Jul 7, 1989 - which is mutagenic (18) and carcinogenic (11) in mammals. Halogenated ... methanotrophs and toluene-oxidizing bacteria degrade TCE.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1989, p. 2723-2725
Vol. 55, No. 10
0099-2240/89/102723-03$02.00/0 Copyright C) 1989, American Society for Microbiology
Toxicity of Trichloroethylene to Pseudomonas putida Fl Is Mediated by Toluene Dioxygenase LAWRENCE P. WACKETT* AND STEVEN R. HOUSEHOLDER Gray Freshwater Biological Institute and Department of Biochemistry, The University of Minnesota, P.O. Box 100, Navarre, Minnesota 55392 Received 17 April 1989/Accepted 7 July 1989
Trichloroethylene was metabolically activated by toluene dioxygenase to produce toxic effects in Pseudomoputida Fl. Cytotoxicity was indicated by growth inhibition and by the covalent modification of cellular molecules in P. putida Fl exposed to ['4C]trichloroethylene. With a toluene dioxygenase mutant, neither growth inhibition nor alkylation of intracellular molecules was observed. nas
Trichloroethylene (TCE) is a widespread groundwater pollutant (17, 20), and it is also a suspected carcinogen (14). Furthermore, anaerobic bacteria that are present in TCEcontaminated water transform TCE to vinyl chloride (22), which is mutagenic (18) and carcinogenic (11) in mammals. Halogenated alkenes (7) and many other classes of compounds (12) require metabolic activation for the manifestation of toxic and/or carcinogenic effects. In many wellstudied cases, bioactivation is mediated by cytochrome P-450 monooxygenases (25). The oxidation of xenobiotic compounds by cytochrome P-450 may lead to the formation of electrophilic products that alkylate DNA, RNA, and proteins (12). For example, TCE and vinyl chloride are oxidized in the mammalian liver to unstable epoxides (7). It is thought that the reaction of these epoxides and of their decomposition products with critical cellular macromolecules underlies the cytotoxic effects of vinyl chloride and TCE. Several aerobic bacteria that oxidize hydrocarbons degrade TCE, and the use of these strains in TCE bioremediation has been proposed (10, 15, 16, 23, 24). For example, methanotrophs and toluene-oxidizing bacteria degrade TCE. In the latter organisms, the specific enzymes that act on TCE are the oxygenases that catalyze the first step in the oxidation of toluene. Thus, toluene monooxygenase, which oxidizes toluene to p-cresol (K. L. Richardson and D. T. Gibson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, K54, p. 156), and toluene dioxygenase, which catalyzes the dioxygenation of toluene to form (+)-cis-1(S),2(R)-dihydroxy3-methylcyclohexa-3,5-diene (5, 26), have been implicated in TCE degradation (23, 24). By analogy to metabolism of chlorinated alkenes by mammalian cytochrome P-450 monooxygenase, bacterial oxygenases might also activate TCE and produce cytotoxic effects. In a previous study, the initial rate of TCE oxidation by Pseudomonas putida Fl declined rapidly, and it was proposed that the formation of toxic intermediates might underlie this phenomenon (23). Thus, it is possible that oxygenase-mediated TCE bioremediation systems might be limited by intracellular destruction wrought by toxic reaction products. In this study, inhibition of cell growth and covalent modification of cellular macromolecules were used to measure the cytotoxic effects of TCE on P. putida Fl. Additionally, the role of toluene dioxygenase in TCE activation was investigated. In the first experiments, the growth rate of toluene*
induced P. putida Fl was markedly inhibited by the addition of TCE vapors supplied from a bulb suspended above the liquid culture. The cultures were grown in mineral medium (19) in 500-ml Erlenmeyer flasks containing 100 ml of 0.2% (wt/vol) L-arginine at a starting optical density at 600 nm of 0.1. The inoculum was supplied from a toluene-containing culture that had been grown to late exponential phase as previously described (23). The doubling time was 5.0 h in the presence of TCE, compared with 1.5 h for a parallel culture that lacked TCE (Table 1). The growth of P. putida Fl in the presence of tetrachloroethylene (PCE) was also examined because growth inhibition could result from solvation of membrane lipids. PCE and TCE have similar solvent properties (8), but PCE was previously shown not to be significantly oxidized by P. putida Fl (23). As demonstrated in Table 1, PCE did not inhibit growth. To develop further insight into the potential role of TCE oxidation in the inhibition of cell growth, P. putida F4 was used. P. putida F4 is a derivative of strain Fl that lacks toluene dioxygenase activity (4), and it was previously shown to be unable to degrade TCE (23). In experiments with this toluene dioxygenase mutant, neither TCE nor PCE inhibited growth (Table 1). These data were consistent with the premise that toluene dioxygenase mediates TCE toxicity in P. putida Fl. The hypothesis that reactive products of TCE oxidation alkylate cellular molecules in P. putida Fl was tested directly. P. putida Fl was grown on 0.2% (wt/vol) L-arginine plus toluene as previously described (23). The cells were harvested, washed, and suspended in 50 ml of minimal medium containing arginine at an optical density of 1.0 at 600 nm in a septum-sealed 120-ml vial. [1,2-14C]TCE (8.1 x 106 cpm) was added by syringe to a final concentration of 20 pLM. The cell suspension was incubated for 10 min at 21°C, after which the septum was removed, and unoxidized TCE was removed by air-stripping in a fume hood. The cells were TABLE 1. Growth rates of P. putida with TCE or PCE added to arginine salts medium Doubling Medium Strain and phenotype time (h) additive
Corresponding author. 2723
Fl (wild type)
None PCE TCE
1.5 1.7 5.0
F4 Toluene dioxygenase mutant
None PCE TCE
1.4 1.5 1.7
2724
APPL. ENVIRON. MICROBIOL.
NOTES
TABLE 2. Amount of 14C incorporated into cellular macromolecules following a 10-min incubation of 1,000 nmol of [1,2-'4C]TCE with toluene-induced cultures of P. putida Fl and P. putida F4 [14C]TCE equivalents Cell fraction incorporated (nmol) by P. putida Fl'
2.01
-
0 0
Protein ................................... 113 Small molecules ............................... .... 33 RNA .................................... .15 DNA ................................... 3 2 Lipid ...................................
1.0
Ec 0.8 0.6
%.) z
0.4
4
a P. putida F4 incorporated
