Microbial Transformation of 2,4,6-Trinitrotoluene in Aerobic Soil Columns

2,4,6-Trinitrotoluene (TNT)-contaminated soil material of a former TNT production plant was percolated aerobically in soil columns. Nineteen days of percolation with a potassium phosphate buffer supplemented with glucose or glucose plus ammonium sulfate caused an over 90% decline in the amount of extractable nitroaromatics in soils containing 70 to 2,100 mg of TNT per kg (dry weight). In the percolation solution, a complete elimination of TNT was achieved. Mutagenicity and soil toxicity were significantly reduced by the percolation process. 4-N-Acetylamino-2-amino-6-nitrotoluene was generated in soil and percolation fluid as a labile TNT metabolite.     

Biotransformation Patterns of 2,4,6-Trinitrotoluene by Aerobic Bacteria

2,4,6-Trinitrotoluene (TNT), a toxic nitroaromatic explosive, accumulates in the environment, making necessary the remediation of contaminated areas and unused materials. Although bioremediation has been utilized to detoxify TNT, the metabolic processes involved in the metabolism of TNT have proven to be complex. The three aerobic bacterial strains reported here (Pseudomonas aeruginosa, Bacillus sp., and Staphylococcus sp.) differ in their ability to biotransform TNT and in their growth characteristics in the presence of TNT. In addition, enzymatic activities have been identified that differ in the reduction of nitro groups, cofactor preferences, and the ability to eliminate-NO₂ from the ring. The Bacillus sp. has the most diverse bioremediation potential owing to its growth in the presence of TNT, high level of reductive ability, and capability of removing-NO₂ from the nitroaromatic ring. Received: 16 May 1997 / Accepted: 19 July 1997     

Initial Stages of 2,4,6-Trinitrotoluene Transformation by Microorganisms

Screening of a wide range of microorganisms (32 strains) isolated from various anthropogenic and natural environments and of a number of collection strains showed that the early stages of 2,4,6-trinitrotoluene (TNT) transformation by the majority of the strains studied resulted in the formation of hydroxylaminodinitrotoluenes (HADNTs). The levels of HADNTs were in a number of cases comparable to the initial TNT level. The alternative reductive attack on TNT through the reduction of the aromatic ring was not characteristic of most of the prokaryotes studied. The susceptibility to the toxic effect of TNT was different for gram-positive and gram-negative bacteria.     

Anaerobic/Aerobic Composting of Soil Contaminated with 2,4,6-Trinitrotoluene

A loam soil from Pennsylvania without a history of exposure to explosives was incubated with 5 g kg^-1 of ¹⁵N-labeled 2,4,6-trinitrotoluene (TNT) and 200 μCi kg^-1 of ¹⁴C-TNT for 3 days and then amended with compost at a 1:2 soil to compost ratio. The compost was prepared by mixing 40% alfalfa hay, 40% grass hay, 10% spent mushroom compost, and 10% municipal biosolids. The mixture of soil and compost was inoculated with methanogens from cattle manure, amended with glucose and starch, and incubated for 37 days under anaerobic conditions. The anaerobic incubation was followed by 26 days of forced aerobic incubation. At the end of the aerobic phase, most of the radioactivity was associated with organic matter; only 8.7% could be extracted with water and methanol, but no TNT was present in the extracts as determined by high-performance liquid chromatography. The unextractable radioactivity was associated with humic acid (40.0±1.0%), fulvic acid (14.3±1.4%), and humin (28.2±0.5%). Radioactive materials associated with humic acid and humin were analyzed by solid-state ¹⁵N-nuclear magnetic resonance (NMR) spectrometry. The NMR spectra indicated that nitro groups of TNT had been reduced to amino groups thatwere subsequently involved in the formation of covalent bonds with soil organic matter.     

Alternative Pathways of the Initial Transformation of 2,4,6-Trinitrotoluene by Yeasts

A new model for the initial transformation of 2,4,6-trinitrotoluene (TNT) by facultatively anaerobic and aerobic yeasts is presented. The model is based on the data that Saccharomyces sp. ZS-A1 was able to reduce the nitrogroups of TNT with the formation of 2- and 4-hydroxyaminodinitrotoluenes (2-HADNT and 4-HADNT) as the major early TNT metabolites (the molar HADNT/TNT ratio reached 0.81), whereas aminodinitrotoluenes (ADNTs) and the hydride-Meisenheimer complex of TNT (H-TNT) were the minor products. Candidasp. AN-L13 almost completely transformed TNT into H-TNT through the reduction of the aromatic ring. Candida sp. AN-L14 transformed TNT through a combination of the two mechanisms described. Aeration stimulated the production of HADNT from TNT, whereas yeast incubation under stationary conditions promoted the formation of HADNT. The transformation of TNT into HADNT led to a tenfold increase in the acute toxicity of the TNT preparation with respect to Paramecium caudatum, whereas the increase in the toxicity was about twofold in the case of the alternative attack at the aromatic ring.     

Biological Degradation of 2,4,6-Trinitrotoluene

Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives. These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms. 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound. Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons. Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes. Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates. Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes. Anaerobic microorganisms can also degrade TNT through different pathways. One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known. Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized. Another pathway has been described in Pseudomonas sp. strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells. It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis. In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils. These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material. These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.     

Aerobic Gram-Positive and Gram-Negative Bacteria Exhibit Differential Sensitivity to and Transformation of 2,4,6-Trinitrotoluene (TNT)

A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 μg TNT ml⁻¹. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 μg TNT ml⁻¹ became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 μg TNT ml⁻¹, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination. Received: 2 December 1996 / Accepted: 3 February 1997     

Biotransformation of 2,4,6-Trinitrotoluene by Pure Culture Ruminal Bacteria

Twenty-one ruminal bacteria species were tested for their ability to degrade 2,4,6-trinitrotoluene (TNT) within 24 h. Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lactobacillus vitulinus, Selenomonas ruminantium, Streptococcus caprinus, and Succinivibrio dextrinosolvens were able to completely degrade 100 mg/L TNT, with <5% of the original TNT recovered as diaminonitrotoluene metabolites. Eubacterium ruminantium, Lactobacillus ruminis, Ruminobacter amylophilus, Streptococcus bovis, and Wolinella succinogenes were able to completely degrade 100 mg/L TNT, with 23–60% of the TNT recovered as aminodinitrotoluene and/or diaminonitrotoluene metabolites. Clostridium polysaccharolyticum, Megasphaera elsdenii, Prevotella bryantii, Prevotella ruminicola, Ruminococcus albus, and Ruminococcus flavefaciens were able to degrade 80–90% of 100 mg/L TNT. Desulfovibrio desulfuricans subsp. desulfuricans, Prevotella albensis, and Treponema bryantii degraded 50–80% of the TNT. Anaerovibrio lipolytica was completely inhibited by 100 mg/L TNT. These results indicate that a variety of rumen bacteria is capable of transforming TNT.     

Accelerated Transformation and Binding of 2,4,6-Trinitrotoluene in Rhizosphere Soil

Enhanced microbial activity and xenobiotic transformations take place in the rhizosphere. Degradation and binding of 2,4,6-trinitrotoluene (TNT) were determined in two rhizosphere soils (RS) and compared to respective unplanted control soils (CS). The rhizosphere soils were obtained after growing corn for 70 d in soils containing 2.8% (Soil A) or 5.9% (Soil B) organic matter. Aerobically agitated soil slurries (3:1, solution/soil) were prepared from RS and CS and amended with 75 mg TNT L^-1 (¹⁴C-labeled). TNT degraded more rapidly and formed more un-extractable bound residue in RS than in CS. In Soil A, total extractable TNT decreased from 225 to 1.0 mg kg^-1 in RS, whereas 11 mg kg^-1 remained in CS after 15 d. Unextractable bound ¹⁴C residues accounted for 40% of the added ¹⁴C-TNT in RS and 28% in CS. The smaller differences in Soil B were attributed partially to the higher organic matter content. The predominant TNT degradation products were monoaminodinitrotoluenes (ADNT), which accumulated and disappeared more rapidly in RS than in CS, and hydroxylaminodinitrotoluenes (HADNT). When sterilized by γ-irradiation, no significant differences between RS and CS were observed in TNT loss or bound residue formation. More rapid TNT degradation and enhanced bound residue formation in the unsterilized RS was attributed to micro-bial-facilitated production and transformation of HADNT and ADNT, which are potential precursors to bound residue formation. If plants can be established on TNT-contaminated soil, these results indicate that the rhizosphere can accelerate reductive transformation of TNT and promote bound residue formation.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K_m of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R² = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.     

Specific Toxic Effects of 2,4,6-Trinitrotoluene on Bacillus subtilisSK1

Using Bacillus subtilis SK1 as an example, it was demonstrated for the first time that 2,4,6-trinitrotoluene (TNT) transformation pathways change with TNT concentration. The growth of cultured B. subtilis SK1, delayed at 20 mg/l TNT (minimum toxic concentration), was resumed following TNT transformation. Aromatic amines were predominant metabolites detected in the culture medium at early stages of TNT transformation. The culture growth was completely inhibited by 200 mg/l TNT. As this took place, nitrites accumulated in the culture medium.     

Fate and Transport of 2,4,6-Trinitrotoluene in Loams at a Former Explosives Factory

Natural attenuation processes affecting 2,4,6-trinitrotoluene (TNT) were determined within loams for two study areas at the former Explosives Factory Maribyrnong, Australia. TNT fate and transport was investigated through spectrophotometric/High Performance Liquid Chromatography (HPLC) analyses of soil and groundwater, adsorption and microcosm testwork. A five tonne crystalline TNT source zone delineated within near surface soils at the base of a TNT process waste lagoon was found to be supplying aqueous TNT loading (7 ppm) to subsurface soils and groundwater. The resultant plume was localized within the loam aquitard due to a combination of natural attenuation processes and hydrogeological constraints, including low hydraulic conductivity and upward hydraulic gradients. Freundlich described sorptive partitioning was the main TNT sink (K_F = 29 mL/g), while transformation rates were moderate (1.01 × 10^-4 h^-1) under the aerobic conditions. Increasing 2-amino-4,6-dinitrotoluene predominance over 4-amino-2,6-dinitrotoluene was discovered with depth (in situ) and time (microcosms). Simplified dissolution rate calculations indicate that without mitigation of the TNT source, contaminant persistence within the vadose zone may approach 2000 years, while ATRANS20 simulations demonstrate that the TNT plume propagates very slowly along the flow path within the aquitard.     

Mass Balance Studies with 14C-Labeled 2,4,6-Trinitrotoluene (TNT) Mediated by an Anaerobic Desulfovibrio Species and an Aerobic Serratia Species

Investigations were carried out to evaluate the level of incorporation of radiolabeled 2,4,6-trinitrotoluene (TNT) and metabolites into the bacterial biomass of two different bacterial species after cometabolically mediated TNT transformation. Biotransformation experiments with ¹⁴C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with the cells. It was found that more than 42% of the initially applied radiolabel was associated with the cell biomass after cometabolic ¹⁴C-TNT transformation with the strictly anerobic Desulfovibrio species strain SHV, whereas with the strictly aerobic Serratia plymuthica species strain B7, 32% of cell-associated ¹⁴C activity was measured. The remainder of the radiolabel was present in the supernatants of the liquid cultures in the form of different TNT metabolites. Under anoxic conditions with the Desulfovibrio species, TNT was ultimately transformed to 2,4,6-triaminotoluene (TAT) and both diaminonitrotoluene isomers, whereas under oxic conditions with the Serratia species, TNT was converted to hydroxylaminodinitrotoluenes and aminodinitrotoluenes, with 4-amino-2,6-dinitrotoluene (4ADNT) being the major end product. In both culture supernatants, small amounts of very polar, radiolabeled, but unidentified metabolites were detected. At the end of the experiments approximately 92% and 96% of the originally applied radioactivity was recovered in the studies with the Serratia and Desulfovibrio species, respectively. Received: 21 May 1998 / Accepted: 6 July 1998     

Biotransformation of 2,4,6-Trinitrotoluene with Phanerochaete chrysosporium in Agitated Cultures at pH 4.5

The biotransformation of 2,4,6-trinitrotoluene (TNT) (175 μM) by Phanerochaete chrysosporium with molasses and citric acid at pH 4.5 was studied. In less than 2 weeks, TNT disappeared completely, but mineralization (liberated ¹⁴CO₂) did not exceed 1%. A time study revealed the presence of several intermediates, marked by the initial formation of two monohydroxylaminodinitrotoluenes (2- and 4-HADNT) followed by their successive transformation to several other products, including monoaminodinitrotoluenes (ADNT). A group of nine acylated intermediates were also detected. They included 2-N-acetylamido-4,6-dinitrotoluene and its p isomer, 2-formylamido-4,6-dinitrotoluene and its p isomer (as acylated ADNT), 4-N-acetylamino-2-amino-6-nitrotoluene and 4-N-formylamido-2-amino-6-nitrotoluene (as acetylated DANT), 4-N-acetylhydroxy-2,6-dinitrotoluene and 4-N-acetoxy-2,6-dinitrotoluene (as acetylated HADNT), and finally 4-N-acetylamido-2-hydroxylamino-6-nitrotoluene. Furthermore, a fraction of HADNTs were found to rearrange to their corresponding phenolamines (Bamberger rearrangement), while another group dimerized to azoxytoluenes which in turn transformed to azo compounds and eventually to the corresponding hydrazo derivatives. After 30 days, all of these metabolites, except traces of 4-ADNT and the hydrazo derivatives, disappeared, but mineralization did not exceed 10% even after the incubation period was increased to 120 days. The biotransformation of TNT was accompanied by the appearance of manganese peroxidase (MnP) and lignin-dependent peroxidase (LiP) activities. MnP activity was observed almost immediately after TNT disappearance, which was the period marked by the appearance of the initial metabolites (HADNT and ADNT), whereas the LiP activity was observed after 8 days of incubation, corresponding to the appearance of the acyl derivatives. Both MnP and LiP activities reached their maximum levels (100 and 10 U/liter, respectively) within 10 to 15 days after inoculation.     

Comparison of 2,4,6-Trinitrotoluene Degradation by Seven Strains of White Rot Fungi

The degradation of 2,4,6-trinitrotoluene (TNT) by seven strains of white rot fungi was examined in two different media containing 50 mg L⁻¹ of TNT. When TNT was added into a nutrient-rich YMG medium at the beginning of the incubation, four of the fungal strains completely removed TNT during several days of incubation and showed higher removal rates than those of Phanerochaete chrysosporium. TNT added into YMG medium after a 5-day preincubation period completely disappeared within 12 hours, and the removal rates were higher than those in N-limited minimal medium. Isomers of hydroxylamino-dinitrotoluene were identified as the first detectable metabolites of TNT. These were transformed to amino-dinitrotoluenes, which also disappeared during further incubation from cultures of Irpex lacteus. During the initial phase of TNT degradation by I. lacteus, dinitrotoluenes were also detected after the transient formation of a hydride-Meisenheimer complex, indicating that I. lacteus used two different pathways of TNT degradation simultaneously. Received: 29 March 2000 / Accepted: 23 May 2000     

Products of Anaerobic 2,4,6-Trinitrotoluene (TNT) Transformation by Clostridium bifermentans

Experiments to elucidate the 2,4,6-trinitrotoluene (TNT)-transforming activity of Clostridium bifermentans LJP-1 identified reductive TNT transformations that ultimately produced as end products triaminotoluene (TAT) and phenolic products of TAT hydrolysis. An adduct of TAT, apparently formed by condensation of TAT and pyruvic aldehyde (methyl glyoxal), was also detected.     

Microbial transformation of 2,4,6-trinitrotoluene

The manufacture and decommissioning of explosives has generated, and continues to generate, large quantities of waste material whose primary toxic and mutagenic component is 2,4,6-trinitrotoluene (TNT). The magnitude of this problem has motivated a great deal of research into treatment processes and environmental fate studies, including characterization of microbial transformations of TNT. This work has encompassed studies with mixed cultures and pure cultures of microorganisms derived from either TNT-exposed or unexposed sources, and studies using microorganisms chosen for their known capacities to degrade other pollutants. Several of these studies are discussed with regard to whether they identified a process that may lead to the complete detoxification or mineralization of TNT. Since oxygen can have a significant influence on the types of biochemical reactions that can occur and on the oxidation of intermediates of TNT transformation processes, studies in which oxygen was not excluded are discussed separately from studies conducted under anaerobic conditions. Received 31 October 1995/ Accepted in revised form 29 March 1996     

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis

The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification.Large amounts of land and water are heavily contaminated by explosives, mainly as a result of the manufacture and military use of munitions. The high financial cost associated with cleaning up these contaminated sites largely precludes the use of many existing remediation technologies, such as soil excavation and incineration or disposal to landfill. There is a great deal of work documenting the global contamination, general toxicity, and microbial metabolism of 2,4,6-trinitrotoluene (TNT) in the environment; however, relatively little is known about the enzymes mediating the detoxification of TNT in plants (for review, see Rylott and Bruce, 2009).Phytoremediation, the use of plants to remove environmental pollutants, offers a low-cost, sustainable alternative to conventional remediation technologies and is attracting considerable attention as a means to clean up sites contaminated with explosives. While TNT is a potent phytotoxin, plants are able to detoxify low levels of TNT. In an effort to determine how plant tolerance could be further improved, we are investigating the biochemistry and enzymology underlying the innate ability of plants to detoxify TNT. The detoxification of xenobiotics has been loosely categorized into three phases (Sandermann, 1992): activation, conjugation, and compartmentation. The proposed route of TNT detoxification follows these phases. The electron-withdrawing properties of the nitro groups of TNT make the aromatic ring electron deficient. This favors reductive transformation reactions in plants, and the TNT molecule is most commonly activated by the reduction of a nitro group to give hydroxylamino and then amino derivatives (Fig. 1, pathway A). Following the introduction of a functional group, more hydrophilic molecules such as Glc are conjugated to the activated TNT molecule (Gandia-Herrero et al., 2008), facilitating transport and subsequent compartmentation or sequestration.Open in a separate windowFigure 1.The transformation of TNT by pentaerythritol tetranitrate reductase. Pathway A shows the transformation of the nitro group to nitroso-dinitrotoluene (NODNT), HADNT, and then ADNT products. Pathway B shows the reduction of the aromatic ring to form hydride and dihydride Meisenheimer complexes, then chemical condensation with HADNT to form diarylamines.Data from both our microarray experiments (Gandia-Herrero et al., 2008) and other expression studies (Ekman et al., 2003; Mezzari et al., 2005) have found that members of the small gene family of oxophytodienoate reductases (OPRs) in Arabidopsis (Arabidopsis thaliana) are up-regulated following exposure to TNT. The Arabidopsis genome contains three characterized OPRs: OPR1, OPR2, and OPR3. In addition, there are three as yet uncharacterized putative OPRs, named here as OPR4, OPR5, and OPR6, with OPR4 and OPR5 being identical. The physiological functions of the OPRs remain obscure, with the exception of OPR3, which is involved in jasmonic acid biosynthesis, converting (9S,13S)-12-oxophytodienoic acid to 3-2(2′(Z)-pentyl)cyclopentane-1-octanoic acid in the peroxisome (Sanders et al., 2000; Stintzi and Browse, 2000; for review, see Wasternack, 2007). OPR3 is located on chromosome II within the Arabidopsis genome and contains a C-terminal Ser-Arg-Leu type 1 peroxisome-targeting sequence. The remaining OPRs are all located on chromosome I and do not possess any known organelle-targeting sequences. The spatial expression of OPR1 and OPR2 across root cells where TNT accumulates (Biesgen and Weiler, 1999; Baerenfaller et al., 2008) favors a role in detoxification.The OPRs share similarity with the Old Yellow Enzyme family, a group of flavoenzymes that has been repeatedly associated with the transformation of explosives (Binks et al., 1996; Schaller and Weiler, 1997; Snape et al., 1997; Basran et al., 1998; French et al., 1998; Blehert et al., 1999; Pak et al., 2000; Fitzpatrick et al., 2003; Williams et al., 2004). Studies also indicate that Old Yellow Enzyme homologs function as antioxidants, detoxifying the breakdown products of lipid peroxidation and other toxic electrophilic compounds (Kohli and Massey, 1998; Williams and Bruce, 2002; Fitzpatrick et al., 2003; Trotter et al., 2006). This oxidative stress could result from exposure to xenobiotics including TNT, wounding, or pathogen attack.Pentaerythritol tetranitrate reductase, an Old Yellow Enzyme homolog isolated from Enterobacter cloacae (Binks et al., 1996), possesses two catalytic activities toward TNT (Fig. 1): nitroreduction of TNT to form hydroxylamino-dinitrotoluene (HADNT) and then amino-dinitrotoluene (ADNT), and aromatic ring reduction of TNT to yield hydride and dihydride (2H⁻-TNT) Meisenheimer TNT adducts (French et al., 1998; Williams et al., 2004). The TNT ring-reduced compounds condense via a nonenzymatic reaction with HADNTs to form diarylamines, with the liberation of nitrite (Wittich et al., 2008). Expression of pentaerythritol tetranitrate reductase in tobacco (Nicotiana tabacum) confers both resistance to, and the ability to transform, TNT (French et al., 1999). OPR1, OPR2, and OPR3 share 43%, 44%, and 36% identity, respectively, with pentaerythritol tetranitrate reductase, and all possess the conserved active site amino acids crucial for TNT transformation by pentaerythritol tetranitrate reductase and other members of the Old Yellow Enzyme family (Snape et al., 1997; French et al., 1998; Khan et al., 2004), suggesting that they are capable of transforming TNT.The OPR4/5 protein is predicted to have reduced activity toward TNT, compared with the other OPRs, owing to a C-terminal truncation that removes residues thought to be important in binding the cofactor NADH, Thus, we investigated OPR1, -2, and -3 as likely candidates for the TNT nitroreduction activity in Arabidopsis.