氏名　北川　航
学位の種類　博士（工学）
学位記番号　博甲第233号
学位授与の日付　平成13年12月31日
学位論文題目　芳香族化合物水酸化オキシゲナーゼの多様性と機能
論文審査委員
　主査　教授　福田　雅夫
　副査　教授　森川　康
　副査　助教授　岡田　宏文
　副査　助教授　政井　英司
　副査　独立行政法人産業技術総合研究所　グループ長　鎌形　洋一

• 論文目次
• 論文要旨

［平成13（2001）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.3
Chapter 1; Diversity of ring-hydroxylation dioxygenase genes in Rhodococcus sp. strain RHA1　p.7
Introduction　p.7
Materials and Methods　p.7
Results　p.10
Discussion　p.14
Chapter 2; Characterization of the new dioxygenase gene A　p.15
Introduction　p.15
Materials and Methods　p.17
Results　p.20
Discussion　p.36
Chapter 3 ; Characterization of the new dioxygenase gene E　p.39
Introduction　p.39
Materials and Methods　p.41
Results　p.42
Discussion　p.54
Chapter 4; Novel 2,4-dichlorophenoxyacetic acid degradation genes from oligotrophic Bradyrhizobium sp. strain HW13 isolated from a pristine environment　p.57
Introduction　p.57
Materials and Methods　p.59
Results　p.63
Discussion　p.75
Concluding Remarks　p.81
Acknowledgments　p.87
References　p.88

In this research, the diverse aromatic ring-hydroxylation dioxygenases （RHDOs） were examined. RHDOs play a key role in the degradation of a variety of aromatic compounds. In the chapter 1 , the diversity of RHDO genes in a gram-positive PCB degrader, Rhodococcus sp. strain RHA1 was investigated using denaturing gradient gel electrophoresis （DGGE）. The DGGE technique has been used preferentially for the detection of a single base mutation, and the separation of 16S rDNAs. In this study, I expanded the application of DGGE to the examination of the diversity of a certain gene family in a single strain, and the targeted cloning of new genes of interest. Then I succeeded in identifying two new genes A and E, which showed identity with benzoate dioxygenase and phthalate dioxygenase, respectively.
In the chapter 2, the new gene A was characterized. In its flanking region, benzoate dioxygenase genes benABC were identified. The BenABC was thought to belong to the class IB RHDO. The benA mutant neither grew on benzoate nor transformed benzoate. It showed diminished growth on biphenyl. These results indicate that the cloned benABCD play an essential role not only in benzoate but also in biphenyl catabolism. This is the first report on the genes for benzoate dioxygenase of a gram-positive bacterium, and also on the role of benzoate dioxygenase in aerobic bacterial degradation of biphenyl.
In the chapter 3, the new RHDO gene E was characterized. In its flanking region, phthalate dixygenase genes padA1A2 were identified. The genes coding electron transport （ET） component of the phthalate dioxygenase were not found in the vicinity of the oxygenase （DO） component genes padA1A2. Pulsed-field gel electrophoresis and Southern hybridization analysis indicated the duplicate padA1 genes are located on pRHL1 and pRHL2 linear plasmids, respectively. The disruption of each padA1 gene suggested that both the padA1 genes on pRHL1 and pRHL2 are functional, and involved in phthalate catabolism.
In the chapter 4, a new RHDO gene, cadABC encoding 2,4-dichlorophenoxyacetic acid （2,4-D） oxygenase was cloned from Bradyrhizobium sp. strain HW13, which was isolated from a buried Hawaiian soil that had never experienced anthropogenic chemicals. This is the first report on 2,4-D oxygenase gene other than the well- characterized tfdA type of gene. Although CadA showed similarity with class IB RHDO, the CadC showed similarity with cytochrome P-450 monooxygenase type of ferredoxin, which has not been identified in class IB RHDO before. The putative reductase gene was not found in the adjacent region.
The results obtained in this study suggest that the phylogenetic relationships of oxygenase protein sequences are not usually reflected in the traditional classification, in which RHDOs have been classified by their ET components. The DO and ET components of RHDOs seem to have evolved independently. Thus, it seems reasonable to classify RHDOs fundamentally by the DO （lSPα） component of RHDOs that is essentially responsible for the function of RHDOs.
In addition, the results obtained in this study are useful for developing microorganisms, which can efficiently degrade PCBs, phthalate esters, and 2,4-D, which are a notorious environmental pollutant all over the world, suspected endocrine disruptors （environmental hormones）, and a well-known environmental pollutant and also a suspected endocrine disruptor, respectively.