Vol. 54, No. 7APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1988, p. 1703-17080099-2240/88/071703-06$02.00/0Copyright © 1988, American Society for Microbiology

Degradation of Trichloroethylene by Toluene Dioxygenase inWhole-Cell Studies with Pseudomonas putida Fl

LAWRENCE P. WACKETT1* AND DAVID T. GIBSON2

Gray Freshwater Biological Institute and Department of Biochemistry, The University of Minnesota, P.O. Box 100,Navarre, Minnesota 55392,1 and Center for Applied Microbiology, The University of Texas at Austin,

Austin, Texas 787122

Received 29 February 1988/Accepted 27 April 1988

Toluene-induced cells of Pseudomonas putida Fl removed trichloroethylene from growth media at a

significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida Fl, the initial degradation rate varied linearly with trichloroethylene concentration over therange of 8 to 80 ,uM (1.05 to 10.5 ppm). At 80 ,uM (10.5 ppm) trichloroethylene and 30°C, the initial rate was

1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strainsderived from P. putida Fl that are defective in the todC gene, which encodes the oxygenase component oftoluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneousrevertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo,and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida Fl, buttetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.

Trichloroethylene (TCE) is an Environmental ProtectionAgency priority pollutant, and 178 million pounds were usedby American industry in 1985 (21). It is a suspected carcin-ogen in rats (15). Thus, the fate of TCE in water supplies isof great interest. Concerns over the presence of TCE ingroundwater are heightened by reports that some anaerobicbacteria biotransform TCE to vinyl chloride. This lattercompound is well documented to be tumorigenic in mam-mals (14).

In light of the above observations, there have been exten-sive efforts to document the biodegradation of trichloroethy-lene by microbes. Consortia of anaerobic microorganismshave been shown to degrade TCE (7, 25). Mixtures ofbacteria that proliferate under aerobic conditions have beenreported to be stimulated in their TCE-degrading activitiesby the addition of methane (28). More recently, three bac-terial pure cultures that catabolize toluene, strain G4, Pseu-domonas putida Fl, and P. putida B5, have been demon-strated to biodegrade TCE (16-18). Furthermore, Nelson etal. (18) used a toluene dioxygenase mutant strain and rever-tant strains to implicate toluene dioxygenase in TCE metab-olism by P. putida Fl. Considerable information is availableon the biochemistry and genetic regulation of toluene diox-ygenase in P. putida Fl (6, 11, 22-24), making this organismthe best choice for making inroads in discovering the molec-ular basis of bacterial TCE degradation. In this study, we useadditional mutants to support the demonstration that toluenedioxygenase catalyzes degradation of TCE, compare rates ofTCE degradation by P. putida Fl with that of a methano-troph, determine whole-cell initial rates as a function of TCEconcentration, and examine the biodegradative potential ofP. putida Fl with the full suite of chlorinated ethylenes.

MATERIALS AND METHODSMaterials. TCE, 1,1-dichloroethylene, cis-1,2-dichloroeth-

ylene, trans-1,2-dichloroethylene, tetrachloroethylene, and1,2-dibromoethane (EDB) were purchased from AldrichChemical Co. (Milwaukee, Wis.). Vinyl chloride was ob-

* Corresponding author.

tained from Fluka Chemical Co. (Ronkonkoma, N.Y.),ethylene was from Matheson Gas Products (Danvers,Mass.), n-pentane was from Mallinckrodt Chemical Co.(Paris, Ky.), toluene and p-dioxane were from Fisher Chem-ical Co. (Fair Lawn, N.J.), and indole was from EastmanKodak Co. (Rochester, N.Y.).Organisms and culture conditions. The strains used in this

study are listed in Table 1. To generate cell mass for TCEtransformation studies, P. putida F strains were grown on amineral salts medium (20) containing 0.2% (wt/vol) L-argi-nine with toluene supplied in the vapor phase as describedpreviously (3). Typically, 100-ml cultures in 500-ml Erlen-meyer flasks were grown at 30°C with shaking at 180 rpm.The revertant strain P. putida F4a was generated by inocu-lating P. putida F4 into mineral salts medium containing0.02% (wt/vol) arginine and toluene supplied as before. Theculture was grown to late exponential phase and transferredinto fresh growth medium until a yellow color appeared inthe culture filtrate, indicating that toluene was being oxi-dized to produce 2-hydroxy-6-oxo-2,4-heptadienoic acid.Organisms were plated onto mineral salts solid mediumcontaining 2% (wt/vol) agar with toluene supplied in thevapor phase as described previously (3). A large colony waspicked and designated P. putida F4a.Methylosinus trichosporium OB3b was grown on Higgins

medium (4) at 30°C with shaking at 180 rpm under anatmosphere of 30% (vol/vol) methane in air.GC. Gas chromatography (GC) analysis was conducted

with a Hewlett-Packard 5790A series gas chromatographequipped with a 30-m RSL-160 polydimethylsiloxane column(Alltech Associates, Deerfield, Ill.) and electron capture orflame ionization detection systems. Samples were appliedwith a Hewlett-Packard 7672A autosampler, and peak inte-grations were obtained with a Hewlett-Packard 3390A inte-grator. The following operating conditions were used; injec-tor temperature, 150°C; detector temperature (electroncapture detector), 250°C; column temperature, 35°C run to150°C at 15°C/min postinjection; hydrogen carrier flow, 6 ml/min. Under these conditions, TCE and EDB (internal stan-dard) had retention times of 5.3 and 6.9 min, respectively.

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TABLE 1. Bacterial strains

Bacterial strain Relevant properties' Reference

Pseudomonas putidaFl Tol+ prototroph 9F4 Tol- todC 6F4a Tol' todC revertant This studyF7 Tol- todABCDEF 6F106 Tol- todC 6F107 Tol- todE B. Finette, Ph.D. thesis, University of Texas

at Austin, 1984Methylosinus trichosporium OB3b Methane+, prototroph 27

a Gene designations: tod, toluene degradation: todC, terminal dioxygenase; todD, cis-toluene dihydrodiol dehydrogenase; todE, 3-methylcatechol 2,3-dioxygenase; todA, ferredoxinTOL reductase; todB, ferredoxinTOL; todF, 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase.

Quantitative determination of TCE degradation. P. putidaF strains were grown as described previously to a turbido-metric density at 600 nm of 1.3 to 1.5. M. trichosporiumOB3b was used for biotransformation after attaining anoptical density at 600 nm of 0.25 to 0.30. Cultures wereharvested by centrifugation at 8,000 x g for 10 min. Cellpellets of P. putida strains were suspended in mineral saltsmedium (19) containing 0.2% (wt/vol) L-arginine to an opticaldensity at 600 nm of 1.0. M. trichosporium OB3b wassuspended in fresh Higgins medium (4) to a turbidometricdensity at 600 nm of 1.2. Addition of methanol, formate, ormethane was observed not to enhance TCE degradation, sothese carbon sources were not added to reaction mixtures inthe experiments described.A 2-ml amount of the respective cell suspensions was

dispensed into 10-ml (total volume) heavy glass vials thatwere crimp-sealed with an inert rubber septum (AmericanScientific Products, Bedford, Mass.). The sealed cultureswere equilibrated at 30°C on a shaker platform operating at180 rpm. A 5-pd sample of an appropriate TCE stock solutionin p-dioxane was added by injection through the septa with agas-tight syringe (Hamilton, Reno, Nev.). After incubationof the inverted vial for a given length of time, reactions werequenched by the addition of 1 ml of n-pentane containing 1ppm EDB. EDB was added as an internal standard to correctfor losses in work-up and for injection imprecision. Thesealed vials were centrifuged at 5,000 x g for 10 min to pelletthe cells and to aid in the separation of the pentane phase.Thereafter, approximately 1 ml of the organic phase wastransferred to a septum-sealed 1-ml autosampling vial. Injec-tions of 1 1LI were routinely made for GC analysis. Typical0-min incubations, which were always performed as con-trols, gave TCE recoveries of 85 to 90%. These zero-timecontrols served as the baseline for monitoring TCE disap-pearance over time. In some experiments, cells that wereboiled for 10 min were also used as an additional control.The data represent an average of triplicate determinations,and standard deviations were always less than +8% in theseexperiments.A difficulty that arises in rate studies on TCE degradation

is the absolute concentration of TCE available to bacterialcells. The application of Henry's Law allows one to estimatethat approximately 30% of the added TCE is in the liquidphase in these experiments. Note that the relevance of thiscalculation is affected by the deviation ofTCE from behavioras an ideal gas, the likelihood that TCE will partion intobacterial cells, which would increase the effective concen-tration in the liquid phase, and the potential for adsorption ofTCE onto the surfaces of the reaction vessel. These param-eters, which pose a difficult analytical problem, prompted usto conduct and report our experiments in the following

manner. First, all incubations were conducted in identical10-ml reaction vials containing 2 ml of liquid and shaken atthe same speed. Second, all concentrations of TCE areexpressed as molar concentrations, with the total TCEadded calculated as being in the 2-ml liquid phase. Since theobserved extent of degradation is far in excess of the initialsoluble TCE concentration, equilibration of TCE betweengas and liquid phases must be occurring during the course ofdegradation. The possible effect of mass transport of TCEinto the liquid phase in limiting biodegradation is furtherexamined in the Results and Discussion sections.

Quantitative determination with other olefinic substrates.Incubations with other substrates were conducted as de-scribed for TCE. All were added from p-dioxane stocksolutions to a final olefine concentration of 20 ,uM, exceptethylene, which was injected directly as a gas to concentra-tions (in a volume of 2 ml) of 20 FM, 200 ,uM, or 2 mM.Work-up and GC were conducted as for TCE, except thatdirect headspace analysis was conducted with ethylene andthe GC was operated with a flame ionization detector.

Protein determinations. The cell suspensions from anincubation mixture were solubilized by diluting 1:1 with 2 NNaOH and heating to 85°C for 10 min. The liberated proteinlevel was determined by the Bradford assay (2) and cali-brated with stock concentrations of carbonic anhydrasewhich had been carried through NaOH and heat treatment inparallel.

RESULTS

TCE metabolism by mutants derived from P. putida Fl. Aprevious study by Nelson et al. (18) implicated toluenedioxygenase in the degradation of TCE by P. putida Fl. Inthat study, the wild-type strain and two mutants wereincubated with TCE for 24 h. A toluene dioxygenase mutant,P. putida F106, showed only a 22% decrease in TCEcompared with that in a control incubation without cells,whereas the wild type displayed complete removal of TCE.We sought to confirm and expand on these data by conduct-ing 0- and 1-h incubations, by using pentane extraction oftotal reaction mixtures to obtain highly reproducible com-parisons, and by examining additional mutant strains. Thestrains used were wild-type P. putida Fl, P. putida F106,two additional todC (toluene dioxygenase) mutants, a rever-tant strain, P. putida 4a, and a catechol 2,3-dioxygenasemutant, P. putida F7 (Table 1 and Fig. 1).

In these experiments, in which extraction efficienciesranged from 85 to 90%, there was no discernible decrease inTCE concentration with heat-killed wild-type cells or withthe three todC mutants (Table 2). Furthermore, the recoveryof TCE from incubations with todC strains was similar to

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TRICHLOROETHYLENE DEGRADATION BY P. PUTIDA Fl

Toluene

CH3

NADH+H+Rd~ ~ ~ ~O+ 02NADH +H+ RdTOL 2FdTOL ISPTOL.,.+ °2

(FAD) NK (red.) (oxd.) jFe2+NAD+RdTOL 2FdTOL ISPTOLH

(FADH2) (oxd.) (red.) OHOHI

I cis-Toluene 3-

DLITE Dihydrodiol

FerredoxinToL FerredoxinToL Iron-Sulfur cis-TolueneReductase ProteinToL Dihydrodiol(ReductaseToL) Dehydrogenase

CH3,,OH

OH

-Methyl 2.Catechol 2,

3-MethylCatechol

2,3- Dioxygenase

tod A

FIG. 1. Initial reactions in the oxidation of toluene by P. putida Fl. The metabolic intermediates, the enzymes or enzyme components,and the corresponding gene designations are shown. RdTOL, FerredoxinTOL reductase; FdTOL, ferredoxinTOL; ISPTOL, iron-sulfur proteinTOL,FAD, flavin adenine dinucleotide; FADH2, reduced form of this coenzyme; red. and oxd., reduced and oxidized forms, respectively, of theproteins.

recoveries of TCE from vials containing sterile mediumalone, ruling out the possibility that sorption to cells makesTCE inaccessible to pentane extraction. In contrast to thecontrols and the todC strains, the wild-type strain, the todEmutant, and the todC revertant all clearly degraded TCE, asindicated by a 51 to 63% decrease in pentane-extractableTCE. Note that strain P. putida F4a was demonstrated toexpress in vivo toluene dioxygenase activity comparable tothat of the wild type by its ability to oxidize indole to indigo,a reaction catalyzed by toluene dioxygenase (5). The threetodC mutants did not form indigo from indole. The completecorrelation of indigo-producing activity with TCE-biode-grading ability points to toluene dioxygenase as the commonenzyme responsible for both transformations.Time course of TCE degradation by P. putida Fl and M.

trichlosporium OB3b. In previous studies with mixed andpure cultures (1, 7, 16-18), TCE degradation has beenaccessed over periods greater than 1 h. Since P. putida Fldegraded a significant quantity of TCE in 1 h, it wasimportant to examine the kinetics of substrate disappearancefurther and to compare the rate with that in another class of

TABLE 2. TCE metabolism and indigo formationby P. putida Fl and derivatives

Mean pentane-extractableP. putida strain TCE' (>M) ± SD % Decrease Indigo

formationOh lh

Fl 17.0 ±0.2 7.5 ± 0.1 56 +Fl (heat killed) 17.2 ± 0.5 18.0 + 0.2 0 -

F4 17.6 0.4 18.1 ± 0.1 0 -F7 17.0± 0.3 17.7 ± 0.9 0 -F106 17.4 ± 0.1 17.8 ± 0.4 0 -F107 17.1 ± 0.3 8.4 ± 0.3 51 +F4a 17.3 ± 0.2 6.4 ± 0.9 63 +

a Reactions were initiated by addition ofTCE to 20 ,uM. TCE was extractedby the addition of pentane at the indicated time and quantitatively determinedby GC as described in Materials and Methods.

b A few crystals of indole were added to a portion of the culture used forTCE degradation studies. Positive cultures turned blue within 15 min.Negative cultures remained colorless, even after several hours.

bacteria, the methanotrophs, which have been implicated inTCE degradation (Table 1). In this experiment, the biodeg-radation ofTCE by P. putida Fl and M. trichosporium OB3bwas followed for 6 h (Fig. 2). Overall, the extent of biodeg-radation of TCE over the time course was greater with P.putida Fl. However, it should also be noted that theprogress curves of TCE disappearance were quite different.With P. putida Fl, the initial rate of degradation was

comparatively great but fell off to less than 2% of the initialrate after 20 min (Fig. 2, inset). In contrast, M. trichospo-rium was slower but more substained in its biodegradativeactivity. Note that a similar decline in the TCE degradationactivity of P. putida Fl over time was observed by monitor-ing the TCE concentration in the headspace of the incuba-tion mixture. This result precluded the possibility that therate decrease was due to very slow equilibration of TCEbetween the gas and liquid phases that would keep TCEunavailable to the organism, which is confined to the liquidphase.

Initial rates of TCE degradation. The rapid initial rate ofdegradation of TCE by P. putida Fl reflects the maximalbiodegradative potential of this organism under the condi-tions used. In order to begin to develop insights into in vivokinetics of TCE disappearance, 5-min assays were con-

ducted at various TCE concentrations. A 5-min samplingpoint, at which time approximately 10 to 20% of the TCE hadbeen consumed, offered the best compromise for accurateanalytical determination of TCE disappearance and for esti-mation of an initial rate at a given substrate concentration(Fig. 2, inset). In this manner, initial rates were determinedat 8, 20, 80, and 320 ,uM TCE. The rate of TCE degradationwas linear with respect to substrate concentration over therange from 8 to 80 ,uM (1.05 to 10.50 ppm) but was belowdetectable limits at 320 ,uM (Fig. 3). The highest observedrate was 1.8 nmol/min per mg of cell protein at a TCEconcentration of 80 ,uM. The degradation rate plummeted toundetectable levels at a fourfold-higher concentration.

Substrate specificity of toluene dioxygenase with chlorinatedolefins. Toluene dioxygenase is known to dioxygenate alkyl-benzenes to cis-dihydrodiols (10), to oxidize indole to indigo

GENEPRODUM

GENEDESIGNATION

CH3

01so&OH

OH

!-Hydroxy-6-oxo4- Heptadienoic

acid

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1706 WACKETT AND GIBSON

16

2i-

z

z

J

12

'u

I--

Lu

z 8LU-j

LU

0

0

I--

1 2 3 4 5 6TIME (h)

FIG. 2. TCE removal from culture medium by P. putida Fl (@)and M. trichosporium OB3b (0) cell suspensions at optical densitiesof 1.0 and 1.2, respectively, at 600 nm. TCE, initially added to a

concentration of 15 ,uM, was extracted with pentane and quantita-tively determined by GC as described in Materials and Methods.Inset: Compressed time scale of TCE degradation by P. putida Fl.

(5), and to catalyze benzylic monooxygenation of indan (26).Thus, it was of interest to determine the range of chlorinatedolefins oxidized by toluene dioxygenase in an effort tounderstand this enzyme better as well as to establish thebiodegradative potential of P. putida Fl with this class ofcompounds. In this context, ethylene and the full set ofchlorinated ethylenes were examined as substrates for deg-radation by this organism (Table 3).

Clearly, TCE and cis-1,2-dichloroethylene were the bestin vivo substrates for biodegradation. In contrast, the twoother isomeric dichloroethylenes were degraded to lesserextents in these 1-h incubations. The 2% decrease in tetra-chloroethylene is within the statistical error of these deter-minations, and thus, degradation of this substrate was insig-nificant under these experimental conditions. Degradation ofvinyl chloride, as determined by pentane extraction anddirect headspace analysis, was not observed. Ethylene dis-appearance was not observed upon incubation with P.putida Fl at 20, 200, or 2,000 ,uM concentrations.

DISCUSSION

Nelson et al. (17) first demonstrated that aromatic com-

pounds induce TCE-degrading activity in certain bacteria.More recently, the same research group presented evidencethat toluene dioxygenase is the enzyme responsible for TCE

z

4_m ._

,,, o0 CL.

jE

o cOE

E

-

2.0

1.6

1.2

0.8

0.4

I

20 40 60 80 320

INITIAL TCE CONCENTRATION (jiM)

FIG. 3. Initial rate of TCE degradation as a function of startingTCE concentration. Rates were determined by measuring disap-pearance of TCE after 5 min of incubation.

degradation by P. putida F1 (18). In the present study, dataobtained with additional mutants and use of the indigo assayto demonstrate in vivo toluene dioxygenase activity supportthe conclusion that toluene dioxygenase is required for TCEdegradation by P. putida Fl. All of the TCE-negative strainswere defective in todC, which encodes the oxygenase(USPTOL) component of toluene dioxygenase (Fig. 1). Itshould be noted that ISPTOL is composed of two distinctsubunits (22), and genetic evidence shows that these are

encoded by separate genes (B. A. Finette, G. J. Zylstra, andD. T. Gibson, unpublished data). At this time it is notpossible to determine whether expression of one or bothgenes is required for TCE degradation. The recent cloning ofboth ISPTOL genes (W. R. McCombie, G. J. Zylstra, andD. T. Gibson, unpublished data) may help to resolve thisissue.

This report also initiates comparisons of the TCE-biode-gradative potential of pure cultures by examining the cata-lytic activities of P. putida Fl and M. trichosporium OB3bunder identical conditions. Previously, all rate studies ofTCE biodegradation have been conducted with soils or

mixed microbial cultures (1, 7, 13, 19, 25, 28). In thosestudies with aerobic mixed cultures and with anaerobicconsortia, it is unclear what proportion of the total biomassis represented by TCE-degrading organisms. Hence, it isdifficult to compare previous kinetic data spanning hours anddays with the rate data determined in the present studyexpressed as nanomoles per minute per milligram of cellprotein. Furthermore, significant differences in the progress

TABLE 3. Substrate specificity displayed by P. putida Fl in thedegradation of ethylene and chlorinated ethylenesSubstrate % Decrease

(20 p.m initial concn) after 1 ha

Tetrachloroethylene ............................................ 2TCE ............................................ 68cis-1,2-Dichloroethylene .......................................... 67trans-1,2-Dichloroethylene ...................................... 101,1-Dichloroethylene ........................................... 18Vinyl chloride ...................... ...................... 0Ethylene ........................................... 0

" Expressed as 100 - [(concn at 1 h/concn at 0 h) x 100]. Concentrationswere obtained as the average of triplicate determinations.

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TRICHLOROETHYLENE DEGRADATION BY P. PUTIDA Fl

curves of TCE disappearance were observed in this studywith P. putida Fl and M. trichosporium OB3b. Clearly,more comparisons with these organisms under various con-ditions and with additional bacterial isolates are germane toour continuing efforts to obtain an optimal system forbacterial TCE degradation.Another point relevant to biodegradation of complex

mixtures of compounds is the overall substrate range oftoluene dioxygenase. In this context, other chlorinated ole-fins were examined to provide a baseline for further studieson the biodegradation of multiple substrates. For example,P. putida Fl oxidizes TCEs and dichloroethylenes (thisstudy), substituted benzenes (10), and 3-methylcyclohexene(29), as well as fused and heterocyclic ring systems (5, 26). Inthe presence of a number of these compounds, the course ofbiodegradation will depend on relative binding affinities andturnover numbers of the substrates with toluene dioxygen-ase and/or transport system(s). Thus, further rate studieswith various substrates of toluene dioxygenase are requiredin order to allow predictions of biodegradation kinetics ofcomplex mixtures by P. putida Fl.The elucidation of TCE degradation rates is rendered

difficult by uncertainties in determining the actual concen-trations of TCE available to cells at a given time duringassay. This physical problem, which is a complex function ofwater and headspace volumes, cell mass, glass reactionvessel surface area, and agitation rates, was dealt with byholding these parameters constant during the course of theexperiments. For ease of presentation and understanding ofthese studies, the TCE concentrations are expressed as if allthe TCE added to the reaction vials were in solution. Notethat we observed up to 95% degradation of TCE over 1 h inthe course of in vitro experiments under conditions similar tothose described here (S. Householder and L. P. Wackett,unpublished data). Clearly, TCE oxidation in the liquidphase results in reequilibration between gas and liquidphases so that all of the TCE ultimately becomes available toliquid-phase enzymes or to bacterial cells. A related issuethat is relevant to rate studies is the rapidity of reequilibra-tion between the gas and liquid phases that could impose ratelimitations on biodegradation. In this context, it should bepointed out that in the 5-min determinations of TCE degra-dation, up to 70% of the initially soluble TCE would havebeen consumed. Thus, if reequilibration is slow, these initialrates may underestimnate the true in vivo biodegradativeabilities of P. putida Fl with TCE. The development of anassay based on product formation rather than TCE disap-pearance would increase assay sensitivity. This would allowmonitoring of the biodegradation at shorter time points andgive more accurate initial rates. Studies are underway todetermine the product(s) of TCE oxidation by toluene diox-ygenase.These studies suggest that TCE may be toxic to P. putida

Fl in two different ways. First, at 320 ,uM TCE, the highestconcentration examined, TCE degradation no longer oc-curred. Note that high concentrations of TCE have previ-ously been shown to disrupt metabolic activity in Photobac-terium phosphoreum, as evidenced by its loss ofbioluminescence (12). A second phenomenon is the decreasein the rate of the reaction that was observed during thecourse of TCE metabolism (Fig. 2). This could not beexplained as being due to slow equilibration between the gasand liquid phases, as headspace monitoring of reactionsshowed the same loss of activity, as was determined in theexperiment shown in Fig. 2 by total TCE extraction withpentane. A more likely explanation for the observed dimi-

nution in biodegradation rate over time is the formation oftoxic products. In this respect, it is of interest that themonooxygenation of TCE by rat liver cytochrome P-450monooxygenase yields an epoxide that can direcily alkylatecellular nucleophiles, rearrange to form a reactive acylchloride, or undergo spontaneous hydration and HCl elimi-nation to yield a different acyl chloride (15). Note that directdioxygenation ofTCE by toluene dioxygenase is comparablein outcome to the epoxidation and oxirane hydration path-way documented in mammalian systems to prbduce agentsthat can alkylate cellular nucleophiles. Furthermore, toluenedioxygenase is known to have both dioxygenase and mono-oxygenase activities (26), so all of the reaction pathwaysobserved in mammalian systems that lead to the formation ofreactive intermediates must be considered. Clearly, an un-derstanding of TCE metabolism at the molecular level willrequire in vitro studies to identify transient, highly reactivechemical intermediates. Studies in this area that may explainthe dramatic diminution in biodegrative rates are in progress.

ACKNOWLEDGMENTS

The skilled technical assistance of Steve Householder and GregBrusseau is gratefully acknowledged. We thank Richard Hanson ofthe Gray Freshwater Biological Institute for kindly providing M.trichosporium OB3b and for helpful discussion.This work was supported in part by Public Health Service grant

GM29909 awarded to D.T.G. by the National Institute of GeneralMedical Science and by grant IN-13 from the American CancerSociety to L.P.W.

LITERATURE CITED1. Barrio-Lage, G., F. Z. Parsons, and R. S. Nassar. 1987. Kinetics

of the depletion of trichloroethene. Environ. Sci. Technol. 21:366-370.

2. Bradford, M. M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Anal. Biochem. 72:248-254.

3. Claus, D., and N. Walker. 1964. The decomposition of tolueneby soil bacteria. J. Gen. Microbiol. 36:107-122.

4. Cornish, A., K. M. Nicholls, D. Scott, B. K. Hunter, W. J. Aston,I. J. Higgins, and J. K. M. Sanders. 1984. In vitro 13C-NMRinvestigations of methanol oxidation by the obligate methano-troph Methylosinus trichosporium OB3b. J. Gen. Microbiol.130:2565-2575.

5. Ensley, B. D,. B. J. Ratzkin, T. D. Osslund, M. J. Simon, L. P.Wackett, and D. T. Gibson. 1983. Expression of naphthaleneoxidation genes in Escherichia coli results in the biosynthesis ofindigo. Science 222:167-169.

6. Finette, B. A., V. Subramanian, and D. T. Gibson. 1984.Isolation and characterization of Pseudotnonas putida PpF1mutants defective in the toluene dioxygenase enzyme system. J.Bacteriol. 160:1003-1009.

7. Fogel, M. M., A. R. Taddeo, and S. Fogel. 1986. Biodegradationof chlorinated ethenes by a methane-utilizing mixed culture.Appl. Environ. Microbiol. 51:720-724.

8. Gibson, D. T., M. Hensley, H. Yoshioka, and T. J. Mabry. 1970.Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry 9:1626-163.0.

9. Gibson, D. T., J. R. Koch, and R. E. Kallio. 1968. Oxidativedegradation of aromatic hydrocarbons by microorganisms. I.Enzymatic formation of catechol from benzene. Biochemistry 7:2653-2662.

10. Gibson, D. T., and V. Subramanian. 1984. Microbial degrada-tion of aromatic hydrocarbons, p. 181-252. In D. T. Gibson(ed.), Microbial degradation of aromatic hydrocarbons. MarcelDekker, Inc., New York.

11. Gibson, D. T., W.-K. Yeh, T.-N. Liu, and V. Subramanian.1982. Toluene dioxygenase: a multicomponent enzyme systemfrom Pseudomonas putida, p. 51-61. In M. Nozaki, S. Yama-

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