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Rebecca E. Parales - One of the best experts on this subject based on the ideXlab platform.
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Three Types of Taxis Used in the Response of Acidovorax sp. Strain JS42 to 2-Nitrotoluene
2015Co-Authors: Christine A. Rabinovitch-deere, Rebecca E. ParalesAbstract:Acidovorax sp. strain JS42 is able to utilize 2-Nitrotoluene (2NT) as its sole carbon, nitrogen, and energy source. We report here that strain JS42 is chemotactic to 2NT and that the response is increased when cells are grown on compounds such as 2NT that are known to induce the first step of 2NT degradation. Assays with JS42 mutants unable to oxidize 2NT showed that the first step of 2NTmetabolism was required for the induced response, but not for a portion of the constitutive response, indicating that 2NT itself is an attractant. The 2NTmetabolite nitrite was shown to be a strong attractant for strain JS42, and sufficient nitrite was produced during the taxis assay to account for a large part of the induced response. Amutant with an inactivated ntdY gene, which is located adjacent to the 2NT degradation genes and codes for a putative methyl-accepting chemotaxis protein, showed a defect in taxis toward 2NT that may involve a reduced response to nitrite. Responses of a mutant defective for the energy-taxis receptor, Aer, indicated that a functional aer gene is required for a substantial part of the wild-type induced response to 2NT. In summary, strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive 2NT-specific che-motaxis to directly sense 2NT, metabolism-dependent nitrite-specific chemotaxis that may be mediated by NtdY, and en-ergy taxis mediated by Aer. Nitroarene compounds are a group of primarily man-madetoxic chemicals known to contaminate the soil and ground-water of various locations around the world, including chemical manufacturing plants and munitions production and detonatio
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selection for growth on 3 nitrotoluene by 2 nitrotoluene utilizing acidovorax sp strain js42 identifies nitroarene dioxygenases with altered specificities
Applied and Environmental Microbiology, 2015Co-Authors: Kristina M Mahan, Juanito V Parales, Joseph T Penrod, Natascia Al Kass, Watumesa A Tan, Richard Truong, Rebecca E. ParalesAbstract: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 α 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.
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Isotope Effects of Enzymatic Dioxygenation of Nitrobenzene and 2‑Nitrotoluene by Nitrobenzene Dioxygenase
2014Co-Authors: Sarah G. Pati, Rebecca E. Parales, Hans-peter E. Kohler, Jakov Bolotin, Thomas B. HofstetterAbstract:Oxygenation of aromatic rings is a frequent initial step in the biodegradation of persistent contaminants, and the accompanying isotope fractionation is increasingly used to assess the extent of transformation in the environment. Here, we systematically investigated the dioxygenation of two nitroaromatic compounds (nitrobenzene and 2-Nitrotoluene) by nitrobenzene dioxygenase (NBDO) to obtain insights into the factors governing its C, H, and N isotope fractionation. Experiments were carried out at different levels of biological complexity from whole bacterial cells to pure enzyme. C, H, and N isotope enrichment factors and kinetic isotope effects (KIEs) were derived from the compound-specific isotope analysis of nitroarenes, whereas C isotope fractionation was also quantified in the oxygenated reaction products. Dioxygenation of nitrobenzene to catechol and 2-Nitrotoluene to 3-methylcatechol showed large C isotope enrichment factors, ϵC, of −4.1 ± 0.2‰ and −2.5 ± 0.2‰, respectively, and was observed consistently in the substrates and dioxygenation products. ϵH- and ϵN-values were smaller, that is −5.7 ± 1.3‰ and −1.0 ± 0.3‰, respectively. C isotope fractionation was also identical in experiments with whole bacterial cells and pure enzymes. The corresponding 13C-KIEs for the dioxygenation of nitrobenzene and 2-Nitrotoluene were 1.025 ± 0.001 and 1.018 ± 0.001 and suggest a moderate substrate specificity. Our study illustrates that dioxygenation of nitroaromatic contaminants exhibits a large C isotope fractionation, which is not masked by substrate transport and uptake processes and larger than dioxygenation of other aromatic hydrocarbons
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three types of taxis used in the response of acidovorax sp strain js42 to 2 nitrotoluene
Applied and Environmental Microbiology, 2012Co-Authors: Christine A Rabinovitchdeere, Rebecca E. ParalesAbstract:Acidovorax sp. strain JS42 is able to utilize 2-Nitrotoluene (2NT) as its sole carbon, nitrogen, and energy source. We report here that strain JS42 is chemotactic to 2NT and that the response is increased when cells are grown on compounds such as 2NT that are known to induce the first step of 2NT degradation. Assays with JS42 mutants unable to oxidize 2NT showed that the first step of 2NT metabolism was required for the induced response, but not for a portion of the constitutive response, indicating that 2NT itself is an attractant. The 2NT metabolite nitrite was shown to be a strong attractant for strain JS42, and sufficient nitrite was produced during the taxis assay to account for a large part of the induced response. A mutant with an inactivated ntdY gene, which is located adjacent to the 2NT degradation genes and codes for a putative methyl-accepting chemotaxis protein, showed a defect in taxis toward 2NT that may involve a reduced response to nitrite. Responses of a mutant defective for the energy-taxis receptor, Aer, indicated that a functional aer gene is required for a substantial part of the wild-type induced response to 2NT. In summary, strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive 2NT-specific chemotaxis to directly sense 2NT, metabolism-dependent nitrite-specific chemotaxis that may be mediated by NtdY, and energy taxis mediated by Aer.
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reconstructing the evolutionary history of nitrotoluene detection in the transcriptional regulator ntdr
Molecular Microbiology, 2009Co-Authors: Juanito V Parales, Rebecca E. ParalesAbstract:Many toxic man-made compounds have been introduced into the environment, and bacterial strains that are able to grow on them are ideal model systems for studying the evolution of metabolic pathways and regulatory systems. Acidovorax sp. strain JS42 is unique in its ability to use 2-Nitrotoluene as a sole carbon, nitrogen, and energy source for growth. The LysR-type transcriptional regulator NtdR activates expression of the 2-Nitrotoluene degradation genes not only when nitroaromatic compounds are present, but also in the presence of a wide range of aromatic acids and analogues. The molecular determinants of inducer specificity were identified through comparative analysis with NagR, the activator of the naphthalene degradation pathway genes in Ralstonia sp. strain U2. Although NagR is 98% identical to NtdR, it does not respond to nitrotoluenes. Exchange of residues that differ between NagR and NtdR revealed that residues at positions 227 and 232 were key for the recognition of nitroaromatic compounds, while the amino acid at position 169 determined the range of aromatic acids recognized. Structural modelling of NtdR suggests that these residues are near the predicted inducer binding pocket. Based on these results, an evolutionary model is presented that depicts the stepwise evolution of NtdR.
David T Gibson - One of the best experts on this subject based on the ideXlab platform.
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expression of the nitroarene dioxygenase genes in comamonas sp strain js765 and acidovorax sp strain js42 is induced by multiple aromatic compounds
Journal of Bacteriology, 2003Co-Authors: Daniel J Lessner, Rebecca E. Parales, Shakti Narayan, David T GibsonAbstract:This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-Nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.
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molecular characterization and substrate specificity of nitrobenzene dioxygenase from comamonas sp strain js765
Applied and Environmental Microbiology, 2002Co-Authors: Daniel J Lessner, Jim C Spain, Rebecca E. Parales, Glenn R Johnson, David T GibsonAbstract:Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase NBZ , Ferredoxin NBZ , Oxygenase NBZα , and Oxygenase NBZβ , with the gene designations nbzAa , nbzAb , nbzAc , and nbzAd , respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-Nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.
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oxidation of nitrotoluenes by toluene dioxygenase evidence for a monooxygenase reaction
Applied and Environmental Microbiology, 1992Co-Authors: J B Robertson, Jim C Spain, John Haddock, David T GibsonAbstract:Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.
Harichandra Z Ninnekar - One of the best experts on this subject based on the ideXlab platform.
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enhanced degradation of 2 nitrotoluene by immobilized cells of micrococcus sp strain smn 1
Chemosphere, 2013Co-Authors: Sikandar I Mulla, Robertcyril S. Hoskeri, Manjunatha P Talwar, Zabin K Bagewadi, Harichandra Z NinnekarAbstract:Abstract Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-Nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate–polyvinyl alcohol (SA–PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-Nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-Nitrotoluene than freely suspended cells and the cells immobilized in SA–PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-Nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1.
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Biodegradation of 2-Nitrotoluene by Micrococcus sp. strain SMN-1
Biodegradation, 2011Co-Authors: Sikandar I Mulla, Robertcyril S. Hoskeri, Yogesh S. Shouche, Harichandra Z NinnekarAbstract:A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-Nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-Nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-Nitrotoluene through 3-methylcatechol by a meta -cleavage pathway, with release of nitrite.
Sikandar I Mulla - One of the best experts on this subject based on the ideXlab platform.
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enhanced degradation of 2 nitrotoluene by immobilized cells of micrococcus sp strain smn 1
Chemosphere, 2013Co-Authors: Sikandar I Mulla, Robertcyril S. Hoskeri, Manjunatha P Talwar, Zabin K Bagewadi, Harichandra Z NinnekarAbstract:Abstract Nitrotoluenes are the toxic pollutants of the environment because of their large scale use in the production of explosives. Biodegradation of such chemicals by microorganisms may provide an effective method for their detoxification. We have studied the degradation of 2-Nitrotoluene by cells of Micrococcus sp. strain SMN-1 immobilized in various matrices such as polyurethane foam (PUF), sodium alginate (SA), sodium alginate–polyvinyl alcohol (SA–PVA), agar and polyacrylamide. The rate of degradation of 15 and 30 mM 2-Nitrotoluene by freely suspended cells and immobilized cells in batches and fed-batch with shaken cultures were compared. The PUF-immobilized cells achieved higher degradation of 15 and 30 mM 2-Nitrotoluene than freely suspended cells and the cells immobilized in SA–PVA, polyacrylamide, SA and agar. The PUF-immobilized cells could be reused more than 24 cycles without loosing their degradation capacity and showed more tolerance to pH and temperature changes than freely suspended cells. These results revealed the enhanced rate of degradation of 2-Nitrotoluene by PUF-immobilized cells of Micrococcus sp. strain SMN-1.
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Biodegradation of 2-Nitrotoluene by Micrococcus sp. strain SMN-1
Biodegradation, 2011Co-Authors: Sikandar I Mulla, Robertcyril S. Hoskeri, Yogesh S. Shouche, Harichandra Z NinnekarAbstract:A bacterial consortium capable of degrading nitroaromatic compounds was isolated from pesticide-contaminated soil samples by selective enrichment on 2-Nitrotoluene as a sole source of carbon and energy. The three different bacterial isolates obtained from bacterial consortium were identified as Bacillus sp. (A and C), Bacillus flexus (B) and Micrococcus sp. (D) on the basis of their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. The pathway for the degradation of 2-Nitrotoluene by Micrococcus sp. strain SMN-1 was elucidated by the isolation and identification of metabolites, growth and enzymatic studies. The organism degraded 2-Nitrotoluene through 3-methylcatechol by a meta -cleavage pathway, with release of nitrite.
Daniel J Lessner - One of the best experts on this subject based on the ideXlab platform.
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expression of the nitroarene dioxygenase genes in comamonas sp strain js765 and acidovorax sp strain js42 is induced by multiple aromatic compounds
Journal of Bacteriology, 2003Co-Authors: Daniel J Lessner, Rebecca E. Parales, Shakti Narayan, David T GibsonAbstract:This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-Nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator.
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molecular characterization and substrate specificity of nitrobenzene dioxygenase from comamonas sp strain js765
Applied and Environmental Microbiology, 2002Co-Authors: Daniel J Lessner, Jim C Spain, Rebecca E. Parales, Glenn R Johnson, David T GibsonAbstract:Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase NBZ , Ferredoxin NBZ , Oxygenase NBZα , and Oxygenase NBZβ , with the gene designations nbzAa , nbzAb , nbzAc , and nbzAd , respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-Nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite.