Dataset Information

NpdR, a repressor involved in 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1.

ABSTRACT: Rhodococcus opacus HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source. The initial attack on picric acid occurs through two hydrogenation reactions. Hydride transferase II (encoded by npdI) and hydride transferase I (encoded by npdC) are responsible for the hydride transfers. Database searches with the npd genes have indicated the presence of a putative transcriptional regulator, npdR. Here, the npdR gene was expressed in Escherichia coli, and the protein was purified and shown to form a complex with intergenic regions between open reading frames A and B and between npdH and npdI within the npd gene cluster. A change in DNA-NpdR complex formation occurred in the presence of 2,4-dinitrophenol, picric acid, 2-chloro-4,6-dinitrophenol, and 2-methyl-4,6-dinitrophenol. By constructing a promoter-probe vector, we demonstrated that both intergenic regions caused the expression of reporter gene xylE. Hence, both of these regions contain promoters. A deletion mutant of R. opacus HL PM-1 was constructed in which part of npdR was deleted. The expression of npdI and npdC was induced by 2,4-dinitrophenol in the wild-type strain, while in the mutant these genes were constitutively expressed. Hence, NpdR is a repressor involved in picric acid degradation.

SUBMITTER: Nga DP

PROVIDER: S-EPMC303439 | biostudies-literature | 2004 Jan

REPOSITORIES: biostudies-literature

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NpdR, a repressor involved in 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1.

Nga Dang P DP Altenbuchner Josef J Heiss Gesche S GS

Journal of bacteriology 20040101 1

Rhodococcus opacus HL PM-1 utilizes 2,4,6-trinitrophenol (picric acid) as a sole nitrogen source. The initial attack on picric acid occurs through two hydrogenation reactions. Hydride transferase II (encoded by npdI) and hydride transferase I (encoded by npdC) are responsible for the hydride transfers. Database searches with the npd genes have indicated the presence of a putative transcriptional regulator, npdR. Here, the npdR gene was expressed in Escherichia coli, and the protein was purified ...[more]

PMID: 14679229

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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.

Project description:The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene expression measurements of selected catabolic genes by RT-PCR. Taken together, this study provides a comprehensive understanding of the genome plasticity and ecological competitiveness of strain M213 likely facilitated by horizontal gene transfer (HGT), bacteriophage attacks and genomic reshuffling- aspects that continue to be understudied and thus poorly understood, in particular for the soil-borne Rhodococcii.

Dataset Information

NpdR, a repressor involved in 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1.

Publications

NpdR, a repressor involved in 2,4,6-trinitrophenol degradation in Rhodococcus opacus HL PM-1.

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