interpreting the NMR spectra, Howard Mayfield, and Chris Ant- worth for the GC-MS analyses, Joe Bernardo for technical assis- tance, and Sharon Gaffney for ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1992, p. 2643-2648
Vol. 58, No. 8
0099-2240/92/082643-06$02.00/0 Copyright C) 1992, American Society for Microbiology
Oxidation of Nitrotoluenes by Toluene Dioxygenase: Evidence for a Monooxygenase Reaction JEFFREY B. ROBERTSON,' JIM C. SPAIN,`* JOHN D. HADDOCK,2 AND DAVID T. GIBSON2 Air Force Civil Engineering Support Agency, Tyndall Air Force Base, Florida 32403,1 and Department of Microbiology and Center for Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 522422 Received 4 February 1992/Accepted 1 June 1992
Pseudomonas putida Fl and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida Fl under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida Fl and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.
Nitrotoluenes are used extensively in the manufacture of azo and sulfur dyes and in the production of explosives (13). They have been detected in wastewater from paper mills and chemical plants, and it is estimated that the levels of 2- and 4-nitrotoluene in wastewater from 2,4,6-trinitrotoluene manufacturing facilities may be as high as 16 and 9.2 ppm, respectively (24). Little is known about the environmental fate and biodegradation of these compounds. It has been suggested that aromatic hydrocarbons with nitro substituents are resistant to electrophilic attack by oxygenases. This is attributed to the electron-withdrawing properties of nitro groups on the aromatic nucleus (3). In contrast, polar nitroaromatic compounds such as nitrophenols (3, 20, 21, 26) and nitrobenzoic acids (4, 5) are readily biodegradable. Several studies have shown that nitrotoluenes can be transformed to aromatic amines by bacteria (7, 13, 16), and a very recent report has shown that the methyl group of m- and p-nitrotoluene can be oxidized by the toluene monooxygenase encoded by the TOL plasmid (6). However, there is little information available on the structure and physiological properties of metabolites that are formed by the bacterial oxidation of nitrotoluenes. The first intermediate in the degradation of toluene by Pseudomonas putida Fl and Pseudomonas sp. strain JS150 is cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) (8, 10). The enzyme catalyzing this reaction, toluene dioxygenase, has a relaxed substrate specificity and is capable of oxidizing a wide range of aromatic compounds (11). We now report novel reactions in the oxidation of isomeric nitrotoluenes by the oxygenases in P. putida Fl and Pseudomonas sp. strain JS150 and by the cloned P. putida Fl toluene dioxygenase in Escherichia coli JM109. MATERIALS AND METHODS Materials. The following were obtained from the sources indicated: toluene, Fisher Scientific Co., Fair Lawn, N.J.; 2-, 3-, and 4-nitrotoluene, 2- and 3-nitrobenzyl alcohol, and *
Corresponding author.
3-nitrobenzoate, Aldrich Chemical Co., Inc., Milwaukee, Wis.; 2-methyl-5-nitrophenol, Pfaltz and Bauer, Stanford, Conn.; 1802, MSD Isotopes, Montreal, Quebec, Canada; isopropyl-,-D-thiogalactopyranoside (IPTG), Boehringer Mannheim Biochemicals, Indianapolis, Ind. Organisms and culture conditions. Pseudomonas sp. strain JS150 (12), P. putida Fl (9), and E. coli JM109(pDTG601) (27) were isolated as described in the references cited. Liquid cultures of JS150 and P. putida Fl were grown in minimal salts medium (MSB) (23) with toluene as described previously (9). Transformation of nitrotoluene by JS150 and P. putida Fl. Pseudomonas strains were grown overnight on toluene, harvested by centrifugation, and suspended in 100 ml of dilute MSB (1:4) to anA6. of 0.4 to 0.75 (0.10 to 0.23 mg of protein per ml). Nitrotoluenes were dissolved in the MSB to a final concentration of 10-4 M before cells were added. Flasks (140 ml) were sealed and incubated at 30°C on a rotary shaker at 250 rpm. At designated intervals, samples (0.45 ml) were removed, mixed with 0.05 ml of methanol, and clarified by centrifugation. All samples were held on ice until analyzed by high-performance liquid chromatography (HPLC). E. coli JM109(pDTG601) was grown at 37°C to an A600 of 0.5 before IPTG was added to a final concentration of 1.0 mM. After 1 h, cells were harvested by centrifugation, washed with 0.05 M KH2PO4 buffer (pH 7.3), and suspended in 0.5 volumes of the same buffer containing 0.01 M sodium pyruvate and 10-4 M nitrotoluene. Flasks (250 ml) containing 25 ml of the amended cell suspension were incubated at 30°C on a rotary shaker at 200 rpm. At intervals, samples (1.0 ml) were removed and acidified with 10 ,u of concentrated phosphoric acid, and the cells were removed by centrifugation. The clear supernatant solution was stored at -70°C until analyzed. Incorporation of 1802 into 4-nitrotoluene. Toluene-grown cells of JS150 were suspended in dilute MSB (1:4) to give an A600 of 1.5 (0.54 mg of protein per ml). The cell suspension (500 ml) was transferred to a 1.0-liter round-bottom flask, sealed with a stopcock, and stirred with a magnetic stirrer at 25°C. The oxygen in the headspace of the flask was adjusted 2643
2644
APPL. ENVIRON. MICROBIOL.
ROBERTSON ET AL. 100 80
60 40-
20
0 100
B 80
80'
CH3
60
&N02
60
._
CM
C
CH20H a)
40
0 0
20
40 0)
&N02
(NO
COOH
CZ
0 --
20
0
0
0
Time (hours)
Time (hours)
FIG. 1. Oxidation of 2-, 3-, and 4-nitrotoluene by toluene-grown cells of JS150. Cell suspensions contained protein concentrations of 0.10 mg/ml (A), 0.23 mg/ml (B), and 0.20 mg/ml (C). Nitrotoluenes were added to a concentration of 10-4 M.
FIG. 2. Oxidation of 2-, 3-, and 4-nitrotoluene by toluene-grown cells of P. putida Fl. Cell suspensions contained protein concentrations of 0.10 mg/ml (A and B) and 0.16 mg/ml (C). Nitrotoluenes were added to a concentration of 10-4 M.
to contain 52% 1602, 46% 1802, and 2% 1602-)802 as described previously (22). 4-Nitrotoluene was added to a final concentration of 10-4 M. A control culture contained air in the headspace. Samples from the control culture were taken as described above and analyzed by HPLC for the disappearance of 4-nitrotoluene and the appearance of metabolites. After complete converison of 4-nitrotoluene (1.75 h), the reaction mixtures from both flasks were centrifuged to remove cells and the clear supernatant solutions were extracted with equal volumes of ethyl acetate. The extracts were dried over anhydrous sodium sulfate and the solvent was removed in vacuo at 40°C. The residues from each flask were dissolved in ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS). Isolation and purification of 3-methyl-6-nitrocatechol. Cells
of JS150 were grown overnight on toluene in 17 liters of MSB in a Virtis model 43-100 20-liter fermentor. Cells were harvested with a Pellicon cassette cell concentrator (Millipore Corporation, Bedford, Mass.). The pore size of the cassette was 0.45 ,um. Cells were washed with MSB and suspended in 6 liters of dilute (1:4) MSB to give an optical density of 1.8 at 600 nm (0.66 mg of protein per ml). 4-Nitrotoluene in acetone was added in two equal portions to give a final concentration of 3.5 x 10-4 M. The suspension was incubated at 30°C until 4-nitrotoluene was no longer detected by HPLC (1.5 h). The cells were removed as described above, and the supernatant solution was extracted twice with 3 liters of methylene chloride. The extract was dried over anhydrous sodium sulfate, and the solvent was
MONOOXYGENATION OF NITROTOLUENES
VOL. 58, 1992
removed to yield a brown oil. This residue was dissolved in 2 ml of hexane and applied to the top of a column (2.8 by 31 cm) of silica gel. The column was eluted with benzene: hexane (6:4), and 250-ml fractions were collected. Fractions 13 to 23 were pooled, and the solvent was removed by flash evaporation to leave yellow crystals (mp, 60 to 61°C). Analysis by HPLC, nuclear magnetic resonance (NMR), and GC-MS revealed a single compound subsequently identified as 3-methyl-6-nitrocatechol (see Results). Analytical methods. HPLC separations were done with ,uBondapak C18 columns (4.6 mm by 25 cm; Waters Associates Inc., Milford, Mass.). For the experiments described in Fig. 1 and 2, UVA210 was monitored with an HP1040A diode array detector (Hewlett-Packard Corp., Palo Alto, Calif.). Compounds were eluted with a mobile phase of methanol, water, and phosphoric acid (370:630:1) at a flow rate of 1.5 ml/min. Retention volumes for nitroaromatic compounds were as follows: 2-nitrotoluene, 16.1 ml; 3-nitrotoluene, 18.3 ml; 4-nitrotoluene, 17.4 ml; 2-nitrobenzoyl alcohol, 6.0 ml; 3-nitrobenzoyl alcohol, 5.6 ml; 3-nitrobenzoic acid, 8.4 ml; 3-methyl-6-nitrocatechol, 11.4 ml; 2-hydroxy-4-nitrotoluene, 9.1 ml. For the experiments shown in Fig. 4, the UV absorbance was monitored with a 990-MS photodiode array detector (Waters Associates Inc.), and the flow rate was 1.0 ml/min. The mobile phase described above was used for 11 min, followed by a convex gradient curve where the methanol concentration was increased to 90% over a 10-min period. Identification and quantification of substrates and metabolites was accomplished by comparison with authentic standards. Identification of nitrobenzoyl alcohols was confirmed by GC-MS. GC-MS analyses were conducted on a Hewlett-Packard model 5890 gas chromatograph equipped with a fused-silica capillary column (0.25 mm by 30 m) with a 1-,um DB-5 stationary phase (J&W Scientific, Folsom, Calif.). Helium was the carrier gas. The oven temperature was maintained at 40°C for 4 min and then increased to 250°C at a rate of 10°C/min. Mass spectra of compounds eluting from the column were obtained with a Hewlett-Packard model 5970 mass selective detector. Proton (1H) and carbon (13C) NMR spectra were obtained with a Bruker AC-300 Fourier transform NMR spectrometer at a field of 7.2 T. The corresponding resonance frequencies for 'H and 13C at this field strength are 300.135 and 75.469 MHz, respectively. Samples were dissolved in acetone-d6 (99.8%). Predicted 1 C chemical shifts were calculated by the method described by Levy et al. (15). The protein concentration in cell suspensions was determined with the bicinchoninic acid assay procedure described by Smith et al. (18), with bovine serum albumin as the standard. RESULTS
Oxidation of nitrotoluenes by Pseudomonas sp. strain JS150 and P. putida Fl. Glucose-grown cells of Pseudomonas sp. strain JS150 and P. putida Fl did not catalyze the oxidation of 2-, 3-, or 4-nitrotoluene. In contrast, toluene-grown cells of Pseudomonas sp. strain JS150 and P. putida Fl catalyzed the almost stoichiometric oxidation of 2-nitrotoluene to 2-nitrobenzyl alcohol (Fig. 1A and 2A). Experiments with 1802 showed the incorporation of one atom of molecular oxygen, indicating that 2-nitrobenzyl alcohol is formed by a monooxygenation of 2-nitrotoluene (data not shown). JS150 also produced small amounts of a transient metabolite (data not shown). The mass spectrum of this compound gave a parent ion at mlz 169 with major fragments at m/z 121 and m/z 65. These properties are consistent with the structure of
15
0
65
A
CH3
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-
2645
169
121
3
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173
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30 50 70
IL
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-
90 110 130 150 170 190 210
m/z FIG. 3. Mass spectra of 3-methyl-6-nitrocatechol formed from 4-nitrotoluene by toluene-grown cells of JS150. 3-Methyl-6-nitrocatechol formed in air (A), and 3-methyl-6-nitrocatechol formed in the presence of 52% 1602 and 46% 1802 (B). Experimental details are given in Materials and Methods. a nitromethylcatechol (see Fig. 3A). However, the positions of the hydroxyl groups in the metabolite were not determined. When 3-nitrotoluene was used as a substrate, the metabolites formed by toluene-grown cells of JS150 were 3-nitrobenzyl alcohol and 3-nitrobenzoate (Fig. 1B). In a separate experiment the same cell preparation oxidized 3-nitrobenzyl alcohol stoichiometrically to 3-nitrobenzoate over a 24-h period. In contrast, P. putida Fl oxidized 3-nitrotoluene to 3-nitrobenzyl alcohol (Fig. 2B) and 3-nitrobenzoate was not detected during the period of the experiment. JS150 and P. putida Fl both oxidized 4-nitrotoluene to two products (Fig. 1C and 2C). The major metabolite formed by P. putida Fl, and the minor metabolite formed by JS150, was identified as 2-methyl-5-nitrophenol by comparison of its HPLC and GC-MS properties with those given by authentic material. The other metabolite formed by both organisms (the major metabolite formed by JS150) gave the mass spectrum shown in Fig. 3A. The spectrum reveals a molecular ion at mlz 169 and a base peak at mlz 65. The major fragments observed are formed by loss of -NO2 (m/z 123) and the subsequent losses either of two protons (m/z 121) or of oxygen (m/z 107). Confirmation of the structure of the metabolite was provided by 'H- and 13C-NMR spectroscopy. The 'H-NMR spectrum showed signals at 2.27 ppm (3H, singlet, methyl), 2.93 ppm (2H, broad singlet, two OH groups), 6.84 ppm ( H, doublet, J4,5 = 8.8 Hz, aromatic) and 7.52 ppm ('H, doublet, J5,4 = 8.8 Hz, aromatic). The 13C-NMR spectrum showed a
2646
APPL. ENvIRON. MICROBIOL.
ROBERTSON ET AL.
100 80
60 40
20 0 ._
a) 0
0 100
CD
0 0
80 60-
40
HH3
-
rrOH NOH
0 0
1
2
3
4
6 0
5
1
2
3
45
6
Time (hours) FIG. 4. Oxidation of 2-, 3-, and 4-nitrotoluene by IPTG-induced cells of E. coli JM109(pDTG601). Cell suspensions contained 0.4 mg of protein per ml (A to C) and 0.72 mg of protein per ml (D). Substrates were added to a final concentration of 10-4 M.
singlet at 16.43 ppm (methyl) and peaks at 115.4, 122.2, 133.6 134.2, 143.8, and 145.9 ppm for six aromatic carbon atoms (17). The NMR spectra along with the mass spectrum confirm that the metabolite formed from 4-nitrotoluene by JS150 is 3-methyl-6-nitrocatechol. Toluene-grown cells of JS150 were incubated with 4-nitrotoluene in the presence of a mixture of 1602 and 1802. The mass spectrum of the 3-methyl-6-nitrocatechol formed under these conditions is shown in Fig. 3B. The results indicate that both atoms of oxygen in 3-methyl-6-nitrocatechol originate from a single oxygen molecule. Oxidation of nitrotoluenes by E. coli JM109(pDTG601A). The preferential oxidation of the methyl group in 2- and 3-nitrotoluene by P. putida Fl and JS150 was unexpected. The possible role of toluene dioxygenase in the formation of nitrobenzyl alcohols was investigated by incubating 2- and 3-nitrotoluene with IPTG-induced cells of E. coli JM109 (pDTG601). This organism contains the toluene dioxygenase genes (todClC2BA) from P. putida Fl in the expression vector pKK223-3, where they are under the control of the tac promoter (27). Expression of the cloned toluene dioxygenase genes in E. coli JM109(pDTG601) yields cells that oxidize 2- and 3-nitrotoluene to 2- and 3-nitrobenzyl alcohol, respectively (Fig. 4). Nitrobenzoates were not detected. The
same induced cell preparations oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol (Fig. 4C). The latter gave a mass spectrum identical to that shown in Fig. 3A for the 3-methyl-6-nitrocatechol formed by JS150. In a separate experiment, induced cells of E. coli JM109 (pDTG601) oxidized 2-methyl-5-nitrophenol to 3-methyl-6nitrocatechol (Fig. 4D). Toluene-grown cells of P. putida Fl gave similar results, whereas cells of JS150 oxidized 2methyl-5-nitrophenol at very low rates (data not shown). In control experiments, E. coli JM109(pKK223-3), which does not contain the todCJC2BA genes, was incubated for 6 h with 2-, 3-, and 4-nitrotoluene. No products were detected by HPLC under conditions where the detection limits were
