氏名　PARICHAT SUKDA
学位の種類　博士（工学）
学位記番号　博甲第511号
学位授与の日付　平成21年3月25日
学位論文題目　Cloning and Characterization of the Genes Involved in the Dibenzofuran Degradation by Nocardioides sp.DF412　（Nocardioides sp.DF412株のジベンゾフラン分解遺伝子の単離と解析）
論文審査委員
　主査　教授　福田　雅夫
　副査　教授　森川　康
　副査　教授　解良　芳夫
　副査　准教授　岡田　宏文
　副査　准教授　政井　英司

• 論文目次
• 論文要旨
• 論文審査の結果の要旨

［平成20（2008）年度博士論文題名一覧］ ［博士論文題名一覧］に戻る．

Contents
Introductory Remarks　p.5
Chapter 1　Isolation of a dibenzofuran degrading bacterium, Nocardioides sp. DF412　p.11
　Materials and Methods　p.11
　Isolation of the bacterium grown on dibenzofuran　p.11
　DNA manipulations and analysis　p.11
　The degradation of chlorodibenzofuran by DF412　p.14
　The degradation of dibenzofuran （DF） and dibenzo-p-dioxin （DD） by DF412　p.14
　Results and Discussion　p.15
　Isolation of DF degrading bacterium　p.15
　Growth of DF412 on DF and various aromatic compounds, and degradation of DF by DF412　p.15
　The degradatio of chlorodibenzofuran by DF412　p.17
　Conclusion　p.20
Chapter 2　Cloning and characterization of dibenzofuran dioxygenase genes　p.21
　Materials and Methods　p.21
　Bacterial strains, plasmids, and culture conditions　p.21
　DNA manipulations and analysis　p.21
　Construction of DNA library　p.24
　Sourthen, Northern, and colony hybridization　p.24
　Reverse transcription （RT）-PCR analysis　p.24
　Expression of gene products　p.24
　Detection of DfdA activity　p.27
　Results and Discussion　p.29
　Cloning of dibenzofuran dioxygenase genes　p.29
　Localizarion of dfdA genes on a plasmid　p.33
　Transcription of dfdA genes　p.33
　Activity of dfdA product　p.36
　Conclusion　p.41
Chapter 3　Cloning and characterization of ring-cleavage dioxygenase genes　p.42
　Materials and Methods　p.42
　Bacterial strains, plasmids, and culture conditions　p.42
　DNA manipulations and analysis　p.42
　Construction of DNA library　p.45
　Isolation of colonies containing ring-cleavage dioxygenase gene　p.45
　Sourthen and Northern hybridization　p.45
　Reverse transcription （RT）-PCR analysis　p.45
　Expression of gene products　p.45
　Detection of DfdB and DfdC activity　p.47
　Results and Discussion　p.48
　Cloning of ring-cleavage dioxygenase gene　p.48
　Localizarion of dfdBC genes on the chromosome　p.52
　Transcription of dfdBC genes　p.52
　Activity of DfdBC proteins　p.54
　Conclusion　p.56
Chapter 4　Cloning and characterization of transcriptional regulatory gene　p.57
　Materials and Methods　p.57
　Bacterial strains, plasmids, and culture conditions　p.57
　DNA manipulations and analysis　p.57
　Plasmid construction　p.60
　Assay for DfdB activity　p.62
　Luciferase assay　p.62
　Primer extention analysis　p.63
　Gel shift analysis　p.65
　Detection of gene product　p.65
　Site directerd mutangenesis　p.66
　Results and Discussion　p.68
　The structure of a transcriptional regulatory gene　p.68
　Influence of dfdS on the expression of dfdB gene　p.68
　Complementation of dfdS deletion　p.70
　Substrate preference of dfdS　p.70
　Promoter analysis of dfdB　p.73
　Transcriptional start site of dfdB and dfdS　p.75
　Site-directerd mutangenesis　p.79
　Gel shift analysis　p.79
　Influence of DfdS on the dfdS promoter　p.84
　Conclusion　p.87
Concluding Remarks　p.88
Acknowledgements　p.90
References　p.91

　Dioxins are a group of compounds containing polychlorinated dibenzofurans, and dibenzo-p-dioxins, and coplanar-polychlorinated biphenyls, and have caused serious environmental problems. Microorganisms, which degrade dioxins to some extent using their metabolic pathway of dibenzofuran （DF）, dibenzo-p-dioxin, or biphenyl, have been reported. Such microorganisms can be used to develop an efficient degradation system to remediate the dioxin-contaminated environments. In this study, a DF-degrading bacterium was isolated, and its degradation enzyme system was characterized toward the development of an efficient degradation system.
　A strain DF412 was selected from DF-degrading bacteria, because it grew exceptionally well on DF. It was identified as Nocardioides sp. based on partial sequence of 16S-rRNA gene and depleted 2,8-dichlorodibenzofuran, suggesting cometabolism of chlorinated DFs through DF metabolic pathway. DF412 produced salicylate （SA） transiently accumulating yellow color intermediate. These results suggested DF412 degrades DF via SA through meta-cleavage pathway. ［Chapter 1］
　The 4.6-kb fragment containing DF dioxygenase gene was subcloned from a charomid clone obtained by colony hybridization using the PCR-amprified dfdA sequence followed by Northern blot hybridization with RNA from the cells grown in the presence of DF. It contained four open reading frames （ORFs）,having identity with the corresponding DF dioxygenase gene sequences of a DF-degrading Terrabacter sp. YK3. These ORFs were designated dfdA1A2A3A4 coding for large and small subunits of a terminal dioxygenase, ferredoxin, and reductase respectively. The results of Southern hybridization suggested the location of dfdA1A2A3A4 on a large plasmid. RT-PCR using primers to amplify inter- and intra-gene sequences of dfdA1A2A3A4 indicated that their transcription is induced in the presence of DF, and that they constitute an operonic structure. Rhodococcus erythropolis IAM399 expressing dfdA1A2A3A4 produced 2,2’,3’-trihydroxybiphenyl （2,2’,3’-THBP） from DF. These results suggest that dfdA1A2A3A4 genes encode a DF angular dioxygenase （DF 4, 4a-dioxygenase）. ［Chapter 2］
　The 2.5-kb fragment containing dfdB gene was subcloned from a clone obtained by screening of clones producing yellow color ring-cleavage product from 2,3-dihydroxybiphenyl （2,3-DHBP） followed by northern dot hybridization. It contained open reading frames having identity with genes for salycilate hydroxylase （ORF1）, TetR-type transcriptional regulator （dfdS）, ring-cleavage dioxygenase （dfdB）, ring-cleavage compound hydrolase （dfdC）, and chlorophenol monooxygenase （ORF5）, respectively. RT-PCR using primers to amplify inter- and intra-gene sequences of ORF1 to ORF5 including dfdBC indicated that transcription of dfdBC and ORF5 is induced in the presence of DF, and that dfdBC and ORF5 constitute an operonic structure. R. erythropolis IAM399 expressing dfdB produced ring-cleavage product, 2-hydroxy-6-oxo-6- phenylhexa-2,4-dienoic acid （HPDA） from 2,3-DHBP, and that expressing dfdC depleted HPDA. These results suggest that dfdBC genes encode a ring-cleavage dioxygenase and a ring-cleavage compound hydrolase that are involved in DF degradation. ［Chapter 3］
　Introduction of a plasmid containing dfdS and dfdB into R. erythropolis IAM399 suggested repression of the dfdB promoter activity by dfdS product, because deletion in dfdS brought about overproduction of dfdB protein and elevated ring-cleavage dioxygenase activity against 2,3-DHBP. This was confirmed by complementation experiments of dfdS gene deletion using intact dfdS gene plasmid, which restored repression. R. erythropolis IAM399 harboring a dfdS plasmid and a plasmid containing the luxAB luciferase genes connected to a dfdB promoter sequence exhibited derepression of luciferase activity in the presence of 2,3-DHBP or 2,2’3-THBP. These results suggest that dfdS protein （DfdS） is a repressor of dfdB promoter, and 2,3-DHBP and 2,2’3-THBP are inducers of DfdS repression.
　Transcriptional starts of dfdB and dfdS are mapped at 35- and 84-bp upstream from the putative initiation codons, respectively, based by a primer extension technique. The region required for repression by DfdS was limited by deletion analysis to the sequence spanning 88-bp upstream from the putative initiation codon of dfdB. The binding of DfdS to the dfdB promoter sequence, which was released in the presence of 2,3-DHBP, was suggested by gel-shift analysis. The dfdB transcriptional start was located in the center of an 22-bp inverted repeat （IR）. Repression by DfdS of luciferase activity in IAM1399 harboring a dfdS plasmid and a plasmid containing luxAB connected to dfdB promoter, and binding of DfdS to the dfdB promoter sequence were canceled by a mutation in this IR. On the other hand, luciferase activity in IAM1399 harboring a dfdS plasmid and a plasmid containing luxAB connected to dfdS promoter suggested an autogeneous repression of dfdS transcription by DfdS itself. These results suggest that repression of dfdB transcription by DfdS is mediated by the binding of DfdS to 22-bp IR in dfdB promoter. The previous reports on TetR-type regulators imply the binding of DfdS to a half site of IR as a dimmer. ［Chapter 4］
　In conclusion, this study provided key findings to illustrate the DF degradation system and its regulatory mechanism in DF and chlorodibenzofuran degrading bacterium, Nocardioides sp. DF412. These findings are estimated to be of great use in the development of efficient degradation systems for dioxins.

　本論文はCloning and Characterization of the Genes Involved in the Dibenzofuran Degradation by Nocardioides sp. DF412（Nocardioides sp. DF412株のジベンゾフラン分解遺伝子の単離と解析）と題して、ダイオキシンのモデル化合物であるジベンゾフランについて微生物における分解酵素系の解析を行った一連の研究結果をまとめている。
　本論文ではまず、ジベンゾフランを効果的に分解して生育する微生物を検索し、ノカルディオイデス属細菌DF412株を取得した。DF412株はサリチル酸を生成しつつジベンゾフランで旺盛に生育し、2,8-二塩化ジベンゾフランも分解した。この結果からサリチル酸を経由してジベンゾフランを代謝することが示唆された。次に、分解の第一段階に予想されるジベンゾフラン・ジオキシゲナーゼの遺伝子断片をDF412株から単離した。同遺伝子断片はdfdA1～A4の4つの遺伝子を含み、ジベンゾフラン分解時に誘導されるオペロンを構成することが示唆された。異種宿主での発現では、ジベンゾフランを2,2’,3’-トリヒドロキシビフェニルに変換するアンギュラー・ジオキシゲナーゼであることが示された。さらに、分解の第二段階に予想される芳香環開裂ジオキシゲナーゼの遺伝子断片を取得した。同遺伝子断片にはTetR型転写制御因子遺伝子dfdS、芳香環開裂ジオキシゲナーゼ遺伝子dfdB、加水分解酵素遺伝子dfdC、モノオキシゲナーゼ遺伝子ORF5が含まれ、dfdB～ORF5がジベンゾフラン分解時に誘導されるオペロンを構成することが示唆された。異種宿主での発現では、dfdB産物が2,3-ジヒドロキシビフェニルをメタ開裂物質に変換し、dfdC産物がメタ開裂物質を代謝し、これらの遺伝子がジベンゾフラン分解に直接関与することが示唆された。最後に、dfdS遺伝子産物がdfdBの発現を抑制することを、dfdS遺伝子の欠失変異体の異種宿主での発現で示唆した。さらに、dfdBプロモーター配列の解析とゲルシフト解析から、dfdS遺伝子産物がdfdBプロモーターの逆方向反復配列に結合してdfdBプロモーターからの転写を抑制し、2,3-ジヒドロキシビフェニルや2,2’,3’-トリヒドロキシビフェニルの存在下ではdfdS産物が解離して転写が誘導されるメカニズムを明らかにした。
　以上、本論文は、ダイオキシンのモデル化合物であるジベンゾフランの新規分解菌を取得し、分解酵素系を構成する遺伝子の構造と機能ならびに酵素誘導機構を明らかにした。本論文で得られた知見は、環境汚染の元凶となるダイオキシンの効率的な分解除去システムを開発する上で意義の大きいものであり、ダイオキシン汚染の浄化における応用に大いに寄与するものと考えられる。よって、本論文は工学上貢献するところが大きく、博士（工学）の学位論文として十分な価値を有するものと認める。