A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101.
p-Nitrophenol (4-NP) is recognized as an environmental contaminant; it is used primarily for manufacturing medicines and pesticides. To date, several 4-NP-degrading bacteria have been isolated; however, the genetic information remains very limited. In this study, a novel 4-NP degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101, was identified and characterized. The deduced amino acid sequences of npcB, npcA, and npcC showed identity with phenol 2-hydroxylase component B (reductase, PheA2) of Geobacillus thermoglucosidasius A7 (32%), with 2,4,6-trichlorophenol monooxygenase (TcpA) of Ralstonia eutropha JMP134 (44%), and with hydroxyquinol 1,2-dioxygenase (ORF2) of Arthrobacter sp. strain BA-5-17 (76%), respectively. The npcB, npcA, and npcC genes were cloned into pET-17b to construct the respective expression vectors pETnpcB, pETnpcA, and pETnpcC. Conversion of 4-NP was observed when a mixture of crude cell extracts of Escherichia coli containing pETnpcB and pETnpcA was used in the experiment. The mixture converted 4-NP to hydroxyquinol and also converted 4-nitrocatechol (4-NCA) to hydroxyquinol. Furthermore, the crude cell extract of E. coli containing pETnpcC converted hydroxyquinol to maleylacetate. These results suggested that npcB and npcA encode the two-component 4-NP/4-NCA monooxygenase and that npcC encodes hydroxyquinol 1,2-dioxygenase. The npcA and npcC mutant strains, SDA1 and SDC1, completely lost the ability to grow on 4-NP as the sole carbon source. These results clearly indicated that the cloned npc genes play an essential role in 4-NP mineralization in R. opacus SAO101.
Kitagawa
Wataru
W
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan. n-kimura@aist.go.jp
Kimura
Nobutada
N
Kamagata
Yoichi
Y
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002733:Chlorophenols / Q000378:metabolism; D003001:Cloning, Molecular; D049308:Dioxygenases; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D009596:Nitrophenols / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D002733:Chlorophenols; D009596:Nitrophenols; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; C041894:hydroxyquinol 1,2-dioxygenase; C022336:phenol 2-monooxygenase; C024564:2,4,6-trichlorophenol