3-Nitrotoluene dioxygenase from Diaphorobacter sp. strains: cloning, sequencing and evolutionary studies

Singh, Deepak; Kumari, Archana; Ramanathan, G.

doi:10.1007/s10532-013-9675-9

Cited by 19 publications

(14 citation statements)

References 48 publications

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“…strain DNT (12), and 3-nitrotolueneutilizing Diaphorobacter species strains (11). Nevertheless, it is not known whether a functional internal promoter containing the 18-bp direct repeat is present for the enhanced expression of catabolism enzymes in these strains, in addition to the expression directed by Nag-like regulators.…”

Section: Discussionmentioning

confidence: 99%

“…strain U2, the catabolism of naphthalene is initiated by naphthalene dioxygenase NagAaAbAcAd (7). This Nag dioxygenase is thought to be the progenitor of nitroarene dioxygenases (collectively called Nag-like dioxygenase) described so far (8), including nitrobenzene dioxygenase (9), 2-nitrotoluene dioxygenase (10), 3-nitrotoluene dioxygenase (11), and 2,4-dinitrotoluene dioxygenase (12,13). The synthetic nitro-and chloroaromatic compound, 2-chloronitrobenzene (2CNB), has been in the environment only for a short period of time.…”

Section: Importancementioning

confidence: 99%

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Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

Gao

Liu

Chao

et al. 2016

Appl Environ Microbiol

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The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment. IMPORTANCEThe release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid evolution in regulatory networks for the catabolism of synthetic compounds. The 2CNB utilizer Pseudomonas stutzeri ZWLR2-1 in this study has adapted itself to the new pollutant by evolving the always-inducible Nag-like dioxygenase into a constitutively expressed enzyme, and its expression has escaped the influence of salicylate. This may facilitate an understanding of how bacteria can rapidly adapt to the new synthetic compounds by evolving its expression system for key enzymes involved in the degradation of a xenobiotic. E volutionary changes in a regulated gene promoter have been heavily studied, particularly changes in promoter sequences in the deployment of the transcriptional factors (1). Horizontal gene transfer can significantly alter the composition of bacterial regulons (2). The promoter sequence in a regulatory system can be changed by point mutation (3) or insertion of a transposable element (4) in order to allow the organism to adapt to environmental change. With the rapid development of the chemical industry, many synthetic compounds have been...

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Section: Discussionmentioning

confidence: 99%

Section: Importancementioning

confidence: 99%

Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

Gao

Liu

Chao

et al. 2016

Appl Environ Microbiol

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“…Interestingly, 3NTDO from Diaphorobacter sp. strain DS2 has an isoleucine at position 204 and a glycine at position 405 (50). Modeling based on the structure of NBDO indicated that 3NTDO from Diaphorobacter has a larger active site than does NBDO (50).…”

Section: Discussionmentioning

confidence: 99%

“…strain DS2 has an isoleucine at position 204 and a glycine at position 405 (50). Modeling based on the structure of NBDO indicated that 3NTDO from Diaphorobacter has a larger active site than does NBDO (50). It is possible that the glycine at position 405 plays a role in determining the larger size of the active site.…”

Section: Discussionmentioning

confidence: 99%

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Mahan

Penrod

Kass

et al. 2015

Appl Environ Microbiol

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Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3-or 4-nitrotoluene. Using both long-and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the ␣ subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome. N itroaromatic compounds are toxic and stable in the environment and are important components in the manufacture of dyes, polymers, explosives, and pesticides (1). The synthesis and widespread use of these products have caused environmental contamination of soil and groundwater (2, 3). In addition, nitroaromatic compounds tend to undergo reduction, resulting in the formation of mutagenic aromatic amines (4). Due to the stability, toxicity, and mutagenicity of these chemicals, the U.S. Environmental Protection Agency has designated several nitroaromatic compounds as priority pollutants (5).Although the majority of nitroarene compounds are anthropogenic, a number of microorganisms have developed the ability to utilize compounds of this chemical class as sources of carbon, nitrogen, and energy (6). Bacterial strains capable of growth on nitrobenzene (7-9), mononitrotoluenes (10-16), and dinitrotoluenes (17-19) have been isolated from various contaminated environments. For example, Acidovorax sp. strain JS42 (formerly Pseudomonas sp. strain JS42) uses nitrobenzene (NB) and 2-nitrotoluene (2NT) as sole sources of carbon, nitrogen, and energy (15,20), while Comamonas sp. strain JS765 is capable of growth on NB and 3-nitrotoluene (3NT) (9, 21). The first enzymes of each degradation pathway, 2-nitro...

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“…Pseudomonas sp., by producing catechol 2, 3-dioxygenase (C23O) can utilize and decomposition polycyclic aromatic hydrocarbons (PAHs). C23O is a nonheme iron dioxygenase and a key enzyme in the biodegradation of PAH compounds that is released into the environment by a large number of an industrial process by catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde (9)(10)(11).…”

Section: Introductionmentioning

confidence: 99%

Application of Genetically Engineered Dioxygenase Producing Pseudomonas putida on Decomposition of Oil from Spiked Soil

Mardani

Mahvi

Hashemzadeh‐Chaleshtori

et al. 2017

Jundishapur J Nat Pharm Prod

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Background: It is well known that bioremediation or using microorganism enzymes plays an important role in decomposition and changing of oil pollutants and PAH compounds into nonhazardous or less-hazardous substances. The genetically manipulated bacteria like Pseudomonas sp. can enhancement the natural biodegradation activity of polycyclic aromatic hydrocarbons (PAHs) in polluted cities. Pseudomonas putida (P. putida) is a saprotrophic soil bacterium that is found throughout various environments such as soil and freshwater environments. Oil and petroleum industries are important exposure of PAHs in environment that strongly associated with the development of human cancers. Objectives: In the present work, P. putida was genetically manipulated for the biodegradation of oil in spiked soil and its activity was measured using the high-performance liquid chromatography (HPLC) method. Methods: The catechol 2,3-dioxygenase (C23O) encoded gene (nahH) was cloned into pUC18 for generating pUC18-nahH. This recombinant vector was transferred into P. putida, successfully, and both wild type and genetically modified P. putida were inoculated in spiked soil with oil for pilot plan preparation. Then, biodegradation activity of this bacterium on the elimination of phenanthrene and pyrene as an oil indicator in spiked soil were evaluated by the HPLC measurement technique. Results:The results showed the biodegradation of these PAH compounds in oil-spiked soil by genetically manipulated P. putida in both groups (containing autoclaved soil and dish containing natural soil) comparing to the inoculated group by wild type of P. putida were statistically significant (P < 0.05). Finally, confirmatory tests (catalase, oxidase, and PCR) were accomplished on isolated bacteria from spiked soil and were indicated that engineered P. putida was alive and functional by producing the C23O enzyme for biodegradation activity of oil. Conclusions: These findings demonstrated a degradable strain of P. putida that was generated in this study by genetic engineering can be useful to assess biodegradation of PAHs compounds in oil and petrochemical pollutions.

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3-Nitrotoluene dioxygenase from Diaphorobacter sp. strains: cloning, sequencing and evolutionary studies

Cited by 19 publications

References 48 publications

Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Application of Genetically Engineered Dioxygenase Producing Pseudomonas putida on Decomposition of Oil from Spiked Soil

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