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A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101.

ABSTRACT: 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.

SUBMITTER: Kitagawa W

PROVIDER: S-EPMC451640 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101.

Kitagawa Wataru W Kimura Nobutada N Kamagata Yoichi Y

Journal of bacteriology 20040801 15

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 compon ...[more]

PMID: 15262926

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Project description:Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus. The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales, the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis, but not for Corynebacterium glutamicum, and the new vector was used to study if mycolic acid is essential for R. opacus. We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. KEY POINTS: • Improved vector for homologous recombination in R. opacus. • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium. • Similar conditional suicide plasmids may be constructed for other bacteria.

Project description:BackgroundBacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o-xylene degradation in R. opacus strain R7 through a genome-based approach.ResultsUsing genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb, phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o-xylene oxidation was evaluated. The involvement of the akb genes in o-xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o-xylene and by the selection of the R7-50 leaky mutant. Although the akb genes are specifically activated for o-xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o-xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o-xylene and R7 growth on dimethylphenols.ConclusionsThese results suggest that R7 strain is able to degrade o-xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o-xylene degradation pathway in R7 strain.

Dataset Information

A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101.

Publications

A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101.

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