Vol. 57, No. 11

Biotransformation of Nitrobenzene by Bacteria ContainingToluene Degradative Pathways

BILLY E. HAIGLER AND JIM C. SPAIN*

Air Force Civil Engineering Support Agency, Tyndall Air Force Base, Florida 32403-6001

Received 15 April 1991/Accepted 21 August 1991

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of theelectron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strainscontaining toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vaporprior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatog-raphy and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonasharboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida Fl andPseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene inthe presence of 1802 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygenin the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putidaFl, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures ofPseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%,respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serveas an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring issubject to initial attack by both mono- and dioxygenase enzymes.

Nitrobenzene is widely used in the manufacture of anilineand pyroxylin compounds, in the refining of lubricating oils,and in the production of soaps and shoe polishes (21). It hasbeen estimated that some 19 million lb (1 lb = 453.59237 g) ofnitrobenzene is released into the environment annually (37).Because of its toxicity, nitrobenzene has been listed as apriority pollutant by the EPA (18) and has recently beenadded to the list of compounds regulated under the ResourceConservation and Recovery Act (13). There is little informa-tion about the fate of nitrobenzene in the environment, andvirtually nothing is known about mechanisms for its biodeg-radation.

Bacteria that metabolize polar nitroaromatic compoundssuch as the mononitrobenzoates (3, 17), mononitrophenols(33, 40), and several of the dinitrophenol isomers (2, 14) havebeen described, but little is known about the degradation ofnonpolar nitroaromatic compounds. Biodegradation of ni-trobenzene has been reported in activated sludge (26, 28), inmunicipal wastewater systems (9), in aniline plant waste-water (26), and in nitrobenzene plant wastewater (27). In onestudy, sewage effluent converted nitrobenzene to anilineunder anaerobic conditions, but under aerobic conditionsnitrobenzene disappeared and no aromatic amines weredetected (12). Bacteria that degrade 1,3-dinitrobenzene (4,22) and transform trinitrotoluene have been described (20),but nothing is known about the degradative pathways.Tennessee River microorganisms that mineralized 1,3-dini-trobenzene were unable to oxidize nitrobenzene (22). TwoNocardia sp. that grew on nitrobenzene as the sole source ofcarbon and energy were described in a preliminary report;however, the degradative pathways were not studied (23).A variety of pathways are known for the initial attack on

toluene by bacteria (Fig. 1). Strains that contain the enzymes

* Corresponding author.

first described on the TOL plasmid oxidize the methyl groupas the first step (24, 25). Pseudomonas putida Fl (8) andsimilar strains that produce toluene dioxygenase carry out aninitial dioxygenase attack at the 2,3- position on the ring.Recently, strains that catalyze an initial monooxygenaseattack at the 2- (6, 30), 3- (16, 19), or 4- (39) position on thering have been described. The toluene dioxygenase in P.putida Fl has a broad substrate specificity that permits it tooxidize a variety of substituted aromatic compounds (8). Theability of toluene dioxygenase to oxidize 3-nitrophenol (32)suggested that it also attacks nitrobenzene. Therefore, wetested the ability of P. putida Fl and other toluene-degradingstrains for their abilities to oxidize nitrobenzene after growthon toluene. In this report we present evidence that bothmonooxygenase and dioxygenase enzymes can attack ni-trobenzene.

MATERIALS AND METHODS

Materials. Chlorobenzene and toluene were from FisherScientific Co., Fairlawn, N.J. Nitrobenzene (>99%), 3-ni-trophenol, 4-nitrophenol, and 4-nitrocatechol were fromAldrich Chemical Co., Inc., Milwaukee, Wis. 3-Nitrocate-chol was prepared from catechol as described by Astle andStephenson (1).Organisms and culture conditions. Bacterial strains used in

this investigation are listed in Fig. 1. Cultures were grown in500-ml Erlenmeyer flasks in 100 or 300 ml of minimal saltsmedium (MSB) (36) at 30°C with shaking at 200 rpm.Toluene, chlorobenzene, or nitrobenzene was supplied inthe vapor phase via a bulb above the culture as previouslydescribed (7). Cultures of P. putida 39/D and P. pickettiiwere supplemented with arginine (0.2%) and lactate (0.2%),respectively. For induction studies the growth medium of P.mendocina and Pseudomonas strain JS150 were supple-

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BIOTRANSFORMATION OF NITROBENZENE 3157

Initial attack on toluene

CH3$:( OH

OH

Reference

10, This study

8

Ppu3P. putida 39/D >,

CH3

P. pickettli PKO1

CHH

CH

OH

CH3

P. cepacia G-4

CH

CH3

CH2()H

CH3,O OH

OH

CHO

t - o~~~~1Pseudomonas sp.(pTN2)

P. mendocina KR1

CH3

OH

CH2OH

OH

6,30

24,25

39

FIG. 1. Bacterial strains used in this study.

mented with glucose (0.2%), and the growth medium of P.putida Fl was supplemented with arginine (0.2%).

Nitrobenzene oxidation. Cells were harvested by centrifu-gation and suspended in 120 ml of dilute (1:4) MSB in a

160-ml serum bottle to an A6. of 0.34 to 0.73 (0.09 to 0.22 mgof protein per ml). Nitrobenzene (10-2 M in water) was

added to a concentration of 10-4 M, and the serum bottleswere sealed with Teflon-backed septa and incubated at 30°Cwith shaking at 200 rpm. Suspensions with P. pickettii weresupplemented with lactate (0.05%). At intervals, 0.5-mlsamples were removed for analysis of metabolites.

Analytical methods. Samples of culture fluids were clari-fied by centrifugation and analyzed by high-performanceliquid chromatography (HPLC). HPLC was performed on a,uBondapak C18 column (10 ,m; 3.9 by 300 mm; WatersAssociates, Inc., Milford, Mass.) with methanol-water-phos-phoric acid (370:630:1) as the mobile phase at a flow rate of1.5 ml/min. Nitrophenols were separated on a Spherisorb C8column (10 ,um; 250 by 4.6 mm; Alltech, Deerfield, Ill.) withtrifluoroacetic acid (13.5 mM)-acetonitrile (70:30) as themobile phase at a flow rate of 1.5 ml/min. Compounds weredetected by their UV A210 with an HP1040A diode arraydetector (Hewlett-Packard Corp., Palo Alto, Calif.). Ni-trobenzene, 3-nitro- and 4-nitrocatechols, and 3- and 4-nitro-phenols were identified and quantitated by comparison toauthentic standards. The identities of nitrophenols and ni-trocatechols were confirmed by gas chromatography-massspectral (GC-MS) analyses as previously described (32).

Methods for measurement of oxygen uptake (34) and extrac-tion of metabolites were described previously (32).

Liquid chromatography-mass spectral (LC-MS) analyseswere performed with an HP1050 liquid chromatographlinked to an HP5987 quadrapole mass spectrometer with athermospray interface (Vestec Corp., Houston, Tex.). Sep-aration was achieved with a Spherisorb C8 column (Alltech)and a mobile phase consisting of methanol-0. 1 M ammoniumacetate (37:63) at a flow rate of 1.4 ml/min. The ammoniumacetate was acidified with 0.6% acetic acid. The thermospraysource was operated without the filament or discharge on.The spectrum was scanned for positive ions between 125 and200 atomic mass units.

Isolation of putative nitrobenzene dihydrodiol. The putativenitrobenzene dihydrodiol was extracted from culture fluidswith ethyl acetate and purified by HPLC on a C18 semi-preparative column (Zorbax ODS; 9.4 by 250 mm; Du Pont,Wilmington, Del.) at flow rate of 3.5 ml/min. The solventsystem was water-acetonitrile in a gradient from 85:15 to75:25 over a period of 5 min. The putative nitrobenzenedihydrodiol which eluted at 4.0 min was collected, and theacetonitrile was removed by flash evaporation. The remain-ing solution was extracted with 3 equal volumes of ethylacetate, the ethyl acetate was removed by flash evaporation,and the residue was stored at -70°C until analyzed byLC-MS.

1802 incorporation. Toluene-grown cells of Pseudomonassp. strain JS150 were harvested by centrifugation, sus-

Strain

Pseudomonassp. strain JS150

andP. putida Fl

CH3 CH

8

16, 19

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3158 HAIGLER AND SPAIN

00

20

C 1 C D

40 1 2 3 4 0 1 2 34 5

Time (hrs)FIG. 2. Metabolism of nitrobenzene by toluene-grown cells of P. putida Fl (0.07 mg of protein per ml) (A), P. mendocina (0.11 mg of

protein per ml) (B), Pseudomonas sp. strain JS150 (0.09 mg of protein per ml) (C), and chlorobenzene-grown Pseudomonas sp. strain JS150(0.22 mg of protein per ml) (D). Experimental details are described in Materials and Methods. The conversion of nitrobenzene (*) to3-nitrocatechol (E), 3-nitrophenol (A), or 4-nitrophenol (0) was followed by HPLC.

pended in dilute MSB (pH 7.2) to an A6. of 0.5 (0.12 mg ofprotein per ml), and incubated with nitrobenzene (10-4 M) inthe presence of 1802 as previously described (35). After 1.5h, the metabolites were extracted from the culture fluid andanalyzed by GC-MS.

RESULTS

All strains tested could grow in the presence of nitroben-zene vapors when glucose (0.2% [wt/vol]) was supplied as analternate carbon source. None of the seven strains grew onnitrobenzene as the sole carbon source. All strains testedexcept P. putida 39/D rapidly oxidized toluene after growthwith toluene. P. putida 39/D is a mutant strain derived fromP. putida Fl which lacks the enzyme necessary for conver-sion of cis-dihydrodiols to catechols (8). Active cultures ofeach strain were tested for the ability to degrade nitroben-zene. Toluene-induced cultures of P. cepacia G4 and Pseu-domonas sp.(pTN2) did not catalyze nitrobenzene disap-pearance. P. putida Fl, P. putida 39/D, Pseudomonas sp.strain JS150, P. mendocina, and P. pickettii PKO1 trans-formed nitrobenzene and, therefore, were examined ingreater detail.

Oxidation of nitrobenzene by P. putida Fl and P. putida39/D. Toluene-grown P. putida Fl rapidly converted ni-trobenzene to a single major product detected by HPLC.

The HPLC retention time (R,) of 4.5 min and spectralproperties of the metabolite were identical to those of3-nitrocatechol. The identity of 3-nitrocatechol was con-firmed by GC-MS analysis of ethyl acetate extracts. Thekinetics of 3-nitrocatechol formation indicated that nitroben-zene conversion was nearly stoichiometric (82% recovered)(Fig. 2A). Two trace metabolites were detected by HPLC.One (R, = 5.5 min) was identified as 3-nitrophenol on thebasis of HPLC and GC-MS analyses of ethyl acetate ex-tracts. The other trace metabolite (R, = 2.1 min), suspectedto be cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene (nitroben-zene dihydrodiol), appeared transiently during the assay.Conversion of substituted aromatic compounds to cate-

chols by P. putida Fl involves an initial attack by a dioxy-genase which results in the formation of cis-dihydrodiols asintermediates (8). After growth in the presence of toluene, P.putida 39/D, a mutant lacking the enzyme required forconversion of cis-dihydrodiols to catechols (8), rapidly con-verted nitrobenzene to two products detectable by HPLC(R, = 2.2 min [compound I] and R, = 5.5 min [compound II])(data not shown). Compound I appeared transiently andslowly disappeared as compound II increased. Compound IIwas identified as 3-nitrophenol by HPLC and GC-MS. Com-pound I and 3-nitrophenol were both converted to 3-nitro-catechol when toluene-grown cells of P. putida Fl wereadded to the spent medium. This suggested that compound I

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100-

50 -

120

157175

126 144

,IIIII .1 ,I . 1. 1III .1. 93- I-

1 130 140 150 1601

170 180

BIOTRANSFORMATION OF NITROBENZENE 3159

2001 90 200

m/zFIG. 3. Positive thermospray LC-MS of compound I formed by

toluene-induced P. putida 39/D.

was the intermediate nitrobenzene dihydrodiol formed dur-ing conversion of nitrobenzene to 3-nitrocatechol. Dihy-drodiols undergo acid catalyzed dehydration to phenols (7).Compound I decomposed to 3-nitrophenol overnight; how-

ever, the rate of conversion to 3-nitrophenol did not increaseunder acid conditions.

Large-scale (6-liter) cultures df toluene-induced P. putida39/D were incubated with nitrobenzene for 3 h, and theproducts were extracted with ethyl acetate. The putativenitrobenzene dihydrodiol wdt frified by HPLC and wasstored at -70°C until analyi8d by LC-MS (Fig. 3). Theisolated compound gave the expected fragment ions at m/z193 [M+ + (NH4)2], m/z 175 (M+ + NH4), and m/z 157 [(M++ NH4) - H20]. The results are consistent with thoseexpected of nitrobenzene dihydrodiol. The compound wasunstable, and further attempts to characterize it were unsuc-cessful.

Oxidation of nitrobenzene by Pseudomonas sp. strain JS150.Toluene- and chlorobenzene-grown cultures of JS150 cata-lyzed the nearly stoichiometric conversion of nitrobenzeneto 3-nitrocatechol (Fig. 2C and D).To establish whether a dioxygenase reaction is responsible

for conversion of nitrobenzene to 3-nitrocatechol, toluene-grown cells of strain JS150 were exposed to nitrobenzene inthe presence of equal amounts (50.1 and 49.9%) of 1802 and1602. GC-MS analysis revealed that 51.4% of the 3-nitrocate-chol contained two atoms of 16Q, 46.1% contained twoatoms of 180, and 2.5% contained one atom of 18Q and oneatom of 160 (Fig. 4). In the 3-nitrocatechol standard 98.2% ofthe molecules contained two atoms of 160, 0.8% contained

50I g

50

107 155

52 7.21, I il .11l1iS. , 1109155 9

39~~~~~~~~~~~I

B111 1

159

10752

81

IJ ~~63 tl____

11I11..I..j.I I I I 12..15 I

150I I I I I I I I I I I I I I I I I I 9 VI-"|70 90 110 130

mlzFIG. 4. MS analysis of 3-nitrocatechol produced from nitrobenzene by cells of toluene-grown strain JS150 in the presence of 1802. Spectra

of 3-nitrocatechol standard (A) and 3-nitrocatechol produced in the 1802-enriched atmosphere (B) are shown.

VOL. 57, 1991

0>0<

C,)

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3160 HAIGLER AND SPAIN

c 20c

0

't o

X 0*

a)

O 800

0 10 20 30 40 50

Time (min)FIG. 51. Metabolism of nitrobenzene dihydrodiol by tolueli-

grown cells of Pseudomonas sp. strain JS150. Experimental detaIlsare described in the text. Conversion of nitrobenze dihydrodiol (Q)to 3-nitrocatechol (*) and 3-nitrophenol (U) in dilute (1:4) MSB with(A) (0.15 mg of protein per ml) and without (B) cells. The concen-tration of nitrobenzene dihydrodiol was estimated from its A340 onthe assumption of complete conversion of nitrobenzene (10-n M) byP. putida 39/D.

two atoms of 180, and 1.0%o contained one atom of 180 andone atom of 160. The results are consistent with a dioxygen-ase reaction in which both atoms of an oxygen molecule areincorporated into the substrate.

In a separate experiment, cells of P. putida 39/D wereallowed to oxidize 10-4 M nitrobenzene. The cells wereremoved by centrifugation, toluene-grown cells of strainJS150 were added to the medium, and the formation ofproducts was followed by HPLC. Before the JS150 cellswere added, the major metabolite was the putative nitroben-zene dihydrodiol (Fig. 5A and B). Minor amounts of 3-nitro-catechol and 3-nitrophenol were present. In the presence ofJS150, the putative nitrobenzene dihydrodiol was rapidlyconverted to 3-nitrocatechol (Fig. 5A). A minor amount of3-nitrophenol was formed -in control and culture fluids,presumably by nonenzymatic- dehydration of the nitroben-zene dihydrodiol. The small amount of 3-nitrocatecholpresent at the beginning of the assay was probably formed byoxidation of 3-nitrophenol by the initial dioxygenase (32).

Oxidation of nitrobenzene by P. mendocina. Toluene-growncultures of P. mendocina converted nitrobenzeine to 3-nitro-phenol (10%) anid 4-nitrophenol (63%) (Fig. 2B). After 5 h,15% of the nitrobenzene remained. In control experimentswithout cells, 93% of the nitrobenzene remained after 5 h. Insome experiments traces of 4-nitrocatechol were detected.

80

060

00J203=

0 1 2 3 4 5

Time (hrs)FIG. 6. Metabolism of nitrobenzene by toluene-induced cells of

P. pickettii PKO1 (0.17 mg of protein per ml). Experimental detailsare described in Materials and Methods. The conversion of ni-trobenzene (*) to 3-nitrocatechol (E), 3-nitrophenol (A), or 4-nitro-catechol (0) was followed by HPLC.

In separate experiments, toluene-induced cells of P. men-docina catalyzed the conversion of 3- and 4-nitrophenol (5 x10-5 M) to 4-nitrocatechQl, which was identified by HPLCand GC-MS after extraction of the culture fluids (data notshown).

Oxidation of nitrobenzene by P. pickettii PKO1. Toluene-induced cultures of P. picketti catalyzed the conversion ofnitrobenzene to 3-. and 4-nitrocatechol (Fig. 6). The transientappearance of 3-nitrophenol and trace amounts of 4-nitro-phenol during the conversion of nitrobenzene suggested thatthese compounds Were intermediates in nitrocatechol forma-tion. In separate experiments toluene-induced cultures of P.pickettii converted 4-nitrdjllhenol (5 x 1O-5 M) to 4-hitro-catechol and converted 3-nitrophenol (5 x 10-5 M) to3-nitrocatechol and trace amounts of 4-nitrocatechol. Thenitrocatechols were- slowly degraded and no metaboliteswere detected by HiPLC.

Induction studies. In order to determine whether nitroben-zene could induce the enzymes necessary for its degrada-tion, cultures of each nitrobenzene-degrading strain weregrown overnight in the presence and absence of toluepe ornitrobenzene. When the cells were tested for the ability totransform nitrobenzene, only cultures grown in the presenicieof toluene were able to remove nitrobenzene at appreciablerates (data not shown). The evidence suggests that nitroben-zene is not an effective inducer in these strains. A slowconversion of nitrobenzene to nitrophenol by glucose-grownP. mendocina suggested that low constitutive levels of thenecessary enzymes were present in this strain. The resultsare similar to those reported for P. putida 39/D, in whichnitrobenzene was found not to induce toluene dioxygenase(38).

DISCUSSION

Nonpolar nitroaromatic compounds are considered resis-tant to microbial attack (5). This resistance is due in part tothe reduction of electron density in the aromatic ring by thenitro groups which can hinder electrophilic attack by oxy-genases and thus prevent aerobic degradation of nitrodro-matic compounds (2). In some systems the nitro groups in

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BIOTRANSFORMATION OF NITROBENZENE 3161

A

NO2

Nitrobenzen

B

NO2

Nitrobenzene

C

NO22

NO

spontaneous

NO

H

e NitrobenzeneDihydrodiol

NO

OH

~~NO

NO

OH0-y

NitrobenzeneNO2-

OOH

FIG. 7. Nitrobenzene degradation by PseudoJS150 and P. putida Fl (A), P. mendocina KR1 (EPKO1 (C). The dashed arrows indicate minor rout

aromatic compounds can be enzymatically rican result in secondary nonenzymatic cobiologically inert compounds (2). In additiotance to biodegradation, many nitroaromatictoxic to microorganisms (2).

Recently, a number of reports have desstrains that can degrade chlorobenzene anddichlorobenzene (31, 34). In most instancesthe oxidation of chloroaromatic compoundsspecific dioxygenase similar to the tolueistudied by Gibson and his colleagues (7, 8)tuents are also electrophilic and might be exlelectrophilic attack by oxygenases. Attack ctuted aromatic compounds by dioxygenasethe electrophilic character of a substituent isto aerobic oxidation. This is supported telectronic factors did not have a strong effe(tion of catechol by Pseudomonas sp. strain ]toluene degradation pathway similar to thEputida Fl (15). Interestingly, nitrobenzerinhibitor to catechol formation in Pseudorn

T-12 (15), suggesting that it either inhibits enzyme inductionor acts directly on the enzymes of catechol formation.Recent observations that P. putida Fl can convert 3-nitro-

H phenol to 3-nitrocatechol (32) prompted us to investigate theability of toluene oxygenases to attack nitrobenzene.The toluene monooxygenases in both P. mendocina and

14 P. pickettii readily oxidized nitrobenzene. Conversion ofNO2 nitrobenzene to 4-nitrophenol by P. mendocina (Fig. 7B) and

OH 3-nitrophenol by P. picketti (Fig. 7C) is analogous to the1 LO° initial oxidation of toluene to the respective cresols by these

OH organisms (16, 39). Formation of small amounts of 3-nitro-3-Nitrocatechol phenol by P. mendocina and 4-nitrophenol by P. pickettii

suggests that the initial attack on nitrobenzene is not veryspecific. The enzymes responsible for addition of the secondhydroxyl group to the nitrophenols to form nitrocatecholshave not been identified. Attack by either toluene monoox-ygenase or a phenol hydroxylase could catalyze the reaction.

NO The further degradation of 3- and 4-nitrocatechol by P.12 pickettii is under investigation.

Pseudomonas sp. strain JS150 was derived from the sameOH parent strain as Pseudomonas sp. strain JS6, which cata-

OH lyzes dioxygenation of toluene and chlorobenzenes (10, 11,4-Nitrocatechol 34). Our unpublished data indicate that it contains a nonspe-

cific toluene dioxygenase similar to that present in P. putidaFl (7, 8). Conversion of nitrobenzene to 3-nitrocatechol bytoluene-grown P. putida Fl and toluene- or chlorobenzene-grown strain JS150 (Fig. 7A) appears to be analogous to the

NO reactions by which toluene is converted to 3-methylcatechol2 via cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene by thesestrains (Fig. 1).The results of our 1802 study clearly demonstrated that

OH both atoms of molecular oxygen were incorporated intonitrobenzene by JS150. This indicates that the initial reaction4-Nitrocatechol is catalyzed by a dioxygenase enzyme. Conversion of ni-

NO2 trobenzene to the putative dihydrodiol by P. putida 39/D andKOH subsequent conversion to 3-nitrocatechol by JS150 strongly01-..||_ support the identity of the nitrobenzene dihydrodiol.

OH The nitrobenzene dihydrodiol undergoes spontaneous de-3-Nitrocatechol composition to form 3-nitrophenol. Previous studies showed

that toluene-induced P. putida Fl transformed 3-nitrophenolm) and P. pickettri to 3-nitrocatechol (32). Pseudomonas sp. strain JS150 alsotes

an P.petais transformed 3-nitrophenol to 3-nitrocatechol (data nottes of metabolism, shown). These observations provide a plausible explanation

for a minor pathway for the conversion of nitrobenzene to3-nitrocatechol (Fig. 7A).

educed, but this Recently, a dioxygenase-mediated denitration of 2,4,5-)upling to yield trichloronitrobenzene was suggested by Sander et al. (29).in to their resis- Our results provide evidence that bacterial oxygenases cancompounds are attack unsubstituted nitroaromatic compounds. Future stud-

ies on regulation of the systems combined with the biochem-,cribed bacterial ical information presented here should provide the basis forI the isomers of construction of strains able to grow on nitrobenzene.the first step ininvolves a non-ne dioxygenaset. Chloro substi-pected to hinder)n chloro-substi-,s suggests thatnot an obstacle

)y findings thatct on the forma-r-12 which has aat present in P.ie acted as anjonas sp. strain

ACKNOWLEDGMENTS

We thank David T. Gibson for providing P. putida 39/D, P. putidaFl, and P. mendocina KR1, Ron Olsen for providing P. pickettiiPKO1, Peter Chapman for Pseudomonas sp. (pTN2), and MichaelNelson for P. cepacia G4. We also thank Michael Henley forGC-MS and LC-MS analyses, Joe Wander for helpful discussions,and Shirley Nishino for reviewing the manuscript.

REFERENCES1. Astle, M. J., and S. P. Stephenson. 1943. Polarographic investi-

gation of hydrogen bonding. III. Nitrodihydroxybenzenes. J.Am. Chem. Soc. 65:2399-2402.

2. Bruhn, C., H. Lenke, and H.-J. Knackmuss. 1987. Nitrosubsti-

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tuted aromatic compounds as nitrogen source for bacteria.Appl. Environ. Microbiol. 53:208-210.

3. Cartwright, N. J., and R. B. Cain. 1959. Bacterial degradation ofthe nitrobenzoic acids. Biochem. J. 71:248-261.

4. Dey, S., P. Kanekar, and S. H. Godbole. 1986. Aerobic microbialdegradation of m-dinitrobenzene. Indian J. Environ. Health29:118-128.

5. Fewson, C. A. 1981. Biodegradation of aromatics with industrialrelevance, p. 141-179. In T. Leisenger, A. M. Cook, R. Huttler,and J. Nuesch (ed.), Microbial degradation of xenobiotics andrecalcitrant compounds. Academic Press, Ltd., London.

6. Folsom, B. R., P. J. Chapman, and P. H. Pritchard. 1990. Phenoland trichloroethylene degradation by Pseudomonas cepacia G4:kinetics and interactions between substrates. Appl. Environ.Microbiol. 56:1279-1285.

7. Gibson, D. T., J. R. Koch, C. L. Schuld, and R. E. Kallio. 1968.Oxidative degradation of aromatic hydrocarbons by microor-ganisms. II. Metabolism of halogenated aromatic hydrocarbons.Biochemistry 7:3795-3802.

8. Gibson, D. T., G. J. Zylstra, and S. Chauhan. 1990. Biotrans-formations catalyzed by toluene dioxygenase from Pseudomo-nas putida Fl. In S. Silver, A. M. Chakrabarty, B. Iglewski, andS. Kaplan (ed.), Pseudomonas. Biotransformations, pathogen-esis, and evolving biotechnology. American Society for Micro-biology, Washington, D.C.

9. Gomolka, E., and B. Gomolka. 1979. Ability of activated sludgeto degrade nitrobenzene in municipal wastewater. Acta Hydro-chim. Hydrobiol. 7:605-622.

10. Haigler, B. E., C. A. Pettigrew, and J. C. Spain. 1990. Multiplepathways for the biodegradation of substituted benzenes in asingle strain of Pseudomonas, abstr. Q-67, p. 299. Abstr. 90thAnnu. Meet. Am. Soc. Microbiol. 1990. American Society forMicrobiology, Washington, D.C.

11. Haigler, B. E., and J. C. Spain. 1989. Degradation of p-chloro-toluene by a mutant of Pseudomonas sp. strain JS6. Appl.Environ. Microbiol. 55:372-379.

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