Bt cotton was the first genetically modified crop approved for use in India. However, only a few ... more Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.
Biochemical and Biophysical Research Communications, 2005
Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited becaus... more Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited because of risks associated with their release in the environment. Recent developments in the area of recombinant DNA technologies have paved the way for conceptualizing ''suicidal genetically engineered microorganisms'' (S-GEMS) to minimize such anticipated hazards and to achieve efficient and safer bioremediation of contaminated sites. Our strategy of designing a novel S-GEM is based on the knowledge of killer-anti-killer gene(s) that would be susceptible to programmed cell death after detoxification of any given contaminated site(s).
Background: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic ... more Background: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO 2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.
The introduction of culture-independent molecular screening techniques, especially based on 16S r... more The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.
Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium ab... more Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H 2 to derive energy for fixation of CO 2 . Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H 2 generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H 2 as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes.
The chlorinated insecticide hexachlorocyclohexane (HCH) has been used extensively in the past, an... more The chlorinated insecticide hexachlorocyclohexane (HCH) has been used extensively in the past, and contaminated sites are present throughout the world. Toward their bioremediation, we isolated a bacterium Pseudomonas aeruginosa ITRC-5 that mediates the degradation of all the four major isomers of HCH under aerobic conditions, both in liquid-culture and contaminated soils. In liquidculture, the degradation of R-and γ-HCH is rapid and is accompanied with the release of 5.6 µmole chloride ions and 4.1 µmole CO 2 µmole -1 HCH-isomer. The degradation of -and δ-isomers is slow, accompanied with the release of 0.9 µmole chloride ions µmole -1 HCH-isomer, and results in a transient metabolite 2,3,4,5,6-pentachlorocyclohexan-1ol. The strain ITRC-5 also mediates the degradation of R-, -, γ-, and δ-isomers in contaminated soils, where degradation of otherwise persistent -and δ-HCH is enhanced severalfold in the presence of R-or γ-HCH. The degradation of soil-applied -and δ-HCH under aerobic conditions has not been reported earlier. The isolate ITRC-5 therefore demonstrates potential for the bioremediation of HCHwastes and contaminated soils.
With the emergence of transgenic technologies, new ways to improve the agronomic performance of c... more With the emergence of transgenic technologies, new ways to improve the agronomic performance of crops for food, feed, and processing applications have been devised. In addition, ability to express foreign genes using transgenic technologies has opened up options for producing large quantities of commercially important industrial or pharmaceutical products in plants. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of genetically modified (GM) crops on the environment. Potential contamination of the environment and food chains has prompted detailed consideration of how such crops and the molecules that they produce can be effectively isolated and contained. One of the reasonable steps after creating a transgenic plant is to evaluate its potential benefits and risks to the environment and these should be compared to those generated by traditional agricultural practices. The precautionary approach in risk management of GM plants may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Effective risk assessment and monitoring mechanisms are the basic prerequisites of any legal framework to adequately address the risks and watch out for new risks. Several agencies in different countries monitor the release of GM organisms or frame guidelines for the appropriate application of recombinant organisms in agro-industries so as to assure the safe use of recombinant organisms and to achieve sound overall development. We feel that it is important to establish an internationally harmonized framework for the safe handling of recombinant DNA organisms within a few years.
Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bi... more Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. Bioremediation of PNP-containing soil was first carried out in pots using immobilized and free cells of Arthrobacter protophormiae RKJ100 in order to ascertain the role of a suitable carrier material. Results showed that stability of the introduced strain was enhanced upon immobilization and that the rate of PNP depletion decreased with increasing depth of soil. Small-scale field studies (in one square meter plots) were then conducted in which PNP-contaminated soil from an agricultural field was bioaugmented with strain RKJ100 under natural environmental conditions. PNP was totally depleted in 5 days by immobilized cells, whereas free cells were able to deplete 75% of PNP in the same time period. The fate of the released strain as monitored by plate counts, hybridization studies, and real-time polymerase chain reaction revealed fairly stable population of the cells upon immobilization on corncob powder throughout the period of study.
Bioremediation of contaminated sites has been accepted as an efficient and cheaper alternative to... more Bioremediation of contaminated sites has been accepted as an efficient and cheaper alternative to physicochemical means of remediation in several cases. Although chemotactic behaviour of many bacteria has been studied earlier and assays have been developed to study bacterial chemotaxis in semi-solid media, this phenomenon has never been demonstrated in soil. For bioremediation application it is important to know whether bacteria actually migrate through the heterogenous soil medium towards a gradient of a particular chemoattractant. In the present study we have successfully demonstrated bacterial chemotaxis of a Ralstonia sp. SJ98 in soil microcosm using qualitative and quantitative plate and tray assays. The migration of bacteria has been established using several methods such as plate counting, vital staining and flow cytometry and slot blot hybridization. A non-chemotactic p-nitrophenol utilizing strain Burkholderia cepacia RKJ200 has been used as negative control. Our work clearly substantiates the hypothesis that chemotactic bacteria may enhance in situ bioremediation of toxic pollutants from soils and sediments.
We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 ... more We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA). During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies. Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7. HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase. In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.
Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pa... more Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featurin... more Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
International Journal of Systematic and Evolutionary Microbiology, 2006
A Gram-positive actinobacterium, strain RKJ300 T , capable of utilizing p-nitrophenol and 2,4dini... more A Gram-positive actinobacterium, strain RKJ300 T , capable of utilizing p-nitrophenol and 2,4dinitrophenol, was isolated from a pesticide-contaminated site in India. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. The DNA G+C content was 72 mol%. Strain RKJ300 T exhibited the highest level of sequence similarity with Rhodococcus wratislaviensis NCIMB 13082 T (99?3 %), followed by Rhodococcus opacus DSM 43205 T (98?8 %), Rhodococcus percolatus MBS1 T (98?6 %) and Rhodococcus koreensis DNP505 T (98?1 %). The low levels of DNA-DNA relatedness (49-58 %) with the above micro-organisms, and the differences in the biochemical and physiological properties, suggest that strain RKJ300 T should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus imtechensis sp. nov. is proposed. The type strain is RKJ300 T (=MTCC 7085 T =JCM 13270 T ).
Bt cotton was the first genetically modified crop approved for use in India. However, only a few ... more Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.
The use of microorganisms is expected to be an effective tool for remediation of environments con... more The use of microorganisms is expected to be an effective tool for remediation of environments contaminated with various aromatic compounds. Aromatic compounds are degraded via diverse ring-cleavage pathways by soil microorganisms. Two bacterial strains isolated from pesticide-contaminated agricultural fields and identified as Arthrobacter protophormiae RKJ100 and Burkholderia cepacia RKJ200 were found to be capable of utilizing p-hydroxybenzoate (PHB) as a sole source of carbon and energy. Thin layer chromatography, gas chromatography and gas chromatography-mass spectrometry of the ethyl acetate extracts from the culture medium showed the presence of protocatechuate. Both the strains follow the ortho-cleavage pathway for the catabolism of PC, since it was found that the terminal aromatic intermediate of the degradation pathway is cleaved to β β-ketoadipate. Plasmid cured derivatives of A. protophormiae and B. cepacia were also investigated to determine the location of the genes involved in PHB degradation.
Bt cotton was the first genetically modified crop approved for use in India. However, only a few ... more Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.
Biochemical and Biophysical Research Communications, 2005
Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited becaus... more Use of genetically modified microorganisms (GEMs) for pollution abatement has been limited because of risks associated with their release in the environment. Recent developments in the area of recombinant DNA technologies have paved the way for conceptualizing ''suicidal genetically engineered microorganisms'' (S-GEMS) to minimize such anticipated hazards and to achieve efficient and safer bioremediation of contaminated sites. Our strategy of designing a novel S-GEM is based on the knowledge of killer-anti-killer gene(s) that would be susceptible to programmed cell death after detoxification of any given contaminated site(s).
Background: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic ... more Background: Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium capable of utilizing CO (carbon monoxide) and fixing CO 2 (carbon dioxide). We previously published the draft genome of this organism and recently submitted the complete genome sequence to GenBank.
The introduction of culture-independent molecular screening techniques, especially based on 16S r... more The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.
Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium ab... more Oligotropha carboxidovorans OM5 T. (DSM 1227, ATCC 49405) is a chemolithoautotrophic bacterium able to utilize CO and H 2 to derive energy for fixation of CO 2 . Thus, it is capable of growth using syngas, which is a mixture of varying amounts of CO and H 2 generated by organic waste gasification. O. carboxidovorans is capable also of heterotrophic growth in standard bacteriologic media. Here we characterize how the O. carboxidovorans proteome adapts to different lifestyles of chemolithoautotrophy and heterotrophy. Fatty acid methyl ester (FAME) analysis of O. carboxidovorans grown with acetate or with syngas showed that the bacterium changes membrane fatty acid composition. Quantitative shotgun proteomic analysis of O. carboxidovorans grown in the presence of acetate and syngas showed production of proteins encoded on the megaplasmid for assimilating CO and H 2 as well as proteins encoded on the chromosome that might have contributed to fatty acid and acetate metabolism. We found that adaptation to chemolithoautotrophic growth involved adaptations in cell envelope, oxidative homeostasis, and metabolic pathways such as glyoxylate shunt and amino acid/cofactor biosynthetic enzymes.
The chlorinated insecticide hexachlorocyclohexane (HCH) has been used extensively in the past, an... more The chlorinated insecticide hexachlorocyclohexane (HCH) has been used extensively in the past, and contaminated sites are present throughout the world. Toward their bioremediation, we isolated a bacterium Pseudomonas aeruginosa ITRC-5 that mediates the degradation of all the four major isomers of HCH under aerobic conditions, both in liquid-culture and contaminated soils. In liquidculture, the degradation of R-and γ-HCH is rapid and is accompanied with the release of 5.6 µmole chloride ions and 4.1 µmole CO 2 µmole -1 HCH-isomer. The degradation of -and δ-isomers is slow, accompanied with the release of 0.9 µmole chloride ions µmole -1 HCH-isomer, and results in a transient metabolite 2,3,4,5,6-pentachlorocyclohexan-1ol. The strain ITRC-5 also mediates the degradation of R-, -, γ-, and δ-isomers in contaminated soils, where degradation of otherwise persistent -and δ-HCH is enhanced severalfold in the presence of R-or γ-HCH. The degradation of soil-applied -and δ-HCH under aerobic conditions has not been reported earlier. The isolate ITRC-5 therefore demonstrates potential for the bioremediation of HCHwastes and contaminated soils.
With the emergence of transgenic technologies, new ways to improve the agronomic performance of c... more With the emergence of transgenic technologies, new ways to improve the agronomic performance of crops for food, feed, and processing applications have been devised. In addition, ability to express foreign genes using transgenic technologies has opened up options for producing large quantities of commercially important industrial or pharmaceutical products in plants. Despite this high adoption rate and future promises, there is a multitude of concerns about the impact of genetically modified (GM) crops on the environment. Potential contamination of the environment and food chains has prompted detailed consideration of how such crops and the molecules that they produce can be effectively isolated and contained. One of the reasonable steps after creating a transgenic plant is to evaluate its potential benefits and risks to the environment and these should be compared to those generated by traditional agricultural practices. The precautionary approach in risk management of GM plants may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Effective risk assessment and monitoring mechanisms are the basic prerequisites of any legal framework to adequately address the risks and watch out for new risks. Several agencies in different countries monitor the release of GM organisms or frame guidelines for the appropriate application of recombinant organisms in agro-industries so as to assure the safe use of recombinant organisms and to achieve sound overall development. We feel that it is important to establish an internationally harmonized framework for the safe handling of recombinant DNA organisms within a few years.
Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bi... more Biodegradation of p-nitrophenol (PNP), a priority pollutant, was studied as a model system for bioremediation of sites contaminated with nitroaromatic/organic compounds. Bioremediation of PNP-containing soil was first carried out in pots using immobilized and free cells of Arthrobacter protophormiae RKJ100 in order to ascertain the role of a suitable carrier material. Results showed that stability of the introduced strain was enhanced upon immobilization and that the rate of PNP depletion decreased with increasing depth of soil. Small-scale field studies (in one square meter plots) were then conducted in which PNP-contaminated soil from an agricultural field was bioaugmented with strain RKJ100 under natural environmental conditions. PNP was totally depleted in 5 days by immobilized cells, whereas free cells were able to deplete 75% of PNP in the same time period. The fate of the released strain as monitored by plate counts, hybridization studies, and real-time polymerase chain reaction revealed fairly stable population of the cells upon immobilization on corncob powder throughout the period of study.
Bioremediation of contaminated sites has been accepted as an efficient and cheaper alternative to... more Bioremediation of contaminated sites has been accepted as an efficient and cheaper alternative to physicochemical means of remediation in several cases. Although chemotactic behaviour of many bacteria has been studied earlier and assays have been developed to study bacterial chemotaxis in semi-solid media, this phenomenon has never been demonstrated in soil. For bioremediation application it is important to know whether bacteria actually migrate through the heterogenous soil medium towards a gradient of a particular chemoattractant. In the present study we have successfully demonstrated bacterial chemotaxis of a Ralstonia sp. SJ98 in soil microcosm using qualitative and quantitative plate and tray assays. The migration of bacteria has been established using several methods such as plate counting, vital staining and flow cytometry and slot blot hybridization. A non-chemotactic p-nitrophenol utilizing strain Burkholderia cepacia RKJ200 has been used as negative control. Our work clearly substantiates the hypothesis that chemotactic bacteria may enhance in situ bioremediation of toxic pollutants from soils and sediments.
We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 ... more We have earlier reported a novel reductive pathway for o-nitrobenzoate (ONB) degradation (at 0.5 mM) in Arthrobacter protophormiae RKJ100, which proceeds via the formation of o-hydroxylaminobenzoate (HABA) and anthranilate (AA). During growth of this organism at 40 times higher concentration (20 mM) of ONB, 3-hydroxyanthranilate (HAA) was identified as an intermediate by thin layer chromatography, gas chromatography and high performance liquid chromatography studies. Crude cell extracts of ONB-grown cells showed HAA 3,4-dioxygenase activity suggesting HAA as a terminal aromatic intermediate of the catabolic energy-yielding pathway as shown before in Pseudomonas fluorescens strain KU-7. HAA is further cleaved to 2-amino-3-carboxymuconic-6-semialdehyde by the action of HAA 3,4-dioxygenase. In this report we propose that ONB degradation occurs via the formation of HABA and the pathway branches at this point to form the two different aromatic intermediates AA and HAA by the action of a reductase and a mutase, respectively.
Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pa... more Microbial degradation studies have pointed toward the occurrence of two distinct PNP catabolic pathways in Gram positive and Gram negative bacteria. The former involves 4-nitrocatechol (4-NC), 1,2,4-benzenetriol (BT), and maleylacetate (MA) as major degradation intermediates, whereas the later proceeds via formation of 1,4-benzoquinone (BQ) and hydroquinone (HQ). In the present study we identified a Gram negative organism viz. Burkholderia sp. strain SJ98 that degrades PNP via 4NC, BT, and MA. A 6.89 Kb genomic DNA fragment of strain SJ98 that encompasses seven putatively identified ORFs (orfA, pnpD, pnpC, orfB, orfC, orfD, and orfE) was cloned. PnpC is benzenetriol dioxygenase belonging to the intradiol dioxygenase superfamily, whereas PnpD is identified as maleylacetate reductase, a member of the Fe-ADH superfamily showing NADH dependent reductase activity. The in vitro activity assays carried out with purified pnpC and pnpD (btd and mar) gene products transformed BT to MA and MA to beta-ketoadipate, respectively. The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies ascertained the involvement of 4-NC, BT, and MA as degradation intermediates of PNP pathway in this strain. This is one of the first conclusive reports for 4-NC and BT mediated degradation of PNP in a Gram negative organism.
Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featurin... more Ring hydroxylating dioxygenases (RHDOs) are one of the most important classes of enzymes featuring in the microbial metabolism of several xenobiotic aromatic compounds. One such RHDO is benzenetriol dioxygenase (BtD) which constitutes the metabolic machinery of microbial degradation of several mono- phenolic and biphenolic compounds including nitrophenols. Assessment of the natural diversity of benzenetriol dioxygenase (btd) gene sequence is of great significance from basic as well as applied study point of view. In the present study we have evaluated the gene sequence variations amongst the partial btd genes that were retrieved from microorganisms enriched for PNP degradation from pesticide contaminated agriculture soils. The gene sequence analysis was also supplemented with an in silico restriction digestion analysis. Furthermore, a phylogenetic analysis based on the deduced amino acid sequence(s) was performed wherein the evolutionary relatedness of BtD enzyme with similar aromatic dioxygenases was determined. The results obtained in this study indicated that this enzyme has probably undergone evolutionary divergence which largely corroborated with the taxonomic ranks of the host microorganisms.
International Journal of Systematic and Evolutionary Microbiology, 2006
A Gram-positive actinobacterium, strain RKJ300 T , capable of utilizing p-nitrophenol and 2,4dini... more A Gram-positive actinobacterium, strain RKJ300 T , capable of utilizing p-nitrophenol and 2,4dinitrophenol, was isolated from a pesticide-contaminated site in India. The morphological and chemotaxonomic properties of the isolate were typical of members of the genus Rhodococcus. The DNA G+C content was 72 mol%. Strain RKJ300 T exhibited the highest level of sequence similarity with Rhodococcus wratislaviensis NCIMB 13082 T (99?3 %), followed by Rhodococcus opacus DSM 43205 T (98?8 %), Rhodococcus percolatus MBS1 T (98?6 %) and Rhodococcus koreensis DNP505 T (98?1 %). The low levels of DNA-DNA relatedness (49-58 %) with the above micro-organisms, and the differences in the biochemical and physiological properties, suggest that strain RKJ300 T should be classified within a novel species of the genus Rhodococcus, for which the name Rhodococcus imtechensis sp. nov. is proposed. The type strain is RKJ300 T (=MTCC 7085 T =JCM 13270 T ).
Bt cotton was the first genetically modified crop approved for use in India. However, only a few ... more Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.
The use of microorganisms is expected to be an effective tool for remediation of environments con... more The use of microorganisms is expected to be an effective tool for remediation of environments contaminated with various aromatic compounds. Aromatic compounds are degraded via diverse ring-cleavage pathways by soil microorganisms. Two bacterial strains isolated from pesticide-contaminated agricultural fields and identified as Arthrobacter protophormiae RKJ100 and Burkholderia cepacia RKJ200 were found to be capable of utilizing p-hydroxybenzoate (PHB) as a sole source of carbon and energy. Thin layer chromatography, gas chromatography and gas chromatography-mass spectrometry of the ethyl acetate extracts from the culture medium showed the presence of protocatechuate. Both the strains follow the ortho-cleavage pathway for the catabolism of PC, since it was found that the terminal aromatic intermediate of the degradation pathway is cleaved to β β-ketoadipate. Plasmid cured derivatives of A. protophormiae and B. cepacia were also investigated to determine the location of the genes involved in PHB degradation.
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Papers by Debarati Paul