Degradation of Nitrobenzene by aPseudomonas pseudoalcaligenes
SHIRLEY F. NISHINO AND JIM C. SPAIN*AL/EQ-OL, Suite 2, 139 Bamnes Drive, Tyndall Air Force Base, Florida 32403-5323
Received 17 March 1993/Accepted 3 June 1993
A Pseudomonas pseudoalkaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energywas isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates ableto support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspen-sions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release ofammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygenuptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions,crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage,which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence ofNAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product wasspontaneously converted to picolinic acid, which was not further metabolized. These results indicate that thecatabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene;each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement convertshydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconicsemialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation.
Nitrobenzene is the primary starting material for themanufacture of aniline, which consumes some 97% of thenitrobenzene produced in the United States (7). It is fre-quently released in the effluent from explosives manufactureas well as in the manufacture of organic chemicals andplastics (14). The pollution potential is great, and nitroben-zene is listed as a U.S. Environmental Protection Agencypriority pollutant (17), yet little is known about its biodegra-dation. Degradation of nitrobenzene has been detected invarious waste streams and sludges (9, 25, 26) and in soilenrichment cultures (13, 21), but the degradative pathwaysremain unknown.Both oxidative pathways that result in the release of nitrite
from the aromatic ring (3, 4, 6, 11, 22, 34, 35, 39) andreductive pathways that liberate ammonia (10, 38, 39) havebeen reported for degradation of nitroaromatic compounds.Recent work has shown that various monooxygenase anddioxygenase enzymes can attack nitrobenzene (12), but theattack leads to dead-end products in the systems studied andthe nitro group remains bound to the ring.We report here the isolation and characterization of a
Pseudomonas pseudoalcaligenes which is able to use nitro-benzene as a sole source of carbon, nitrogen, and energy.We present evidence for an initial partial reduction tohydroxylaminobenzene, which undergoes a novel rearrange-ment to 2-aminophenol and then meta ring cleavage.
MATERIALS AND METHODSIsolation and growth of bacteria. Soil and groundwater
samples were obtained from a nitrobenzene-manufacturingfacility in Pascagoula, Miss. One milliliter of a slurry of thesoil and water was inoculated into 125 ml of minimal medium(MSB) (36). Nitrobenzene was provided in the vapor phaseas the sole carbon source via a glass bulb suspended througha foam plug in the neck of a 250-ml shake flask. Cultures
* Corresponding author.
were incubated at 30°C with shaking at 200 rpm in aGyrotory shaker. Nitrobenzene concentrations in the MSBwere monitored by high-performance liquid chromatography(HPLC; see below). Transfers to fresh MSB were madewhen concentrations of nitrobenzene in the culture mediumdecreased, and the cultures became turbid. After 2 monthsof enrichment, samples were spread on MSB agar platessupplemented with yeast extract (200 mg/liter). Nitroben-zene was provided in the vapor phase via tubes (5 by 60 mm)plugged with cotton and placed in the lids of the petri plates.The plates were then sealed with Parafilm M (AmericanNational Can, Greenwich, Conn.).
Strains were characterized by standard procedures (29)and with GN Microplates (Biolog, Inc., Hayward, Calif.).The ability of nitrobenzene-degrading strains to use otheraromatic compounds as growth substrates was screened byauxanography (23), or, for volatile compounds, the substratewas provided in the vapor phase.
Cultures were maintained on an agar-solidified, nitrogen-free medium (BLK) as previously described by Bruhn et al.(2), with nitrobenzene provided as described above.For experiments with induced cells, cultures containing
nitrobenzene (2.5 mM) in BLK (125 ml) were grown for 24 hat 30°C with shaking (200 rpm). These cultures were inocu-lated into 1.5 liters of BLK containing nitrobenzene (2.5mM) and incubated with shaking. After 16 h, a secondaliquot of nitrobenzene (2.5 mM) was added to the culture,and incubation was continued for 2 to 3 h until all of thenitrobenzene disappeared. Cells were harvested by centrif-ugation and washed twice with fresh phosphate buffer (0.02M, pH 7.0) before use in subsequent experiments. Unin-duced cells were grown overnight in BLK supplementedwith succinate (20 mM) and NH4C1 (9.4 mM).
Preparation of cell extracts. Washed cells were broken bytwo passages through a French pressure cell at 20,000 lb/in2,and the exudate was divided. Halfwas centrifuged at 100,000x g for 60 min at 4°C, and half was centrifuged at 26,000 x
g for 60 min at 4°C; the pellets were discarded, and thesupematant fluids were stored on ice until used.Enzyme assays. Catechol 1,2-dioxygenase and catechol
2,3-dioxygenase were measured as described previously(33). Nitrobenzene reductase activity was measured spectro-photometrically by monitoring the decrease in A340 with theconversion of NADPH to NADP. Reaction mixtures con-tained 0.1 ,umol of nitrobenzene, NADPH (0.1 to 0.3 ,umol),sodium phosphate (9.5 to 9.8 ,umol, pH 7.0), and cell extract(0.1 to 0.3 mg of protein) in a final volume of 1 ml.Nitrosobenzene reductase, hydroxylaminobenzene in-
tramolecular transferase (mutase), and 2-aminophenol 1,6-dioxygenase activities were measured by monitoring theincrease in the A380 of the 2-aminophenol ring cleavageproduct. The molar extinction coefficient (E380 = 15.1 mM-1cm-1) was estimated by assuming complete conversion of2-aminophenol to the ring cleavage product under conditionsof excess enzyme. Reaction mixtures for nitrosobenzenereductase contained 0.1 ,umol of nitrosobenzene, NADPH(0.05 to 0.2 ,umol), sodium phosphate (9.5 to 9.8 ,mol, pH7.0), and cell extract (0.1 to 0.3 mg of protein) in a finalvolume of 1 ml. Hydroxylaminobenzene mutase reactionmixtures contained 0.1 ,mol of hydroxylaminobenzene, 9.8,mol of sodium phosphate (pH 7.0), and cell extract (0.01 to0.3 mg of protein) in a final volume of 1 ml. Hydroxylami-nobenzene mutase activity was also measured by monitoringthe conversion of hydroxylaminobenzene to 2-aminophenolby HPLC.
Reaction mixtures for 2-aminophenol 1,6-dioxygenasecontained 0.1 ,mol of 2-aminophenol, sodium phosphate(9.5 to 9.8 ,umol, pH 7.0), and cell extract (0.1 to 0.4 mg ofprotein) in a final volume of 1 ml. Some cell extracts weredialyzed for 3 h against three changes of phosphate buffer(0.02 M, pH 7.0) before use. Cell extracts were preincubatedwith ferrous sulfate (50 ,M) for 5 min prior to the assay.Specific activities are expressed as micromoles of substratetransformed per minute per milligram of protein.Anaerobic experiments. Some experiments were per-
formed in an anaerobic chamber (Coy Laboratory Products,Inc., East Lansing, Mich.) under an atmosphere of 80% N2,10% CO2, and 10% H2.
Analytical methods. Intermediates and products were iden-tified by HPLC by comparison of retention times and spectrawith authentic standards. HPLC analyses were performedon a Spherisorb C8 column (Alltech, Deerfield, Ill.) with agradient of acetonitrile and water as the mobile phase.Trifluoroacetic acid was added to both the acetonitrile andthe water (6.75 and 13.5 mM, respectively). Two elutionprograms were used: program A started with 60% water and40% acetonitrile and changed to 40% water and 60% aceto-nitrile over 6 min; program B started with 80% water and20% acetonitrile, changed to 60% water and 40% acetonitrileover 7 min, and then changed to 40% water and 60%acetonitrile over 5 min. Flow rates were 1.5 ml/min. Com-pounds were monitored at a UVA230 with an HP1040A diodearray detector (Hewlett-Packard Co., Palo Alto, Calif.).Spectrophotometric analyses were performed on a Cary 3EUV-VIS spectrophotometer (Varian Associates, Sunnyvale,Calif.); authentic standards were used to identify products ofreactions monitored spectrophotometrically. Protein wasmeasured by the bicinchoninic acid method (30). Nitrite (29)and ammonia (24) releases were measured by standardmethods.
Chemicals. Hydroxylaminobenzene was prepared by thereduction of nitrobenzene with zinc (8) and then purified byrecrystallization from benzene. Nitrobenzene, nitrosoben-
1200
=L 800
I 600z
400z
200
o 2 4 6 8 1hours
200 - 0.4B
150 0.3
100 0.2
L. 50, >/ - 0.1
0 2 4 6 8 10
hoursFIG. 1. Growth on nitrobenzene. Triplicate cultures were inoc-
ulated with nitrobenzene-grown cells. (A) Disappearance of nitro-benzene (NB; 0) and release of ammonia (NH3; *) into culturemedium; (B) increase in protein (0) and optical density (U).
zene, 2-, 3-, and 4-aminophenol, picolinic acid, and catecholwere from Aldrich Chemical Co., Inc., Milwaukee, Wis.Catechol was purified by vacuum sublimation. All otherchemicals were of the highest purity commercially available.
RESULTS
Isolation and identification of nitrobenzene-degrading bac-teria. After 2 months of enrichment, several strains ofbacteria able to use nitrobenzene as a sole carbon andnitrogen source were isolated. The isolate selected for fur-ther study was an oxidase- and catalase-positive, nonfluo-rescent, nonmotile, gram-negative rod that does not utilizeglucose. On the basis of these characteristics and the BiologGN array, this isolate was identified as a Pseudomonaspseudoalcaligenes and designated strain JS45.
Growth on nitrobenzene. When nitrobenzene was providedas a sole carbon, nitrogen, and energy source, washednitrobenzene-grown cells removed nitrobenzene from cul-ture fluid rapidly after a slight lag, with the concomitantrelease of about one-third of the available nitrogen as am-monia and a sharp increase in the amount of total cell proteinduring the period of rapid nitrobenzene disappearance (Fig.1). There were no changes in protein or optical density incontrol cultures incubated without nitrobenzene. No nitriterelease was detected in any culture that degraded nitroben-zene. No metabolites other than traces of 4-aminophenol
a Oxygen uptake was measured polarographically as described previously(32). Substrates were added at 100 F.M.
b Numbers in parentheses indicate stoichiometry (moles of 02 per mole ofsubstrate).
c The rate for nitrosobenzene was determined at 2.5 x 10-5 M because ofthe multiphasic rates at higher substrate concentrations.
d ND, not determined.
were detected by HPLC in any culture grown on nitroben-zene.Growth on other substrates. The isolate grew on nitroben-
zene, hydroxylaminobenzene, and 2-aminophenol. It did notgrown on 1,2-, 1,3-, or 1,4-dinitrobenzene, 2-, 3-, or 4-ni-trobenzoate, 2-, 3-, or 4-nitrotoluene, 2-, 3- or 4-nitrophenol,catechol, 3- or 4-nitrocatechol, 3- or 4-aminophenol, aniline,benzene, phenol, toluene, nitrosobenzene, or picolinic acid.
Cultures inoculated with uninduced cells grew on 2-ami-nophenol only when 2-aminophenol was provided as solidcrystals to cultures on agar plates. 2-Aminophenol wasunstable when dissolved in the medium, whether agar orliquid, and induction did not occur until much of the sub-strate had decomposed or polymerized.
Respirometry. Nitrobenzene, nitrosobenzene, hydroxy-laminobenzene, and 2-aminophenol stimulated the rapiduptake of oxygen in washed suspensions of nitrobenzene-grown cells (Table 1). The stoichiometries of oxygen con-sumption were similar for these four compounds. The muchlower activities in succinate-grown cells indicated that thenitrobenzene degradation pathway is inducible. Attempts toinduce JS45 with 2-aminophenol in liquid cultures were notsuccessful because of the instability of 2-aminophenol.Nitrobenzene, nitrosobenzene, hydroxylaminobenzene,
and 2-aminophenol stimulated rapid oxygen consumption inextracts of nitrobenzene-grown cells (data not shown). Sto-ichiometries of oxygen consumption for these substrateswere all approximately 1. NADPH was required for stimu-lation of oxygen consumption by nitrobenzene and ni-trosobenzene but not by hydroxylaminobenzene or 2-ami-nophenol. Complete disappearance of nitrobenzene required2 mol of NADPH per mol of substrate, whereas reduction ofnitrosobenzene required only equimolar amounts ofNADPH.
Reduction of nitrobenzene to 2-aminophenol. Enzymes inextracts of nitrobenzene-grown cells catalyzed the rapid
TABLE 2. Enzyme activities in cell extracts
Enzyme assayeda Sp act(assay substrate)' (pmol/min/mg of protein)
a Assay details are given in Materials and Methods.b Substrates were provided at 100 AM. NADPH was provided at 200 AM to
assays with nitrobenzene or nitrosobenzene as the substrate.
removal of nitrobenzene from mixtures of phosphate bufferand NADPH (Table 2). Replacing NADPH with NADHresulted in a fourfold decrease in the reaction rate (data notshown). When cell extracts were frozen and thawed beforeuse, 2-aminophenol accumulated in the reaction mixtures.Two moles of NADPH was required to produce 1 mol of2-aminophenol (data not shown). Nitrosobenzene was con-verted to 2-aminophenol with the oxidation of 1 mol ofNADPH. Under anaerobic conditions, with excess NADPH,cell extracts produced stoichiometric amounts of 2-ami-nophenol from nitrobenzene (data not shown).
Conversion of hydroxylaminobenzene to 2-aminophenol.Hydroxylaminobenzene was converted to 2-aminophenol inthe presence of cell extract. No cofactors were required, andthe rates were proportional to the protein concentration (Fig.2). No activity was detected in extracts that had been heatedto 60°C for 10 min. This result indicates that the rearrange-ment of hydroxylaminobenzene to 2-aminophenol is enzymecatalyzed.Ring cleavage of 2-aminophenol. Freshly prepared extracts
of nitrobenzene-grown cells catalyzed the cleavage of 2-ami-nophenol with the rapid appearance and disappearance of ayellow intermediate with a maximum UV A380 followed bythe appearance of a compound with a maximum A264 whose
250
200-
c 150|
E 100-
50-
0 50 100 150 200 250
Protein (tg)
FIG. 2. Dependence of the rate of hydroxylaminobenzene (0)disappearance and appearance of 2-aminophenol (-) on proteinconcentration. Frozen and thawed cell extract was incubated withexcess hydroxylaminobenzene and incubated at 25°C for 2 min.Reaction mixtures were centrifuged for 1 min and then immediatelyanalyzed by HPLC. Rates are based on a 3-min reaction time.
FIG. 3. Spectral changes during metabolism of 2-aminophenolby cell extracts. (A) The reaction was initiated by the addition of2-aminophenol (100 F±M) to phosphate buffer and cell extract. Thereaction mixture was scanned at intervals of 30 s (spectra 1 to 6).Arrows indicate the direction of spectral changes. (B) The condi-tions were the same as those described for panel A, but NAD wasadded (100 pM) to the reaction mixture.
UV spectrum and HPLC retention time matched those ofpicolinic acid (Fig. 3A). When NAD was included in thereaction mixture, the yellow metabolite appeared transientlybut was not converted to picolinic acid. Instead, a compoundwith a maximum A340 accumulated and then slowly disap-peared (Fig. 3B). When cell extracts that had been centri-fuged at 100,000 x g were used, the maximum A340 wasstable. Attempts to extract the yellow product resulted in theformation of picolinic acid. Dialysis of the cell extractsslowed the rate of the conversion of 2-aminophenol to theyellow product by 80%; addition of ferrous ions enhancedthe reaction rate by 420% above that of undialyzed extracts.Heating of the cell extracts to 50°C for 5 min reduced thereaction rate by 90%, and heating to 60°C abolished theactivity.The compound with a maximum A340 appeared only in
reaction mixtures that included NAD. The addition of lacticdehydrogenase (1 U) and pyruvate (0.2 ,mol) to reactionmixtures prevented the accumulation of the compound withA340. NADH added to control mixtures without substrateshowed the same UV spectrum, which slowly disappearedwith cell extracts centrifuged at 26,000 x g but not withextracts centrifuged at 100,000 x g. These results providestrong evidence that the compound with A340 was NADH.Multiple additions of 2-aminophenol could be made to mix-tures containing cell extracts centrifuged at 26,000 x gwithout requiring additional NAD to prevent formation ofpicolinate. In contrast, mixtures containing cell extracts
centrifuged at 100,000 x g produced increasing amounts ofboth NADH and picolinate with each addition of 2-ami-nophenol. These results indicate that some type of mem-brane-associated NADH oxidase reoxidized the NADH.
DISCUSSION
The accumulation of ammonia but not nitrite in media ofnitrobenzene-grown cultures of P. pseudoalcaligenes JS45suggested that the initial attack on the nitro group wasreductive rather than oxidative as has been reported for 2-and 4-nitrophenol (32, 40), 2,4-dinitrotoluene (35), and 1,3-dinitrobenzene (6, 22). The further observation that hydrox-ylaminobenzene but not aniline served as a growth substratesuggested that the degradation of nitrobenzene proceeded bya partial reductive pathway similar to that demonstrated in aComamonas acidovorans for 4-nitrobenzoate (10). The re-sults of the simultaneous adaptation studies provided addi-tional evidence for a reductive pathway. The C. acidovorans(10) contained a single oxygen-insensitive nitrobenzoatereductase which required 2 mol of NADPH to convert4-nitrobenzoate to 4-hydroxylaminobenzoate via 4-ni-trosobenzoate. The requirement of 2 mol of NADPH forconversion of nitrobenzene to hydroxylaminobenzene alsosuggests similar initial reactions; however, the subsequentreactions for degradation of hydroxylaminobenzene by JS45are different from those used for oxidation of hydroxylami-nobenzoate (10). Our results indicate that hydroxylami-nobenzene undergoes an enzyme-catalyzed rearrangementto 2-aminophenol. A Rhodosporidium sp. was reported toproduce both ortho- and para-aminophenols from chlorohy-droxylaminobenzene (5). The reactions are similar to eachother and are analogous to the Bamberger rearrangement. Ina nonenzymatic chemical rearrangement, thepara isomer isthe expected product (28, 31). Traces of 4-aminophenol wereoccasionally found in culture fluids from nitrobenzene-grown JS45; however, those traces never disappeared fromthe culture fluids, and induced cells did not degrade 4-ami-nophenol. 2-Aminophenol was the only product detected inmixtures of frozen-and-then-thawed extracts of nitroben-zene-grown cells and nitrobenzene, nitrosobenzene, or hy-droxylaminobenzene. These results indicate that the en-zyme-catalyzed reaction favors the formation of the orthoisomer. An analogous enzyme catalyzed rearrangement of1-hydroxy-1-phenyl-3-methylurea to 1-(o-hydroxyphenyl)-3-methylurea has been reported in rat liver extracts (37).Although there have been reports of organisms able to
degrade 2-aminophenol, the pathway remains unknown (1,18). In our studies, freshly prepared extracts of nitroben-zene-grown cells caused the rapid disappearance of 2-ami-nophenol and the concomitant production of picolinic acidwhich was strongly reminiscent of the formation of quinoli-nate from 3-hydroxyanthranilate during the biosynthesis ofnicotinic acid (19, 20) (Fig. 4). In the nicotinic acid pathway,3-hydroxyanthranilate is cleaved in the presence of ferrousiron and 02 to form 2-amino-3-carboxymuconic semialde-hyde, which spontaneously rearranges and dehydrates toform quinolinate. We propose a similar sequence in JS45where 2-aminophenol is cleaved to form 2-aminomuconicsemialdehyde, which then spontaneously converts to picoli-nate.Que (27) reported the extradiol cleavage of 2-aminophenol
by catechol 1,2-dioxygenase prepared from benzoate-grownPseudomonas arvilla C-1. In that system, 95% of the prod-ucts of 2-aminophenol cleavage resulted from extradiolcleavage, but the rate of the reaction of the catechol dioxy-
Quinolinic acid Picolinic acidFIG. 4. Proposed pathway for conversion of 2-aminophenol to
picolinic acid. The pathway on the left is the analogous portion ofthe nicotinamide biosynthesis pathway which converts 3-hydroxy-anthranilate to quinolinic acid (19).
NH2
COOH
CHO
4 NAD
I NH3
2-Aminomuconic semialdehyde
FIG. 5. Proposed pathway for the biodegradation of nitrobenzene.
genase with 2-aminophenol was 1,000-fold slower than thereaction of the enzyme with the physiological substrate. The2-aminomuconic semialdehyde gave a transient maximumA380. Similarly, a catechol 1,2-dioxygenase from Pseudo-monas aeruginosa 2x (15) was reported to cleave 2-ami-nophenol in an extradiol fashion to form 2-aminomuconicsemialdehyde with subsequent dehydration to picolinate, butthe rate of the reaction with 2-aminophenol was lower thanthat for catechol cleavage although still sufficient for growthon 2-aminophenol (16). The similarity of the sequence ofevents in these two systems to that for JS45 supports ourinterpretation of the ring cleavage mechanism. The transientA380 reported by Que for 2-aminomuconic semialdehyde is inagreement with our findings. However, in contrast to thesesystems, the rate of cleavage of 2-aminophenol is 60 timesfaster than the rate of cleavage of catechol by JS45, suggest-ing that 2-aminophenol and not catechol is the physiologicalsubstrate for the JS45 enzyme.The addition of NAD to mixtures of 2-aminophenol and
cell extracts prevented the formation of picolinate from2-aminomuconic semialdehyde. In the presence of NAD, thesemialdehyde disappeared and NADH accumulated before itwas slowly recycled. Growth studies with whole cells dem-onstrated the liberation of ammonia. These findings and thefailure of induced cells to utilize picolinate suggest that
picolinate is not directly on the 2-aminophenol degradativepathway but is the product of a nonenzymatic reaction. Theproducts of the NAD-dependent reaction have not beenidentified. They show no UV absorbance, which suggeststhe loss of the conjugated double bonds.On the basis of our results and analogy with other sys-
tems, we propose the following pathway for the initial stepsin the degradation of nitrobenzene (Fig. 5). Nitrobenzene isreduced to hydroxylaminobenzene via nitrosobenzene withthe concomitant oxidation of 2 mol of NADPH. An enzyme-catalyzed rearrangement of hydroxylaminobenzene resultsin the formation of 2-aminophenol. Ring cleavage produces2-aminomuconic semialdehyde, which then is further de-graded via NAD-dependent reactions. Ammonia is liberatedfollowing ring cleavage. An effort is currently being made todetermine the steps leading to the release of ammonia.
ACKNOWLEDGMENTSWe thank D. T. Gibson for helpful discussions and B. E. Haigler
and L. Nadeau for reviewing the manuscript.
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