1999 Fiscal Year Final Research Report Summary
Detection and quantification of petroleum hydrocarbon degrading bacteria targeting catechol cleavage genes
| Project/Area Number |
10680544
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
環境保全
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| Research Institution | Yamanashi University (1999)
Osaka University (1998) |
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Principal Investigator |
MORI Kazuhiro Faculty of Eng., Lecturer, 工学部, 講師 (90294040)
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| Co-Investigator(Kenkyū-buntansha) |
IKE Michihiko Graduate School of Eng., Osaka Univ., Associate Prof., 大学院・工学研究科, 助教授 (40222856)
FUJITA Masanori Graduate School of Eng., Osaka Univ., Professor, 大学院・工学研究科, 教授 (70029289)
KONO Tetsuro Faculty of Eng., Associate Prof., 工学部, 助教授 (50111779)
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| Project Period (FY) |
1998 – 1999
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| Keywords | PCR / hybridization / aromatic compounds / catechol 1,2-dioxygenase / catechol 2,3-dioxygenase / universal primer / MPN-PCR |
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| Research Abstract |
For the general detection of bacterial populations capable of degrading aromatic compounds, two PCR primer sets were designed which can, respectively, amplify specific fragments from a wide variety of catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenasc (C23O) genes. The C12O-targeting primer set (C12O primers) was designed based on the homologous regions of 11 C12O genes listed in the GenBank, while the C23O-targeting one (C23O primers) was designed based on those of 17 known C23O genes. Oligonucleotide probes (C12Op and C23Op) were also designed from the internal homologous regions to identify the amplified fragments. The specificity of the primer sets and probes was confirmed using authentic bacterial strains known to carry the C12O and/or C23O genes used for the primer and probe design. PCR with the C12O primers amplified DNA fragments of the expected sizes from 5 of the 6 known C12O-carrying bacterial strains tested, and positive signals were obtained from 4 of the 5 ampli
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fied fragments on Southern hybridization with the C12Op. The C23O primers amplified DNA fragments of the expected size from all the 11 tested C230-carrying bacterial strains used for their design, while the C23Op detected positive signals in the amplified fragments from 9 strains. On the other hand, no DNA fragments were amplified from the negative controls. To evaluate the applicability of the designed primers and probes for the general detection of aromatic compound-degrading bacteria, they were applied to wild-type phenol- and/op benzoate-degrading bacteria newly isolated from a various environment samples. The C12O and/or C23O primers amplified DNA fragments of the expected size from 69 of the 106 wild-type strains tested, while the C120p and/or C23Op detected positive signals in the amplfied fragemnts from 63 strains. These results suggest that our primer and probe systems can detect a considerable proportion of bacteria which can degrade aromatic compounds via catechol cleavage pathways. And finally we also developed the quantifcation method for the genes coding catechol cleavage in the environmental samples. Using MPN-PCR with stepdown PCR, target genes were successfully quantified against various environmental samples. These techniques investigated in this work would be applicable to the bioremediation. Less
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Research Products
(2 results)
