｘｏｎｏｍｉｃ ｓｔｕｄｙ ｏｆ ａｒｏｍａｔｉｃ−ｄｅｇｒａｄｉｎｇ ｂａｃｔｅｒｉａ ｆｒｏｍ ｄｅｅｐ−ｔｅｒｒｅｓｔｒｉａｌ−ｓｕｂｓｕｒｆａｃｅ ｓｅｄｉｍｅｎｔｓ ａｎｄ ｄｅｓｃｒｉｐｔｉｏｎ ｏｆ Ｓｐｈｉｎｇｏｍｏｎａｓ ａｒｏｍａｔｉｃｖｏｒａｎｓ ｓｐ． ｎｏｖ．， Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｕｂｔｅｒｒａｎｅａ ｓｐ． ｎｏｖ． ａｎｄ Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｔｙｇｉａ ｓｐ． ｎｏｖ．， Ｉｎｔ． Ｊ． Ｓｙｓｔ． Ｂａｃｔｅｒｉｏｌ．， ４７， １９１−２０１， １９９７）。
【００１５】
本発明のＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ菌株は、日本海重油流出事故で汚染された沿岸海域の海水試料から分離された微生物で、いずれもアントラセン（ＡＮ）やフェナントレン（ＰＨＥ）の分解能を有していることから、現場油濁海域の浄化に働いているものと推察された。また、ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ菌株の近縁種Ｓｐｈｉｎｇｏｍｏｎａｓｓｕｂａｒｃｔｉａはポリクロロフェノール分解細菌であることが報告されている（Ｎｏｈｙｎｅｋ， Ｌ． Ｊ．， Ｎｕｒｍｉａｈｏ−Ｌａｓｓｉｌａ， Ｅ． Ｌ．， Ｓｕｈｏｎｅｎ， Ｅ． Ｌ．， Ｂｕｓｓｅ， Ｈ． Ｊ．， Ｍｏｈａｍｍａｄｉ， Ｍ．， Ｈａｎｔｕｌａ， Ｊ．， Ｒａｉｎｅｙ， Ｆ． ａｎｄ Ｓａｌｋｉｎｏｊａ−Ｓａｌｏｎｅｎ， Ｍ．Ｓ： Ｄｅｓｃｒｉｐｔｉｏｎ ｏｆ ｃｈｌｏｒｏｐｈｅｎｏｌ−ｄｅｇｒａｄｉｎｇ Ｐｓｅｕｄｏｍｏｎａｓ ｓｐ． ｓｔｒａｉｎｓ ＫＦ１， ＫＦ３， ａｎｄ ＮＫＦ１ ａｓ ｎｅｗ ｓｐｅｃｉｅｓ ｏｆ ｔｈｅ ｇｅｎｕｓ Ｓｐｈｉｎｇｏｍｏｎａｓ， Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｕｂａｒｃｔｉｃａ ｓｐ． ｎｏｖ． Ｉｎｔ． Ｊ． Ｓｙｓｔ． Ｂａｃｔｅｒｉｏｌ．， ４６， １０４２−１０５５， １９９６）。
【００１６】
上記のことから、本発明のＳｐｈｉｎｇｏｍｏｎａｓ属に属する新種の微生物は、ＰＡＨを含む石油や有害物質の分解能を有し、これらにより汚染された環境の浄化に利用できるきわめて有用な微生物である。また、Ｓｐｈｉｎｇｏｍｏｎａｓ属細菌は上述のように非常に広範な環境汚染物質に対する強力な分解能を有することから、本発明のＳｐｈｉｎｇｏｍｏｎａｓ属の新種の微生物もＰＡＨ 分解能以外に環境ホルモン、有機塩素化合物、農薬、合成高分子化合物などの有害環境汚染物質分解能を有するものということができ、極めて有用な微生物であり、有害物質で汚染された環境浄化に利用することができる。
【００１７】
〔他の微生物〕
次ぎに、油濁環境や有害物質汚染環境の浄化に本微生物を利用する場合は、本発明の微生物を単独で使用してもよく、また本発明の微生物と例えば脂肪族炭化水素分解細菌Ａｌｃａｎｉｖｏｒａｘ属細菌や公知の炭化水素分解微生物、また有害環境汚染物質分解能を有する微生物と混合して環境汚染物質分解微生物コンソーシアとして利用することもできる。さらに、石油等有害汚染物質の分解効率を促進するため、本発明の微生物を含む分解微生物コンソーシアに、有害汚染物質の中間分解物や代謝産物を分解する非有害汚染物質分解微生物を加えた複合微生物系を構築し、利用することもできる。
【００１８】
〔培地〕
本発明の微生物や本発明の微生物を含む環境汚染物質分解微生物コンソーシアの培養に用いる培地は、これらの微生物が良好に増殖し、かつＰＡＨ分解能や環境汚染物質分解能が発現できる培地であれば、いかなる組成の培地でもよい。炭素源としては、ＰＡＨを唯一の炭素源・エネルギー源として用いることができるが、ＰＡＨを含む原油、灯油、軽油、重油等の石油製品、船舶や工場からの流出油なども利用することができる。さらに、炭水化物、ピルビン酸のような有機酸、廃糖蜜なども用いることができる。これらはＰＡＨや石油類と混合して用いると効果的な場合もある。また、疎水性のＰＡＨや石油類を微生物によって分解され易くするため、これらにＴｗｅｅｎ ８０のよう界面活性剤を添加し、さらに超音波発生機にて乳化・分散させる場合もある。ＰＡＨを微量なエタノールやアセトンに溶解して添加することもできる。窒素源としては、微生物に利用される有機・無機化合物であればよい。有機窒素源としてはペプトン、肉エキス、コンステイプリカー、脱脂大豆、カゼンインなどが、無機窒素源としてはアンモニウム塩、硝酸塩、尿素などが利用できる。無機塩類としては、各種のリン酸塩、塩化ナトリウム、マグネシウム、鉄、マンガン、カルシウム、亜鉛、モリブデンなどを添加してもよい。また、増殖因子として、ビタミン類、アミノ酸類があり、肉エキス、ペプトン、酵母エキス、コンステイプリカーなど前記栄養因子を含有する天然有機栄養物を添加してもよい。
なお、これらの微生物を効率よく大量に培養するときは、必ずしもＰＡＨ等環境汚染物質を含む培地で培養する必要はなく、Ｍａｒｉｎｅ Ｂｒｏｔｈ （Ｄｉｆｃｏ） やＮｕｔｒｉｅｎｔＢｒｏｔｈ （Ｄｉｆｃｏ） のような有機栄養培地でもよい。
【００１９】
〔培養方法〕
培養は好気的条件、例えば振とう培養法、通気撹拌培養法が好適であるが、
適宜液体静置培養を組み合わせてもよいし、また液体静置培養でもよい。培地のｐＨは５−９、好ましくは６−８であればよい。培養温度は１５−３７℃、好ましくは２０−３０℃であればよい。
【００２０】
〔本発明の微生物を用いた環境汚染物質の分解手段〕
本発明の微生物および本発明の微生物を含有する石油等環境汚染物質分解微生物コンソーシアを用いた浄化方法としては、ＰＡＨを含む石油等の環境汚染物質に汚染された海洋、湖沼、河川、廃液などに、これらの微生物の培養液、生菌体、凍結乾燥菌体を散布すればよい。この場合、有機または無機の窒素、リンなどの栄養源とこれらの微生物を混合した栄養・微生物製剤として、また本発明者らが開示したアルギン酸を用いた栄養源含有固定化担体（特開２００１−３７４６６、公開日：２００１．１２．１３）、ポリアクリルアミドゲル、ポリウレタンフォームなど公知の微生物固定化担体を用いて、これらの微生物を固定化した各種の微生物製剤を利用することができる。この場合栄養源とこれらの微生物を同時に固定化した方が好ましい。
【００２１】
〔環境汚染物質分解微生物の検出、定量、スクリーニング、同定及び環境評価方法〕
次ぎに、核酸プローブやそれを用いた石油等有害環境汚染物質分解微生物の検出・定量方法、スクリーニング方法、同定方法、およびこの検出・定量法を用いた有害汚染物質汚染のモニタリング、解析・評価方法などについて説明する。
先に述べた汚染物質分解微生物を用いた環境調和型の生物学的環境浄化技術、すなわちバイオレメディエーション技術には，一般に海等の汚染環境に欠乏している窒素（Ｎ），リン（Ｐ）などの栄養製剤を散布し，現場に生息している土着分解微生物の活性を高める方法（バイオスティミュレーション，Ｂｉｏｓｔｉｍｕｌａｔｉｏｎ）と分解微生物製剤等を散布する方法がある（バイオオーギュメンテーション，Ｂｉｏａｕｇｍｅｎｔａｔｉｏｎ）（Ｒ． Ｍ． Ａｔｌａｓ ａｎｄ Ｒ． Ｂａｒｔｈａ： Ｈｙｄｒｏｃａｒｂｏｎ Ｂｉｏｄｅｇｒａｄａｔｉｏｎ ａｎｄ Ｏｉｌ Ｓｐｉｌｌ Ｂｉｏｒｅｍｅｄｉａｔｉｏｎ． Ａｄｖａｎｃｅｓ ｉｎ Ｍｉｃｒｏｂｉａｌ Ｅｃｏｌｏｇｙ （ｅｄ． Ｋ．Ｃ． Ｍａｒｓｈａｌｌ）， ＰｌｅｎｕｍＰｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ， １２， ２８７−３３８，１９９２、Ｋ． Ｌｅｅ ｅｔ ａｌ．： Ｂｉｏａｕｇｍｅｎｔａｔｉｏｎ ａｎｄ ｂｉｏｓｔｉｍｕｌａｔｉｏｎ： ａ ｐａｒａｄｏｘ ｂｅｔｗｅｅｎ ｌａｂｏｒａｔｏｒｙ ａｎｄ ｆｉｅｌｄ ｒｅｓｕｌｔｓ． Ｉｎ Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ １９９７ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｏｉｌ Ｓｐｉｌｌ Ｃｏｎｆｅｒｅｎｃｅ， ｐ ６９７−７０５， ＡｍｅｒｉｃａｎＰｅｔｅｒｏｌｅｕｍ Ｉｎｓｔｉｔｕｔｅ，Ｗａｓｈｉｎｇｔｏｎ．Ｄ．Ｃ．， １９９７ ）。この環境浄化技術を確立するためには、栄養源や微生物を散布した場合、現場環境における石油等有害物質分解微生物を定性的、定量的に把握し、汚染浄化に関与する微生物群集の挙動や機能を解明する必要がある。これまでに、石油で汚染された沿岸海域や航路海域に石油分解微生物が最も多く分布していることが報告されている（Ａｔｌａｓ， Ｒ． Ｍ．： Ｍｉｃｒｏｂｉａｌ ｄｅｇｒａｄａｔｉｏｎ ｏｆ ｐｅｔｒｏｌｅｕｍ ｈｙｄｒｏｃａｒｂｏｎｓ： ａｎ ｅｎｖｉｒｏｎｍｅｎｔａｌ ｐｅｒｓｐｅｃｔｉｖｅ． Ｍｉｃｒｏｂｉａｌ． Ｒｅｖ．， ４５， １８０−２０９， １９８１）。
例えば、一般に非汚染海域に分布する炭化水素分解微生物の割合は全微生物数の１％以下であるが、油濁海域ではその比率がしばしば１０％以上なるといわれている（Ｒ． Ｍ． Ａｔｌａｓ： Ｐｅｔｒｏｌｅｕｍ ｂｉｏｄｅｇｒａｄａｔｉｏｎ ａｎｄ ｏｉｌ ｓｐｉｌｌ ｂｉｏｒｅｍｅｄｉａｔｉｏｎ， Ｍａｒｉｎｅ Ｐｏｌｌｕｔｉｏｎ Ｂｕｌｌｔｅｔｉｎ， ３１， １７８−１８２， １９９５、Ｒ． Ｍ． Ａｔｌａｓ ａｎｄ Ｒ． Ｂａｒｔｈａ： Ｈｙｄｒｏｃａｒｂｏｎ Ｂｉｏｄｅｇｒａｄａｔｉｏｎ ａｎｄ Ｏｉｌ Ｓｐｉｌｌ Ｂｉｏｒｅｍｅｄｉａｔｉｏｎ． Ｉｎ： Ａｄｖａｎｃｅｓ ｉｎ Ｍｉｃｒｏｂｉｏｌｂｉａｌ Ｅｃｏｌｏｇｙ， Ｅｄ． Ｋ． Ｃ． Ｍａｒｓｈａｌｌ， ＰｌｅｎｕｍＰｒｅｓｓ， Ｎｅｗ Ｙｏｒｋ， １２， ２８７−３３８， １９９２）。 一般に微生物群集全体に占める分解微生物の割合は石油等有害物質汚染の程度を反映し、その指標になるといわれている（Ａｔｌａｓ， Ｒ． Ｍ．： Ｍｉｃｒｏｂｉａｌ ｄｅｇｒａｄａｔｉｏｎ ｏｆ ｐｅｔｒｏｌｅｕｍ ｈｙｄｒｏｃａｒｂｏｎｓ： ａｎ ｅｎｖｉｒｏｎｍｅｎｔａｌ ｐｅｒｓｐｅｃｔｉｖｅ． Ｍｉｃｒｏｂｉａｌ． Ｒｅｖ．， ４５， １８０−２０９， １９８１）。
【００２２】
また、木材処理施設から排出されるクレオソート（約８５％の多環芳香族炭化水素 ［ＰＡＨｓ］ を含有する）で汚染された港湾の堆積物中のＰＡＨ分解菌数が調査されている（Ａ． Ｄ． Ｇｅｉｓｅｌｂｒｅｃｈｔ ｅｔ ａｌ： Ｅｎｕｍｅｒａｔｉｏｎ ａｎｄ Ｐｈｙｌｏｇｅｎｅｔｉｃ Ａｎａｌｙｓｉｓ ｏｆ Ｐｏｌｙｃｙｃｌｉｃ Ａｒｏｍａｔｉｃ Ｈｙｄｒｏｃａｒｂｏｎ−Ｄｅｇｒａｄｉｎｇ Ｍａｉｒｎｅ Ｂａｃｔｅｒｉａ ｆｒｏｍ Ｐｕｇｅｔ Ｓｏｕｎｄ Ｓｅｄｉｍｅｎｔ， Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ６２， ３３４４−３３４９， １９９６）。それによると、ＭＰＮ培養計数法により計数された、ＰＡＨ分解菌数は、汚染サイトで１０４〜１０７ ＭＰＮ／ｇ （乾燥重量）、非汚染サイトで１０３〜１０４ ＭＰＮ／ｇ （乾燥重量）であった。一方、堆積物中の全菌数は１０９ ／ｇ （乾燥重量）程度で、汚染サイトと非汚染サイト間でほとんど差がみられなかったという。すなわち、クレオソート汚染サイトでは、ＰＡＨ分解菌が非汚染サイトの１０−１０００倍多いというだけでなく、全微生物群集に占める割合も１％程度にまで増大していた。このように、環境が汚染されたことを反映して微生物群集に占める分解菌群の割合が増大していることが見出されており、分解菌の比率は汚染環境のよい指標になると考えられる。
【００２３】
しかし、従来の石油やＰＡＨ等の有害物質分解微生物の計数法は、上記報告のように平板培養法やＭＰＮ（最確数）法を用いた培養法によるものである。この培養法は、いずれも多大な労力と時間を要する。また、寒天平板培地を用いる計数法では， 炭化水素無添加の対照培地において，試料中の細菌が寒天中の不純物を利用して増殖し， コロニーを形成する。微量の増殖因子として酵母エキス（０．０１％）を添加した寒天平板培地を用いた計数では、炭化水素培地と炭化水素無添加培地の両培地において微小なコロニーが生成し，その数やコロニーの大きさで炭化水素分解細菌と非分解細菌の違いを明らかにすることができなかったといわれている（Ｔ． Ｈｉｇａｓｈｉｈａｒａ， Ａ． Ｓａｔｏ ａｎｄ Ｕ． Ｓｈｉｍｉｄｕ： Ａｎ ＭＰＮ ｍｅｔｈｏｄ ｆｏｒ ｔｈｅ ｅｎｕｍｅｒａｔｉｏｎ ｏｆ ｍａｒｉｎｅ ｈｙｄｒｏｃａｒｂｏｎ ｄｅｇｒａｄｉｎｇ ｂａｃｔｅｒｉａ， Ｂｕｌｌ． Ｊａｐａｎ． Ｓｏｃ． Ｓｃｉ． Ｆｉｓｈ．， ４４， １１２７−１１３４， １９７８）。以上のことから，炭化水素無添加の対照培地においても，寒天中の微量な有機物を利用してコロニーを形成する細菌が存在するため，寒天平板培地で特定の炭化水素分解細菌を選択的かつ正確に計数することは困難である。
加えて特定分解微生物を検出するには、寒天平板法にて目的微生物を分離し、分類・同定（属、種レベル）を行う必要があり、さらに長時間を要し多数の分離微生物を分類・同定することは困難である。さらに、自然界に生息する微生物の内、これら従来の分離・培養法で検出できる微生物の数は極めて少ない。すなわち、蛍光ＤＮＡ染色剤で染色し顕微鏡下で計数する直接顕微鏡計数法（たとえば、Ｊ． Ｅ． Ｈｏｂｂｉｅ， Ｒ．Ｊ． Ｄａｌｅｙ，ａｎｄ Ｓ． Ｊａｓｐｅｒ： Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ． ３３：１２２５−１２２８， １９７７やＫ． Ｇ． Ｐｏｒｔｅｒ ａｎｄ Ｙ． Ｓ． Ｆｅｉｇ： Ｌｉｍｎｏｌ． Ｏｃｅａｎｏｇｒ． ２５： ９４３−９４８，１９８０）で得られた全菌数と比較して、分離・培養可能な微生物の割合は１％以下でしかないと考えられる（Ｒ． Ｉ． Ａｍａｎｎ， Ｗ． Ｌｕｄｗｉｇ ａｎｄ Ｋ−Ｈ． Ｓｃｈｌｅｉｆｅｒ： Ｐｈｙｌｏｇｅｎｅｔｉｃ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ａｎｄ Ｉｎ Ｓｉｔｕ Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｉｎｄｉｖｉｄｕａｌ Ｍｉｃｒｏｂｉａｌ Ｃｅｌｌ ｗｉｔｈｏｕｔ Ｃｕｌｔｉｖａｔｉｏｎ， Ｍｉｃｒｏｂｉａｌ． Ｒｅｖ．， ５９， １４３−１６９， １９９５）。したがって、従来の培養法では現場環境中に生息する微生物の１％程度を対象とした特定分解微生物や微生物相の調査しかできず、環境微生物群集中の分解微生物が十分に反映されていないという大きな欠点があった。
【００２４】
近年 、分子生物学的手法に基づく分子微生物生態学が発展し 、従来のように分離・培養法に依存せず 、分子・細胞レベルで 、環境中の微生物群集構造や多様性の解析が可能になってきている（Ｉ． Ｍ． Ｈｅａｄ， Ｊ．Ｒ． Ｓａｕｎｄｅｒｓ ａｎｄ Ｒ． Ｗ． Ｐｉｃｋｕｐ： Ｍｉｃｒｏｂｉａｌ ｅｖｏｌｕｔｉｏｎ， ｄｉｖｅｒｓｉｔｙ，ａｎｄ ｅｃｏｌｏｇｙ： ａ ｄｅｃａｄｅ ｏｆ ｒｉｂｏｓｏｍａｌ ＲＮＡ ａｎａｌｙｓｉｓ ｏｆ ｕｎｃｕｌｔｉｖａｔｅｄ ｍｉｃｒｏｏｒｇａｎｉｓｍｓ， Ｍｉｃｒｏｂ． Ｅｃｏｌ．， ３５， １−２１， １９９８． 渡辺一哉， 二又裕之：環境中で働く微生物， 化学と生物， ３８，２３０−２３６， ２０００、丸山明彦：海洋微生物の分子・細胞レベルでの解析，海洋微生物，月刊海洋，号外Ｎｏ．２３， １６２−１７０， ２０００、
浦川秀敏，大和田紘一：核酸を用いた培養に依存しない微生物群集解析手法，海洋微生物，月刊海洋，号外Ｎｏ．２３， １７６−１８２， ２０００）。この分子生物学的手法による微生物群集解析手法は 、石油等の有害物質汚染環境やバイオレメディエーション技術による環境修復過程の分解微生物群の挙動や微生物群集構造の変遷を把握するために必要不可欠であり、最近それらの技術が開発されつつある。
最近、全微生物や環境汚染物質分解微生物等の特定微生物を対象として、それらに特異的なＤＮＡプローブを用い、従来の分離・培養法に依存しない分子遺伝学的な検出および定量化が試みられている。 例えば、分離・培養法によらず細胞レベルで分解微生物等の特定微生物を検出する場合には、蛍光ｉｎ ｓｉｔｕハイブリダイゼーション法（ＦＩＳＨ法：ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ）が用いられている（Ｒ． Ｉ． Ａｍａｎｎ， Ｗ． Ｌｕｄｗｉｇ ａｎｄ Ｋ−Ｈ． Ｓｃｈｌｅｉｆｅｒ： Ｐｈｙｌｏｇｅｎｅｔｉｃ Ｉｄｅｎｔｉｆｉｃａｔｉｏｎ ａｎｄ Ｉｎ Ｓｉｔｕ Ｄｅｔｅｃｔｉｏｎ ｏｆ Ｉｎｄｉｖｉｄｕａｌ Ｍｉｃｒｏｂｉａｌ Ｃｅｌｌ ｗｉｔｈｏｕｔ Ｃｕｌｔｉｖａｔｉｏｎ， Ｍｉｃｒｏｂｉａｌ． Ｒｅｖ．， ５９， １４３−１６９， １９９５）。すなわち、ＤＮＡプローブを用いて、ＦＩＳＨを行うことにより 、微生物群集中の特定の分解微生物細胞のみを特異的に検出・計数することができる。直接顕微鏡計数法で求めた全菌数と比較することにより 、全微生物群集中の 特定微生物の定量的比率を算出することができる。さらに、水環境試料を対象とし、細胞レベルで全群集に占める分解微生物等の特定微生物の割合を解析する場合には、ＦＩＳＨ−ＤＣ法が有効である（Ａ． Ｍａｒｕｙａｍａａｎｄ Ｍ． Ｓｕｎａｍｕｒａ： Ｓｉｍｕｌｔａｎｅｏｕｓ ｄｉｒｅｃｔ ｃｏｕｎｔｉｎｇ ｏｆ ｔｏｔａｌ ａｎｄ ｓｐｅｃｉｆｉｃ ｍｉｃｒｏｂｉａｌ ｃｅｌｌｓ ｉｎ ｓｅａｗａｔｅｒ， ｕｓｉｎｇ ａ ｄｅｅｐ−ｓｅａ ｍｉｃｒｏｂｅ ａｓ ｂｉｏｍａｒｋｅｒ． Ａｐｐｌｉｅｄａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ， ６６： ２２１１−２２１５， ２０００）。また、ＤＮＡプローブを用いた分子レベルでの特定分解微生物の定量的解析手法としては 、核酸ハイブリダイゼーション法がある（Ｄ． Ａ． Ｓｔａｈｌ， Ｂ． Ｆｌｅｓｈｅｒ， Ｈ． Ｒ． Ｍａｎｓｆｉｅｌｄ ａｎｄ Ｌ． Ｍｏｎｔｇｏｍｅｒｙ ： Ｕｓｅ ｏｆ ｐｈｙｌｏｇｅｎｅｔｉｃａｌｌｙ ｂａｓｅｄ ｈｙｂｒｉｄｉｚａｔｉｏｎ ｐｒｏｂｅｓ ｆｏｒ ｓｔｕｄｉｅｓ ｏｆ ｒｕｍｉｎａｌ ｍｉｃｒｏｂｉａｌ ｅｃｏｌｏｇｙ． Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ５４， １０７９−１０８４， １９８８）。この方法は環境試料や微生物試料から核酸を抽出し 、核酸試料をナイロン膜フィルター上に固定し 、次ぎに放射性同位体（ＲＩ）等で標識したＤＮＡプローブを加えて 、ハイブリダイゼーションを行い 、膜上に固定さている核酸と相補的に結合した標識プローブの放射能強度等を測定し 、プローブと特異的に結合した核酸濃度から特定微生物の定量化をはかる方法である。
【００２５】
最近 、丸山らはＲＩを用いない蛍光ドットブロットハイブリダイゼーション法による相対分子定量法を開発している（Ａ．Ｍａｒｕｙａｍａ， Ｋ， Ｋｉｔａｍｕｒａ， Ｈ． Ｉｓｈｉｗａｔａ， Ｍ． Ｍａｔｓｕｏ， Ｔ． Ｈｉｇａｓｈｉｈａｒａ， ａｎｄ Ｒ． Ｋｕｒａｎｅ： Ｍｏｌｅｃｕｌａｒ ｓｐｅｃｉｆｉｃａｔｉｏｎ ａｎｄ ｑｕａｎｔｉｆｉｃａｔｉｏｎ ｏｆ ｐｒｅｄｏｍｉｎａｎｔ ｍｉｃｒｏｂｅｓ ｉｎ ｏｉｌ ｃｏｎｔａｍｉｎａｔｅｄ ｓｅａｗａｔｅｒ． ９ｔｈＩｎｔｅｒｎａｔｉｏｎａｌ Ｓｙｍｐｏｓｉｕｍ ｏｎ Ｍｉｃｒｏｂｉａｌ Ｅｃｏｌｏｇｙ（Ａｍｓｔｅｒｄａｍ）， Ａｂｓｔｒａｃｔ， ｐ．３７５， ２００１、丸山明彦：分離培養困難な環境微生物へのアプローチ，バイオサイエンスとインダストリー， ６０， ３１−３４， ２００２）。
以上のようなことから、本発明においては、本発明の上記新規微生物を特異的に検出・計数することが可能なＤＮＡプローブを新たに作製している。以下ＤＮＡプローブを作製する工程について説明する。
【００２６】
〔プローブ〕
ＡＮＩ７Ａ菌株または／およびＡＮＩ７Ｐ 菌株の各１６Ｓ ｒＤＮＡ（ＡＮＩ７Ａ菌株；配列番号１、ＡＮＩ７Ｐ 菌株；配列番号２）の塩基配列情報に基づいて、種々の用途に適したＲＮＡおよびＤＮＡプローブを設計することができる。プローブの塩基配列および長さは検出、定量、スクリーニング、あるいは同定の対象とするＳｐｈｉｎｇｏｍｏｎａｓ 属微生物の範囲に応じて適宜選択すればよい。例えば、本発明の上記新種の微生物のみをスクリーニングしたい場合には、該微生物の１６Ｓ ｒＤＮＡの特異的部分の塩基配列によりプローブを設計すればよく、さらに近縁種をも含めてスクリーニング範囲を広げたいときには、近縁種の１６Ｓ ｒＤＮＡと共通な塩基配列部分を含むよう例えば塩基配列の長さを短縮する等プローブを設計する、また、塩基配列部分の選択あるいはその長さを調節することによりさらに、プローブの菌株特異性を低下させれば、さらに広い範囲の有用細菌をスクリーニングすることができる。
本発明のプローブは 例えばＦＩＳＨ法（ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ）により、試料（例えば、石油等有害物質で汚染された海、河川、湖沼、排水・廃液などの環境試料水）中、あるいは多数の微生物群の中から、Ｓｐｈｉｎｇｏｍｏｎａｓ属に属する、本発明の上記新種微生物、その近縁種、あるいは該近縁種の石油分解細菌を検出および／または定量したり、また、スクリーニングするためには、例えば、配列番号１の塩基配列の塩基番号 ５６５−６１５の領域（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉの１６Ｓ ｒＤＮＡの塩基配列における５’末端からの位置（ナンバーリングシステム）では、６３０−６８０の領域）などから選択される領域に対応する塩基長１０−５０ｂｐ、好ましくは塩基長１５−２５ｂｐのプローブを設計するとよい。一例として以下のプローブを挙げることができる。
（１）５’−ＣＧＣＣＴＣＴＣＣＡＡＧＡＴＴＣＴＡＧＣＧＡＣＣＴ−３’（ＳＰＧ６４５＊， ２５ｍｅｒ）（配列番号３）
３’−ＧＣＧＧＡＧＡＧＧＴＴＣＴＡＡＧＡＴＣＧＣＴＧＧＡ −５’
（２）５’−ＣＣＡＡＧＡＴＴＣＴＡＧＣＧＡＣＣ−３’（ＳＰＧ６４３＊， １７ｍｅｒ）（配列番号４）
３’−ＧＧＴＴＣＴＡＡＧＡＴＣＧＣＴＧＧ −５’
（３）５’−ＴＡＧＣＧＡＣＣＴＡＧＴＴＴＣＡＡＡＧＧ −３’（ＳＰＧ６３４＊， ２０ｍｅｒ）（配列番号５）
３’−ＡＴＣＧＣＴＧＧＡＴＣＡＡＡＧＴＴＴＣＣ −５’
（なお、＊（数字）はＥｓｃｈｅｒｉｃｈｉａ ｃｏｌｉの１６Ｓ ｒＤＮＡ塩基配列における５’末端からの位置（ナンバーリングシステム）を示す（Ｎｏｌｌｅｒ Ｈ． Ｆ． ａｎｄ Ｃ． Ｒ． Ｗｏｅｓｅ， １９８１． Ｓｃｉｅｎｃｅ， ２１２：４０３−４１１）。
プローブは、公知の方法、例えば、ホスホルアミド法またはトリエステル法により合成することができる。あるいは、ＤＮＡ自動合成機により合成してもよい。
【００２７】
また、プローブは、アイソトープ（３２Ｐ、３５Ｓなど）、蛍光色素（ビオチン／アビジン、ジゴキシゲニン／抗ジゴキシゲニン−ローダミン、
Ｆｌｕｏｒｅｓｃｅｉｎ−ｉｓｏｔｈｉｏｃｙａｎａｔｅ （ＦＩＴＣ）、ＬｕｃｉｆｅｒＹｅｌｌｏｗ ＣＨ、Ｒｈｏｄａｍｉｎｅ １２３、Ａｃｒｉｄｉｎｅ ｏｒａｎｇｅ、Ｐｙｒｏｎｉｎ Ｙ、Ｅｔｈｉｄｉｕｍ ｂｒｏｍｉｄｅ、Ｐｒｏｐｉｄｉｕｍ ｉｏｄｉｄｅ、Ｅｔｈｉｄｉｕｍ ｈｏｍｏｄｉｍｅｒ、ＢＯＢＯ−１、ＰＯＰＯ−１、ＴＯＴＯ−１、ＹＯＹＯ−１、Ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ ｄｉａｃｅｔａｔｅ （ＣＦＤＡ）、Ｆｌｕｏｒｅｓｃｅｉｎ ｄｉａｃｅｔａｔｅ （ＦＤＡ）、Ｃａｒｂｏｘｙｆｌｕｏｒｅｓｃｅｉｎ ｄｉａｃｅｔａｔｅ−ａｃｅｔｏｘｙｍｅｔｈｙｌｅｓｔｅｒ （ＣＦＤＡ−ＡＭ）、５−ｃｙａｎｏ−２，３０ｄｉｔｏｌｙｌ ｔｅｔｒａｚｏｌｉｕｍ ｃｈｌｏｒｉｄｅ （ＣＴＣ）、Ｔｅｔｒａｍｅｔｈｙｌｒｈｏｄａｍｉｎｅ ｉｓｏｔｈｉｏｃｙａｎａｔｅ（ＴＲＩＴＣ）、Ｓｕｌｆｏｒｈｏｄａｍｉｎｅ １０１ ａｃｉｄ ｃｈｌｏｒｉｄｅ （Ｔｅｘａｓ Ｒｅｄ）、Ｃｙ３、Ｃｙ５、Ｃｙ７、２−ｈｙｄｒｏｘｙ−３−ｎａｐｈｔｏｉｃ ａｃｉｄ−２’−ｐｈｅｎｙｌａｎｉｌｉｄｅｐｈｏｓｐｈａｔｅ （ＨＮＰＰ）など）、化学発光などで標識するとよい。
【００２８】
〔Ｓｐｈｉｎｇｏｍｏｎａｓ属の有用細菌のスクリーニング及び検出、定量〕
本発明のＲＮＡまたはＤＮＡプローブを用い、種々のハイブリダイゼーション法（サザンブロット法、ノーザンブロット法、コロニーハイブリダイゼーション、ドットハイブリダイゼーション、ｉｎ ｓｉｔｕハイブリダイゼーション（例えば、ＦＩＳＨ法）などにより、Ｓｐｈｉｎｇｏｍｏｎａｓ属に属する、本発明の新種微生物、その近縁種、あるいは該近縁種の石油分解細菌種の石油分解細菌を検出及び／または定量したり、スクリーニングすることができる。
本発明のＤＮＡプローブを用いて、石油や環境汚染物質で汚染された現場の水や海水から石油等汚染物質分解微生物を検出・定量する方法の一例について以下に説明する。有害物質汚染現場から水や海水試料を採取し、この試料中に存在する微生物をフィルター（孔径０．２μｍ）に固定し、これを蛍光色素等で標識した配列番号３の塩基配列を有するＤＮＡプローブとハイブリダイズさせ、プローブを洗い落とした後、蛍光顕微鏡で観察して、ＤＮＡプローブとハイブリダイズし、標識した蛍光を呈している特定の分解微生物を選択的に検出または計数を行う。
上記したように、環境が汚染されれば、分解菌群の割合が増大してくるのでこれにより、環境汚染の指標とすることが可能となる。
【００２９】
また、本発明のＤＮＡプローブを用い、コロニーハイブリダイゼーション手法、ブロットハイブリダイゼーション手法、フローサイトメトリー法などにより、多数の微生物群の中からＳｐｈｉｎｇｏｍｏｎａｓ属の本発明の新種の微生物およびその近縁種、およびＳｐｈｉｎｇｏｍｏｎａｓ属の石油分解細菌、とくにＳｐｈｉｎｇｏｍｏｎａｓ属の本発明の新種の微生物およびその近縁種の石油分解細菌をスクリーニングすることができる。
【００３０】
〔Ｓｐｈｉｎｇｏｍｏｎａｓ属菌の同定〕
さらに、配列番号１または２の塩基配列情報や配列番号３、４または５を用いて、Ｓｐｈｉｎｇｏｍｏｎａｓ属の本発明の新種の微生物およびその近縁種、およびＳｐｈｉｎｇｏｍｏｎａｓ属の本発明の新種の微生物およびその近縁種の石油等有害物質分解細菌を同定することができる。例えば、配列番号１または配列番号２の塩基配列との相同性、または請求項５〜７いずれかに記載のＲＮＡまたはＤＮＡプローブを用いたＤＮＡ／ＤＮＡまたはＤＮＡ／ＲＮＡハイブリダイゼーションにより同種の菌であることが同定できる。さらに、上記プローブの塩基配列（ＤＮＡ断片）をプライマーとして用いて、ＰＣＲを行うことによって菌種の同定を行うこともできる。すなわち、同定の対象となる菌体を溶菌して、上記プローブの塩基配列をもつＤＮＡ断片をプライマーとして添加した後、ＰＣＲ増幅する。そのＰＣＲ産物を電気泳動等により１６Ｓ ｒＤＮＡの増幅が確認されれば、対象とした菌には、用いたＤＮＡ断片に相補的な遺伝子部分を有していることになる。すなわち、同種の菌であることが特定できる。
【００３１】
〔有害物質汚染環境のモニタリング、解析・評価〕
石油等の有害物質汚染環境の指標となる特定の分解微生物の挙動、およびそれが全微生物群集に占める割合（優占度）を、簡単、迅速にモニタリングすることが可能になれば、汚染の程度、および汚染環境の修復、回復の程度などを、その汚染環境の診断が高精度かつ早期に可能になる。 例えば、環境中に石油分解菌やＰＣＢ分解菌がある時期に優占度が上昇していれば、その環境は石油やＰＣＢで汚染されている可能性が高いと判断できるし、その微生物群集全体として石油やＰＣＢ分解能が高まっていると判定できる。さらに、その優占度の変化を長期間にモニタリングし、その変遷の周期性や季節性を把握しておけば、その変化が突発的なものかどうか、その負荷が船舶事故や工場排水の流入など人為的なものかどうかを推定できる。 本発明のＤＮＡプローブを用いて、油濁環境中の全微生物群集中のＰＡＨ分解微生物の優占度を調べることにより、環境中の炭化水素成分、ＰＡＨの比率、濃度および消長など汚染の度合を把握でき、汚染物質の自然浄化過程やバイオ環境修復過程の解析・評価が可能になる。
【００３２】
【実施例】以下本発明を実施例によって具体的に説明する。
【実施例１】
１）ＰＡＨ分解微生物の分離源試料
日本海重油流出事故で石川県沿岸域で最も汚染された地点、Ｓｔｎ．１９（珠洲西海海岸、長橋）で１９９８年６月１１日に採取された海水試料に無機栄養塩を加え系（ＳＷ＋Ｎ＋Ｐ）（表２−Ａ）に０．５％Ｃ重油を添加して、２０℃で６５日培養し、分解試験を行った。
この６５日の分解試験培養液中の分解細菌数のＭＰＮ計数培養（培地：表２−Ｃ）で、Ｃ重油で良好な増殖を示した希釈段階のもっとも高い試験管の培養液を、０．５％Ｃ重油を含む（ＳＷ＋Ｎ＋Ｐ）培地に接種し、２０℃にて培養期間１２−１７日で３回集積培養を繰り返した後、さらにＣ重油を加えたＮＳＷ培地（表２−Ｂ）を用いて、前記同様に、培養期間９−１７日で４回集積培養を繰り返した培養液を下記ＡＮ分解細菌の集積培養の種菌として用いた。
【表２】

２） ＰＡＨ分解細菌の集積培養
前記Ｃ重油集積培養液０．１ｍｌを種菌として、０．１％ （ｗ／ｖ） ＡＮを添加したＮＳＷ培地（表２−Ｂ参照）１０ｍｌを含む大型試験管に接種し、２０℃で８日間振とう培養によりＡＮ分解細菌の集積培養を行った。
３） ＰＡＨ分解細菌の分離に用いた平板培地と培養法
ＡＮ 分解細菌およびＰＨＥ分解細菌の分離は、０．１％（ｗ／ｖ）ＡＮをＮＳＷ寒天培地に添加した（ＮＳＷ＋ＡＮ）平板培地（表２−Ｄ）、２％寒天ＮＳＷ下層培地にＡＮまたはＰＨＥのエタノール溶液を添加した１％アガロースＮＳＷ上層培地を被せた二層平板培地（表２：ＮＳＷ＋ＡＮ二層培地、ＮＳＷ＋ＰＨＥ二層培地）およびＭａｒｉｎｅ Ａｇａｒ ２２１６ （Ｄｉｆｃｏ製） （ＭＡ）平板培地を用いて、２０℃で平板培養により行った。なお、平板培地に形成されたコロニー形態は実体顕微鏡で観察した。
また、使用したＮＳＷ＋ＡＮ二層平板培地は、以下の２％寒天ＮＳＷ下層培地（１）に（シャーレ、直径９ｃｍ）、アントラセン（ＡＮ）を加えた１％アガロースＮＳＷ上層培地（２）５ｍｌ（約５０℃保温）をすばやく添加して、下層培地表面全体に上層培地を均一に広げて調製したものである。
（１）． ２％寒天ＮＳＷ下層培地
表２ＮＳＷ培地に２％濃度の寒天を添加した培地１５−２０ｍｌを直径９ｃｍのシャーレに加えて平板培地を調製する。
（２）． ＡＮ添加１％アガロースＮＳＷ上層培地
１％アガロースを添加したＮＳＷ培地５ｍｌを含む試験管をオートクレーブ滅菌後、約５０℃に保温し、その試験管にろ過滅菌した０．１％ＡＮエタノール（９９．５％、脱水エタノール）溶液０．２ｍｌを添加し、すばやく撹拌して均一に分散させる。
なお、本二層平板培地の調製法は下記文献に記載されている方法に準じて行った。
Ｂｏｇａｒｄｔ， Ａ． Ｈ． ａｎｄ Ｈｅｍｍｉｎｇｓｅｎ， Ｂ． Ｂ．： Ｅｎｕｍｅｒａｔｉｏｎ ｏｆ ｐｈｅｎａｎｔｈｒｅｎｅ−ｄｅｇｒａｄｉｎｇ ｂａｃｔｅｒｉａ ｂｙ ａｎ ｏｖｅｒｌａｙｅｒ ｔｅｃｈｎｉｑｕｅ ａｎｄ ｉｔｓ ｕｓｅ ｉｎ ｅｖａｌｕａｔｉｏｎ ｏｆ ｐｅｔｒｏｌｅｕｍ−ｃｏｎｔａｍｉｎａｔｅｄ ｓｉｔｅｓ． Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ．， ５８， ２５７９−２５８２（１９９２）．
ＮＳＷ培地：文献参照（Ｔ． Ｈｉｇａｓｈｉｈａｒａ， Ａ． Ｓａｔｏ ａｎｄ Ｕ． Ｓｈｉｍｉｚｕ： Ａｎ ｍｅｔｈｏｄ ｆｏｒｔｈｅ ｅｎｕｍｅｒａｔｉｏｎ ｏｆ ｍａｒｉｎｅ ｈｙｄｒｏｃａｒｂｏｎ ｄｅｇｒａｄｉｎｇ ｂａｃｔｅｒｉａ， Ｂｕｌｌｅｔｉｎ ｏｆ Ｊａｐａｎｅｓｅ Ｓｏｃｉｅｔｙ ｏｆ Ｓｃｉｅｎｔｉｆｉｃ Ｆｉｓｈｉｅｒｅｉｅｓ， ４４， １１２７−１１３４， １９７８）
【００３３】
４）ＰＡＨ分解細菌の分離
前記２）項のＡＮ分解細菌の集積培養液を（ＮＳＷ＋ＡＮ）平板培地に塗抹し、２０℃、８日間培養した。この平板培養培地に形成されたコロニーを釣菌し、さらにその分離菌株を （ＮＳＷ＋ＡＮ）平板培地とＭＡ平板培地を用いて、平板分離培養を２回繰り返し、１６菌株を分離した。なお、平板培養は２０℃、１６日間行った。
さらに、これらの分離菌株を、０．１％ＡＮを添加したＮＳＷ培地５ｍｌを加えたキャップ付ねじ口試験管（直径１８ｍｍ）にて４０日振とう培養を行った結果、ＡＮの部分分解物と推定されるｐａｌｅ ｐｕｒｐｌｉｓｈ ｐｉｎｋ系の色素を生成し、ＡＮ分解性が示唆されるＡＮＩ７菌株を見出した。なお、本菌株はＮＳＷ＋ＡＮ平板培養培地からの分離菌株である。
【００３４】
５） ＡＮＩ７菌株の純粋分離
上記ＡＮＩ７菌株を０．１％ＡＮまたは０．１％ＰＨＥ を添加したＮＳＷ培地を用いて、約１ヶ月− １．５ヶ月間、振とう培養後、この培養液をＭＡ平板培地に塗抹し、培養した結果、いずれの培養液においてもＹｅｌｌｏｗ系の色調の濃いコロニー（Ａタイプ）と色調の薄いコロニー（Ｐ タイプ）の２種類のコロニーが混在していた。そこで、本菌株の純粋分離を行った。純粋分離は、ＭＡ平板培地、ＮＳＷ＋ＡＮ平板培地およびＮＳＷ＋ＡＮ二層培地を用いた平板培養と０．１％ＡＮまたは０．１％ＰＨＥ を添加したＮＳＷ培地を用いた液体培養を数回繰り返すことにより行った。なお、ＡタイプはＡＮ添加ＮＳＷ培地、Ｐ タイプはＰＨＥ添加ＮＳＷ培地を用いた液体培養系から、それぞれの純培養株としてＡＮＩ７Ａ菌株（受託番号ＦＥＲＭ Ｐ−１９０９５）とＡＮＩ７Ｐ菌株（（受託番号ＦＥＲＭ Ｐ−１９０９６）の分離に成功した。なお、分離菌株の純粋性試験には上記の平板培地以外に、ＮＳＷ＋ＰＨＥ二層培地も用いた。
また、純粋分離したＡＮＩ７Ａ菌株とＡＮＩ７Ｐ菌株は、いずれもＡＮ添加ＮＳＷ培地を用いた液体培養でＡＮ の部分酸化物由来と推定されるＰａｌｅ ｐｉｎｋ−Ｐａｌｅ ｐｕｒｐｌｉｓｈ ｐｉｎｋ系の色素を、またＰＨＥ添加ＮＳＷ培地ではＤｕｌｌ ｙｅｌｌｏｗ−ｐａｌｅ ｙｅｌｌｏｗ ｏｒａｎｇｅ系の色素を生成した。さらに、両菌株のＰＡＨ分解性を調べた結果、いずれもＡＮやＰＨＥを分解することが確認された（実施例２参照）。また、ＡＮＩ７Ａ菌株については、ビフェニール（ＢＰ）を添加したＮＳＷ培地を用いた液体培養でＢＰの部分酸化物と推定されるＰａｌｅ ｙｅｌｌｏｗ ｇｒｅｅｎ−Ｐａｌｅ ｙｅｌｌｏｗ系の色素を生成した。
【００３５】
【実施例２】
ＡＮＩ７Ａ菌株およびＡＮＩ７Ｐ菌株のＰＡＨ分解試験には、炭素源として０．１％（ｗ／ｖ） ＡＮまたは０．１％（ｗ／ｖ） ＰＨＥを添加したＮＳＷ培地（表２−Ｂ）を用いた。このＡＮまたはＰＨＥを添加したＮＳＷ培地５ ｍｌをキャップ付ネジ口試験管（直径１８ｍｍ）に、または前記ＮＳＷ培地１０ｍｌをシリコセン付Ｌ字型試験管（直径１８ｍｍ）に加え、２０℃で振とう培養（４５ ｒｐｍ）を行った。
培養液中のＡＮおよびＰＨＥの定量は培養液と等量のジクロロメタンで２回抽出後、２５ｍｌ定容量とした。この抽出液の一定量を下記条件のガスクロマトグラフィー（ＧＣ）により分析した。なお、内部標準物質としてはｎ−ヘキサデカンを用いた。
ＧＣの分析条件（本体：島津ＧＣ−１７Ａ）
１． カラム
液相： ＴＣ−７０（ＧＬサイエンス製キャピラリーカラム）； 長さ：３０ｍｘ０．２５ｍｍ；
液相の膜厚：０．２５μｍ
２． キャリヤガス（Ｎ２）
流速：１ｍｌ／ｍｉｎ
３． 測定条件
試料注入口温度：２５０ ℃
イニシャル温度：１２０ ℃
昇温速度：１０ ℃／ｍｉｎ （２６０ ℃まで）；２６０ ℃で８ｍｉｎ保持
検出器： ＦＩＤ
炭化水素の分解率は同一条件で振とう培養した菌無接種の対照培地中の残存炭化水素量を基準に求めた。なお、分解率は同一培養条件の試験管３本または４本の各分解率の平均値で示した。
【００３６】
シリコセン付Ｌ字型試験管（直径１８ｍｍ）にて２０℃で６０日振とう培養を行った場合、ＡＮＩ７Ａ菌株のＡＮおよびＰＨＥの分解率は８％と２１％であった。また、キャップ付ネジ口試験管（直径１８ｍｍ）を用いて、２０℃、３４日振とう培養を行った場合、ＡＮＩ７Ａ菌株のＰＨＥの分解率は ２２％であった。さらに、後者の培養条件下におけるＡＮＩ７Ｐ菌株のＰＨＥ分解率は１９％であり、ＡＮＩ７Ａ菌株とほぼ同程度であった。また、両菌株からなる微生物コンソーシアのＰＨＥ分解率は、後者の培養条件で２３％で、各単一菌株の分解率と大差はなく、両菌株を組み合わせたコンソーシアによるＰＨＥ分解の促進効果はみられなかった。ＡＮＩ７Ａ菌株とＡＮＩ７Ｐ菌株はコロニー色調は異なるが、分子系統解析からＳｐｈｉｎｇｏｍｏｎａｓ 属の同一種とみなされたことから、ＰＨＥ分解能にも顕著な差はみられなかったものと推察された。
また、ＡＮＩ７Ａ菌株は、０．１％（ｗ／ｖ） ＢＰを添加したＮＳＷ培地を用いたシリコセン付Ｌ字型試験管による前者の培養条件で、培養１日目からＢＰの部分酸化物と推定されるＰａｌｅ ｙｅｌｌｏｗ ｇｒｅｅｎ −Ｐａｌｅ ｙｅｌｌｏｗの色素を生成した。、ＡＮＩ７Ｐ菌株についてはこのＢＰ分解試験を行わなかったが、上記したようにＡＮ１７Ａ菌株とＡＮ１７Ｐ菌株とは、同じ細菌種で、ともにＰＨＥを分解することから、ＡＮ１７Ｐ菌株もＢＰ分解能を有すると考えられる。
【００３７】
【実施例３】
Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ．ＡＮＩ７Ａ菌株検出用ＤＮＡプローブの調製
石川県流出油汚染沿岸域より採取した試料から、集積培養を経て純粋分離に成功したＰＡＨ分解細菌Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ． ＡＮＩ７Ａ菌株（ＦＥＲＭ Ｐ−１９０９５）の１６Ｓ ｒＤＮＡ塩基配列情報（配列番号１）の中から、Ｓｔａｈｌ ａｎｄ Ａｍａｎｎ （Ｄｅｖｅｌｏｐｍｅｎｔ ａｎｄ Ａｐｐｌｉｃａｔｉｏｎ ｏｆ Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｐｒｏｂｅｓ． Ｉｎ： Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｔｅｃｈｎｉｑｕｅｓ ｉｎ Ｂａｃｔｅｒｉａｌ Ｓｙｓｔｅｍａｔｉｃｓ． Ｅｄ．： Ｅ． Ｓｔａｃｋｅｂｒａｎｄｔ ａｎｄ Ｍ． Ｇｏｏｄｆｅｌｌｏｗ， Ｊｏｈｎ Ｗｉｌｅｙ ａｎｄ Ｓｏｎｓ， Ｃｈｉｃｈｅｓｔｅｒ， ｐｐ． ２０５−２４８， １９９１）により示された高次構造による障害が見られないと考えられる領域から、この菌種に特異的な配列を選抜し、該配列を有するオリゴヌクレオチドを合成し、その５‘末端をＴＲＩＴＣや Ｃｙ ５等の蛍光色素により標識化し、最終的に配列番号３示した配列を有する比放射性標識ＤＮＡプローブ（ＳＰＧ６４５）、配列番号４に示す比放射性標識ＤＮＡプローブ（ＳＰＧ６４３）及び配列番号５に示す比放射性標識ＤＮＡプローブ（ＳＰＧ６３４）を作製した。
【００３８】
【実施例４】
実施例３記載のＤＮＡプローブを用いたＳｐｈｉｎｇｏｍｏｎａｓ属微生物の検出、計数法
実施例３の上記各ＤＮＡプローブの使用にあたっては、Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ． ＡＮＩ７Ａ菌株を標的微生物、Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｕｂｔｅｒｒａｎｅａ ＩＦＯ１６０８６（標準菌株）などを対象微生物とし、Ｍａｒｕｙａｍａ ａｎｄ Ｓｕｎａｍｕｒａ（Ｓｉｍｕｌｔａｎｅｏｕｓ ｄｉｒｅｃｔ ｃｏｕｎｔｉｎｇ ｏｆ ｔｏｔａｌ ａｎｄ ｓｐｅｃｉｆｉｃ ｍｉｃｒｏｂｉａｌ ｃｅｌｌｓ ｉｎ ｓｅａｗａｔｅｒ， ｕｓｉｎｇ ａ ｄｅｅｐ−ｓｅａ ｍｉｃｒｏｂｅ ａｓｂｉｏｍａｒｋｅｒ． Ａｐｐｌｉｅｄ ａｎｄ Ｅｎｖｉｒｏｎｍｅｎｔａｌ Ｍｉｃｒｏｂｉｏｌｏｇｙ， ６６： ２２１１−２２１５， ２０００）に記載した装置及び手法を用い、ＦＩＳＨ法にて実際にその有効性を確認した。供試菌株の培養、固定及びハイブリダイゼーションの方法については、特開２０００−３３３６８０に準じた。ただしこの際のハイブリダイゼーションは、５０％ ホルムアミド存在下で４２℃で行い、洗浄処理は４２℃で行った。 試料の蛍光顕微鏡観察によるプローブの有効性を表３に示す。
【表３】

【００３９】
今回デザインしたプローブＳＰＧ６４５、 ＳＰＧ６４３， およびＳＰＧ６３４の場合、ＵＶ励起では各細胞中 ＤＮＡにＤＡＰＩが普遍的に結合した結果として、ＤＡＰＩ由来の青色蛍光が ＡＮＩ７Ａ菌および対照菌株として供した２種類Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｕｂｔｅｒｒａｎｅａ （ＩＦＯ１６０８６） 、Ｐｓｙｃｈｒｏｂａｃｔｅｒ ｐａｃｉｆｉｃｅｎｓｉｓ ＮＩＢＨ Ｐ２Ｋ１８）とも観察することができた。しかし、同視野をＧ励起で観察すると、ＡＮＩ７Ａ菌株のみがプローブＳＰＧ６４５、ＳＰＧ６４３、およびＳＰＧ６３４ と相補的な配列を持つため、プローブの５’末端をラベルしたＴＲＩＴＣ由来の赤色蛍光を発した。
一方、Ｂａｃｔｅｒｉａドメインに特異的なプローブＥＵＢ３３８の場合は、Ｂ励起での観察においても、ＡＮＩ ７Ａおよび２種類の対照菌株とも、それぞれＥＵＢ３３８の５’末端をラベルしたＦＩＴＣ由来の緑色蛍光を発した。
以上のことから、今回、油濁環境由来の微生物１６Ｓ ｒＤＮＡ塩基配列からデザインした３つのプローブは、ＡＮＩ７Ａ菌株またはその近縁種を特異的に検出する上で、その高次構造に起因する結合上の妨害も見られず、実際に大変有効であることが示された。
【００４０】
【発明の効果】
本発明は、環境汚染物質、特に石油、廃棄物あるいはこれらに含まれるＰＡＨ等有害環境汚染物質の分解能を有するＳｐｈｉｎｇｏｍｏｎａｓ属に属する新規微生物を提供するものであり、本発明の新規微生物を利用して、上記環境汚染物質で汚染された海洋、湖沼、河川、廃液などを効率よく浄化することができる。また、本発明によれば、上記新規微生物の１６Ｓ ｒＤＮＡ／ＲＮＡ から調製したプローブにより、Ｓｐｈｉｎｇｏｍｏｎａｓ属に属する上記環境汚染物質分解能を有する細菌の検出・定量を簡便迅速に行うことが可能となり、この細菌の検出・定量方法により、世界的な環境問題になっているＰＡＨ等有害物質汚染環境のモニタリング、解析・評価、診断、ならびにバイオレメディーエション技術によるＰＡＨ等有害物質汚染環境の浄化・修復過程を解析・評価することができるきわめて有益な技術を提供できた。
【００４１】
【配列表】

【図面の簡単な説明】
【図１】Ｓｐｈｉｎｇｏｍｏｎａｓ ｓｐ．ＡＮ１７Ａ株、及び同ＡＮ１７Ｐ株の各１６ＳｒＤＮＡの分子系統解析に基づき作成された分子系統樹である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a new bacterium belonging to the genus Sphingomonas, which is capable of decomposing and removing environmental pollutants. The present invention relates to a method for purifying water or soil contaminated with harmful environmental pollutants such as polycyclic aromatic hydrocarbons (PAH) using the bacteria. In addition, the present invention relates to a method for detecting and quantifying a specific bacterium belonging to the genus Sphingomonas and a bacterium having a capability of degrading harmful environmental pollutants. Furthermore, the present invention relates to a method for monitoring, analyzing, evaluating, and diagnosing environments contaminated with harmful substances such as petroleum, and a method for analyzing and evaluating purification and restoration processes of environments contaminated with harmful substances.
[0002]
[Prior art]
In recent years, the pollution of the ocean by petroleum has become a global environmental problem because it has an adverse effect on marine ecosystems and marine products.
Oil spilled into the ocean can be physically recovered using an oil fence, an oil recovery machine, an oil adsorbent, or the like, or chemically processed using an oil processing agent such as an oil gelling agent or an emulsifying dispersant (see Non-Patent Document 1). .
Physical treatment seems to have little effect on marine ecosystems, but chemical treatment requires careful consideration of the toxicity of dispersants and other ecosystems such as marine life. However, such physical and chemical removal and treatment are not complete, and unrecovered oil spills evaporate and undergo physical and chemical changes due to light and oxygen. It is degraded by the self-cleaning action of natural ecosystems based on the biodegradability of microorganisms in seawater and marine sediments (see Non-Patent Document 2).
In recent years, development of an environmentally harmful biological oil spill purification technology (bioremediation) using such microorganisms has attracted attention (see Non-Patent Documents 3 and 4). This bioremediation technology is a technology for decomposing and removing contaminants by accelerating a biodegradation process by microorganisms operating in nature.
Therefore, in order to develop bioremediation technology, the distribution and type of microorganisms that decompose harmful environmental pollutants such as petroleum in the on-site environment, their resolution, etc. are investigated, and the estimation of the natural purification ability for harmful material polluted environments and its mechanism are investigated. It needs to be clarified. Furthermore, in order to apply bioremediation technology, it is necessary to monitor, analyze and evaluate microflora and harmful substance-degrading microorganisms in toxic substance-contaminated environments, and to properly purify and remediate toxic substance-contaminated environments using bioremediation technology. A method for analysis and evaluation is required.
For example, crude oil is a complex mixture of thousands of different types of hydrocarbons, and the hydrocarbons present in petroleum can be divided into saturated hydrocarbons and aromatic hydrocarbons according to their chemical structures. N-paraffins and branched paraffins are classified into cycloparaffins (monocyclic and polycyclic), and the latter are classified into monocyclic and polycyclic aromatic hydrocarbons (see Non-Patent Document 5).
Petroleum can also be classified into saturated components (SA), aromatic components (AR), resin components (RE), and asphaltenes (AS) depending on the hydrocarbon composition (see Non-Patent Document 6).
Degradability of these hydrocarbons by microorganisms decreases in the order of n-alkane> branched alkane> low molecular weight aromatic hydrocarbon> cycloalkane (see Non-Patent Document 7).
Microbial degradation of petroleum spills to the site environment first degrades readily degradable saturated components and low-molecular-weight aromatic hydrocarbons, and very high-molecular-weight aromatic hydrocarbons, RE and AS are difficult to decompose. It is said that the decomposition rate is extremely slow (see Non-Patent Document 8).
In addition, according to the investigations by the inventors, the degradability of heavy oil by microbial communities in seawater at the site contaminated by the oil spill accident in the Sea of Japan indicates that SA in heavy oil is relatively well degraded, It was recognized that AR containing a ring-aromatic hydrocarbon (PAH) and the like was little decomposed, and RE and AS having a condensed aromatic ring structure and high molecular weight were hardly decomposed (see Non-Patent Documents 9 and 10).
PAHs contained in heavy oils such as heavy oil have carcinogenicity, mutagenicity, and are hardly decomposable, so microorganisms that degrade them efficiently in a short period of time and safe environmentally friendly biological Establishment of decomposition / removal technology is required. As a polycyclic aromatic hydrocarbon (PAH) -degrading bacterium isolated from the marine environment, Sphingomonas sp. AJ1 (Non-Patent Document 11) Cycloplasticus pugetii, see Non-Patent Document 12), Neptunomonas naphovorans (Non-Patent Document 13), Lutibacterium anuloederans (Non-Patent Document 14), and some non-patent documents such as Vibrio Cyclotic 15 There is. However, none of them have excellent PAH decomposition activity and can be put to practical use. Although the above has mainly explained petroleum pollution, urgent measures must also be taken to deal with environmental spills caused by spills of chemicals, factory effluents, and waste dumping. Also in this case, the importance of the technology for effectively decomposing the above-mentioned PAHs or environmental hormones is increasing more and more, and the search for useful bacteria for this purpose is also being actively conducted.
In search of useful microorganisms in these environmental pollution purification technologies, a search technology capable of performing the search quickly, simply, and accurately is desired.
[Non-Patent Document 1] Response to Marine Oil Spills, The International Tanker Owners Pollution Federation Ltd. 1993 Oil Federation, p.I3-I5, Tokyo, 1997.
[Non-Patent Document 2] “Monthly Ocean”, Vol. 30, No. 10, 1998 (pp. 613-621)
[Non-Patent Document 3] "Marine Pollution Bulletin", Vol. 26 (1993), pp. 476-481.
[Non-Patent Document 4] "Crit. Rev. Microbiol." Vol. 19 (1993) pp. 217-242.
[Non-Patent Document 5] "Petroleum and microorganisms" No. 1 (1968) pp. 2-24 "
[Non-Patent Document 6] "Quality and Standards of Petroleum Products" (1997) pp. 142-153 "
[Non-Patent Document 7] "Microbiol. Rev." 54 (1990) 305-315.
[Non-Patent Document 8] "Advances in Microbial Ecology (ed. KC Marshall), PlenumPress, New York", 12th (1992), pages 287-338.
[Non-Patent Document 9] “1998 Environmental Conservation Research Results Collection, Environmental Research and Technology Division, Planning and Coordination Bureau, Environment Agency” (1999) 48-II, 1-9, pp.
[Non-Patent Document 10] Proceedings of the 14th Annual Meeting of the Japanese Society for Microbial Ecology, 41, 1998
[Non-Patent Document 11] "J. Fer. Bioeng." Vol. 82 (1996), pp. 570-574.
[Non-Patent Document 12] "Int. J. Syst. Bacteriol., Vol. 45 (1995), pp. 116-123"
[Non-Patent Document 13] "Appl. Environ. Microbiol. 65 (1999) pp. 251-259"
[Non-Patent Document 14] "Appl. Environ. Microbiol." 67 (2001) pp. 5585-5592.
[Non-Patent Document 15] "Int. J. Syst. Evol. Microbiol., Vol. 51 (2001), pp. 61-66"
[0003]
[Problems to be solved by the invention]
The present invention particularly relates to a new bacterium belonging to the genus Sphingomonas, which has the ability to decompose environmental pollutants such as PAH contained in spilled oil or waste, and a method for purifying water or soil contaminated with the environmental pollutants. To provide. Further, the present invention provides a method for detecting and quantifying the above-mentioned new bacterium belonging to the genus Sphingomonas, or a useful bacterium having the ability to decompose environmental pollutants such as PAH, monitoring, analyzing, evaluating, and diagnosing an environment contaminated with harmful substances such as petroleum and waste. It is intended to provide a method and a method for analyzing and evaluating a process of purifying and remediating an environment contaminated with harmful substances by bioremediation technology.
[0004]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in view of the above-mentioned problems of the conventional technology, and as a result, have found a new species of bacteria belonging to the genus Sphingomonas having PAH resolution from seawater in the coastal sea area contaminated with heavy oil spilled from the Sea of Japan, and further identified the present bacteria. The present invention was completed by successfully producing a gene probe that can be specifically detected and quantified.
That is, the present invention provides a novel microorganism, a method for purifying an environment contaminated with harmful substances such as petroleum using the novel microorganism, and a method for detecting and quantifying the novel microorganism or a useful bacterium belonging to the genus Sphingomonas.
The gist of the present invention is as follows.
[0005]
(1) A novel microorganism belonging to the genus Sphingomonas, wherein the base sequence of the 16S rRNA gene (16S rDNA) shows 97% or more homology with the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
(2) Sphingomonas sp. ANI7A strain or Sphingomonas sp. ANI7P strain
(3) The microorganism according to (1) or (2), which has a polycyclic aromatic hydrocarbon resolution.
(4) 16S rDNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
(5) An RNA or DNA probe having a part of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a part of a riboxynucleotide sequence corresponding thereto and having a base length of 10 to 50 bp.
(6) The probe according to (5), which specifically hybridizes with the microorganism-derived RNA or DNA according to any one of (1) to (3).
(7) The RNA or DNA probe according to (5) or (6), having the base sequence of SEQ ID NO: 3, 4, or 5, or the corresponding riboxynucleotide sequence.
(8) The RNA or DNA probe according to any one of (5) to (7), which is used for detecting or quantifying an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas.
(9) The RNA or DNA probe according to any one of (5) to (7), wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium.
(10) Sphingomonas sp. ANI7A strain, or Sphingomonas sp. The RNA or DNA probe according to (6) or (7), which is used for detecting and / or quantifying the ANI7P strain.
(11) A method for detecting and / or quantifying a bacterium belonging to the genus Sphingomonas using the RNA or DNA probe according to any of (5) to (7).
(12) The method according to claim 11, wherein the bacterium to be detected or quantified is an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas.
(13) The method according to (12), wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium.
(14) The method according to (11), wherein the bacterium to be detected and / or quantified is the microorganism according to any of (1) to (3).
(15) A method for screening a useful bacterium belonging to the genus Sphingomonas using the RNA or DNA probe according to any of (5) to (7).
(16) The method according to (15), wherein the bacterium to be screened is an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas.
(17) The method according to (16), wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium.
(18) The method according to (15), wherein the bacterium to be screened is the microorganism according to any one of (1) to (3).
(19) Homology with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or DNA / DNA or DNA / RNA hybridization using the RNA or DNA probe according to any of (5) to (7), or The method for identifying a bacterium belonging to the genus Sphingomonas according to any one of (1) to (3), wherein PCR is performed using the DNA probe as a primer.
(20) The method according to (19), wherein the identified bacteria are environmental pollutant-degrading bacteria belonging to the genus Sphingomonas.
(21) The method according to (20), wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium.
(22) A method for purifying a polluted environment, comprising treating the environment contaminated with an environmental pollutant with the microorganism according to any one of (1) to (3).
(23) The method according to (22), wherein the environmental pollutant is petroleum or petroleum-derived.
(24) A method for monitoring, analyzing, evaluating and diagnosing a toxic substance-contaminated environment using the method according to any one of (11) to (14).
(25) A method for analyzing and evaluating a process of purifying and / or restoring a toxic substance-contaminated environment using the method according to any one of (11) to (14).
[0006]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
(Microorganisms)
The microorganism of the present invention is based on being isolated from a seawater sample at the most contaminated site on the coast of Ishikawa Prefecture, which is one of the coastal waters contaminated with heavy oil spilled from the Sea of Japan. The strains thus obtained are specifically two strains: ANI7A strain (Accession number FERMP-19095) and ANI7P strain (Accession number FERMP-19096). These strains use natural seawater, preferably seawater collected aseptically, as a microbial source, for example, PAW as the sole carbon and energy source in NSW medium (Table 2; T. Higashihara, A. Sato and U. Shimizu: An). Bulletin of Japan Society of Scientific of Scientific Fisheries, which was obtained by the method for the enumeration of Marine Hydrocarbon Degrading Bacteria. A detailed separation method is shown in Example 1.
[0007]
Both the ANI7A strain and the ANI7P strain can degrade PAHs such as anthracene (AN) and phenanthrene (PHE). Hereinafter, classification and identification of these strains by phenotypes and molecular phylogenetic analysis based on the base sequence of 16S rDNA will be described.
Table 1 shows the bacteriological properties of the ANI7A strain.
[Table 1]

Based on these mycological properties, Bergey's Manual of Systematic Bacteriology, Volume 1 (1984) (Krieg, NR, and Holt, J.G .: Gasoline Biochemistry. & Wilkins, Maryland, 1984) and Bergey's Manual of Determinative Bacteriology, Ninth Edition (1994) (Holt, G., Kryeg, N.R., S.T.A., S.T.R. and Williams, ST (eds.): Bergey's Manual of Deter minimal Bacteriology (9th ed.) Williams and Wilkins, Maryland, 1994).
As a result, the ANI7A strain was identified as a bacterium belonging to the genus Sphingmonas.
[0008]
Next, the ANI7A strain and the ANI7P strain will be described.
A pure culture obtained from an A-type colony (see Example 1), the colony morphology of the ANI7A strain (NSW + AN two-layer medium (Table 2), cultured at 20 ° C. for 26 days), were colony shape: circular, large Length: 2.0 mm, surface: smooth, raised state: semi-lens (convex), peripheral edge: whole edge (entire), color tone: light brown (color tone at the center of the colony was darker than the edge) . On the other hand, the colony morphology of the pure culture strain and the ANI7P strain from the P-type colony under the above culture conditions was as follows: colony shape: circular, size: 1.8 mm, surface: smooth, raised state: Semi-lens-like (convex), margin: entire, hue: light brown (colony hue is homogeneous and shiny throughout). Thus, although the colony morphology of the ANI7A strain and the ANI7P strain were similar, the color tone and luster of the colonies were different, and depending on the type of culture medium and the culturing period, the prominence of the colonies of both strains was different.
[0009]
As described above, the colony morphology of the ANI7A strain and the ANI7P strain was slightly different. However, as a result of molecular phylogenetic analysis based on the base sequence of 16S rDNA of both strains, the homology of the base sequence of 16S rDNA of both strains was 99.7%. Therefore, the ANI7A strain and the ANI7P strain were of the same species. It was estimated. The nucleotide sequences of the 16S rDNA of the ANI7A strain and the ANI7P strain are shown in SEQ ID NOs: 1 and 2 in the sequence listing. FIG. 1 shows the molecular phylogenetic tree of the ANI7A strain and the ANI7P strain.
[0010]
The classification / identification based on the phenotype is performed by the “heavy oil decomposition method” disclosed by the present inventors (Japanese Patent Application Laid-Open No. 2001-37466 (publication date: 2001.1.213) and RM Smibert and N.M. R. Kreig: Phenotypic Characterization.Methods for General and Molecular Bacteriology (P. Gerhardt, R.G.E.M.R.E., M.O.R.i.e., R.O.E.G.R.i.g. The sequencing was performed according to the method described in Washington, DC (1994), and the 16S rDNA was sequenced using the method described in "Detecting novel psychrotrophic bacteria" disclosed by the present inventors. DNA probe for DNA "(Japanese Patent Application Laid-Open No. 2000-333680 (published on 2001.2.5)). Further, the base sequences of the genus Sphingomona and representative microorganisms obtained from a DNA database Were aligned in parallel to remove incomparable gaps, and molecular phylogenetic analysis was performed by the NJ method.The accuracy of each branch of the obtained phylogenetic tree was calculated by 100 times bootstrap analysis ( Maruyama, A., D. Honda, H. Yamamoto, K. Kitamura and T. Higashihara: Phylogenetic analysis of psychrophilic bacteria isolated phenolic isamately isolated professional italy isolated professional chemistry. luding a description of the deep-sea species Psychrobacter pacificensis sp. nov. International Journal of Systematic and Evolutionary Microbiology. 50, 835-846, 2000).
[0011]
The positions of the molecular phylogenetic trees of the ANI7A strain and the ANI7P strain were shown to belong to Sphingomonas, as were the classification positions by phenotype. Furthermore, the homology between Sphingomonas subarctia, which is most closely related to the ANI7A strain in molecular phylogeny, and the 16S rDNA of this strain was 95.8%.
On the other hand, it is defined as "a bacterial species is a strain that systematically exhibits approximately 70% or more DNA-DNA homology" (Report of the Special Committee of the International Commission on Nomenclature for Bacterial Classification, LG Wayne). , DJ J. Brenner, RR Colwell, PA Grimmont, O. Kandler, MI Krichevsky, L.H. Moore, W.E.C. Moor, R.G.E. Murray, E. Stackebrandt, MP Starr and H. G. Trooper: Report of the ad hoc committee on reconciliation of stakeholder agreements. c Bacteriology, 37, 463-464, 1987), Stackerbrandt et al. described the relationship between DNA-DNA homology and 16S rDNA homology in the above definition by comparing DNA-DNA homology and 16S rDNA homology. Those having a DNA homology of 70% or more and those having a homology of 16S rDNA of 97% or more correspond to each other, and those having a homology of 16S rDNA of 97% or more are regarded as the same species (Stackebrandt, E. and Goebel, B.M .: Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definitions. in bacteriology. Int. J. Syst. Bacteriol., 44, 846-849, 1994).
[0012]
From the above, the ANI7A strain and the ANI7P strain were named as Sphingomonas genus ANI7A and ANI7P strains as new species of the genus Sphingomonas.
These strains have been deposited at the National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary under the accession number FERMP-19095 (Sphingomonas sp. ANI7A strain) and the accession number FERMP-19096 (Sphingomonas sp. ANI7P strain).
In view of the relationship between the 16S rDNA homology and the species, if the ANI7A strain or the ANI7P strain has 97% or more homology with the 16S rDNA strain, these strains are considered to be the same species as these strains and included in the present invention. Is done.
[0013]
[Function of Sphingmonas microorganism]
The genus Sphingmonas is a new genus proposed by Yabuuchi et al. In 1990. It has glycosphingolipids in the cell membrane, the quinone type is ubiquinone Q10, and it is characterized by producing a yellow carotenoid pigment (Nostoxanthin). (Yabuchi, E., Yano, I., Oyaizu, H., Hashimoto, Y., Ezaki, T., and Yamamoto, H .: Proposal of s. Pingomospamubospaimopsia. sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhae iva sp. nov., Sphingomonas capsulata comb. nov., and two genospecies of the genus Sphingomonas. Microbiol. Immunol., 34, 99-119, 1990).
Recently, bacteria of the genus Sphingmonas include environmental hormones such as biphenyl (BP), organic chlorine compounds such as chlorophenol and hexachlorocyclohexane, aromatic hydrocarbons such as xylene, naphthalene, and phenanthrene, pesticides such as herbicides, and synthetic glycols such as polyethylene glycol. It has been shown that it has a strong resolution for a very wide variety of environmental pollutants such as molecular compounds, and is expected to find new resolution of environmental pollutants. Special issue, "The genus Sphingomonas", which summarizes the latest knowledge of Sphingmonas bacteria, Journal of Industrial Microbiology & Biotechnology, 23 (No. 4/5), 231-445. 1999). In Japan as well, sphingmonas bacteria have an extremely strong ability to degrade harmful environmental pollutants and are a group of bacteria that have attracted attention as a trump card for bioremediation. Therefore, research using the ability of sphingmonas bacteria for environmental purification Ecology (structure and function), resolution (enzymes and genes, molecular breeding), usage (introduction of bacteria or supply of nutrients to pollutants), engineering aspects (growth control, environmental control, complex Research on proposals for comprehensive research such as microbial control (Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research, specific area research) is being conducted (Symposium Scientific Research Grants-in-Aid for Scientific Research (C) ( Planning survey) "Environmental purification system construction platform using sphingomonas bacteria-sphingomonas bacteria and bioremediation Summary of Lectures, 2001). The symposium led to the establishment of the Environmental Microorganisms Research Group (Sphingomonas Research Group).
[0014]
Examples of the PAH-degrading bacteria of the genus Sphingomonas isolated from the marine environment include PHE-degrading bacteria (Komukai-Nakamura, S., Sugiura, K., Yamauchi-Inomata, Y., Toki, H., Venkaswar, Y.Kawashima, Venkaswar. J., S., Tanaka, H., and Harayama, S .: Construction of Bacterial Consortia that degrad Arabian light crude oil, J. Ferd., Bior. , Gallagher, E. and Shiaris, MP: Spatial and tem. The bacterial bacterium is known to have the following: bacterial variation of phenanthrene-degarding bacteria in interpartal seeds. Appl. Environ. Microbiol., 64, 2560-2565, 1998, and 2-methyatheniaz. Budzinski, H., Doumenq, P., Michote, V., and Bertrand, J.C .: Isolation and Characterization of a Marine Bacterium Pharmaceuticals. microbiol. Biotechnol., 48, 528-533 1997). In addition, among the bacteria belonging to the genus Sphingomonas obtained from the marine environment, bacteria decomposing various hydrocarbons such as aromatic hydrocarbons such as toluene and xylene in addition to PAH have been reported (R. Cazicchiol, F. Fegatala, M. Ostrowski, M. Eguchi, J. Gottschal: Sphingomonas from marine environmentals, Journal of Industrial Microbiology & Biotechnology, 72, 72-72, Biotechnology & Biotechnology, 72-68. Furthermore, Sphingomonas aromaticiborans F199 strain decomposes toluene, o, m, p-xylene, p-cresol, naphthalene, biphenyl (BP), dibenzothiophene, fluorene, salicylic acid, benzoic acid, etc., and is a single strain of various harmful substances. It has a resolution (JK Fredrickson, FJ Brockman, DJ Workman, SW Liand TO Stevens: Isolation and characterization of a gastrophysical sacrifice sabsorption professiona surafacturabacturaçafurasafurafuraçafurasafurafurafurafurafushifurasuka) Naphthalene, and other aromatic compounds, Appl. n. Microbiol., 57, 796-803, 1991; JK Fredrickson, DL Balkwill, GR Drake, MF Romine, DB Ringelberg and D.C.R. -Degrading Sphingomonas isolates from the deep subsurface, Appl. Environ. Microbiol., 61, 1917-1922, 1995; D.L.K.R.D.R.K.R.D.K.R.D.R.K.R.D. C. White, D. C. Ringelberg, D. P. Chandler, MF Romane, D. W. Ke nedy and C. M. Spadoni, Taxonomic study of aromatic-degrading bacteria from deep-terrestrial-subsurface sediments and description of Sphingomonas aromaticvorans sp. nov., Sphingomonas subterranea sp. nov. and Sphingomonas stygia sp. nov., Int. J. Syst. Bacteriol., 47, 191-201, 1997).
[0015]
The ANI7A strain and the ANI7P strain of the present invention are microorganisms isolated from a seawater sample in a coastal sea area contaminated by a spill of the Japan Sea oil spill, and both have the resolution of anthracene (AN) and phenanthrene (PHE). Therefore, it was inferred that they were working on the purification of oil spilled waters on site. It has also been reported that the ANI7A strain and the closely related species of the ANI7P strain, Sphingomonassubarctia, are polychlorophenol-degrading bacteria (Nohynek, LJ, Nurniaho-Lassila, E.L., Suhonen, EL. Busse, HJ, Mohammadi, M., Hantula, J., Rainey, F. and Salkinoja-Salonen, MS: Description of epson chlorop-n-Fond. genus Sphingomonas, Sphingomonas subarticas p. nov. Int. J. Syst. Bacteriol., 46, 1042-1055, 1996).
[0016]
From the above, the new microorganism belonging to the genus Sphingomonas of the present invention has the resolution of petroleum and harmful substances including PAH, and is a very useful microorganism that can be used for purification of the environment contaminated by these. In addition, since the Sphingomonas bacterium has a strong ability to decompose an extremely wide range of environmental pollutants as described above, the microorganisms of the sphingomonas genus of the present invention also have environmental hormones, organochlorine compounds, pesticides, synthetics, in addition to the PAH resolution. It can be said that it has the ability to degrade harmful environmental pollutants such as polymer compounds, and is an extremely useful microorganism, which can be used for environmental purification contaminated with harmful substances.
[0017]
[Other microorganisms]
Next, when the microorganism of the present invention is used for purification of an oily environment or an environment contaminated with harmful substances, the microorganism of the present invention may be used alone, or the microorganism of the present invention and, for example, the genus Alcanivorax of the aliphatic hydrocarbon-degrading bacterium may be used. It can also be used as an environmental pollutant-degrading microorganism consortia by mixing with bacteria, known hydrocarbon-degrading microorganisms, and microorganisms having the ability to degrade harmful environmental pollutants. Furthermore, in order to promote the efficiency of decomposing petroleum and other harmful pollutants, a composite microorganism obtained by adding a non-harmful pollutant-degrading microorganism that decomposes intermediate degradation products and metabolites of harmful pollutants to a degrading microbe consortium containing the microorganism of the present invention. A system can be constructed and used.
[0018]
〔Culture medium〕
The medium used for culturing the microorganism of the present invention or the environmental pollutant-degrading microbial consortia containing the microorganism of the present invention may be any medium as long as these microorganisms can be satisfactorily grown and can express PAH resolution or environmental pollutant resolution. A medium having a composition may be used. As a carbon source, PAH can be used as the only carbon source and energy source, but petroleum products including PAH, such as crude oil, kerosene, light oil, and heavy oil, and oil spills from ships and factories can also be used. . Further, carbohydrates, organic acids such as pyruvic acid, molasses and the like can also be used. These may be effective when used in combination with PAH or petroleum. In order to easily decompose hydrophobic PAHs and petroleums by microorganisms, a surfactant such as Tween 80 may be added to these, and then they may be emulsified and dispersed by an ultrasonic generator. PAH can be dissolved in a small amount of ethanol or acetone and added. The nitrogen source may be any organic / inorganic compound used for microorganisms. Examples of the organic nitrogen source include peptone, meat extract, constable liquor, defatted soybean, and kazenin, and examples of the inorganic nitrogen source include ammonium salts, nitrates, and urea. As the inorganic salts, various phosphates, sodium chloride, magnesium, iron, manganese, calcium, zinc, molybdenum and the like may be added. In addition, as growth factors, there are vitamins and amino acids, and natural organic nutrients containing the above nutritional factors such as meat extract, peptone, yeast extract, and constell liquor may be added.
When these microorganisms are efficiently cultured in large quantities, it is not always necessary to culture them in a medium containing environmental contaminants such as PAH, and an organic nutrient medium such as Marine Broth (Difco) or Nutrient Broth (Difco) may be used.
[0019]
(Culture method)
The culture is preferably performed under aerobic conditions, for example, a shaking culture method or an aeration stirring culture method.
Liquid static culture may be combined as appropriate, or liquid static culture may be used. The pH of the medium may be 5-9, preferably 6-8. The culture temperature may be 15-37 ° C, preferably 20-30 ° C.
[0020]
(Means for decomposing environmental pollutants using the microorganism of the present invention)
As a purification method using the microorganism of the present invention and a microbial consortia for decomposing environmental pollutants such as petroleum containing the microorganism of the present invention, there are methods for purifying oceans, lakes, marshes, rivers, waste liquids, etc., which are contaminated with environmental pollutants such as petroleum including PAH. A culture solution, viable cells, and freeze-dried cells of these microorganisms may be sprayed. In this case, a nutrient / microbial preparation obtained by mixing an organic or inorganic nutrient such as nitrogen or phosphorus with these microorganisms, or a nutrient-containing immobilized carrier using alginic acid disclosed by the present inventors (JP-A-2001-2001) 37466, published date: 2001.12.13), various microbial preparations in which these microorganisms are immobilized using known microbial immobilization carriers such as polyacrylamide gels and polyurethane foams can be used. In this case, it is preferable to simultaneously immobilize the nutrient source and these microorganisms.
[0021]
[Detection, quantification, screening, identification and environmental evaluation methods of environmental pollutant-degrading microorganisms]
Next, nucleic acid probes, methods for detecting and quantifying microorganisms degrading harmful environmental pollutants such as petroleum using them, screening methods, identification methods, and monitoring, analysis and evaluation methods for harmful pollutant contamination using these detection and quantification methods Will be described.
The aforementioned environmentally harmonious biological environment purification technology using pollutant-decomposing microorganisms, ie, bioremediation technology, includes nitrogen (N), phosphorus (P), etc., which are generally lacking in polluted environments such as the sea. (Biostimulation, Biostimulation) and a method of spraying a biodegradable microbial preparation or the like (Bioaugmentation, R) M. Atlas and R. Bartha: Hydrocarbon Biodegradation and Oil Spill Bioremediation. Advances in Microbiological Economy (ed. K. C. Marshall Pall). ess, New York, 12, 287-338,1992, K Lee et al .: Bioaugmentation and biostimulation:.. a paradox between laboratory and field results In Proceedings of the 1997 International Oil Spill Conference, p 697-705, AmericanPeteroleum Institute, Washington, DC, 1997). In order to establish this environmental purification technology, when nutrient sources and microorganisms are sprayed, qualitatively and quantitatively ascertain the microorganisms that decompose harmful substances such as petroleum in the on-site environment, and analyze the behavior and functions of the microbial communities involved in the purification. Need to be clarified. It has been reported that petroleum-degrading microorganisms are most frequently distributed in coastal waters and seaway waters contaminated with petroleum (Atlas, RM: Microbial degradation of petroleum hydrocarbons: an environmental perspective.). Rev., 45, 180-209, 1981).
For example, the ratio of hydrocarbon-degrading microorganisms generally distributed in non-polluted seas is less than 1% of the total number of microorganisms, but it is said that the ratio is often 10% or more in oily waters (RM Atlas: Petroleum). biodegradation and oil spill bioremediation, Marine Pollution Bulltetin, 31, 178-182, 1995, R M. Atlas and R. Bartha:.. Hydrocarbon Biodegradation and Oil Spill Bioremediation In:. Advances in Microbiolbial Ecology, Ed K. C. Marshall, PlenumPress, New York, 12, 87-338, 1992). In general, it is said that the ratio of degrading microorganisms in the entire microbial community reflects the degree of contamination of harmful substances such as petroleum and serves as an index (Atlas, RM: Microbial degradation of petroleum hydrocarbons: an environmental perspective. , 45, 180-209, 1981).
[0022]
The number of PAH-degrading bacteria in harbor sediments contaminated with creosote (containing about 85% polycyclic aromatic hydrocarbons [PAHs]) discharged from wood processing facilities has been investigated (A D. Geiselbrecht et al, Enumeration and Phylogenetic Analysis of Polycyclic Aromatic Hydrocarbon-Degrading Maine Bacteria from Pounds. According to this, the number of PAH-degrading bacteria counted by the MPN culture counting method was 104 to 107 MPN / g (dry weight) at the contaminated site and 103 to 104 MPN / g (dry weight) at the non-contaminated site. . On the other hand, the total number of bacteria in the sediment was about 109 / g (dry weight), and there was little difference between the contaminated site and the non-contaminated site. That is, at the creosote-contaminated site, not only the number of PAH-degrading bacteria than that of the non-contaminated site was 10 to 1000 times higher than that of the non-contaminated site, but the ratio of the PAH-degrading bacterium to the total microbial community increased to about 1%. Thus, it has been found that the ratio of degrading bacteria in the microbial community is increasing, reflecting that the environment has been polluted, and the ratio of degrading bacteria is considered to be a good indicator of the polluted environment. .
[0023]
However, the conventional method of counting microorganisms decomposing harmful substances such as petroleum and PAH is based on a culture method using a plate culture method or an MPN (most probable number) method as described above. All of these culture methods require a great deal of labor and time. In addition, in the counting method using an agar plate medium, bacteria in a sample grow on the control medium containing no hydrocarbon by utilizing impurities in the agar to form colonies. In counting using an agar plate medium supplemented with yeast extract (0.01%) as a trace amount of growth factors, minute colonies were formed in both the hydrocarbon medium and the medium without hydrocarbon, and the number and the number of colonies were small. It is said that it was not possible to clarify the difference between hydrocarbon-degrading bacteria and non-degrading bacteria in terms of size (T. Higashihara, A. Sato and U. Shimidu: An MPN method for the enumeration of marine hydracrobatic hydracrodiabatica). Bull. Japan. Soc. Sci. Fish., 44, 1127-1134, 1978). Based on the above, even in the control medium containing no hydrocarbon, there are bacteria that form colonies using a trace amount of organic matter in the agar. Therefore, specific hydrocarbon-degrading bacteria can be selectively and accurately determined on the agar plate medium. Is difficult to count.
In addition, in order to detect specific degrading microorganisms, it is necessary to separate the target microorganisms by the agar plate method and perform classification and identification (genus, species level). It is difficult to identify. Furthermore, among microorganisms that live in nature, the number of microorganisms that can be detected by these conventional separation and culture methods is extremely small. That is, a direct microscope counting method of staining with a fluorescent DNA stain and counting under a microscope (for example, JE Hobbie, RJ Daley, and S. Jasper: Appl. Environ. Microbiol. 33: 1225-1228, 1977 and KG Porter and YS Feig: Limnol. Oceanogr. 25: 943-948, 1980), the ratio of microorganisms that can be separated and cultured is 1% or less. (RI Amann, W. Ludwig and KH. Schleifer: Phylogenetic Identification and In Situ Detection of Individual Microbial). out Cultivation, Microbial. Rev., 59, 143-169, 1995). Therefore, the conventional cultivation method can only investigate about 1% of the microorganisms inhabiting the on-site environment for specific decomposed microorganisms and microflora, and the degraded microorganisms concentrated on environmental microorganisms are not sufficiently reflected. There were drawbacks.
[0024]
In recent years, the development of molecular microbial ecology based on molecular biological techniques has enabled the analysis of microbial community structure and diversity in the environment at the molecular and cellular levels without relying on isolation and culture methods as in the past. (IM Head, JR Saunders and RW Pickup: Microbial evolution, diversity, and economization: a large amount of general anatomical RNA analysis. 21, 1998. Kazuya Watanabe, Hiroyuki Futama: Microorganisms in the Environment, Chemistry and Biology, 38, 230-236, 2000, Akihiko Maruyama: Analysis of Marine Microorganisms at the Molecular and Cellular Levels, Marine Microorganisms, Monthly Ocean Extra edition No.23, 162-170, 2000,
Hidetoshi Urakawa, Koichi Owada: Culture-independent microbial community analysis using nucleic acids, marine microorganisms, Monthly Ocean, Extra. 23, 176-182, 2000). The microbial community analysis method using this molecular biological technique is indispensable for understanding the behavior of degraded microbial communities and changes in the microbial community structure during the environment remediation process using toxic substances such as petroleum and bioremediation technology. Recently, these technologies are being developed.
Recently, molecular genetic detection and quantification that does not depend on conventional separation and culture methods has been attempted for specific microorganisms such as whole microorganisms and environmental pollutant-degrading microorganisms using specific DNA probes. I have. For example, in the case of detecting a specific microorganism such as a degrading microorganism at a cell level regardless of the separation / culture method, a fluorescence in situ hybridization (FISH method) is used (R.I. Amann, W. Ludwig and KH Schleifer: Phylogenetic Identification and In Situ Detection of Individual, Microbial Cellular, Multicultural Inc., 14th ed. That is, by performing FISH using a DNA probe, it is possible to specifically detect and count only specific degraded microbial cells concentrated in a microbial community. By comparing with the total number of bacteria obtained by the direct microscope counting method, it is possible to calculate the quantitative ratio of the specific microorganism in the total concentration of all the microorganisms. Furthermore, when analyzing the ratio of specific microorganisms such as degrading microorganisms in the entire community at the cell level for water environment samples, the FISH-DC method is effective (A. Maruyamaand M. Sunamura: Simultaneous direct counting). of total and specific microbiological cells in seawater, using a deep-sea microbeas as a biomarker. Applied and Environmental Microbiology 21; As a quantitative analysis technique of specific degrading microorganisms at a molecular level using a DNA probe, there is a nucleic acid hybridization method (DA Stahl, B. Flesher, HR Mansfield and L. Montgomery: Use). Appl. Environ. Microbiol., 54, 1079-1084, of phylogenetically based hybridization probes for studies of ruminal microbiology. In this method, nucleic acid is extracted from an environmental sample or a microorganism sample, the nucleic acid sample is immobilized on a nylon membrane filter, and then a DNA probe labeled with a radioisotope (RI) or the like is added, and hybridization is performed. This is a method for measuring the radioactivity of a labeled probe complementary to a nucleic acid immobilized on a probe, and quantifying a specific microorganism from the concentration of the nucleic acid specifically bound to the probe.
[0025]
Recently, Maruyama et al. Have developed a relative molecular quantification method using a fluorescent dot blot hybridization method without using RI (A. Maruyama, K, Kitamura, H. Ishiwata, M. Matsuuo, T. Higashihara, and R. Kurane). : Molecular specification and quantification of predominant microbes in oil contaminated seawater, 9th International Symposium, Microbial Ecosystem, Microbiology and Biotechnology, 9th International Symposium on Microbiology. , 60, 31-34, 2002).
From the above, in the present invention, a DNA probe capable of specifically detecting and counting the above novel microorganism of the present invention is newly produced. Hereinafter, the step of preparing a DNA probe will be described.
[0026]
〔probe〕
RNA and DNA probes suitable for various uses can be designed based on the base sequence information of each 16S rDNA of the ANI7A strain and / or the ANI7P strain (ANI7A strain; SEQ ID NO: 1, ANI7P strain; SEQ ID NO: 2). . The nucleotide sequence and length of the probe may be appropriately selected according to the range of microorganisms of the genus Sphingomonas to be detected, quantified, screened, or identified. For example, when it is desired to screen only the above-mentioned novel microorganism of the present invention, a probe may be designed based on the nucleotide sequence of a specific portion of the 16S rDNA of the microorganism, and the screening range is to be expanded to include closely related species. Sometimes, by designing a probe such as shortening the length of the base sequence to include a base sequence portion common to 16S rDNA of a related species, and further by selecting the base sequence portion or adjusting the length thereof, If the strain specificity of the probe is reduced, a wider range of useful bacteria can be screened.
The probe of the present invention can be used, for example, by a FISH method (fluorescence in situ hybridization) in a sample (for example, an environmental sample water such as sea, river, lake, marsh, wastewater or wastewater contaminated with toxic substances such as petroleum), or a large number of microorganisms. For detecting and / or quantifying the novel microorganism of the present invention, a closely related species thereof, or a petroleum-degrading bacterium of the closely related species belonging to the genus Sphingomonas, or screening, for example, the sequence It corresponds to the region selected from the region of base number 565-615 of the base sequence of No. 1 (the position from the 5 ′ end in the base sequence of 16S rDNA of Escherichia coli (region of 630-680 in the numbering system)). Base length of 10-50 bp, preferably base length 5-25bp of the probe may be designed. As an example, the following probes can be mentioned.
(1) 5'-CGCCTCTCCCAAGATTCTAGCGACCT-3 '(SPG645 *, 25mer) (SEQ ID NO: 3)
3'-GCGGAGAGGGTCTTAAGATCGCTGGA-5 '
(2) 5'-CCAAGATTCTAGCGACC-3 '(SPG643 *, 17mer) (SEQ ID NO: 4)
3′-GGTTCTAAGATCGCTGG-5 ′
(3) 5'-TAGCGACCTAGTTTCAAGG-3 '(SPG634 *, 20mer) (SEQ ID NO: 5)
3'-ATCGCTGGATCAAAGTTTCC-5 '
(Note that * (number) indicates the position (numbering system) from the 5 ′ end in the 16S rDNA base sequence of Escherichia coli (Noler HF and CR Woese, 1981. Science, 212: 403-). 411).
The probe can be synthesized by a known method, for example, a phosphoramide method or a triester method. Or you may synthesize | combine by a DNA automatic synthesizer.
[0027]
Probes include isotopes (32P, 35S, etc.), fluorescent dyes (biotin / avidin, digoxigenin / anti-digoxigenin-rhodamine,
Fluorescein-isothiocyanate (FITC), LuciferYellow CH, Rhodamine 123, Acridine orange, Pyronin Y, Ethidium bromide, Propidium iodide, Ethidium homodimer, BOBO-1, POPO-1, TOTO-1, YOYO-1, Carboxyfluorescein diacetate (CFDA), Fluorescein diacetate (FDA), Carboxyfluorescein diacetate-acetoxymethylester (CFDA-AM), 5-cyano-2,30 ditolyltetrazolium chloride (CTC), Tet amethylrhodamine isothiocyanate (TRITC), Sulforhodamine 101 acid chloride (Texas Red), Cy3, Cy5, etc. Cy7,2-hydroxy-3-naphtoic acid-2'-phenylanilidephosphate (HNPP)), may be labeled with a chemiluminescent.
[0028]
[Screening, detection and quantification of useful bacteria of the genus Sphingomonas]
Using the RNA or DNA probe of the present invention, various hybridization methods (Southern blotting, Northern blotting, colony hybridization, dot hybridization, in situ hybridization (for example, FISH method), etc.) belong to the genus Sphingomonas. The novel microorganism of the present invention, its closely related species, or petroleum-degrading bacteria of the closely related petroleum-degrading bacteria species can be detected and / or quantified or screened.
An example of a method for detecting and quantifying microorganisms that degrade pollutants such as petroleum from on-site water or seawater contaminated with petroleum or environmental pollutants using the DNA probe of the present invention will be described below. A DNA probe having a base sequence of SEQ ID NO: 3 obtained by collecting a water or seawater sample from a harmful substance contamination site, fixing microorganisms present in the sample on a filter (pore size 0.2 μm), and labeling this with a fluorescent dye or the like. After washing off the probe, the probe is observed with a fluorescence microscope to hybridize with the DNA probe and selectively detect or count specific degraded microorganisms exhibiting labeled fluorescence.
As described above, if the environment is polluted, the ratio of the decomposing bacteria group increases, so that it can be used as an index of environmental pollution.
[0029]
In addition, using the DNA probe of the present invention, a colony hybridization technique, a blot hybridization technique, a flow cytometry method, etc., from a large number of microorganisms, a new microorganism of the present invention of the genus Sphingomonas and its closely related species, and It is possible to screen for a petroleum-degrading bacterium of the genus Sphingomonas, particularly a new microorganism of the present invention belonging to the genus Sphingomonas and a petroleum-degrading bacterium related thereto.
[0030]
[Identification of Sphingomonas sp.]
Furthermore, using the nucleotide sequence information of SEQ ID NO: 1 or 2 or SEQ ID NO: 3, 4 or 5, a new microorganism of the present invention belonging to the genus Sphingomonas and its related species, and a new microorganism of the present invention belonging to the genus Sphingomonas and its It is possible to identify closely related bacteria that degrade toxic substances such as petroleum. For example, they are homologous bacteria by homology with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or by DNA / DNA or DNA / RNA hybridization using the RNA or DNA probe according to any one of claims 5 to 7. Can be identified. Furthermore, bacterial species can also be identified by performing PCR using the nucleotide sequence (DNA fragment) of the probe as a primer. That is, the cells to be identified are lysed, and a DNA fragment having the nucleotide sequence of the probe is added as a primer, followed by PCR amplification. If the amplification of 16S rDNA is confirmed by electrophoresis or the like of the PCR product, the target bacterium has a gene portion complementary to the DNA fragment used. That is, it can be specified that they are the same kind of bacteria.
[0031]
[Monitoring, analysis, and evaluation of pollutant environments]
If it becomes possible to easily and quickly monitor the behavior of specific degrading microorganisms, which are indicators of the environment contaminated with petroleum and other harmful substances, and their proportion in the total microbial community (predominance), the degree of contamination will be improved. The diagnosis of the polluted environment, such as the degree of restoration and recovery of the polluted environment, can be performed with high accuracy and at an early stage. For example, if the prevalence of petroleum-degrading bacteria and PCB-degrading bacteria increases in the environment, it can be judged that the environment is highly likely to be contaminated with petroleum or PCB, and that the microbial community as a whole It can be determined that the resolution of petroleum and PCB has increased. Furthermore, if the change in dominance is monitored over a long period of time and the periodicity and seasonality of the change are grasped, whether the change is sudden or not, the load is a ship accident or the inflow of factory drainage. It can be estimated whether it is artificial or not. By examining the dominance of PAH-degrading microorganisms in the concentration of all microorganisms in an oily environment using the DNA probe of the present invention, it is possible to determine the degree of contamination such as the hydrocarbon component, PAH ratio, concentration, and fate in the environment. It is possible to analyze and evaluate the natural purification process of pollutants and the bio-environmental restoration process.
[0032]
The present invention will be described below in detail with reference to examples.
Embodiment 1
1) Source sample of PAH-degrading microorganism
The most polluted point on the coastal area of Ishikawa Prefecture in the Japan Sea heavy oil spill, Stn. At 19 (Suzu West Coast, Nagahashi), mineral nutrients were added to a seawater sample collected on June 11, 1998, and 0.5% C heavy oil was added to the system (SW + N + P) (Table 2-A), and The cells were cultured at 65 ° C. for 65 days to perform a decomposition test.
In the 65-day MPN counting culture of the number of degrading bacteria in the culture for culture of degradation test (medium: Table 2-C), the culture of the test tube with the highest dilution stage, which showed good growth in heavy oil C, was added to 0.1 ml. After inoculating the medium containing 5% C heavy oil (SW + N + P) and repeating the enrichment culture three times at 20 ° C. for a culture period of 12 to 17 days, the NSW medium further containing C heavy oil (Table 2-B) was used. In the same manner as described above, a culture solution obtained by repeating the enrichment culture four times in a culture period of 9 to 17 days was used as a seed for the enrichment culture of the following AN-degrading bacteria.
[Table 2]

2) Enrichment culture of PAH-degrading bacteria
Using 0.1 ml of the C-heavy oil-enriched culture as a seed bacterium, inoculate a large test tube containing 10 ml of NSW medium (see Table 2-B) supplemented with 0.1% (w / v) AN, and then incubate at 20 ° C. for 8 days. An enrichment culture of AN-degrading bacteria was performed by shaking culture.
3) Plate medium used for isolation of PAH-degrading bacteria and culture method
Separation of AN-degrading bacteria and PHE-degrading bacteria was carried out by adding 0.1% (w / v) AN to NSW agar medium (NSW + AN) plate medium (Table 2-D), and adding AN or PHE to 2% agar NSW underlayer medium. Using a two-layer plate medium (Table 2: NSW + AN two-layer medium, NSW + PHE two-layer medium) and a Marine Agar 2216 (manufactured by Difco) (MA) plate medium covered with a 1% agarose NSW upper medium supplemented with an ethanol solution of The plating was performed at 20 ° C. In addition, the morphology of the colony formed on the plate medium was observed with a stereoscopic microscope.
The NSW + AN two-layer plate medium used was the following 2% agar NSW lower layer medium (1) (dish, 9 cm in diameter) and 1 ml of agarose NSW upper layer medium (2) 5 ml (about 50%) supplemented with anthracene (AN). (Preserved at ℃) was quickly added to uniformly spread the upper medium over the entire surface of the lower medium.
(1). 2% agar NSW lower layer medium
Table 2 A plate medium is prepared by adding 15-20 ml of a medium obtained by adding 2% concentration of agar to an NSW medium to a petri dish having a diameter of 9 cm.
(2). 1% agarose NSW upper layer medium with AN
A test tube containing 5 ml of NSW medium supplemented with 1% agarose was sterilized in an autoclave, kept at about 50 ° C., and sterilized in a 0.1% AN ethanol (99.5%, dehydrated ethanol) solution sterilized by filtration in the test tube. Add 2 ml and stir quickly to disperse evenly.
The method for preparing the present two-layer plate medium was performed according to the method described in the following literature.
Bogardt, A .; H. and Hemmingsen, B .; B. : Enumeration of phenanthrene-degrading bacteria by an overlayer technology and and its uses in evaluation of petroleum-contained sites. Appl. Environ. Microbiol. , 58, 2579-2582 (1992).
NSW medium: See literature (T. Higashihara, A. Sato and U. Shimizu: An method for the enumeration of Marine Hydrocarbon degrading bacterium, Contracting Jatar proofing bacteria, Bureau of Petrochemicals, Bureau of Biotechnology, Australia).
[0033]
4) Isolation of PAH-degrading bacteria
The enriched culture solution of the AN-degrading bacterium of the item 2) was spread on a (NSW + AN) plate medium, and cultured at 20 ° C. for 8 days. The colonies formed in the plate culture medium were picked, and the isolated strains were subjected to plate separation culture twice using (NSW + AN) plate medium and MA plate medium to isolate 16 strains. The plate culture was performed at 20 ° C. for 16 days.
Furthermore, these isolated strains were shake-cultured for 40 days in a screw-cap test tube (18 mm in diameter) to which 5 ml of NSW medium supplemented with 0.1% AN was added. An ANI7 strain which produced a presumed pale purplish pink dye and was suggested to have AN degradability was found. This strain is a strain isolated from the NSW + AN plate culture medium.
[0034]
5) Pure isolation of ANI7 strain
The above ANI7 strain was shake-cultured for about 1 to 1.5 months using an NSW medium supplemented with 0.1% AN or 0.1% PHE, and the culture was spread on an MA plate medium. As a result of the culturing, two types of colonies, a yellow-colored colony (A type) and a pale-colored colony (P type), were mixed in any of the culture solutions. Therefore, pure isolation of the present strain was performed. The pure separation is performed by repeating several times the plate culture using the MA plate medium, the NSW + AN plate medium, and the NSW + AN bilayer medium and the liquid culture using the NSW medium supplemented with 0.1% AN or 0.1% PHE. Was. In addition, A type ANI7A strain (Accession No. FERM P-19095) and ANI7P strain ((Accession No. FERM P) were used as pure cultures from a liquid culture system using AN-supplemented NSW medium and PHE-supplemented NSW medium. In addition, in addition to the above-mentioned plate medium, a NSW + PHE two-layer medium was also used for the purity test of the isolated strain.
In addition, the purely isolated ANI7A strain and ANI7P strain are both a pale pink-Pale purplish pink dye which is presumed to be derived from a partial oxide of AN in liquid culture using an NSW medium supplemented with AN, and a NSW medium supplemented with PHE. Produced a Yellow-Pale Yellow Orange dye. Furthermore, as a result of examining the PAH degradability of both strains, it was confirmed that both strains decomposed AN and PHE (see Example 2). In addition, for the ANI7A strain, a pale yellow green-Pale yellow dye, which is estimated to be a partial oxide of BP, was produced by liquid culture using an NSW medium supplemented with biphenyl (BP).
[0035]
Embodiment 2
For the PAH degradation test of the ANI7A strain and the ANI7P strain, an NSW medium (Table 2-B) supplemented with 0.1% (w / v) AN or 0.1% (w / v) PHE as a carbon source was used. . Add 5 ml of this NSW medium supplemented with AN or PHE to a screw-cap test tube with a cap (diameter: 18 mm), or add 10 ml of the NSW medium to an L-shaped test tube with a silicocene (diameter: 18 mm), and culture at 20 ° C. with shaking. (45 rpm).
The quantitative determination of AN and PHE in the culture solution was performed by extracting twice with the same amount of dichloromethane as the culture solution, and then adjusting the volume to 25 ml. A certain amount of this extract was analyzed by gas chromatography (GC) under the following conditions. In addition, n-hexadecane was used as an internal standard substance.
GC analysis conditions (Main body: Shimadzu GC-17A)
1. column
Liquid phase: TC-70 (capillary column manufactured by GL Science); Length: 30 mx 0.25 mm;
Liquid phase thickness: 0.25 μm
2. Carrier gas (N2)
Flow rate: 1 ml / min
3. Measurement condition
Sample inlet temperature: 250 ° C
Initial temperature: 120 ° C
Heating rate: 10 ° C / min (up to 260 ° C); Hold at 260 ° C for 8 minutes
Detector: FID
The hydrocarbon degradation rate was determined based on the amount of residual hydrocarbons in a control medium without inoculation of the bacteria cultured under shaking under the same conditions. In addition, the decomposition rate was shown by the average value of the respective decomposition rates of three or four test tubes under the same culture conditions.
[0036]
When shaking culture was performed at 20 ° C. for 60 days in an L-shaped test tube (18 mm in diameter) with silicocene, the degradation rates of AN and PHE of the ANI7A strain were 8% and 21%. When shaking culture was performed at 20 ° C. for 34 days using a screw-cap test tube (18 mm in diameter) with a cap, the degradation rate of PHE of the ANI7A strain was 22%. Furthermore, the PHE degradation rate of the ANI7P strain under the latter culture conditions was 19%, which was almost the same as that of the ANI7A strain. The PHE degradation rate of the microbial consortia composed of both strains was 23% under the latter culture condition, and there was no significant difference from the degradation rate of each single strain, and the effect of promoting the PHE degradation by the consortia combining both strains was observed. Did not. Although the ANI7A strain and the ANI7P strain differ in the color of the colony, they were considered to be the same species of the genus Sphingomonas from molecular phylogenetic analysis, and it was inferred that there was no significant difference in PHE resolution.
The ANI7A strain was estimated to be a partial oxide of BP from day 1 of culture under the former culture conditions in an L-shaped test tube with silicocene using NSW medium supplemented with 0.1% (w / v) BP. A pale yellow green-Pale yellow dye was produced. , ANI7P strain was not subjected to this BP degradation test. However, as described above, the AN17A strain and the AN17P strain are both of the same bacterial species and degrade PHE. Therefore, it is considered that the AN17P strain also has BP degradability. .
[0037]
Embodiment 3
Sphingomonas sp. Preparation of DNA probe for detecting ANI7A strain
A PAH-degrading bacterium, Sphingomonas sp., Which was successfully isolated purely through enrichment culture from a sample collected from a coastal area contaminated with oil spills from Ishikawa Prefecture. From the 16S rDNA nucleotide sequence information (SEQ ID NO: 1) of the ANI7A strain (FERM P-19095), Stahl and Amann (Development and Application of Nucleic Acids Acid Probes. In: Nucleic Acid Essentials. and M. Goodfellow, John Wiley and Sons, Chichester, pp. 205-248, 1991), and select a sequence specific to this bacterial species from a region in which no disorder due to higher-order structures is considered to be observed. And synthesizing an oligonucleotide having the sequence, and adding a 5′-terminal fluorescent dye such as TRITC or Cy5. Specific radioactively labeled DNA probe (SPG645) having the sequence shown in SEQ ID NO: 3 and finally specific radioactively labeled DNA probe (SPG643) shown in SEQ ID NO: 4 and SPG634 (SPG634) ) Was prepared.
[0038]
Embodiment 4
Detection and counting method of Sphingomonas microorganism using the DNA probe described in Example 3
In using each of the above DNA probes of Example 3, Sphingomonas sp. . ANI7A strains the target microorganisms, Sphingomonas subterranea IFO16086 (standard strain) as the target microorganisms such as, Maruyama and Sunamura (Simultaneous direct counting of total and specific microbial cells in seawater, using a deep-sea microbe asbiomarker Applied and Environmental Microbiology, 66: 2211-2215, 2000), and its effectiveness was actually confirmed by the FISH method. The method of culturing, fixing, and hybridizing the test strain was based on JP-A-2000-333680. However, the hybridization at this time was performed at 42 ° C. in the presence of 50% formamide, and the washing treatment was performed at 42 ° C. Table 3 shows the effectiveness of the probe by observing the sample with a fluorescence microscope.
[Table 3]

[0039]
In the case of the probes SPG645, SPG643, and SPG634 designed this time, as a result of DAPI being universally bound to DNA in each cell by UV excitation, blue fluorescence derived from DAPI showed two types of Sphingomonas subterranea (ANI7A and control strains) that served as control strains. IFO16086) and Psychobacter pacificensis NIBH P2K18). However, when the same visual field was observed with G excitation, only the ANI7A strain had a sequence complementary to the probes SPG645, SPG643, and SPG634, and thus emitted red fluorescence derived from TRITC that labeled the 5 ′ end of the probe.
On the other hand, in the case of the probe EUB338 specific to the Bacteria domain, the ANI 7A and the two kinds of control strains emitted green fluorescence derived from FITC labeled with the 5 'end of EUB338, respectively, even when observed under B excitation.
From the above, the three probes designed from the 16S rDNA base sequence of the microorganism derived from the oil spill environment were used to specifically detect the ANI7A strain or its closely related species, and to determine the binding due to its higher-order structure. No interference was seen, indicating that it was actually very effective.
[0040]
【The invention's effect】
The present invention provides a novel microorganism belonging to the genus Sphingomonas, which has the resolution of environmental pollutants, particularly petroleum, waste, or harmful environmental pollutants such as PAH contained therein, and utilizes the novel microorganism of the present invention. It is possible to efficiently purify oceans, lakes, marshes, rivers, waste liquids and the like contaminated with the environmental pollutants. Further, according to the present invention, a probe prepared from the 16S rDNA / RNA of the novel microorganism makes it possible to easily and quickly detect and quantify a bacterium belonging to the genus Sphingomonas having the ability to degrade environmental pollutants. Monitoring and analysis / evaluation / diagnosis of PAHs and other harmful substances, which have become a global environmental issue, through the detection and quantification methods of the environment, and the purification and restoration process of PAHs and other harmful substances using bioremediation technology. A very useful technology that can be analyzed and evaluated was provided.
[0041]
[Sequence list]

[Brief description of the drawings]
FIG. 1. Sphingomonas sp. It is a molecular phylogenetic tree created based on the molecular phylogenetic analysis of each 16S rDNA of the AN17A strain and the AN17P strain.

Claims (25)

A novel microorganism belonging to the genus Sphingomonas, wherein the base sequence of the 16S rRNA gene (16S rDNA) shows 97% or more homology with the base sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. Sphingomonas sp. ANI7A strain or Sphingomonas sp. ANI7P strain 3. The microorganism according to claim 1, which has a polycyclic aromatic hydrocarbon resolution. 16S rDNA having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. An RNA or DNA probe having a part of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a part of a corresponding riboxynucleotide sequence and having a base length of 10 to 50 bp. The probe according to claim 5, which specifically hybridizes with the microorganism-derived RNA or DNA according to any one of claims 1 to 3. 7. The RNA or DNA probe according to claim 5, which has the nucleotide sequence of SEQ ID NO: 3, 4, or 5, or a riboxynucleotide sequence corresponding thereto. The RNA or DNA probe according to any one of claims 5 to 7, which is used for detecting or quantifying an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas. The RNA or DNA probe according to any one of claims 5 to 7, wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium. Sphingomonas sp. ANI7A strain, or Sphingomonas sp. The RNA or DNA probe according to claim 6 or 7, which is used for detecting and / or quantifying the ANI7P strain. A method for detecting and / or quantifying bacteria belonging to the genus Sphingomonas using the RNA or DNA probe according to any one of claims 5 to 7. The method according to claim 11, wherein the bacterium detected or quantified is an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas. The method according to claim 12, wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium. The method according to claim 11, wherein the bacterium to be detected and / or quantified is the microorganism according to any one of claims 1 to 3. A method for screening a useful bacterium belonging to the genus Sphingomonas using the RNA or DNA probe according to any one of claims 5 to 7. The method according to claim 15, wherein the bacterium to be screened is an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas. The method according to claim 16, wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium. The method according to claim 15, wherein the bacterium to be screened is the microorganism according to any one of claims 1 to 3. DNA / DNA or DNA / RNA hybridization using the RNA or DNA probe according to any one of claims 5 to 7, or a primer using the DNA probe as a homology to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method for identifying a bacterium belonging to the genus Sphingomonas according to any one of claims 1 to 3, wherein PCR is performed using the bacterium. The method according to claim 19, wherein the bacterium to be identified is an environmental pollutant-degrading bacterium belonging to the genus Sphingomonas. The method according to claim 20, wherein the environmental pollutant-degrading bacterium belonging to the genus Sphingomonas is a petroleum-degrading bacterium. A method for purifying a polluted environment, comprising treating an environment contaminated with an environmental pollutant with the microorganism according to claim 1. 23. The method according to claim 22, wherein the environmental pollutant is petroleum or petroleum-derived. A method for monitoring, analyzing, evaluating and diagnosing a toxic substance-contaminated environment using the method according to any one of claims 11 to 14. A method for analyzing and evaluating a process of purifying and / or restoring a toxic substance-contaminated environment using the method according to any one of claims 11 to 14.

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