Trichloroethylene oxidation by toluene dioxygenase.

Trichloroethylene was oxidized by purified toluene dioxygenase obtained from recombinant E. coli strains. The major oxidation products were formic acid and glyoxylic acid. Other potential products, dichloroacetic acid, chloral, phosgene, carbon monoxide, and carbon dioxide, were not detected. [14C]trichloroethylene became covalently attached to protein components and NADPH suggesting non-specific alkylation by reactive products. Oxidation of deuterated trichloroethylene yielded 50.2% deuterated formate. Oxidation of trichloroethylene in D2O yielded 43.7% deuterated formate. These data indicate that both carbon atoms are giving rise to formic acid. The results are consistent with a mechanism of TCE oxygenation not involving epoxide, dioxetane, or dihydroxy intermediates and indicate significant differences from those previously proposed for cytochrome P-450 (Miller, R.E. & Guengerich, F.P. (1982) Biochemistry 21, 1090-1097) or methane monooxygenase (Fox, B.G., Borneman, B.G., Wackett, L.P., & Lipscomb, J.D. (1990) Biochemistry 29, 6419-6227).     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida

Toluene dioxygenase, a multicomponent enzyme system known to oxidize mononuclear aromatic hydrocarbons to cis-dihydrodiols, oxidized indene and indan to 1-indenol and 1-indanol, respectively. In addition, the enzyme catalyzed dioxygen addition to the nonaromatic double bond of indene to form cis-1,2-indandiol. The oxygen atoms in 1-indenol and cis-1,2-indandiol were shown to be derived from molecular oxygen, whereas 70% of the oxygen in 1-indanol was derived from water. All of the isolated products were optically active as demonstrated by 19F NMR and HPLC discrimination of diastereomeric esters and by chiroptic methods. The high optical purity of (-)-(1R)-indanol (84% enantiomeric excess) and the failure of scavengers of reactive oxygen species to inhibit the monooxygenation reaction supported the contention that the monooxygen insertion is mediated by an active-site process. Experiments with 3-[2H]indene indicated that equilibration between C-1 and C-3 occurred prior to the formation of the carbon-oxygen bond to yield 1-indenol. Naphthalene dioxygenase also oxidized indan to 1-indanol, which suggested that benzylic monoxygenation may be typical of this group of dioxygenases.     

Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.

Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees 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 F1 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 F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.     

Expression and substrate specificity of the toluene dioxygenase of Pseudomonas putida NCIMB 11767

 Pseudomonas putida NCIMB 11767 oxidized phenol, monochlorophenols, several dichlorophenols and a range of alkylbenzenes (C₁–C₆) via an inducible toluene dioxygenase enzyme system. Biphenyl and naphthalene were also oxidized by this enzyme. Growth on toluene and phenol induced the meta-ring-fission enzyme, catechol 2,3-oxygenase, whereas growth on benzoate, which did not require expression of toluene dioxygenase, induced the ortho-ringcleavage enzyme, catechol 1,2-oxygenase. Monochlorobenzoate isomers and 2,3,5-trichlorophenol were gratuitous inducers of toluene dioxygenase, whereas 3,4-dichlorophenol was a fortuitous oxidation substrate of the enzyme. The organism also grew on 2,4- and 2,5-dichloro isomers of both phenol and benzoate, on 2,3,4-trichlorophenol and on 1-phenylheptane. During growth on toluene in nitrogen-limited chemostat culture, expression of both toluene dioxygenase and catechol 2,3-oxygenase was positively correlated with increase in specific growth rate (0.11–0.74 h^-1), whereas the biomass yield coefficient decreased. At optimal dilution rates, the predicted performance of a 1-m³ bioreactor supplied with 1 g nitrogen l^-1 for removal of toluene was 57 g day^-1 and for removal of trichloroethylene was 3.4 g day^-1. The work highlights the oxidative versatility of this bacterium with respect to substituted hydrocarbons and shows how growth rate influences the production of competent cells for potential use as bioremediation catalysts. Received: 26 June 1995 / Received revision: 4 September 1995 / Accepted: 20 September 1995     

Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1

Toluene dioxygenase oxidizes toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. This reaction is catalyzed by a multienzyme system that is induced in cells of Pseudomonas putida F1 during growth on toluene. One of the components of toluene dioxygenase has been purified to homogeneity and shown to be an iron-sulfur protein that has been designated ferredoxinTOL. The molecular weight of ferredoxinTOL was calculated to be 15,300, and the purified protein was shown to contain 2 g of atoms each of iron- and acid-labile sulfur which appear to be organized as a single [2Fe-2S]cluster. Solutions of ferredoxinTOL were brown in color and showed absorption maxima at 277, 327, and 460 nm. A shoulder in the spectrum of the oxidized protein was discernible at 575 nm. Reduction with sodium dithionite or NADH and ferredoxinTOL reductase resulted in a decrease in visible absorbance at 460 and 575 nm, with a concomitant shift in absorption maxima to 382 and 438 nm. The redox potential of ferredoxinTOL was estimated to be -109 mV. In the oxidized state, the protein is diamagnetic. However, upon reduction it exhibited prominent electron paramagnetic resonance signals with anisotropy in g values (gx = 1.81, gy = 1.86, and gz = 2.01). Anaerobic reductive titrations revealed that ferredoxinTOL is a one-electron carrier that accepts electrons from NADH in a reaction that is mediated by a flavoprotein (ferredoxinTOL reductase). The latter is the first component in the toluene dioxygenase system. Reduced ferredoxinTOL can transfer electrons to cytochrome c or to a terminal iron-sulfur dioxygenase (ISP-TOL) which catalyzes the incorporation of molecular oxygen into toluene and related aromatic substrates.     

Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system.

Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems.     

Regulated toluene cis-glycol production by recombinant Escherichia coli strains constructed by PCR amplification of the toluene dioxygenase genes from Pseudomonas putida

The toluene dioxygenase genes from Pseudomonas putida NCIMB 11767 were isolated by PCR amplification from recombinant plasmid, p1/1. The genes were subcloned into pUC18 and pKK223-3 and expressed under the lac and tac promoters, respectively. In both cases, toluene cis-glycol was produced, with higher levels of product formation when the genes were expressed from the tac promoter.     

The measurement of toluene dioxygenase activity in biofilm culture of Pseudomonas putida F1

Toluene dioxygenase (Tod) enzyme activity can be measured by the conversion of indole to indigo. Indigo is measured spectrophotometrically at 600 nm. However, this method is inadequate to measure the whole-cell enzyme activity when interference by suspended biomass is present. Indoxyl is a highly fluorescent intermediate in the conversion of indole to indigo by Tod. A fluorescence-based assay was developed and applied to monitor Tod activity in whole cells of Pseudomonas putida F1 biofilm from a continuously operated biofilter. Suspended growth studies with pure cultures indicated that indoxyl, as measured by fluorescence, correlated with indigo production (r(2)=0.89) as measured by spectrophotometry. Whole-cell enzyme activity was followed during growth on a minimal medium containing toluene. The maximum normalized whole cell enzyme activity of 19+/-1.5x10(-4) mg indigo (mg protein)(-1) min(-1) was reached during early stationary phase. P. putida F1 cells from a biofilm grown on vapor phase toluene had a normalized whole-cell enzyme activity of 5.0+/-0.2x10(-4) mg indigo (mg protein)(-1) min(-1). The half-life of whole-cell enzyme activity was estimated to be between 5.5 and 8 h in both suspended and biofilm growth conditions.     

Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol.     

Cloning and expression in Escherichia coli of the toluene dioxygenase gene from Pseudomonas putida NCIB11767

The genes encoding toluene dioxygenase, toluene cis-glycol dehydrogenase and catechol 2.3-oxygenase from Pseudomonas putida NCIB 11767 were cloned and expressed in Escherichia coli HB101 on a 20 kb fragment. The recombinant strain produced indigo and a variety of other coloured products. Although the enzymes were expressed in the absence of inducers, further induction was observed in the presence of toluene or benzene, implying the presence of regulatory elements on the 20 kb insert.     

Cyclodextrin-enhanced degradation of toluene and p-toluic acid by Pseudomonas putida.

Degradation of an immiscible aromatic solvent, toluene, and a water-soluble aromatic compound, p-toluic acid, by a Pseudomonas putida strain in the presence of beta-cyclodextrin (beta-CD) was investigated. The ability of CDs to interact with hydrophobic organics and form inclusion compounds was exploited in this study to remove or alleviate the toxicities of substrates and consequently to enable or enhance degradation. Liquid toluene was found to be highly toxic to P. putida. However, this phase toxicity was removed when crystalline beta-CD-complexed toluene was provided as the substrate. The latter was fully degraded at a concentration of up to 10 g/liter. Degradation of toluene vapors was enhanced in the presence of beta-CD as a result of reduced molecular toxicity and facilitated absorption of the gaseous substrate. Similarly, beta-CD alleviated the inhibitory effect of p-toluic acid on P. putida. This protective effect of CD was remarkably more prominent when the microbial culture was shock loaded with an otherwise toxic dose of p-toluic acid (1.8 g/liter).     

Water stress effects on toluene biodegradation by Pseudomonas putida

We quantified the effects of matric and solute waterpotential on toluene biodegradation by Pseudomonasputida mt-2, a bacterial strain originally isolated fromsoil. Across the matric potential range of 0 to – 1.5 MPa,growth rates were maximal for P. putida at – 0.25MPa and further reductions in the matric potentialresulted in concomitant reductions in growth rates.Growth rates were constant over the solute potential range0 to – 1.0 MPa and lower at – 1.5 MPa. First ordertoluene depletion rate coefficients were highest at0.0 MPa as compared to other matric water potentialsdown to – 1.5 MPa. Solute potentials down to – 1.5 MPadid not affect first order toluene depletion ratecoefficients. Total yield (protein) and carbon utilizationefficiency were not affected by water potential, indicatingthat water potentials common to temperate soils were notsufficiently stressful to change cellular energyrequirements. We conclude that for P. putida: (1)slightly negative matric potentials facilitate faster growthrates on toluene but more negative water potentials resultin slower growth, (2) toluene utilization rate per cell massis highest without matric water stress and is unaffected bysolute potential, (3) growth efficiency did not differ acrossthe range of matric water potentials 0.0 to – 1.5 MPa.     

Induction of the tod Operon by Trichloroethylene in Pseudomonas putida TVA8

Bioluminescence, mRNA levels, and toluene degradation rates in Pseudomonas putida TVA8 were measured as a function of various concentrations of toluene and trichloroethylene (TCE). TVA8 showed an increasing bioluminescence response to increasing TCE and toluene concentrations. Compared to uninduced TVA8 cultures, todC1 mRNA levels increased 11-fold for TCE-treated cultures and 13-fold for toluene-treated cultures. Compared to uninduced P. putida F1 cultures, todC1 mRNA levels increased 4.4-fold for TCE-induced cultures and 4.9-fold for toluene-induced cultures. Initial toluene degradation rates were linearly correlated with specific bioluminescence in TVA8 cultures.     

Formate-stimulated oxidation of methanol by Pseudomonas putida 9816

It has been reported that Pseudomonas putida 9816 is able to grow on methanol, but it does not have methanol dehydrogenase or oxidase activity. To utilize methanol it requires yeast extract. The utilization of methanol can be accelerated by adding formate, which obviously helps oxidize methanol and win biologically useful energy. This pseudo-oxidation is catalyzed by a reverse formaldehyde dismutase. Thus, methanol can be both assimilated and dissimilated. Formate alone cannot replace yeast extract. The strain is auxotrophic.     

Regulation of alkane oxidation in Pseudomonas putida.

We have studied the appearance of whole-cell oxidizing activity for n-alkanes and their oxidation products in strains of Pseudomonas putida carrying the OCT plasmid. Our results indicate that the OCT plasmid codes for inducible alkane-hydroxylating and primary alcohol-dehydrogenating activities and that the chromosome codes for constitutive oxidizing activities for primary alcohols, aliphatic aldehydes, and fatty acids. Mutant isolation confirms the presence of an alcohol dehydrogenase locus on the OCT plasmid and indicated the presence of multiple alcohol and aldehyde dehydrogenase loci on the P. putida chromosome. Induction tests with various compounds indicate that inducer recognition has specificity for chain length and can be affected by the degree of oxidation of the carbon chain. Some inducers are neither growth nor respiration substrates. Growth tests with and without a gratuitous inducer indicate that undecane is not a growth substrate because it does not induce alkane hydroxylase activity. Using a growth test for determining induction of the plasmid alcohol dehydrogenase it is possible to show that heptane induces this activity in hydroxylase-negative mutants. This suggests that unoxidized alkane molecules are the physiological inducers of both plasmid activities.     

Production of toluene cis-glycol by Pseudomonas putida in glucose feb-batch culture

Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene Cis-Glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. Putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25h) of the process or the final concentration of TCG achieved. The rate of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. These changes were a consequence of TCG formation and a cooperative toxic effect was demonstrated for toluene and TCG. Adenylate energy charge values decreased from ca. 0.8 to 0.2 over the course of the biotransformation but were maintained above 0.5 in the absence of TCG. Similarly, cellular AMP levels increased dramatically during biotransformation, presumably as a consequence of RNA degradation, but were maintained at low levels in the absence of TCG. The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.     

Plasmolysis induced by toluene in a cyoB mutant of Pseudomonas putida

The cyoABCDE gene cluster of Pseudomonas putida DOT-T1E encodes a terminal cytochrome oxidase. A 500-bp 'cyoB' DNA fragment was cloned in pCHESI Omega Km and used to generate a cyoB knock-out mutant in vivo. The mutant strain was not limited in the generation of proton-motif force, although when grown on minimal medium with glucose or citrate, the CyoB mutant exhibited a slight increase in duplication time with respect to the wild-type strain. This effect was even more pronounced when toluene was supplied in the gas phase. In consonance with the negative effect of toluene on the growth was the finding that the CyoB mutant was hypersensitive to sudden 0.3% (v/v) toluene shocks, in contrast with the wild-type strain. This effect was particularly exacerbated in cells that reached the stationary phase. The increased sensitivity to solvents of the CyoB mutant did not appear to be related to the inability of the cells to strengthen the membrane package or to induce the efflux pumps in response to the solvent, but rather to solvent-induced plasmolysis that may be triggered by wrinkles in the cytoplasmic membrane at the poles of the mutant cells, and invagination of the outer membranes, which eventually lead to cell death.     

Propane and n-butane oxidation by Pseudomonas putida GPo1

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.