Geobacillus thermoleovorans B23の三連型アルカン酸素添加酵素に関する遺伝子クローニングと機能解析

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23 which was isolated from a deep subterranean oil reservoir at Niigata, Japan, is capable of degrading broad range alkanes (C11-C32) at 70℃ by terminal oxidation pathway, followed by β-oxidation pathway. Whole genome sequence analysis revealed that B23 did not have alkB-type alkane monooxygenases genes like most alkane degrading bacteria but it carried three gene homologs namely ladAαB23, ladAβB23 and ladBB23 on its chromosome in a unique region, namely “ladAB gene island”. The amino acid sequences deduced from these three gene homologs were significantly similar to FMN- dependent alkane monooxygenase, LadA, encoded in a plasmid of Geobacillus thermodetrificans NG 80-2 with 50%, 33% and 21% identity, respectively. I also found a gene encoding FMN reductase in the upstream of ladAB gene island, which provides reduced flavin for the monooxygenase enzyme to activate oxygen molecule in alkane oxidation reaction.In order to confirm that the three gene products are active alkane monooxygenases, heterologous expression of ladAαB23, ladAβB23 and ladBB23 have been performed in Pseudomonas fluorescens KOB2Δ1. The strain KOB2Δ1 is a mutant of alkane degrading P. fluorescens CHA0 with alkB1 deletion which no longer grows in medium containing C12 - C16 of n-alkanes as sole carbon sources. It was found that all these gene homologs functioned in P. fluorescens KOB2Δ1 and partially restored alkane degradation activity. Alkane degradation test at 35℃ for 8 days revealed that all the recombinants harboring either ladAαB23, ladAβB23 or ladAγB23 degraded more C12-C23 alkanes than vector only. Alkane degradation activity of the three recombinant strains was ladAαB23< ladAβB23 < ladBB23. It was also found that the ladAB gene structure is widely shared in Geobacillus strains. Here, I report that a group of G. thermoleovorans utilizes multiple LadA-type alkane monooxygenases for degradation of wide range alkanes.即効性のある環境保全対策のひとつとして、有害な化学汚染物質の浄化技術開発は不可欠である。ここで生物作用を利用するいわゆる生物環境修復技術は省エネかつ環境への負荷が少ない技術として注目されている。原油は油田採掘から輸送の過程において大規模な漏出事故が起こりやすく、これまでも多くの環境汚染を引き起こしてきた。この漏出原油汚染浄化に有効な生物がアルカン分解微生物群であり、その生理生態を理解することは生物環境修復技術開発の重要な科学基盤となる。1895年にわが国の植物学者三好 学によって、ブドウ果実表皮のパラフィンを分解するカビが発見されて以来、さまざまな環境からアルカン分解微生物が見つかっている。 Geobacillus thermoleovorans B23 は、新潟県の深度地下油田から発見した高度好熱性アルカン分解細菌である。本細菌のユニークな特徴は70℃という高温において炭素数11から32程度までの広範囲にわたる鎖長のアルカンを分解できることである。本論文は、B23株の新規アルカン分解機構を解明することを目的として実施した研究についてまとめたものである。まず第一章において本研究の背景とその意義について論述した後、B23株のほぼ全長ゲノム（3.35Mb）の塩基配列を決定した。その結果、3,349個のタンパク質コード遺伝子を見出した。これらを同属のアルカン非分解細菌ゲノム配列と詳細に比較することによって、B23株に特徴的な11.8Kb領域内にフラビン依存性 LadA 型のアルカン分解遺伝子（アルカン酵素添加酵素遺伝子）が３つ連続（三連）して存在する（IadAαb23, ladAβB23, ladAγB23）ことを見出した。また、その酵素活性発現に不可欠なフラビンモノヌクレオチド還元酵素遺伝子も同じ領域内に見出すことに成功した。さらに、Geobacillus属 細菌群におけるアルカン分解遺伝子群の多様性を系統解析した結果、鉄依存性 AlkB 型遺伝子とフラビン依存性 LadA 型遺伝子の分布は種を問わないことを発見した。　次に、第二章ではこれらの遺伝子の機能を確認するために、それぞれの遺伝子を発現ベクターにクローニング後、アルカン分解活性を有さない常温性細菌　Pseudomonas fluorescens KOB2Δ1 に移入した。KOB2Δ1は70℃で生育できないため、通常生育温度の30℃と最高生育温度である35℃において各遺伝子の発現ならびに活性を比較評価した。その結果、ladAαB23, ladAβB23, ladAγB23 遺伝子が基質となるアルカンの鎖長ごとに役割を分担している訳ではないことがあ示唆された。ただし、これは反応温度が、35℃であるが原因かも知れない。さらに興味深いことに、これら３種類の遺伝子のなかでは、既知の ladA と進化系統的に遠縁の ladAγB23が最も高い活性を有していることが判明した。第三章ではB23株と同属である高度好熱性アルカン非分解菌 Geobacillus kaustophilus MK93における異種遺伝子発現の試みについて記述した。第四章では、本研究成果を総括すると共に今後の展望について述べた。本論文では、これまでプラスミド遺伝子としてしか報告のなかったフラビン依存性 LadA 型のアルカン酸素添加酵素遺伝子が機能を持った状態で一つの細胞のゲノム内に三連で存在することを初めて明らかにし、当該遺伝子の進化過程の推定に重要な知見をもたらした。また、高度好熱性 Geobacillus属 細菌群において従来型の鉄依存性 AlkB 型遺伝子と同程度に広く分布することも実証した。　以上の成果は、深度地下 1,000～2,000mに棲息する極限環境微生物のアルカン分解遺伝子群の分布多様性と進化過程ならびにその機能を明らかにしただけではなく、長鎖アルカンを多く含む難分解性化学物質である漏出原油に対する生物環境修復技術基盤の開発にも貢献するものである

Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 A degrees C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladA alpha(B23), ladA beta(B23), and ladB (B23), was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2 Delta 1. It was found that all three genes were functional in P. fluorescens KOB2 Delta 1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes

Biocalcifying Potential of Ureolytic Bacteria Isolated from Soil for Biocementation and Material Crack Repair

Microbially induced calcium carbonate precipitation (MICP) has been highlighted for its application in civil engineering, and in the environmental and geotechnical fields. Ureolytic activity is one of the most promising bacterial mechanisms in terms of inducing calcium carbonate formation. In this study, four bacterial isolates with high-yield urease production capabilities were obtained from two-step screening using a high-buffered urea medium. The highest urease activity and calcium carbonate formation was observed in Lysinibacillus fusiformis 5.1 with 4.40 &times; 103 unit/L of urease and 24.15 mg/mL of calcium carbonate, followed by Lysinibacillus xylanilyticus 4.3 with 3.93 &times; 103 unit/L of urease and 22.85 mg/mL of calcium carbonate. The microstructure of the precipitated crystalline calcium carbonate was observed using scanning electron microscopy. X-ray diffraction analysis confirmed that the main polymorph of the calcium carbonate particle obtained from both isolates was calcite. Examination of the material-crack filling in mortar specimens showed that calcite layers had formed along the crack edges and inside after 10 days, and gradually filled the cracks up to the upper surface. These results showed that these two isolates presented robust characteristics of potential MICP-inducing bacteria for civil engineering and material engineering applications

Isolation and Characterization of a Thermotolerant Ammonia-Oxidizing Bacterium Nitrosomonas sp. JPCCT2 from a Thermal Power Station

A thermotolerant ammonia-oxidizing bacterium strain JPCCT2 was isolated from activated sludge in a thermal power station. Cells of JPCCT2 are short non-motile rods or ellipsoidal. Molecular phylogenetic analysis of 16S rRNA gene sequences demonstrated that JPCCT2 belongs to the genus Nitrosomonas with the highest similarity to Nitrosomonas nitrosa Nm90 (100%), Nitrosomonas sp. Nm148 (99.7%), and Nitrosomonas communis Nm2 (97.7%). However, G+C content of JPCCT2 DNA was 49.1 mol % and clearly different from N. nitrosa Nm90, 47.9%. JPCCT2 was capable of growing at temperatures up to 48°C, while N. nitrosa Nm90 and N. communis Nm2 could not grow at 42°C. Moreover, JPCCT2 grew similarly at concentrations of carbonate 0 and 5 gL−1. This is the first report that Nitrosomonas bacterium is capable of growing at temperatures higher than 37°C. Key words: Nitrosomonas, thermotolerant ammonia-oxidizing bacterium, activated sludge Chemolithoautotrophic ammonia-oxidizing bacteria (AOB), which convert ammonium to nitrite, play an important role in the global cycling of nitrogen (19, 22). Isolation of AOB was first reported in 1890 (2, 28), and since then a con-siderable number of AOB within the Betaproteobacteria and Gammaproteobacteria have been obtained from various envi