1990 Fiscal Year Final Research Report Summary
Cloning of Polycyclic Aromatic Hydrocarbon Dioxygenase Genes and Their Gene Analysis
| Project/Area Number |
01560129
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | Okayama University of Science, Faculty of Engineering |
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Principal Investigator |
KIYOHARU Hohzoh Okayama University of Science, Faculty of Engineering, Profesor, 工学部, 教授 (50068904)
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| Co-Investigator(Kenkyū-buntansha) |
TAKIZAWA Noboru Okayama University of Science, Faculty of Engineering, Lecturer, 工学部, 講師 (50179579)
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| Project Period (FY) |
1989 – 1990
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| Keywords | phenanthrene dioxygenase gene / naphthalene dioxygenase gene / Pseudomonas putida / cosmid vector / NAH7 plasmid / open reading frame / Shine-Dalgarno sequence / polycyclic aromatic hydrocarbons |
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| Research Abstract |
To genetically elucidate abilities of bacteria to oxidize polycyclic aromatic hydrocarbons (PAHSs), phenanthrene dioxygenase gene (phnA) was cloned and the nucleotide sequence was determined. Abacterial atrial (OUS82 Strain) that was able to utilize phenanthrene and naphthalene as a sole carbon source was screened from strains maintained in our laboratory, and identified as Pesudomonas putida. From Sal I-partial digests of the total OUS82 DNA, DNA fragments [around 25 kilo-base (kb)] forming indigo from indole were cloned in Escherichia coli HB101 onto a broad-host range cosmid vector constructed in this study. All fragments commonly contained 9.5-kb Sal I-fragments. Then, the fragments were sub-cloned onto pUC119 in E. coli JM109 and onto pMFY43 in P. putida PpY101. Clones obtained in JM109 expressed indigo formation, but in PpY101 did phenantrene oxidation but not indigo formation. phnA Gene was localized on the 9.5-kb fragment by deletion analysis, and a 2,500-base pair (bp) range i
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n its nucleotides were sequenced by dideoxy chain terminator method using dideoxy NTPs labeled with fluorescent dyes. The 2,855-bp range sequenced was found to contain a promoter gene and three open reading frames (ORFs) which were 315b bp [104 amino acids (aa)], 1,350 bp (449 aa) and 585 bp (194 aa). These frames were designated PdoA, B, C. which had Shine-Dalgorno sequence upstream them. The pdoABC showed 90% homology with the naphthalene dioxygenase, ndoABC, of P. putida NCIB9816, and hybridized strongly to nahA gene in NAH7 plasmid.
All Nah^- mutants of OUS82 that deleted in naphthalene utilization by the treating with 1-chloronaphthalene lost phenanthrene utilization (Phn^-), and Nah^-Sal^- mutants that deleted in salicylate (SA) utilization by the treatment with 1-fluorosalicylate retained the utilization by the treatment with 1-fluorosalicylate retained the utilization of 1-hydroxy-2-naphthoate (1H2NA), an intermediate of phenanthrene catabolism. The facts suggested that both later steps in naphthalene- and phenanthrene-degradative pathways of OUS82 were different. The resting cell suspension with anthracene and biphenyl, which were unable to utilize as growth substrates, gave the corresponding oxidized products.
Through the research, the conclusions were obtained as followed ; (1) early steps up to o-hydroxy-carboxylate (e. g,. SA and 1H2NA) in the degradative pathways of both hydrocarbons proceed by a same emzyme system including an initial dioxygenase that has broad specificity, and (2) utilizabilies of PAHs were determined by the presence of later degradative pathways below o-hydroxy-carboxylate. Less
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