Apr 27, 1988 - WACKETT AND GIBSON. TABLE 1. Bacterial strains. Bacterial strain. Relevant properties'. Reference. Pseudomonas putida. Fl.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1988, p. 1703-1708
Vol. 54, No. 7
0099-2240/88/071703-06$02.00/0 Copyright © 1988, American Society for Microbiology
Degradation of Trichloroethylene by Toluene Dioxygenase in Whole-Cell Studies with Pseudomonas putida Fl 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, LAWRENCE P. WACKETT1*
AND
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 tolueneinduced P. putida Fl, the initial degradation rate varied linearly with trichloroethylene concentration over the range 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 strains derived from P. putida Fl that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant 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, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.
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 Chemical Co. (Fair Lawn, N.J.), and indole was from Eastman Kodak Co. (Rochester, N.Y.). Organisms and culture conditions. The strains used in this study are listed in Table 1. To generate cell mass for TCE transformation studies, P. putida F strains were grown on a mineral salts medium (20) containing 0.2% (wt/vol) L-arginine with toluene supplied in the vapor phase as described previously (3). Typically, 100-ml cultures in 500-ml Erlenmeyer flasks were grown at 30°C with shaking at 180 rpm. The revertant strain P. putida F4a was generated by inoculating P. putida F4 into mineral salts medium containing 0.02% (wt/vol) arginine and toluene supplied as before. The culture was grown to late exponential phase and transferred into fresh growth medium until a yellow color appeared in the culture filtrate, indicating that toluene was being oxidized to produce 2-hydroxy-6-oxo-2,4-heptadienoic acid. Organisms were plated onto mineral salts solid medium containing 2% (wt/vol) agar with toluene supplied in the vapor phase as described previously (3). A large colony was picked and designated P. putida F4a. Methylosinus trichosporium OB3b was grown on Higgins medium (4) at 30°C with shaking at 180 rpm under an atmosphere of 30% (vol/vol) methane in air. GC. Gas chromatography (GC) analysis was conducted with a Hewlett-Packard 5790A series gas chromatograph equipped with a 30-m RSL-160 polydimethylsiloxane column (Alltech Associates, Deerfield, Ill.) and electron capture or flame ionization detection systems. Samples were applied with a Hewlett-Packard 7672A autosampler, and peak integrations were obtained with a Hewlett-Packard 3390A integrator. The following operating conditions were used; injector temperature, 150°C; detector temperature (electron capture detector), 250°C; column temperature, 35°C run to 150°C at 15°C/min postinjection; hydrogen carrier flow, 6 ml/ min. Under these conditions, TCE and EDB (internal standard) had retention times of 5.3 and 6.9 min, respectively.
Trichloroethylene (TCE) is an Environmental Protection Agency priority pollutant, and 178 million pounds were used by American industry in 1985 (21). It is a suspected carcinogen in rats (15). Thus, the fate of TCE in water supplies is of great interest. Concerns over the presence of TCE in groundwater are heightened by reports that some anaerobic bacteria biotransform TCE to vinyl chloride. This latter compound is well documented to be tumorigenic in mammals (14). In light of the above observations, there have been extensive efforts to document the biodegradation of trichloroethylene by microbes. Consortia of anaerobic microorganisms have been shown to degrade TCE (7, 25). Mixtures of bacteria that proliferate under aerobic conditions have been reported to be stimulated in their TCE-degrading activities by the addition of methane (28). More recently, three bacterial pure cultures that catabolize toluene, strain G4, Pseudomonas putida Fl, and P. putida B5, have been demonstrated to biodegrade TCE (16-18). Furthermore, Nelson et al. (18) used a toluene dioxygenase mutant strain and revertant strains to implicate toluene dioxygenase in TCE metabolism by P. putida Fl. Considerable information is available on the biochemistry and genetic regulation of toluene dioxygenase in P. putida Fl (6, 11, 22-24), making this organism the best choice for making inroads in discovering the molecular basis of bacterial TCE degradation. In this study, we use additional mutants to support the demonstration that toluene dioxygenase catalyzes degradation of TCE, compare rates of TCE degradation by P. putida Fl with that of a methanotroph, determine whole-cell initial rates as a function of TCE concentration, and examine the biodegradative potential of P. putida Fl with the full suite of chlorinated ethylenes. MATERIALS AND METHODS Materials. TCE, 1,1-dichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, tetrachloroethylene, and 1,2-dibromoethane (EDB) were purchased from Aldrich Chemical Co. (Milwaukee, Wis.). Vinyl chloride was ob*
Corresponding author. 1703
1704
APPL. ENVIRON. MICROBIOL.
WACKETT AND GIBSON TABLE 1. Bacterial strains Bacterial strain
Relevant properties'
Reference
Pseudomonas putida
Fl F4 F4a F7 F106 F107
Methylosinus trichosporium OB3b
Tol+ prototroph Tol- todC Tol' todC revertant Tol- todABCDEF Tol- todC Tol- todE
Methane+, prototroph
9 6 This study
6 6 B. Finette, Ph.D. thesis, University of Texas at Austin, 1984 27
a Gene designations: tod, toluene degradation: todC, terminal dioxygenase; todD, cis-toluene dihydrodiol dehydrogenase; dioxygenase; todA, ferredoxinTOL reductase; todB, ferredoxinTOL; todF, 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase.
Quantitative determination of TCE degradation. P. putida F strains were grown as described previously to a turbidometric density at 600 nm of 1.3 to 1.5. M. trichosporium OB3b was used for biotransformation after attaining an optical density at 600 nm of 0.25 to 0.30. Cultures were harvested by centrifugation at 8,000 x g for 10 min. Cell pellets of P. putida strains were suspended in mineral salts medium (19) containing 0.2% (wt/vol) L-arginine to an optical density at 600 nm of 1.0. M. trichosporium OB3b was suspended in fresh Higgins medium (4) to a turbidometric density at 600 nm of 1.2. Addition of methanol, formate, or methane was observed not to enhance TCE degradation, so these carbon sources were not added to reaction mixtures in the experiments described. A 2-ml amount of the respective cell suspensions was dispensed into 10-ml (total volume) heavy glass vials that were crimp-sealed with an inert rubber septum (American Scientific Products, Bedford, Mass.). The sealed cultures were equilibrated at 30°C on a shaker platform operating at 180 rpm. A 5-pd sample of an appropriate TCE stock solution in p-dioxane was added by injection through the septa with a gas-tight syringe (Hamilton, Reno, Nev.). After incubation of the inverted vial for a given length of time, reactions were quenched by the addition of 1 ml of n-pentane containing 1 ppm EDB. EDB was added as an internal standard to correct for losses in work-up and for injection imprecision. The sealed vials were centrifuged at 5,000 x g for 10 min to pellet the cells and to aid in the separation of the pentane phase. Thereafter, approximately 1 ml of the organic phase was transferred to a septum-sealed 1-ml autosampling vial. Injections of 1 1LI were routinely made for GC analysis. Typical 0-min incubations, which were always performed as controls, gave TCE recoveries of 85 to 90%. These zero-time controls served as the baseline for monitoring TCE disappearance over time. In some experiments, cells that were boiled 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 these
experiments. A difficulty that arises in rate studies on TCE degradation is the absolute concentration of TCE available to bacterial cells. The application of Henry's Law allows one to estimate that approximately 30% of the added TCE is in the liquid phase in these experiments. Note that the relevance of this calculation is affected by the deviation of TCE from behavior as an ideal gas, the likelihood that TCE will partion into bacterial cells, which would increase the effective concentration in the liquid phase, and the potential for adsorption of TCE onto the surfaces of the reaction vessel. These parameters, which pose a difficult analytical problem, prompted us to conduct and report our experiments in the following
todE, 3-methylcatechol 2,3-
manner. First, all incubations were conducted in identical 10-ml reaction vials containing 2 ml of liquid and shaken at the same speed. Second, all concentrations of TCE are expressed as molar concentrations, with the total TCE added calculated as being in the 2-ml liquid phase. Since the observed extent of degradation is far in excess of the initial soluble TCE concentration, equilibration of TCE between gas and liquid phases must be occurring during the course of degradation. The possible effect of mass transport of TCE into the liquid phase in limiting biodegradation is further examined in the Results and Discussion sections. Quantitative determination with other olefinic substrates. Incubations with other substrates were conducted as described for TCE. All were added from p-dioxane stock solutions to a final olefine concentration of 20 ,uM, except ethylene, which was injected directly as a gas to concentrations (in a volume of 2 ml) of 20 FM, 200 ,uM, or 2 mM. Work-up and GC were conducted as for TCE, except that direct headspace analysis was conducted with ethylene and the GC was operated with a flame ionization detector. Protein determinations. The cell suspensions from an incubation mixture were solubilized by diluting 1:1 with 2 N NaOH and heating to 85°C for 10 min. The liberated protein level was determined by the Bradford assay (2) and calibrated with stock concentrations of carbonic anhydrase which had been carried through NaOH and heat treatment in
parallel. RESULTS TCE metabolism by mutants derived from P. putida Fl. A previous study by Nelson et al. (18) implicated toluene dioxygenase in the degradation of TCE by P. putida Fl. In that study, the wild-type strain and two mutants were incubated with TCE for 24 h. A toluene dioxygenase mutant, P. putida F106, showed only a 22% decrease in TCE compared 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 conducting 0- and 1-h incubations, by using pentane extraction of total reaction mixtures to obtain highly reproducible comparisons, and by examining additional mutant strains. The strains used were wild-type P. putida Fl, P. putida F106, two additional todC (toluene dioxygenase) mutants, a revertant strain, P. putida 4a, and a catechol 2,3-dioxygenase mutant, P. putida F7 (Table 1 and Fig. 1). In these experiments, in which extraction efficiencies ranged from 85 to 90%, there was no discernible decrease in TCE concentration with heat-killed wild-type cells or with the three todC mutants (Table 2). Furthermore, the recovery of TCE from incubations with todC strains was similar to
VOL. 54, 1988
TRICHLOROETHYLENE DEGRADATION BY P. PUTIDA Fl
1705
Toluene CH3
~
~
~ ISPTOL.,.+ ~O+ 02 °2 (oxd.) jFe2+ ISPTOLH OH
NADH Rd NADH 2FdTOL RdTOL +H++H+ NK
(red.)
(FAD)
+
NAD
2FdTOL
RdTOL
(FADH2)
(red.)
(oxd.)
CH3 ,,OH OH
OHI
cis-Toluene
I
Dihydrodiol
METABCDLITE
3--Methyl Catechol
CH3
01so &OH OH
2.!-Hydroxy-6-oxo
2, 4- Heptadienoic acid
FerredoxinToL Reductase
GENE PRODUM
FerredoxinToL
Iron-Sulfur
ProteinToL
(ReductaseToL)
GENE
cis-Toluene
Dihydrodiol
Dehydrogenase
3-Methyl Catechol 2,3- Dioxygenase
tod A
DESIGNATION
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 the proteins.
recoveries of TCE from vials containing sterile medium alone, ruling out the possibility that sorption to cells makes TCE inaccessible to pentane extraction. In contrast to the controls and the todC strains, the wild-type strain, the todE mutant, and the todC revertant all clearly degraded TCE, as indicated by a 51 to 63% decrease in pentane-extractable TCE. Note that strain P. putida F4a was demonstrated to express in vivo toluene dioxygenase activity comparable to that of the wild type by its ability to oxidize indole to indigo, a reaction catalyzed by toluene dioxygenase (5). The three todC mutants did not form indigo from indole. The complete correlation of indigo-producing activity with TCE-biodegrading ability points to toluene dioxygenase as the common enzyme responsible for both transformations. Time course of TCE degradation by P. putida Fl and M. trichlosporium OB3b. In previous studies with mixed and pure cultures (1, 7, 16-18), TCE degradation has been accessed over periods greater than 1 h. Since P. putida Fl degraded a significant quantity of TCE in 1 h, it was important to examine the kinetics of substrate disappearance further and to compare the rate with that in another class of TABLE 2. TCE metabolism and indigo formation
by P. putida Fl and derivatives Mean pentane-extractable P. putida strain
TCE'
(>M) ± SD
F106 F107 F4a
17.0 ±0.2 17.2 ± 0.5 17.6 0.4 17.0± 0.3 17.4 ± 0.1 17.1 ± 0.3 17.3 ± 0.2
formation
56 0 0 0 0 51 63
+
lh
Oh
Fl Fl (heat killed) F4 F7
Indigo
% Decrease
7.5 18.0 18.1 17.7 17.8 8.4 6.4
± 0.1
+ 0.2 ± 0.1 ± 0.9 ± 0.4 ± 0.3 ± 0.9
-
-
+ +
a Reactions were initiated by addition of TCE to 20 ,uM. TCE was extracted by the addition of pentane at the indicated time and quantitatively determined by GC as described in Materials and Methods. b A few crystals of indole were added to a portion of the culture used for TCE degradation studies. Positive cultures turned blue within 15 min. Negative cultures remained colorless, even after several hours.
bacteria, the methanotrophs, which have been implicated in TCE degradation (Table 1). In this experiment, the biodegradation of TCE by P. putida Fl and M. trichosporium OB3b was followed for 6 h (Fig. 2). Overall, the extent of biodegradation of TCE over the time course was greater with P. putida Fl. However, it should also be noted that the progress 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 initial rate after 20 min (Fig. 2, inset). In contrast, M. trichosporium was slower but more substained in its biodegradative activity. Note that a similar decline in the TCE degradation activity of P. putida Fl over time was observed by monitoring the TCE concentration in the headspace of the incubation mixture. This result precluded the possibility that the rate decrease was due to very slow equilibration of TCE between the gas and liquid phases that would keep TCE unavailable to the organism, which is confined to the liquid phase. Initial rates of TCE degradation. The rapid initial rate of degradation of TCE by P. putida Fl reflects the maximal biodegradative potential of this organism under the conditions used. In order to begin to develop insights into in vivo kinetics of TCE disappearance, 5-min assays were conducted at various TCE concentrations. A 5-min sampling point, at which time approximately 10 to 20% of the TCE had been consumed, offered the best compromise for accurate analytical determination of TCE disappearance and for estimation of an initial rate at a given substrate concentration (Fig. 2, inset). In this manner, initial rates were determined at 8, 20, 80, and 320 ,uM TCE. The rate of TCE degradation was linear with respect to substrate concentration over the range from 8 to 80 ,uM (1.05 to 10.50 ppm) but was below detectable limits at 320 ,uM (Fig. 3). The highest observed rate was 1.8 nmol/min per mg of cell protein at a TCE concentration of 80 ,uM. The degradation rate plummeted to undetectable levels at a fourfold-higher concentration. Substrate specificity of toluene dioxygenase with chlorinated olefins. Toluene dioxygenase is known to dioxygenate alkylbenzenes to cis-dihydrodiols (10), to oxidize indole to indigo
APPL. ENVIRON. MICROBIOL.
WACKETT AND GIBSON
1706
z
2.0 4 _m
16
._
1.6
,,, o 0 CL.
2
jE
i-
o c
1.2
0.8
OE
z
z
E
12
0.4
-
'u I
JI--
20
Lu
z
40
60
80
320
INITIAL TCE CONCENTRATION (jiM) 8
FIG. 3. Initial rate of TCE degradation as a function of starting TCE concentration. Rates were determined by measuring disappearance of TCE after 5 min of incubation.
LU -j
LU
0 0
I--
1
2
3
4
5
6
TIME (h)
FIG. 2. TCE removal from culture medium by P. putida Fl (@) and M. trichosporium OB3b (0) cell suspensions at optical densities of 1.0 and 1.2, respectively, at 600 nm. TCE, initially added to a concentration of 15 ,uM, was extracted with pentane and quantitatively 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 chlorinated olefins oxidized by toluene dioxygenase in an effort to understand this enzyme better as well as to establish the biodegradative potential of P. putida Fl with this class of compounds. In this context, ethylene and the full set of chlorinated ethylenes were examined as substrates for degradation by this organism (Table 3). Clearly, TCE and cis-1,2-dichloroethylene were the best in vivo substrates for biodegradation. In contrast, the two other isomeric dichloroethylenes were degraded to lesser extents in these 1-h incubations. The 2% decrease in tetrachloroethylene is within the statistical error of these determinations, and thus, degradation of this substrate was insignificant under these experimental conditions. Degradation of vinyl chloride, as determined by pentane extraction and direct headspace analysis, was not observed. Ethylene disappearance 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 compounds induce TCE-degrading activity in certain bacteria. More recently, the same research group presented evidence that toluene dioxygenase is the enzyme responsible for TCE
degradation by P. putida F1 (18). In the present study, data obtained with additional mutants and use of the indigo assay to demonstrate in vivo toluene dioxygenase activity support the conclusion that toluene dioxygenase is required for TCE degradation by P. putida Fl. All of the TCE-negative strains were defective in todC, which encodes the oxygenase (USPTOL) component of toluene dioxygenase (Fig. 1). It should be noted that ISPTOL is composed of two distinct subunits (22), and genetic evidence shows that these are encoded by separate genes (B. A. Finette, G. J. Zylstra, and D. T. Gibson, unpublished data). At this time it is not possible to determine whether expression of one or both genes is required for TCE degradation. The recent cloning of both ISPTOL genes (W. R. McCombie, G. J. Zylstra, and D. T. Gibson, unpublished data) may help to resolve this issue.
This report also initiates comparisons of the TCE-biodegradative potential of pure cultures by examining the catalytic activities of P. putida Fl and M. trichosporium OB3b under identical conditions. Previously, all rate studies of TCE biodegradation have been conducted with soils or mixed microbial cultures (1, 7, 13, 19, 25, 28). In those studies with aerobic mixed cultures and with anaerobic consortia, it is unclear what proportion of the total biomass is represented by TCE-degrading organisms. Hence, it is difficult to compare previous kinetic data spanning hours and days with the rate data determined in the present study expressed as nanomoles per minute per milligram of cell protein. Furthermore, significant differences in the progress TABLE 3. Substrate specificity displayed by P. putida Fl in the degradation of ethylene and chlorinated ethylenes Substrate (20 p.m initial concn)
% Decrease after 1 ha
cis-1,2-Dichloroethylene .......................................... trans-1,2-Dichloroethylene ......................................
2 68 67 10
1,1-Dichloroethylene ........................................... Vinyl chloride ...................... ......................
18 0
Tetrachloroethylene ............................................ TCE ............................................
Ethylene ...........................................
0
" Expressed as 100 - [(concn at 1 h/concn at 0 h) x 100]. Concentrations were obtained as the average of triplicate determinations.
VOL. 54, 1988
TRICHLOROETHYLENE DEGRADATION BY P. PUTIDA Fl
curves of TCE disappearance were observed in this study with P. putida Fl and M. trichosporium OB3b. Clearly, more comparisons with these organisms under various conditions and with additional bacterial isolates are germane to our continuing efforts to obtain an optimal system for bacterial TCE degradation. Another point relevant to biodegradation of complex mixtures of compounds is the overall substrate range of toluene dioxygenase. In this context, other chlorinated olefins were examined to provide a baseline for further studies on the biodegradation of multiple substrates. For example, P. putida Fl oxidizes TCEs and dichloroethylenes (this study), substituted benzenes (10), and 3-methylcyclohexene (29), as well as fused and heterocyclic ring systems (5, 26). In the presence of a number of these compounds, the course of biodegradation will depend on relative binding affinities and turnover numbers of the substrates with toluene dioxygenase and/or transport system(s). Thus, further rate studies with various substrates of toluene dioxygenase are required in order to allow predictions of biodegradation kinetics of complex mixtures by P. putida Fl. The elucidation of TCE degradation rates is rendered difficult by uncertainties in determining the actual concentrations of TCE available to cells at a given time during assay. This physical problem, which is a complex function of water and headspace volumes, cell mass, glass reaction vessel surface area, and agitation rates, was dealt with by holding these parameters constant during the course of the experiments. For ease of presentation and understanding of these studies, the TCE concentrations are expressed as if all the TCE added to the reaction vials were in solution. Note that we observed up to 95% degradation of TCE over 1 h in the course of in vitro experiments under conditions similar to those described here (S. Householder and L. P. Wackett, unpublished data). Clearly, TCE oxidation in the liquid phase results in reequilibration between gas and liquid phases so that all of the TCE ultimately becomes available to liquid-phase enzymes or to bacterial cells. A related issue that is relevant to rate studies is the rapidity of reequilibration between the gas and liquid phases that could impose rate limitations on biodegradation. In this context, it should be pointed out that in the 5-min determinations of TCE degradation, up to 70% of the initially soluble TCE would have been consumed. Thus, if reequilibration is slow, these initial rates may underestimnate the true in vivo biodegradative abilities of P. putida Fl with TCE. The development of an assay based on product formation rather than TCE disappearance would increase assay sensitivity. This would allow monitoring of the biodegradation at shorter time points and give more accurate initial rates. Studies are underway to determine the product(s) of TCE oxidation by toluene dioxygenase. These studies suggest that TCE may be toxic to P. putida Fl in two different ways. First, at 320 ,uM TCE, the highest concentration examined, TCE degradation no longer occurred. Note that high concentrations of TCE have previously been shown to disrupt metabolic activity in Photobacterium phosphoreum, as evidenced by its loss of bioluminescence (12). A second phenomenon is the decrease in the rate of the reaction that was observed during the course of TCE metabolism (Fig. 2). This could not be explained as being due to slow equilibration between the gas and liquid phases, as headspace monitoring of reactions showed the same loss of activity, as was determined in the experiment shown in Fig. 2 by total TCE extraction with pentane. A more likely explanation for the observed dimi-
1707
nution in biodegradation rate over time is the formation of toxic products. In this respect, it is of interest that the monooxygenation of TCE by rat liver cytochrome P-450 monooxygenase yields an epoxide that can direcily alkylate cellular nucleophiles, rearrange to form a reactive acyl chloride, or undergo spontaneous hydration and HCl elimination to yield a different acyl chloride (15). Note that direct dioxygenation of TCE by toluene dioxygenase is comparable in outcome to the epoxidation and oxirane hydration pathway documented in mammalian systems to prbduce agents that can alkylate cellular nucleophiles. Furthermore, toluene dioxygenase is known to have both dioxygenase and monooxygenase activities (26), so all of the reaction pathways observed in mammalian systems that lead to the formation of reactive intermediates must be considered. Clearly, an understanding of TCE metabolism at the molecular level will require in vitro studies to identify transient, highly reactive chemical intermediates. Studies in this area that may explain the dramatic diminution in biodegrative rates are in progress. ACKNOWLEDGMENTS The skilled technical assistance of Steve Householder and Greg Brusseau is gratefully acknowledged. We thank Richard Hanson of the 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 General Medical Science and by grant IN-13 from the American Cancer Society to L.P.W. LITERATURE CITED 1. 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 the quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72:248-254. 3. Claus, D., and N. Walker. 1964. The decomposition of toluene by 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-NMR investigations of methanol oxidation by the obligate methanotroph 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 naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science 222:167-169. 6. Finette, B. A., V. Subramanian, and D. T. Gibson. 1984. Isolation and characterization of Pseudotnonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system. J. Bacteriol. 160:1003-1009. 7. Fogel, M. M., A. R. Taddeo, and S. Fogel. 1986. Biodegradation of 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-4,6diene from toluene by Pseudomonas putida. Biochemistry 9: 1626-163.0.
9. Gibson, D. T., J. R. Koch, and R. E. Kallio. 1968. Oxidative degradation 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 degradation of aromatic hydrocarbons, p. 181-252. In D. T. Gibson
(ed.), Microbial degradation of aromatic hydrocarbons. Marcel Dekker, Inc., New York. 11. Gibson, D. T., W.-K. Yeh, T.-N. Liu, and V. Subramanian. 1982. Toluene dioxygenase: a multicomponent enzyme system from Pseudomonas putida, p. 51-61. In M. Nozaki, S. Yama-
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13. 14. 15.
16. 17.
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