Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor |
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Authors: | Takaya N Shoun H |
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Institution: | (1) Institute of Applied Biochemistry and TARA (Tsukuba Advanced Research Alliance) Center, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Tel/Fax: +81-298-534603 E-mail: p450nor@sakura.cc.tsukuba.ac.jp, JP |
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Abstract: | The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never
been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated
mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of
F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that
they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous
oxide (N2O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory
nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene
was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced β-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor.
These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N2O, the last step in denitrification by F. oxysporum.
Received: 28 June 1999 / Accepted: 22 December 1999 |
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Keywords: | Denitrification Cytochrome P450 Nitric oxide reductase Fusarium oxysporum |
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