INTRODUCTION

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Toxic nitroaromatic compounds tend to be reduced by biological systems in the environment due to electron deficiencies on the nitrogen atom or the benzene ring (6, 24, 37, 45). Arylhydroxylamine is one of the common intermediate products during nitro group reduction. Hydroxylamines are both reductants and oxidants that attack biomolecules and have highly toxic, carcinogenic, and mutagenic effects on biological systems and human tissues (12, 22).

The previously described routes for metabolism of arylhydroxylamines, which are involved in nitroreductase-initiated degradation of nitroaromatic compounds, include (i) a two-electron reduction process that produces dead end amines, (ii) the Bamberger rearrangement-like reaction which leads to production of 2-aminophenol (2-AP) or 4-AP (31, 36, 41), and (iii) conversion into a 1,2-dihydroxyl aromatic product by hydroxylaminolyase (19, 20, 25, 38).

Only ammonia release from nitroaromatic compounds avoids production of potentially toxic amines in the environment. The following two ammonia release processes during nitroreductase-initiated aerobic degradation of nitroaromatic compounds have been described: (i) ammonia release via ring fission of AP and (ii) ammonia release before ring fission (conversion of arylhydroxylamine into 1,2-dihydroxyl aromatic compounds by proposed hydrolytic hydroxylaminolyases). Nishino and Spain (31) observed the first process in the nitrobenzene (NB) degradation pathway of Pseudomonas pseudoalcaligenes, and Groenewegen et al. (19) observed the second process in the 4-nitrobenzoate degradation pathway in Comamonas acidovorans NBA-10.

Two pathways have been described for degradation of 3-nitrophenol (3-NP), and both of them are initiated by nitroreductases. Meulenberg et al. (26) reported that Pseudomonas putida B2 converts 3-NP to 1,2,4-benzenetriol and ammonia and proposed that a hydroxylaminolyase activity is responsible for this process. Schenzle et al. (41) observed a Bamberger rearrangement type of conversion of 3-hydroxylaminophenol to aminohydroquinone during degradation of 3-NP in Ralstonia eutropha JMP 134 and did not investigate the ammonia release mechanism further. We isolated P. putida 2NP8 growing on 2-NP and 3-NP. 3-NP-grown cells of this strain aerobically released ammonia from both the growth substrate, 3-NP, and a cometabolizing substrate, NB. We observed hydroxylaminobenzene (HAB) production during NB transformation by 3-NP-grown cells of P. putida 2NP8. To shed light on the ammonia release mechanism in this strain, HAB transformation was investigated because of the instability of the metabolites of the growth substrate, 3-NP. In this study, we characterized products obtained from HAB and AP transformation by 3-NP-grown cells of strain 2NP8 and obtained evidence that ammonia was released via oxidation of aminophenolic intermediates to imines and subsequent hydrolysis.

	MATERIALS AND METHODS

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Sources of chemicals. Benzoquinone, hydroquinone, and catechol were obtained from Sigma (St. Louis, Mo.); 2-AP, 4-AP, N-acetyl-2-AP, and N-acetyl-4-AP were obtained from Aldrich (Milwaukee, Wis.); and high-performance liquid chromatography (HPLC) grade methanol was obtained from EM Science (Gibbstown, N.J.). HAB was prepared by using a previously described method (17). Other reagents were 99% pure.

Media. 3-NP was dissolved in methanol to obtain a concentration of 10 mg/ml. We have previously described the basic salts medium used (56). 3-NP basic salts medium contained 20 mg of 3-NP per liter in the basic salts medium. The latter medium was supplemented with 0.1% yeast extract (YE) to obtain 3-NP/YE basic salts medium. YPS medium contained (per liter) 10 g of YE, 10 g of Bacto Peptone, and 5 g of NaCl. 3-NP, sterile trace metal solution, and YE were added to autoclaved liquid media. Agar media contained 2% agar. Media were autoclaved at 120°C for 30 min.

Preparation of cells grown in the presence of 3-NP and degradation of HAB and AP. P. putida 2NP8, a strain isolated by members of our group from municipal activated sludge (Waterloo, Ontario) (56), was maintained on YPS agar. Unless otherwise noted, the strain was grown in 250-ml clear glass Erlenmeyer flasks at 26°C and 200 rpm on an orbital shaker. A fresh YPS culture (5 ml) was inoculated into 50 ml of 3-NP/YE basic salts medium, grown overnight, and then transferred into 375 ml of 3-NP/YE basic salts medium in a 2-liter flask. After 5 h 3-NP (20 mg/liter) and YE (0.1%) were added. After another 2 h 3-NP (20 mg/liter) was added, and the preparation was incubated for 1 h. The final optical density at 600 nm (OD₆₀₀) (1-cm light path) was 1.6. Cells were harvested by centrifugation at 16,300 × g for 15 min and were washed with 100 ml of sterile phosphate buffer (1 g of KH₂PO₄ per liter, 7 g of Na₂HPO₄ · 12H₂O per liter; pH 7.35). The cells were used immediately for biotransformation of HAB and AP. The bottles used for HAB and/or AP biodegradation experiments were 40-ml amber glass bottles with Teflon-silicone septum-lined caps. Freshly grown cells that were suspended at an OD₆₀₀ of 3.5 (1-cm light path) in phosphate buffer containing different concentrations of NB were incubated on an orbital shaker at 200 rpm and 26°C. The caps of the bottles were loosened to maintain aerobic conditions.

Preparation of N-acetyl-2-AP and 2-aminophenoxazine-3-one (APX) from 2-AP. Five grams (wet weight) of P. putida 2NP8 cells grown on 3-NP was harvested from 1.2 liters of 3-NP/YE medium as described above. During incubation for 22 h, YE and 3-NP were added at the following times: 0.2% YE and 42 mg of 3-NP per liter at 7 h; 0.1% YE and 25 mg of 3-NP per liter at 17 h; 0.05% YE and 25 mg of 3-NP per liter at 20 h; and 25 mg of 3-NP per liter at 21 h. Washed cells were suspended in 1 liter of sodium phosphate buffer (25 mM, pH 7.3) supplemented with 150 mg of 2-AP in a 2-liter foam-plugged clear glass flask and incubated at 26°C on an orbital shaker at 200 rpm for 24 h. A yellow color appeared after 4 h and developed into a brown color as incubation proceeded. A brown precipitate formed at a later stage, and 2-AP had completely disappeared from the medium after 24 h. The colored compound was separated from both the supernatant and the cell pellet by centrifugation of biotransformation medium at 16,300 × g for 15 min.

Analysis of metabolites. HAB and its metabolites were analyzed by using a ZORBAX SB-C18 HPLC column (4.6 by 250 mm; Chromatographic Specialties, Brockville, Ontario, Canada). We have previously described the HPLC instruments, general procedures, and methods used for NB and 3-NP analysis (56). For HAB and AP and their metabolites, biotransformation samples were centrifuged at 9,000 × g for 3 min, and 15-µl portions of supernatant were injected and eluted with methanol and MilliQ water. Compounds were monitored at 254 nm.

	RESULTS

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3-NP-induced transformation of NB. 3-NP-grown cells of P. putida 2NP8, which were used throughout this study, transformed 3-NP and NB, at rates of 280 and 230 µM · h¹ (pH 7.3; OD₆₀₀, 3.5), respectively, with ammonia release. Uninduced cells grown on glucose and ammonium sulfate exhibited lower rates of activity with 3-NP (60 µM · h¹) and no activity with NB. No transformation activity with either NB or 3-NP was observed in cells grown on YE alone. These results demonstrated that the transformation activity with 3-NP and NB was induced by 3-NP. Our preliminary experiments established that 3-NP-grown cells metabolized NB to ammonia via HAB.

Transformation of HAB into AP. We used an approach similar to the approach described by Schenzle et al. (41) to investigate aerobic transformation of HAB by resting cells of P. putida 2NP8 grown on 3-NP. The extracellular metabolites of HAB transformation were analyzed by HPLC. By comparing the UV spectra and HPLC retention times with the UV spectra and HPLC retention times of authentic compounds, we found that 2-AP and 4-AP were initial metabolites of HAB, and this finding was similar to the finding of Schenzle et al. (41). We also observed 4-benzoquinone, 4-hydroquinone, and catechol in the HAB transformation medium (Table 2). We observed decomposition of HAB in phosphate buffer containing no cells or dead cells (pH 7.3). Mulvey and Waters (27) reported that the disappearance of HAB could be due to disproportionation. We observed no peaks under our experimental HPLC conditions. We did not find the metabolites produced from cell-mediated transformation of HAB in the decomposing HAB phosphate buffer that did not contain live cells.

Biotransformation of AP. To investigate how ammonia is released, we characterized transformation products of AP formed by 3-NP-grown cells. Rapid appearance of a yellow color and accumulation of a dominant product with an HPLC retention time of 8.6 min indicated that transformation of 2-AP occurred. The initial rate of removal of 2-AP was 220 µM · h¹ (OD₆₀₀, 3.5; pH 7.3). In the control medium containing dead cells, little removal of 2-AP and no yellowish color were observed after 6 h, even though prolonged (48-h) incubation did result in a light yellowish color. Using the HPLC retention time and UV spectrum of this compound, we identified it as a transformation product formed from HAB and NB (Table 2); this suggested that the compound is a common metabolite.

Time course for quantitative metabolite production from HAB. The time course for production of metabolites from 459 µM HAB is shown in Fig. 1. Based on the initial HAB concentration of 459 µM, the yield (on a mole equivalent basis) of 4-AP and its derivatives (including N-acetyl-4-AP and 4-benzoquinone) was 13%, the yield of 2-AP and its derivatives (including N-acetyl-2-AP and APX) was 10%, and the yield of ammonia was 30%. A trace amount of nitrosobenzene, an oxidation product of HAB, was also detected in media with or without live cells. 4-Benzoquinone and N-acetyl-2-AP were major metabolites of HAB transformation formed by resting cells grown on 3-NP. Even though 4-AP is the first product of HAB transformation, production of 4-AP appeared to occur later than production of 4-benzoquinone and N-acetyl-4-AP. This might have been due to the instability of 4-AP in the aerobic transformation medium and to rapid conversion of 4-AP into its derivatives. Instability during aerobic analytical tests might also have contributed to the observed delay in 4-AP production.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Quantitative analyses of metabolites produced from HAB by cells of P. putida 2NP8 grown on 3-NP. The reaction medium contained 50 mg of HAB per liter, cells (OD600, 3.5), and 20 ml of 50 mM phosphate buffer (pH 7.30). Biotransformation was performed in a 40-ml screw-cap amber vial on a rotary shaker at 150 rpm and 26°C. The cap was loosened to maintain aerobic conditions.

Quantitative transformation time course for 2-AP and NB. To quantitatively characterize biotransformation of AP and NB, time courses for transformation of 2-AP and NB were determined. During biotransformation of 2-AP, one-half of the substrate was converted into ammonia, and the rest was converted into N-acetyl-2-AP (Fig. 2A). While a strong yellow color was produced, quantitative analysis revealed that only 0.1% (mole equivalent) of 2-AP was converted into APX. The initial rates of ammonia and APX production were 73 and 0.10 µM · h¹, respectively. Release of ammonia was 730 times faster than formation of APX.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Quantitative analyses of metabolites obtained from transformation of 2-AP (A) and NB (B) by cells of P. putida 2NP8 grown on 3-NP. The biotransformation conditions were the same as those described in the legend to Fig. 1.

	DISCUSSION

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Based on identification of the transformation products of HAB and AP, we propose a pathway leading to ammonia release from HAB by 3-NP-grown cells of P. putida 2NP8 (Fig. 3). Our results revealed a new mechanism of ammonia release through oxidation of AP to imine, followed by hydrolysis, for transformation of nitroaromatic compounds.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Proposed route of HAB biotransformation in P. putida 2NP8 cells grown on 3-NP. BQMI, benzoquinone monoimine. Brackets indicate unidentified compounds.

Corbett (7-11) reported that 4-AP was oxidized to 4-benzoquinone monoimine, but the monoimine product could not be isolated because it was rapidly hydrolyzed in the aqueous buffer into quinone. We observed both 4-benzoquinone and 4-AP in the HAB and NB transformation media, and this indicated that oxidation of 4-AP led to release of ammonia. Compared to 4-AP, 2-AP is relatively stable in aqueous solutions, and oxidation of 2-AP requires a stronger oxidant. Chemical (23, 43, 47) or enzymatic (1, 2, 46, 55) oxidation of 2-AP with production of APX has been described in many reports, and it has been proposed that 2-benzoquinone monoimine is the first oxidation product of 2-AP, which leads to production of APX. Nogami et al. (32) investigated reactions of 2-benzoquinone monoimine in aqueous media and observed the following two reactions involving the imine: (i) hydrolysis into ammonia and 2-benzoquinone and (ii) coupling with another molecule of 2-AP and formation of APX through two addition reactions and two oxidation reactions. These authors reported that the optimal pH for hydrolysis was 6 to 8. The pH of the 2-AP biotransformation medium in our tests (pH 7.3) fell in this range. Because both imine and 2-AP are needed for formation of APX, only the presence of an excess amount of 2-AP favors APX formation, which means that hydrolysis is a dominant reaction at low concentrations of 2-AP. This is consistent with our finding that less than 0.1% APX was produced in the 2-AP biotransformation medium and a much lower yield of APX was obtained in the NB and HAB media (Fig. 1 and 2B) than in the 2-AP medium (Fig. 2A). Therefore, we propose that 2-AP oxidation to imine and the subsequent hydrolysis are the dominant reactions during NB metabolism.

Corbett (11) reported that 4-benzoquinone disappeared rapidly from an aqueous medium, and this explained the low yield of 4-benzoquinone. Many authors (5, 29, 32, 49) have found that 2-benzoquinone is very reactive and more unstable than 4-benzoquinone, especially at a low concentration. This explains our failure to identify 2-benzoquinone in the reaction media. The catechol and 4-hydroquinone detected in the transformation media are reduction products of benzoquinones.

The link between formation of APX and 2-AP oxidation via imine has been established in various studies. Chemical oxidation of 2-AP has been reported to produce either APX (42, 47, 55) or the azo product 2,2'-dihydroxyazobenzene as the sole product (3, 13) or a mixture of APX and the azo product (23, 43). The mechanisms leading to formation of the azo product or APX have been reported to be different (3, 13, 42, 47, 55). A specific phenoxazinone synthase that catalyzes formation of APX and APX analogs from aminophenols is present in microorganisms, plants, animals, and humans (2, 34, 35, 39). Enzymes with phenoxazinone synthase activity have been identified by other researchers as oxidative enzymes; these enzymes include catalase (2, 34), hemoglobin in human erythrocytes (51), tyrosinase (52), and a copper-containing oxidase (2). In all studies of production of APX from 2-AP, imine was considered the first intermediate during chemical or enzymatic oxidation of 2-AP. In this study, 3-NP-grown cells transformed 2-AP at a rate of 220 µM · h¹, and ammonia was released simultaneously at a rate of 73 µM · h¹. Based on the APX concentration in the extracellular aqueous phase, the initial rate of APX production (0.10 µM · h¹) was as much as 2,200 and 730 times slower than the disappearance of 2-AP and the release of ammonia, respectively. Killed cells did not transform 2-AP. This indicated that biological oxidation of 2-AP into imine occurred along with subsequent hydrolysis as the dominant reactions. The reaction in which APX is formed is probably a spontaneous reaction that accompanies oxidation of 2-AP, and we could not conclude that a specific phenoxazinone synthase is involved. P. putida 2NP8 is an oxidase- and catalase-positive strain, and oxidase and catalase may play a role in oxidation of 2-AP and formation of APX. This ammonia release mechanism is different from the mechanisms observed for 2-AP metabolism in Pseudomonas sp. strain AP-3, as described by Takenaka et al. (48), and in P. pseudoalcaligenes JS45, as described by Nishino and Spain (31); in these organisms ammonia is released after dioxygenase cleavage of the aromatic ring.

Our results also provided information concerning the mechanism of ammonia release from 3-NP, a growth substrate and inducer of NB transformation activity in strain 2NP8. Cells induced by 3-NP transformed 3-NP, NB, and 2-AP at similar initial rates (280, 230, and 220 µM · h¹, respectively). Uninduced cells grown on glucose-ammonium sulfate exhibited activity toward 3-NP of 60 µM · h¹. Uninduced cells grown on YE alone exhibited no activity toward 3-NP. Neither of these types of cells transformed NB. These observations indicated that the enzyme(s) that transformed NB was induced by 3-NP. Our conclusion was also supported by the results of Schenzle et al. (41), who reported that 3-NP-induced cells of R. eutropha JMP 134 converted both 3-hydroxylaminophenol and HAB via a Bamberger rearrangement. We propose a parallel 3-NP degradation pathway in which all of the possible intermediates are postulated based on HAB transformation (Fig. 4). 3-Hydroxylaminophenol, the reduction product produced by 3-NP nitroreductase, would be converted to two possible products, aminohydroquinone and 4-aminocatechol, via ortho and para Bamberger rearrangements, respectively. Both aminohydroquinone and 4-aminocatechol should be oxidized into imines more easily than AP is oxidized into imines because of the presence of an additional hydroxyl group (7-11). Only 1,2,4-benzenetriol can be expected if hydrolysis of imines and subsequent reduction of the quinones occur. Meulenberg et al. (26) identified 1,2,4-benzenetriol as an intermediate of nitroreductase-initiated 3-NP transformation by P. putida B2 under anaerobic conditions. Schenzle et al. (41) described aminohydroquinone as an intermediate of 3-NP nitroreductase-initiated 3-NP transformation by R. eutropha JMP134 under anaerobic conditions. All of these results are consistent with our proposed 3-NP degradation mechanism. Our proposed mechanism for 3-NP degradation, which was based on evidence obtained from transformation of the 3-NP analog NB, needs to be confirmed by direct studies of 3-NP metabolism, and we are currently exploring ways to do this.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Proposed route of 3-NP biotransformation in cells of P. putida 2NP8. All intermediates were postulated based on HAB biotransformation by 3-NP-grown cells.

AP is toxic to bacteria (4, 28, 30, 50), and detoxification activity in P. putida 2NP8 was clearly indicated by the presence of acetylated amines. These compounds are known to be important in microbial detoxification and have been widely observed during nitroreductase-initiated degradation of nitroaromatic compounds (18, 33, 36, 40, 41, 53). APX is an analog of the toxic compound actinomycin, which combines with DNA and inhibits RNA synthesis (21). The effect of APX on growth has toxicological significance.