The Experts below are selected from a list of 618 Experts worldwide ranked by ideXlab platform
Magnus Steigedal - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Benzoic Acid-Inducible Gene Expression in
2016Co-Authors: Marte S. Dragset, Amy K. Barczak, Nisha Kannan, Mali Mærk, Trude H. Flo, Svein Valla, Eric J. Rubin, Magnus SteigedalAbstract:Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression inMycobacterium smegmatis andMycobacterium tuber-culosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducerm-Toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expres-sion tools, especially when low basal expression is of particular importance
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benzoic acid inducible gene expression in mycobacteria
PLOS ONE, 2015Co-Authors: Amy K. Barczak, Nisha Kannan, Mali Mærk, Svein Valla, Eric J. Rubin, Magnus Steigedal, Marte Singsas DragsetAbstract:Conditional expression is a powerful tool to investigate the role of bacterial genes. Here, we adapt the Pseudomonas putida-derived positively regulated XylS/Pm expression system to control inducible gene expression in Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of human tuberculosis. By making simple changes to a Gram-negative broad-host-range XylS/Pm-regulated gene expression vector, we prove that it is possible to adapt this well-studied expression system to non-Gram-negative species. With the benzoic acid-derived inducer m-Toluate, we achieve a robust, time- and dose-dependent reversible induction of Pm-mediated expression in mycobacteria, with low background expression levels. XylS/Pm is thus an important addition to existing mycobacterial expression tools, especially when low basal expression is of particular importance.
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Induction of Pm with m-Toluate is robust, time- and dose-dependent.
2015Co-Authors: Marte S. Dragset, Amy K. Barczak, Nisha Kannan, Mali Mærk, Trude H. Flo, Svein Valla, Eric J. Rubin, Magnus SteigedalAbstract:Msmeg transformed with the expression vectors pMDX-luc or the empty vector pMDX (no reporter gene) treated with increasing concentrations of m-Toluate (induced) or ethanol carrier (uninduced). Cells were incubated at 30°C, and luciferase expression was determined at 2.5, 5.5, 11, 23, 31 and 49 hours after addition of m-Toluate. (A) Fold induction of RLU in induced samples compared to uninduced samples. (B) Growth of uninduced and induced samples of pMDX-luc corresponding to samples in (A). (C) Time course of luciferase induction from pMDX-luc with 1.5 mM m-Toluate or ethanol carrier. (D) Maximal induction of pMH109 and pMDX-luc-transformed Msmeg induced with 2 mM m-Toluate. (E) Amount of luciferase produced as determined by the activity of known luciferase concentrations (0, 0.1, 0.2, 0.4 0.6, 0.8 and 1 μg/ml luciferase) in mid log phase or (F) stationary phase. Luciferase fraction of total bacterial protein shown in brackets. RLUs were normalized to the OD600 of the samples before luciferase assay. All results are representative of two or more independent experiments.
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Regulation of the zeocin resistance gene in the presence of zeocin.
2015Co-Authors: Marte S. Dragset, Amy K. Barczak, Nisha Kannan, Mali Mærk, Trude H. Flo, Svein Valla, Eric J. Rubin, Magnus SteigedalAbstract:(A) Msmeg transformed with pMDX-zeo was grown to OD600 0.05–0.1 before addition of 1.5 mM m-Toluate (induced) or ethanol carrier (uninduced) was added. Cells were then incubated for 5 hours at 30°C. The cells were normalized by OD600, serially diluted, and spotted on plates containing increasing amounts of zeocin and 1.5 mM m-Toluate (induced) or ethanol (uninduced) and incubated at 30°C for 2 days. (B) Msmeg transformed with pMDX-zeo was pre-induced for 5 hours as described above, then diluted to OD600 0.005 and grown in triplicates in micro-plate wells in the presence of increasing concentrations of zeocin (0, 0.5, 2.5, 5, 7.5, 10, 15, 25, 50, 100, 150, 200 and 250 μg/ml) and 1.5 mM m-Toluate (induced) or ethanol carrier (uninduced) shaking at 37°C. Growth was monitored by Bioscreen, registering OD600 every other hour. The samples are presented by the OD600 of uninduced or induced Msmeg pMDX-zeo in increasing concentrations of zeocin, when the respective sample grown in the absence of zeocin reached mid log phase. Error bars represent standard deviations and the results represent three independent experiments.
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Benzoic acid-inducible expression system, XylS/Pm, for regulation of genes in mycobacteria.
2015Co-Authors: Marte S. Dragset, Amy K. Barczak, Nisha Kannan, Mali Mærk, Trude H. Flo, Svein Valla, Eric J. Rubin, Magnus SteigedalAbstract:The inducible Pm promoter and its activator XylS regulate the expression of “your favorite gene” (YFG). XylS is constitutively expressed under control of Ptet in the absence of anhydro-tetracycline (atc) and binds the Pm promoter in the presence of the inducer m-Toluate. This facilitates expression of YFG and leaves the expression system ON (upper panel). In the absence of m-Toluate, XylS is not activated, leaving the expression system OFF, as expression from Pm is not induced (middle panel). Reverse TetR is constitutively expressed by Psmyc, and binds the operator in Ptet in the presence of atc blocking transcription of xylS. Addition of atc leaves the system in a more fully OFF mode as potential basal Pm-mediated transcription caused by excessive levels of XylS is abolished (lower panel).
Leena Suominen - One of the best experts on this subject based on the ideXlab platform.
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rhizosphere effect of galega orientalis in oil contaminated soil
Soil Biology & Biochemistry, 2006Co-Authors: Anna H Kaksonen, Minna M. Jussila, Kristina Lindstrom, Leena SuominenAbstract:Abstract Randomized lysimeters in an oil-contaminated field contained the following treatments: (1) Galega orientalis seeds inoculated with Rhizobium galegae HAMBI 540, (2) bioaugmentation with Pseudomonas putida PaW85, and (3) R. galegae -inoculated G. orientalis seeds plus bioaugmentation with P. putida PaW85. The bacterial abundance and diversity were analysed in composite samples after one growing season. A total of 208 m-Toluate tolerating bacteria were isolated and screened with m-Toluate tolerance and utilization tests, and the catechol test. Seventy-nine isolates were characterized with (GTG)5-PCR genomic fingerprinting and 16S rRNA gene PCR-RFLP ribotyping. Only 10% of the isolated strains were able to degrade m-Toluate. Most of the m-Toluate utilizing bacteria were catechol positive indicating the existence of a TOL plasmid. Rhizosphere effect of G. orientalis was manifested in oil-contaminated soil. G. orientalis and Pseudomonas bioaugmentation increased the amount of bacteria in oil-contaminated soil. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-Toluate utilizing and catechol positive bacteria in the soil samples indicating an increase in degradation potential. The rhizosphere of G. orientalis increased also the diversity of bacteria. More ribotypes were found in soils treated with G. orientalis and P. putida PaW85 compared to the untreated soil, but the diversity of the m-Toluate utilizing bacteria did not significantly increase.
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genetic diversity of culturable bacteria in oil contaminated rhizosphere of galega orientalis
Environmental Pollution, 2006Co-Authors: Minna M. Jussila, Kristina Lindstrom, German Jurgens, Leena SuominenAbstract:A collection of 50 indigenous meta-Toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-Toluate also degraded m-Toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-Toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-Toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-Toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.
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potential of the galega rhizobium galegae system for bioremediation of oil contaminated soil
Food Technology and Biotechnology, 2003Co-Authors: Kristina Lindstrom, Jyrki Pitkajarvi, Minna M. Jussila, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Leena SuominenAbstract:Summary Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence.
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Potential of the Galega – Rhizobium galegae System for Bioremediation of Oil-Contaminated Soil
University of Zagreb, 2003Co-Authors: Minna M. Jussila, Leena Suominen, Jyrki Pitkajarvi, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Kristina LindstromAbstract:Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence
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evaluation of the galega rhizobium galegae system for the bioremediation of oil contaminated soil
Environmental Pollution, 2000Co-Authors: Leena Suominen, Minna M. Jussila, Katri Mäkeläinen, Martin Romantschuk, Kristina LindstromAbstract:The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-Toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-Toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-Toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-Toluate-free medium. Nodulation was blocked under the influence of m-Toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-Toluate. Thus, this legume system has good potential for use on oil-contaminated sites
Kristina Lindstrom - One of the best experts on this subject based on the ideXlab platform.
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rhizosphere effect of galega orientalis in oil contaminated soil
Soil Biology & Biochemistry, 2006Co-Authors: Anna H Kaksonen, Minna M. Jussila, Kristina Lindstrom, Leena SuominenAbstract:Abstract Randomized lysimeters in an oil-contaminated field contained the following treatments: (1) Galega orientalis seeds inoculated with Rhizobium galegae HAMBI 540, (2) bioaugmentation with Pseudomonas putida PaW85, and (3) R. galegae -inoculated G. orientalis seeds plus bioaugmentation with P. putida PaW85. The bacterial abundance and diversity were analysed in composite samples after one growing season. A total of 208 m-Toluate tolerating bacteria were isolated and screened with m-Toluate tolerance and utilization tests, and the catechol test. Seventy-nine isolates were characterized with (GTG)5-PCR genomic fingerprinting and 16S rRNA gene PCR-RFLP ribotyping. Only 10% of the isolated strains were able to degrade m-Toluate. Most of the m-Toluate utilizing bacteria were catechol positive indicating the existence of a TOL plasmid. Rhizosphere effect of G. orientalis was manifested in oil-contaminated soil. G. orientalis and Pseudomonas bioaugmentation increased the amount of bacteria in oil-contaminated soil. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-Toluate utilizing and catechol positive bacteria in the soil samples indicating an increase in degradation potential. The rhizosphere of G. orientalis increased also the diversity of bacteria. More ribotypes were found in soils treated with G. orientalis and P. putida PaW85 compared to the untreated soil, but the diversity of the m-Toluate utilizing bacteria did not significantly increase.
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genetic diversity of culturable bacteria in oil contaminated rhizosphere of galega orientalis
Environmental Pollution, 2006Co-Authors: Minna M. Jussila, Kristina Lindstrom, German Jurgens, Leena SuominenAbstract:A collection of 50 indigenous meta-Toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-Toluate also degraded m-Toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-Toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-Toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-Toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.
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potential of the galega rhizobium galegae system for bioremediation of oil contaminated soil
Food Technology and Biotechnology, 2003Co-Authors: Kristina Lindstrom, Jyrki Pitkajarvi, Minna M. Jussila, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Leena SuominenAbstract:Summary Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence.
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Potential of the Galega – Rhizobium galegae System for Bioremediation of Oil-Contaminated Soil
University of Zagreb, 2003Co-Authors: Minna M. Jussila, Leena Suominen, Jyrki Pitkajarvi, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Kristina LindstromAbstract:Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence
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evaluation of the galega rhizobium galegae system for the bioremediation of oil contaminated soil
Environmental Pollution, 2000Co-Authors: Leena Suominen, Minna M. Jussila, Katri Mäkeläinen, Martin Romantschuk, Kristina LindstromAbstract:The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-Toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-Toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-Toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-Toluate-free medium. Nodulation was blocked under the influence of m-Toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-Toluate. Thus, this legume system has good potential for use on oil-contaminated sites
Minna M. Jussila - One of the best experts on this subject based on the ideXlab platform.
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rhizosphere effect of galega orientalis in oil contaminated soil
Soil Biology & Biochemistry, 2006Co-Authors: Anna H Kaksonen, Minna M. Jussila, Kristina Lindstrom, Leena SuominenAbstract:Abstract Randomized lysimeters in an oil-contaminated field contained the following treatments: (1) Galega orientalis seeds inoculated with Rhizobium galegae HAMBI 540, (2) bioaugmentation with Pseudomonas putida PaW85, and (3) R. galegae -inoculated G. orientalis seeds plus bioaugmentation with P. putida PaW85. The bacterial abundance and diversity were analysed in composite samples after one growing season. A total of 208 m-Toluate tolerating bacteria were isolated and screened with m-Toluate tolerance and utilization tests, and the catechol test. Seventy-nine isolates were characterized with (GTG)5-PCR genomic fingerprinting and 16S rRNA gene PCR-RFLP ribotyping. Only 10% of the isolated strains were able to degrade m-Toluate. Most of the m-Toluate utilizing bacteria were catechol positive indicating the existence of a TOL plasmid. Rhizosphere effect of G. orientalis was manifested in oil-contaminated soil. G. orientalis and Pseudomonas bioaugmentation increased the amount of bacteria in oil-contaminated soil. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-Toluate utilizing and catechol positive bacteria in the soil samples indicating an increase in degradation potential. The rhizosphere of G. orientalis increased also the diversity of bacteria. More ribotypes were found in soils treated with G. orientalis and P. putida PaW85 compared to the untreated soil, but the diversity of the m-Toluate utilizing bacteria did not significantly increase.
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genetic diversity of culturable bacteria in oil contaminated rhizosphere of galega orientalis
Environmental Pollution, 2006Co-Authors: Minna M. Jussila, Kristina Lindstrom, German Jurgens, Leena SuominenAbstract:A collection of 50 indigenous meta-Toluate tolerating bacteria isolated from oil-contaminated rhizosphere of Galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16S rDNA partial sequencing showed the presence of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. Only one-fifth of the strains that tolerated m-Toluate also degraded m-Toluate. The inoculum Pseudomonas putida PaW85 was not found in the rhizosphere samples. The ability to degrade m-Toluate by the TOL plasmid was detected only in species of the genus Pseudomonas. However, a few Rhodococcus erythropolis strains were found which were able to degrade m-Toluate. A new finding was that Pseudomonas migulae strains and a few P. oryzihabitans strains were able to grow on m-Toluate and most likely contained the TOL plasmid. Because strain specific differences in degradation abilities were found for P. oryzihabitans, separation at the strain level was important. For strain specific separation (GTG)5 fingerprinting was the best method. A combination of the single locus ribotyping and the whole genomic fingerprinting techniques with the selective partial sequencing formed a practical molecular toolbox for studying genetic diversity of culturable bacteria in oil-contaminated rhizosphere.
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potential of the galega rhizobium galegae system for bioremediation of oil contaminated soil
Food Technology and Biotechnology, 2003Co-Authors: Kristina Lindstrom, Jyrki Pitkajarvi, Minna M. Jussila, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Leena SuominenAbstract:Summary Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence.
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Potential of the Galega – Rhizobium galegae System for Bioremediation of Oil-Contaminated Soil
University of Zagreb, 2003Co-Authors: Minna M. Jussila, Leena Suominen, Jyrki Pitkajarvi, Katri Mäkeläinen, Hannamari Hintsa, Anna H Kaksonen, Lenna Mokelke, Kristina LindstromAbstract:Bioremediation potential of the nitrogen-fixing leguminous plant Galega orientalis Lam. and its microsymbiont Rhizobium galegae was evaluated in microcosm and mesocosm scale in oil and BTEX (benzene, toluene, ethylbenzene, xylene) contaminated soils, with m-Toluate serving as a model for the latter group. G. orientalis and Rhizobium galegae remained viable in m-Toluate fractions up to 3000 ppm. Plant growth and nodulation were inhibited in 500 ppm m-Toluate, but were restored when plants were transferred to clean medium. In soil, G. orientalis nodulated and showed good growth in 2000 ppm m-Toluate as well as in diesel-contaminated soil in the field, where the plant was stimulating bacterial growth in the rhizosphere. A collection of 52 indigenous m-Toluate-tolerating bacteria isolated from oil-contaminated rhizosphere of G. orientalis was characterised and identified by classical and molecular biological methods. 16SrDNA PCR-RFLP and (GTG)5-PCR genomic fingerprinting combined with partial sequencing indicated the presence of five major lineages of the Bacteria domain. A TOL plasmid-specific xylE-PCR was developed in order to detect both active and potential degraders of m-Toluate. The ability to degrade m-Toluate in the presence of the gene xylE was detected only within the genus Pseudomonas. The isolates were tested for capacity to grow on m-Toluate as their sole carbon and energy source. In laboratory experiments, the best rhizosphere isolates performed equally well to the positive control strain and are good candidates for inoculant production in the future. They have been tagged with marker genes for further studies on colonisation and persistence
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evaluation of the galega rhizobium galegae system for the bioremediation of oil contaminated soil
Environmental Pollution, 2000Co-Authors: Leena Suominen, Minna M. Jussila, Katri Mäkeläinen, Martin Romantschuk, Kristina LindstromAbstract:The bioremediation potential of a nitrogen-fixing leguminous plant, Galega orientalis, and its microsymbiont Rhizobium galegae was evaluated in BTX (benzene, toluene, xylene)-contaminated soils in microcosm and mesocosm scale. To measure the intrinsic tolerance of the organisms to m-Toluate, a model compound representing BTX, G. orientalis and R. galegae were cultivated under increasing concentrations of m-Toluate alone and in association with Pseudomonas putida pWWO, a bacterial strain able to degrade toluene-derived compounds. The test plants and rhizobia remained viable in m-Toluate concentrations as high as 3000 ppm. Plant growth was inhibited in concentrations higher than 500 ppm, but restituted when plants were transferred into m-Toluate-free medium. Nodulation was blocked under the influence of m-Toluate, but was restored after the plants were transferred into the non-contaminated media. In the mesocosm assay the Galega plants showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 ppm m-Toluate. Thus, this legume system has good potential for use on oil-contaminated sites
Shigeaki Harayama - One of the best experts on this subject based on the ideXlab platform.
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construction of chimeric catechol 2 3 dioxygenase exhibiting improved activity against the suicide inhibitor 4 methylcatechol
Applied and Environmental Microbiology, 2004Co-Authors: Akiko Okuta, Kouhei Ohnishi, Shigeaki HarayamaAbstract:Catechol 2,3-dioxygenase (C23O; EC 1.3.11.2), exemplified by XylE and NahH, catalyzes the ring cleavage of catechol and some substituted catechols. C23O is inactivated at an appreciable rate during the ring cleavage of 4-methylcatechol due to the oxidation of the Fe(II) cofactor to Fe(III). In this study, a C23O exhibiting improved activity against 4-methylcatechol was isolated. To isolate this C23O, diverse C23O gene sequences were PCR amplified from DNA which had been isolated from mixed cultures of phenol-degrading bacteria and subcloned in the middle of a known C23O gene sequence (xylE or nahH) to construct a library of chimeric C23O genes. These chimeric C23O genes were then introduced into Pseudomonas putida possessing some of the toluene catabolic genes (xylXYZLGFJQKJI). When a C23O gene (e.g., xylE) is introduced into this strain, the transformants cannot generally grow on p-Toluate because 4-methylcatechol, a metabolite of p-Toluate, is a substrate as well as a suicide inhibitor of C23O. However, a transformant of this strain capable of growing on p-Toluate was isolated, and a chimeric C23O (named NY8) in this transformant was characterized. The rate of enzyme inactivation by 4-methylcatechol was lower in NY8 than in XylE. Furthermore, the rate of the reactivation of inactive C23O in a solution containing Fe(II) and ascorbic acid was higher in NY8 than in XylE. These properties of NY8 might allow the efficient metabolism of 4-methylcatechol and thus allow host cells to grow on p-Toluate.
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nucleotide sequences of the acinetobacter calcoaceticus benabc genes for benzoate 1 2 dioxygenase reveal evolutionary relationships among multicomponent oxygenases
Journal of Bacteriology, 1991Co-Authors: Ellen L Neidle, C Hartnett, L N Ornston, Amos Marc Bairoch, M Rekik, Shigeaki HarayamaAbstract:The nucleotide sequences of the Acinetobacter calcoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent Toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the alpha-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(II) ion. The less conserved beta-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the Toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.
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a pseudomonas putida strain able to degrade m Toluate in the presence of 3 chlorocatechol
Nature Biotechnology, 1991Co-Authors: Alain Wasserfallen, M Rekik, Shigeaki HarayamaAbstract:Growth on m-Toluate of Pseudomonas putida carrying the TOL plasmid is inhibited by 3-chlorocatechol. From a P. putida strain, carrying cloned TOL genes responsible for the oxidation of methylbenzoates, we isolated a derivative able to grow on m-Toluate in the presence of 3-chlorobenzoate. A point mutation in xylE, the structural gene for catechol 2,3-dioxygenase, which produced the substitution of valine-291 for isoleucine-291, was responsible for the growth characteristics of the isolate. The mutant catechol 2,3-dioxygenase has (i) decreased affinity for 3-chlorocatechol, (ii) increased affinity for 3-methylcatechol, and (iii) increased efficiency in oxidizing 4-ethylcatechol, compared to the wild-type enzyme. This strain may be useful for the decomposition of m-Toluate in industrial waste containing both 3-chlorobenzoate and m-Toluate.
