Bioremediation of 2,4,6-trinitrotoluene by bacterial nitroreductase-expressing transgenic aspen

НазваниеBioremediation of 2,4,6-trinitrotoluene by bacterial nitroreductase-expressing transgenic aspen
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Bioremediation of 2,4,6-trinitrotoluene by bacterial nitroreductase-expressing transgenic aspen

Pieter van Dillewijn1, José L. Couselo 2, Elena Corredoria2, Antonio Delgado3, Rolf-Michael Wittich1, Antonio Ballester2, and Juan L. Ramos1*

1Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Department of Environmental Protection, E-18008 Granada, Spain

2Instituto de Investigaciones Agrobiológicas de Galicia, Consejo Superior de Investigaciones Científicas, Apdo. Correos 122, E-15780 Santiago de Compostela, Spain

3Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Department of Environmental Geochemistry, E-18008 Granada, Spain

*Corresponding author: Juan L. Ramos


C/ Prof. Albareda 1

18008 Granada


Phone: +34 958 181608

Fax: +34 958 135740



Trees belonging to the genus Populus are often used for phytoremediation (dendroremediation) due to their deep root formation, fast growth and high transpiration rates. Here, we study the capacity of transgenic hybrid aspen (Populus tremula x tremuloides var. Etropole) which express the bacterial nitroreductase, PnrA, to tolerate and take-up greater amounts of the toxic and recalcitrant explosive, 2,4,6-trinitrotoluene (TNT) from contaminated liquid media and soil. Transgenic aspen tolerate up to 57 mg TNT/L in hydroponic media and more than 1000 mg TNT/kg soil while the parental aspen could not endure in hydroponic culture with more than 11 mg TNT/L or soil with more than 500 mg TNT/kg. Likewise, the phytotoxicological limit for transgenic plants to a constant concentration of TNT was 20 mg TNT/L while wild type plants only tolerated 10 mg TNT/L. Transgenic plants also showed improved uptake of TNT over wild type plants when the original TNT concentration was above 35 mg TNT/L in liquid media or 750 mg TNT/kg in soil. Assays with 13C-labeled TNT show rapid adsorption of TNT to the root surface followed by a slower entrance rate into the plant. Most of the 13C- carbon from the labeled TNT taken up by the plant (>95%) remains in the root with little translocation to the stem. Altogether, transgenic aspen expressing PnrA are highly interesting for phytoremediation applications on contaminated soil and underground aquifers.


Improper handling, production, storage and decommissioning of the polynitroaromatic explosive 2,4,6-trinitrotoluene (TNT) has led to extensive contamination of soil and groundwater (1-3). This xenobiotic is toxic to humans, animals, plants and microorganisms and is recalcitrant to degradation (1, 3). To clean up contaminated sites, phytoremediation with plants is receiving increased interest (4, 5). For effective phytoremediation, one should make use of plant species that tolerate contaminants, and have the ability to remove large amounts of target chemicals. The use of tree species for phytoremediation (dendroremediation) has several advantages over smaller plants such as large biomass, long life cycle, low nutrient requirements and intrinsic resistance to many pollutants (6). Trees belonging to the genus Populus are especially useful for phytoremediation because of their deep extensive root system, high water uptake, and rapid growth (5, 8). This genus has also successfully been engineered genetically for phytoremediation purposes (9, 10). Moreover, poplar trees have been shown to resist up to 5 mg TNT/L and remove this xenobiotic from the medium (11-14).

In general, plants appear to deal with TNT as a ’green liver’, whereby the contaminant is detoxified and sequestered within plant tissues rather than mineralized to carbon dioxide and nitrogen (15-17). Detoxification occurs by transforming the chemical, conjugating it to plant metabolites and then sequestering the resulting macromolecules into vacuoles or polymers such as lignin (18-21). As a result, in most terrestrial plants TNT and its derivatives accumulate in the roots and to a lesser extent are transported to the stem and leaves. Similarly in poplar plants, experiments conducted with 14C-TNT showed that about 75% of the radiolabeled carbon remained in roots and lower stems while only about 10% was translocated to the upper stem and leaves (11, 14).

TNT transformation by plants consists mainly of the sequential reduction of the nitro side groups of the molecule by the plant’s endogenous nitroreductases to hydroxylamine intermediates and then to amino derivatives. In poplar plants, aminodinitrotoluenes and as yet unidentified polar metabolites have been detected (11). The latter polar products are probably the result of reduced TNT derivatives conjugated to plant metabolites. Subramanian et al. (22) determined in Arabidopsis thaliana that the conversion of TNT to hydroxylamine derivatives is rate limiting, leading them to suggest that the most promising enzymes for speeding up TNT removal in plants include those, which catalyze this reduction.

Microorganisms have a whole arsenal of nitroreductases, which efficiently reduce the nitro side groups of TNT to different isomers of aminonitrotoluenes. This transformation of TNT to aminonitrotoluenes is additionally interesting because the reduced products can bind irreversibly to clay and organic material in the soil (23, 24). Moreover, many of these enzymes also attack other nitroaromatic compounds, which could be present in contaminated sites. Therefore, in order to improve the phytoremediation capability of plants, bacterial nitroreductases could be engineered into plant genomes. This has been performed with success by Hannink et al. (25) who obtained transgenic tobacco expressing the nitroreductase gene, nfsI, from Enterobacter cloacae. Similarly, Kurumata et al. (26) engineered Arabidopsis thaliana plants to express the nitroreductase, nfsA, of Escherichia coli. In either case, the transgenic plants could resist higher concentrations of TNT as well as take up greater quantities of the xenobiotic from the medium than its wild type counterparts. Here, we improve upon this theme by using a tree species as the target plant. In fact, we describe transgenic aspen (Populus tremula × tremuloides var. Etropole) which express the gene encoding the nitroreductase PnrA of Pseudomonas putida JLR11 (27). This nitroreductase reduces TNT to 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) at a very high rate and also exhibits a relatively broad substrate specificity so that it can reduce other nitroaromatic compounds (28). The aim of this work was to study the characteristics of these transgenic plants compared to the parent wild type plants in different media contaminated with TNT.

Experimental Section

Chemicals. TNT was obtained from Unión Española de Explosivos (Madrid, Spain) and was more than 99% pure. 2-hydroxylamino-4,6-dinitrotoluene (2HADNT), 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 2-amino-4,6-dinitrotoluene (2ADNT), and 4-amino-2,6-dinitrotoluene (4ADNT) were obtained from AccuStandard (New Haven, CT, USA). 13C-TNT was synthesized from >98% pure [ring-U-13C] toluene using the method described by Michels and Gottschalk (29).

Bacterial strains and culture conditions. Pseudomonas putida JLR11, and Agrobacterium tumefaciens C58C1 strains were grown routinely at 28 ºC in Luria Broth (30). Preparation of chromosomal and plasmidic DNA, digestion with restriction enzymes and electrophoresis were carried out using standard methods (30, 31).

pBIpnrA binary plasmid. As described previously, the nitroreductase encoding gene, pnrA, was obtained by amplifying this gene from P. putida JLR11 chromosomal DNA by PCR and cloning the resulting product into pUC19 to obtain pNAJ (28). This plasmid was digested with restriction enzymes XbaI and SacI and the resulting 1 kb fragment was ligated into the binary vector pBI121 (Stratagene, Madrid, Spain), also cut with XbaI and SacI, to yield pBIpnrA. In this way, pnrA was cloned downstream from the cauliflower mosaic virus 35 S promoter.

Plant material. Stock shoot cultures of Populus tremula x tremuloides var. Etropole subcultured at 6-week intervals on MS medium (32), were used as a source for explants. Shoot multiplication of both wild type and transgenic explants were carried out according to Couselo and Corredoira (33). All cultures were kept in a growth chamber with 16 h of light provided by cool-white fluorescent lamps, a temperature of 25 ºC and a photon flux density of 50-60 mol·m-2·s-1. In vitro rooted plants were subjected to acclimatization in a tunnel in a greenhouse and after approximately 9-10 weeks were used for hydroponic and soil experiments.

Plant transformation and molecular analyses. Internodal segments of in vitro micropropagated aspen shoots were transformed by co-culture with A. tumefaciens C58C1 bearing pBIpnrA using the method described by Couselo and Corredoira (33). To confirm the presence of the pnrA genes, PCR and Southern blot hybridization were carried out using standard methods (30) using DNA isolated from leaves of both transgenic and untransformed plants with the DNeasy plant kit (Qiagen). Oligonucleotide primer pairs for pnrA were 5'-AGCCAGCTAACTTACCTGC-3' and 5'-CTCATCCTTCGGTCATAGG-3'. Each successfully transformation event constituted a transgenic line. Two-step real-time RT-PCR was used to determine expression of the transgene in different tissues. Total RNA was extracted with the RNeasy plant mini kit (Qiagen) from leaves, stems and roots of in vitro rooted plants and treated with RNase-free DNase Set (DNase I; Qiagen). cDNA was synthesized using Omniscript reverse transcriptase (Qiagen) and oligo-dT primers according to the manufacturer's protocol. SYBR-Green based quantitative assays were performed in an iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Madrid, Spain). 25 µL reactions consisted of 2.5 µL of cDNA from each sample, 1x SYBR Green PCR Supermix (Bio-Rad, Laboratories, Madrid) and oligonucleotide primer pairs for pnrA: 5'-TCAAGACGAAGCACTCAAAGCC-3'; 5'-GGCACGTACTGATCGATGCTGC-3', and for 18S 5'-AATTGTTGGTCTTCAACGAGGAA-3'; 5'-AAAGGGCAGGGACGTAGTCAA-3' (34). The PCR conditions were: one cycle at 95 °C for 5 min, followed by 50 cycles at 95 °C for 20 sec, 56 ºC for 30 sec, and 72 °C for 30 sec. Each PCR reaction was performed in triplicate together with controls lacking template. The entire assay, including both the RT and real time PCR steps, was repeated two times from two RNA extractions. Data normalization with 18S was performed using the ΔΔCT method (35). The specificity of the amplifications was verified at the end of the PCR run by using melting-curve analysis and by analyzing PCR products with agarose gel electrophoresis.

Hydroponic assays. To determine the concentration of TNT tolerated by wild type and transgenic plants, opaque covered containers containing 2.5 L of Hoagland medium (36) and an aeration system were used. To determine the uptake and toxicity of TNT to wild type and transgenic plants, the effect on transpiration was measured in a gravitational hydroponic system consisting of 250 mL graduated cylinders covered with aluminum foil (to protect roots against light) and filled with Hoagland medium. The roots of acclimatized 10-15 cm long plants were rinsed gently and introduced into either containers or cylinders before fastening with foam rubber plugs. The plants were allowed to acclimatize in the growth chamber for 2 days prior to each experiment. Growth chamber conditions consisted of 60% humidity, 14:10h light: dark photoperiod, 24:18 °C day:night temperature. Each experiment was initiated by replacing the Hoagland medium with medium spiked with different concentrations of TNT. The tolerance assay consisted of exposing four plants to Hoagland medium spiked with a range of TNT concentrations from 0 - 57 mg TNT/L and measuring plant length at a regular basis for 28 days and finally dry weights when the experiment had terminated. The concentrations of TNT and its metabolites were measured (see below) regularly as well. For the uptake experiment, three to four plants were exposed to Hoagland medium spiked with 20, 35 or 50 mg TNT/L and the concentrations of TNT measured at regular time intervals as described below for 48 h. In these experiments the TNT half life was calculated using the slope of the curve multiplied by half the value of the original TNT concentration. For the toxicity experiments, the transpiration of four plants was monitored volumetrically for 2 weeks. These plants were exposed to a relatively constant concentration of TNT by replacing the Hoagland medium spiked with 0, 5, 10, 15, 20, 35 or 50 mg TNT/L every two days. In this experiment, the relative transpiration, RT, was calculated as described in Equation 1 by which the amount of medium transpired by each plant (in mL) at each time point (Tr) was compared to the mean amount (in mL) transpired by plants of the same line but in control uncontaminated medium (Tr0) at the same time point. For comparison purposes the RT of plants in uncontaminated medium was calculated at each time point (mean RT always equals 1) and the largest standard error (p< 0.05) used as upper and lower limits to obtain the grey bars in Figure 4.

To determine TNT and its transformation products in plant organs, at least six plants were exposed for 48 h to Hoagland medium saturated with TNT crystals (approximately 113.5 mg TNT/L). After this period the plants were removed, roots separated from the shoots, and each plant part weighed before freezing at 80 ºC. Prior to freezing, roots were washed three times for 5 minutes with 20% (v/v) methanol/water to remove TNT or its metabolites which had adsorbed to the root surface, in order to obtain accurate incorporation data/rates and exclude or minimize false-positive results.
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