Degradation of Triphenyltin by a Fluorescent Pseudomonad

Abstract

Triphenyltin (TPT)-degrading bacteria were screened by a simple technique using a post-column high-performance liquid chromatography using 3,3′,4′,7-tetrahydroxyflavone as a post-column reagent for determination of TPT and its metabolite, diphenyltin (DPT). An isolated strain, strain CNR15, was identified as Pseudomonas chlororaphis on the basis of its morphological and biochemical features. The incubation of strain CNR15 in a medium containing glycerol, succinate, and 130 μM TPT resulted in the rapid degradation of TPT and the accumulation of approximately 40 μM DPT as the only metabolite after 48 h. The culture supernatants of strain CNR15, grown with or without TPT, exhibited a TPT degradation activity, whereas the resting cells were not capable of degrading TPT. TPT was stoichiometrically degraded to DPT by the solid-phase extract of the culture supernatant, and benzene was detected as another degradation product. We found that the TPT degradation was catalyzed by low-molecular-mass substances (approximately 1,000 Da) in the extract, termed the TPT-degrading factor. The other fluorescent pseudomonads, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa ATCC 15692, also showed TPT degradation activity similar to strain CNR15 in the solid-phase extracts of their culture supernatants. These results suggest that the extracellular low-molecular-mass substance that is universally produced by the fluorescent pseudomonad could function as a potent catalyst to cometabolite TPT in the environment.

Organotins are the most widely used organometallic compounds, which are employed mainly as polyvinyl chloride stabilizers. Recent estimates suggested that the annual world production of organotins may be close to 50,000 tons (12). Trisubstituted organotin compounds have wide-ranging toxicological properties, and their biocidal uses (∼8,000 tons/year) have been reported to have detrimental environmental impacts (9, 12). Of particular importance to the environment is the high toxicity of tributyl and triphenyl derivatives. Tributyltin (TBT) has been extensively used as an active component in antifouling paints, which have been widely employed on boat hulls. Triphenyltin (TPT) has been employed as a cotoxicant with TBT (14), but the major use of TPT lies in agriculture as fungicides to protect crops. These compounds are directly introduced into aquatic systems via leaching from the antifouling paints and runoff from agricultural fields (12, 14, 22, 34). Therefore, they have been detected in the biota, water, and sediments from both freshwater and marine areas, and their toxic effects have been observed on a variety of nontarget organisms, such as plankton (11, 25), gastropods (7, 19), and fish (15, 20, 35). These reports indicate the importance of clarifying the fate and behavior of organotins loaded into the aquatic environment.

The disappearance of organotin compounds from the environment is attributed to their biodegradation, photolysis, biological uptake, sedimentation, and flux (9, 17, 39). It has been reported that the mixed function oxygenase system in fish (13, 26) and rat liver (24) metabolizes TBT with hydroxylation, whereas TPT is more resistant to the analogous monooxygenase attack even though it undergoes dephenylation in rats (24). Diphenyltin (DPT) and monophenyltin are the abiotic and biotic degradation products of TPT in the environment. Photodegradation appears to be the major factor affecting the fate of TPT in soils (22), whereas there is some evidence to show that microorganisms can slowly degrade TPT into inorganic tin via di- and monophenyltins. Microbial interactions with tin are important because microbes are at the base of the food web and because they have the potential for bioremediation (37). An early study showed a half-life of 140 days for the mineralization of radiolabeled TPT to CO₂ in soil samples (3). Kannan and Lee reported that only 5% of the radiolabeled TPT in the soil or sediment were biodegraded to DPT, monophenyltin, and CO₂ after 14 days (22). The half-life of TPT in soil varied between 47 and 140 days, depending on the organic matter content in the soil (28). TPT in estuarine water samples was scarcely degraded (15%) during a 60-day incubation (16). Visoottiviseth et al. have reported the degradation of TPT by Pseudomonas putida no.C under pure culture conditions (36). While these investigations suggest that microorganisms play an important role in the TPT mineralization in the environment, little is known about the degradation mechanism of TPT and other organotins, and it remains also questionable whether the microbial degradation of organotin is an enzymatic reaction (4).

The present study was undertaken to reveal the microbial degradation mechanism of TPT in the environment. In this study, we have isolated and characterized a TPT-degrading isolate, Pseudomonas chlororaphis CNR15. The TPT degradation reaction by strain CNR15 was catalyzed by low-molecular-mass substances (about 1,000 Da), which are secreted into the culture medium, and involved the accumulation of DPT and benzene as a metabolite. In addition, we report that some fluorescent pseudomonad strains are also able to degrade TPT by a mechanism similar to that used by strain CNR15.

MATERIALS AND METHODS

Chemicals.

All chemicals were used without additional purification. TPT chloride (98% purity) was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). DPT dichloride (96% purity) was from Aldrich. 3,3′,4′,7-Tetrahydroxyflavone (fisetin) was obtained from Wako Pure Chemical Industries. All other chemicals used were of analytical grade.

Bacterial strains and culture conditions.

P. chlororaphis CNR15, which was isolated in this study, P. chlororaphis ATCC 9446, Pseudomonas fluorescens ATCC 13525, P. putida ATCC 12633, and Pseudomonas aeruginosa ATCC 15692 were grown on succinate-glycerol (SG) medium made up of 1.0 g of K₂HPO₄, 1.0 g of KH₂PO₄, 1.0 g of (NH₄)₂SO₄, 0.4 g of MgCl₂, 0.5 g of yeast extract, 4.0 g of succinate, and 1.0 ml of glycerol per liter and adjusted to pH 6.8 by adding the required volume of 2 N NaOH prior to sterilization. The growth responses of the individual strains and the degradation of TPT were determined in 500-ml Erlenmeyer flasks, each containing 100 ml of the SG media. A 1-ml volume of the preculture in the late log phase was used to inoculate the medium. The TPT stock solution (13.0 mM) was prepared in ethanol and added to the autoclave-sterilized medium at a final concentration of 2.6 to 260 μM, when necessary. The TPT stock solution was kept in the dark at 4°C. Bacterial growth was spectrophotometrically monitored (model Ubest-30; JASCO, Tokyo, Japan) by measuring the optical density at 600 nm. Selection of the TPT-degrading microorganisms was conducted in a screening medium, which contained the following: 1.0 g of K₂HPO₄, 1.0 g of KH₂PO₄, 1.0 g of (NH₄)₂SO₄, 0.4 g of MgCl₂, 0.125 g of yeast extract, and 1.0 ml of glycerol per liter and 130 μM TPT (supplemented after autoclave sterilization of the medium) (pH 6.8). All cultures were aerobically grown with shaking (∼110 rpm) at 28°C in the dark.

Isolation and identification of TPT-degrading organism.

Twenty-eight coastal soil or sediment samples were cultured in the screening medium for 10 days (0.1 g of soil or sediment per 5 ml of medium). These cultures (0.2 ml) were subcultured into 5 ml of fresh medium every 10 days for a month. The final transfers of the enriched cultures were assayed for their degradation activity (see below), and the cultures capable of degrading TPT were streaked onto 1.5% agar plates containing the screening medium. Single colonies were picked and transferred onto the new plates to obtain pure cultures. Several isolates were cultured in the screening medium to confirm their TPT degradation capability. The positive isolates were restreaked onto the plates, and the purification procedure was repeated. Only one type of colony was obtained. The TPT-degrading microorganism was identified by Gram staining, morphology, motility, and other physiological and biochemical tests following the standard procedures described in Bergey's Manual of Systematic Bacteriology (31).

Preparation of resting cells and cell-free culture supernatants.

Strain CNR15 was grown in SG medium with or without 130 μM TPT. After 24 h of growth, the cells were harvested (20 min, 10,000 × g, 4°C) and their supernatants were filtered through a polyether sulfone filter membrane (0.25-μm pore size; Nalgen) to yield a cell-free solution. The pelleted cells were washed twice with 20 mM potassium phosphate buffer (pH 7.2) and resuspended in a small volume of the same buffer to the final optical density of 5 at 600 nm.

Preparation of solid-phase extracts.

The solid-phase extract of the culture supernatant was prepared by using Sep-Pack C₁₈ Vac 6cc containing 500 mg of octadecylsilane (Waters Association Co. Ltd.). The CNR15 and ATCC strains were grown in 100 ml of SG medium without TPT for 72 h. Each 50 ml of the cell-free supernatant filtered through the polyether sulfone filter membrane was applied to Sep-Pack C₁₈ Vac 6cc, whose conditioning was followed by methanol and water, and was extracted with 20 ml of 50% (vol/vol) methanol. The extract was concentrated to 5 ml in a water bath (30°C) by using a rotary evaporator and stored at 4°C until needed for use. A part of the concentrated extract (0.5 ml) was lyophilized to a constant for 3 days using an FD-80 freeze-dryer (EYELA, Tokyo, Japan) connected to a vacuum pump (model PD-135; Sinku Kiko Co., Ltd., Yokohama, Japan). The concentration of the TPT-degrading factor (TPT-DF) in the extract was estimated from the total weight of the lyophilized samples.

TPT degradation assays.

The TPT degradation activity was measured by monitoring the decrease in TPT and increase in its metabolite, DPT, by post-column high-performance liquid chromatography (HPLC) as described below. To evaluate the TPT degradation activity in the culture medium, 50 μl of the culture was mixed in methanol (400 μl) and 6 N HCl (50 μl) at various intervals. The sample solution (20 μl) was directly injected into the post-column HPLC system without centrifugation when 130 μM TPT was supplemented in the culture medium.

In all the TPT degradation assays, TPT was added at the final concentration of 130 μM. The assay using the resting cell or cell-free supernatant (4 ml) was performed with a total volume of 5 ml in 20 mM potassium phosphate buffer (pH 7.2). The reaction mixtures were incubated at 28°C with shaking (∼110 rpm), and 50 μl of the mixtures were analyzed at various intervals as described for the culture medium assay. The TPT-DF activity of the solid-phase extract was determined with a total volume of 400 μl in 10 mM potassium phosphate buffer (pH 7.2). Normally, the reaction mixture containing about 0.5 mg of extract/ml in a microtube was incubated at 30°C for 30 min in a water bath, and the reaction was terminated by the addition of 6 N HCl (400 μl) and methanol (400 μl). The terminated-reaction mixture (150 μl) was diluted with 350 μl of methanol, and then 20 μl of the sample was injected. The optimal pH conditions for the TPT degradation activities were determined for the reaction mixtures containing MES (morpholineethanesulfonic acid) (pH 5.5 to 6.5), MOPS (morpholinepropanesulfonic acid) (pH 6.5 to 7.5), Tris-HCl (pH 7.5 to 8.5), and CHES [(2-cyclohexylamino)ethanesulfonic acid] (pH 8.5 to 9.5) buffers at a final concentration of 20 mM each.

The apparent initial concentration of TPT for the half-maximal rate of DPT production by the strain CNR15 culture and the apparent K_m value of TPT-DF in the solid-phase extract were estimated from the intercepts of the Lineweaver-Burk plots. All experiments were done in triplicate. After a 40-h incubation in the strain CNR15 cultures containing various initial concentrations of TPT (2.6, 13, 26, 52, 130, and 260 μM), the total amounts of DPT production were measured as described above. The TPT-DF activity was measured as previously described using 0.5 mg of extract/ml in the reaction mixture over a period of 30 min. The concentrations of the TPT substrate used were 5.2, 13, 26, 52, 130, and 260 μM.

Analysis of TPT and its metabolites.

The concentrations of TPT and DPT in the culture medium and the other fractions were determined by post-column HPLC using fisetin as the post-column fluorogenic reagent (8). HPLC was performed using a GULLIVER series system (JASCO). The separation of TPT and DPT by HPLC were performed with modification of the method of Kadokami et al. (21). The TSK gel ODS-80 Ts QA analytical column (4.6-mm inner diameter [i.d.] by 25 cm; TOSOH, Tokyo, Japan) with a guard column (TSKguardgel OSD-80 Ts, 4.6-mm i.d. by 1.5 cm; TOSOH) was used in this system. The mobile phase used was a 4:3:2:1 (vol/vol/vol/vol) mixture of tetrahydrofuran, doubly-deionized water, methanol, and glacial acetate, and the flow rate was 0.6 ml/min with a 40°C column temperature. The post-column reagent containing 70 mM sodium succinate buffer (pH 6.5), 0.0005% fisetin (wt/vol), and 1.5% Triton X-100 (vol/vol) was pumped at 2.0 ml/min using a PU-980 (JASCO). The post-column reaction was achieved at room temperature in a low-dead-volume T-piece with a 50-cm-long reaction coil (0.5-mm i.d.). The fluorescence intensity of the TPT and DPT complexes formed with fisetin was detected at 506 nm using an excitation wavelength of 410 nm by a GULLIVER FP-920S (JASCO). The standard solutions (20 μl) of TPT and DPT in methanol–0.6 N HCl were analyzed, and the calibration graphs established from the peak areas were linear over the ranges of 0.26 to 26 μM (TPT) and 0.14 to 5.8 μM (DPT).

Simultaneous analyses of TPT, DPT, and benzene were done with the same HPLC system and column and were detected using an MD-1510 photodiode array UV-visible light detector (215 to 650 nm) (JASCO). The mobile phase used was a 3:5:1:2 (vol/vol/vol/vol) mixture of tetrahydrofuran, doubly-deionized water, methanol, and glacial acetate, and the flow rate was 0.6 ml/min with a 40°C column temperature. The reaction mixture consisting of the solid-phase extract of the strain CNR15 culture supernatant (1 mg/ml), 10 mM potassium phosphate buffer (pH 7.2), and 260 μM TPT in a final volume of 300 μl was incubated in a microtube at 30°C. After a 5-h incubation, methanol (800 μl) and 6 N HCl (100 μl) were added to the reaction mixture, and then 50 μl of the sample was injected into the HPLC system. The calibration graphs established from the peak areas were linear over the ranges of 1.5 to 130 μM (TPT), 2.0 to 145 μM (DPT), and 9.5 to 168 μM (benzene), respectively, when 50 μl of the mixed standard solutions (containing 300 μl of 10 mM potassium phosphate buffer [pH 7.2], 800 μl of methanol, and 100 μl of 6 N HCl) were analyzed in the system. The standard solutions and the products in the reaction mixture were also eluted under three different chromatographic conditions (tetrahydrofuran-doubly deionized water-methanol-glacial acetate at ratios of 4:5:1:1, 3.5:5:1:2, and 3:5:2:1 [vol/vol/vol/vol]) in order to compare their retention times.

Molecular-mass determination and partial purification of TPT-degrading factor.

The molecular mass of TPT-DF was determined by gel filtration through a Supredex Peptide HR 10/30 (10 mm [i.d.] × 30 cm; exclusion limit, 20,000; Amersham Pharmacia Biotech) equilibrated with 10 mM potassium phosphate buffer (pH 7.2) containing 0.1 M NaCl at a flow rate of 0.5 ml/min. The gel filtration chromatography was performed with an ÄKTA purifier HPLC system (Amersham Pharmacia Biotech), and TPT-DF was simultaneously detected at 214 and 398 nm. The calibration curve was plotted using the following standard peptides (Sigma): Gastrin I (M_r = 2,126), Substance P (M_r = 1,348), glycine hexamer (M_r = 360), and glycine trimer (M_r = 189).

The TPT-DF fractions from the gel filtration chromatography were concentrated by using Sep-Pack C₁₈ Vac 6cc and a rotary evaporator as already described, and the sample was then applied to a Resource S cation-exchange column (6 ml; Amersham Pharmacia Biotech) equilibrated with 20 mM MES–1 EDTA buffer (pH 5.5). TPT-DF was eluted by a linear gradient using buffer B, the same buffer containing 0.5 M NaCl, followed by 0 to 10% (12 ml), 10 to 60% (12 ml), and 60% buffer B (6 ml) with an ÄKTA purifier HPLC system, and was simultaneously detected at 214 and 398 nm. TPT-DF was eluted off the column around 40 mM NaCl (peak F-I) and 180 mM NaCl (peak F-II) as two major peaks. Each peak fraction was collected and concentrated by using the Sep-Pack C₁₈ Vac 6cc and rotary evaporator as already described. The absorption spectra of F-I and F-II were measured in 20 mM MES buffer (pH 5.5) and 20 mM potassium phosphate buffer (pH 7.2) with a model U-3000 spectrophotometer (Hitachi Co., Ltd., Tokyo, Japan).

RESULTS AND DISCUSSION

Determination of TPT and DPT in the culture medium.

A simple analysis method using post-column HPLC was developed for the determination of TPT and DPT in a medium to isolate the TPT-degrading microorganism. The use of fisetin as a post-column reagent, which was reported to determine TPT and TBT in harbor water (8), was found to allow the fluorimetric detection of TPT and DPT separated on a reversed-phase C₁₈ column, and the post-column derivatization was optimized as described in Materials and Methods. The DPT- and TPT-fisetin complexes were specifically detected at retention times of 6.5 and 8.1 min, respectively, without interference by the medium and cells. Selected excitation wavelengths (λ_ex) and emission wavelengths (λ_em) were 410 and 506 nm, respectively, which correspond to values between the TPT-fisetin complex (λ_ex = 405 nm, λ_em = 510 nm) and DPT-fisetin complex (λ_ex = 417 nm, λ_em = 502 nm) maxima obtained with their optimum spectra under the post-reaction conditions. Detection limits were 260 and 140 nM for TPT and DPT, respectively, when 20 μl of the sample solution was injected.

Isolation and identification of the TPT-degrading microorganism.

Many samples from coastal sediment and soil were able to grow in the medium supplemented with 130 μM TPT. As a result of the TPT degradation assay using the post-column HPLC, strain CNR15 was isolated from an enrichment culture and a new HPLC peak of the TPT metabolite was detected in the culture (Fig. 1A). The TPT metabolite was identified as DPT, since it was consistent with authentic DPT with respect to both retention time of the peak and emission spectrum (λ_ex = 410 nm) for the on-flow scanning measurement. Strain CNR15 accumulated 16 μM DPT when cultured for 72 h in the screening medium.

FIG. 1.

TPT biodegradation by P. chlororaphis CNR15 in SG medium supplemented with TPT. (A) Chromatograms of post-column HPLC analysis (λ_ex = 410 nm, λ_em = 506 nm) of TPT and DPT in culture medium at various intervals. (B) TPT (▴) and DPT (●) concentrations were determined by post-column HPLC analysis. Growth (○) was measured as the optical density at 600 nm (OD₆₀₀). Similar results were obtained in three independent experiments.

The isolated organism was a gram-negative, motile rod with polar flagella, and it had the following additional characteristics: gelatin hydrolysis, levan formation, the production of fluorescent pigments (mainly pyoverdine and chlororaphin), denitrification was not observed, and the predominant ubiquinone was ubiquinone Q9. In addition, this strain utilized glucose, trehalose, 2-ketogluconate, meso-inositol, l-valine, β-alanine, and dl-arginine as carbon sources. On the basis of these characteristics, the TPT-degrading isolate was identified as P. chlororaphis CNR15.

Growth and TPT degradation by P. chlororaphis CNR15.

To optimize the culture condition of strain CNR15 for TPT degradation, the effects of several environmental parameters (carbon source, growth temperature, pH, salinity, and so on) were investigated. The total amounts of DPT production after a 40-h incubation were evaluated as the TPT degradation activity in strain CNR15 cultures. It was found that the combination of glycerol and succinate as the carbon sources increased the TPT degradation activity several times compared with individual use. No degradation activity was observed in nutrient medium such as the L-broth. Finally, we decided to use the culture condition with SG medium (see Materials and Methods).

The growth of strain CNR15 cells, the consumption of 130 μM TPT, and the formation of DPT were monitored in the SG medium (Fig. 1). During the incubations, the medium turned yellow with or without TPT, and the peak of the yellow substance (3.95 min) could be detected by the post-column HPLC analysis (Fig. 1A) or the HPLC analysis connected to a diode array detector (215 to 650 nm) in the void volume. The pH value in the medium increased to pH 8.3 during the incubation for 40 h. TPT was rapidly degraded to DPT during the log phase of the growth in the strain CNR15 culture and reduced by 47% of the initial amount in the 48-h culture medium (Fig. 1B). Accumulation of DPT reached a maximum concentration (40 μM) after 48 h, but subsequently slowly decreased, although the degradation of TPT hardly occurred in the stationary phase of growth (Fig. 1B). The amount of DPT production was only slightly affected by supplementation of the TPT, carbon sources, and yeast extract and by neutralization of the pH value in the culture medium after a 40-h incubation.

Strain CNR15 was resistant to 260 μM TPT in the SG medium, and the growth was 73% when its optical density was compared with the optical density for the 40-h culture without TPT. Ethanol, which was used to dissolve TPT, had no effect on the TPT degradation and the growth of cells until the final concentration of 2%. The rate of DPT production depended on the initial concentration of TPT in the culture medium (2.6 to 260 μM). The apparent initial concentration of TPT for a half-maximal rate of DPT production (V_1/2max = 0.87 nmol h⁻¹ ml of culture⁻¹) by strain CNR15 culture was estimated as 133 μM during the 40-h incubation.

TPT and DPT were found to be easily adsorbed on the glass wall during incubation in the control (the medium without cells), since both levels estimated by the post-column HPLC analysis time-dependently decreased. However, it was confirmed that no degradation occurred in the control when washed with methanol–0.6 N HCl solution.

TPT degradation by strain CNR15 culture supernatant.

To our knowledge, the biochemical mechanism of organotin degradation by a microorganism has not been investigated in detail, but microbial degradation of organotin by water samples (16, 17), soils (3, 22, 28), and isolated bacteria (4, 23, 36) have been demonstrated. To clarify the TPT degradation mechanism by strain CNR15, we prepared a TPT-grown resting-cell suspension and its cell-free culture supernatant and investigated the localization of the TPT degradation activities.

TPT detected in the culture medium after a 24-h incubation was concentrated with the resting-cell fraction having an optical density of 5 at 600 nm, suggesting adsorption by the cells or debris, whereas most of the produced DPT in the culture was observed in the supernatant fraction (Fig. 2). We found that the degradation activity was present in the culture supernatant but not the resting-cell suspension (Fig. 2). In addition, the sonicated cells of the crude extract also did not exhibit any degradation activity (data not shown). Interestingly, the cell-free supernatant prepared from the culture grown without TPT exhibited a TPT degradation activity similar to that of the TPT-grown culture supernatant (Fig. 2). When the supernatant from the culture grown without TPT was prepared at various growth stages, the degradation activity was observed from the log phase to stationary phase of the growth. A similar result was also obtained with the TPT degradation in the TPT-grown culture medium (Fig. 1B). An ultrafiltered solution (<3,000 Da) of the culture supernatant exhibited the TPT degradation activity. Thus, these results suggest that the TPT degradation by strain CNR15 is due to an extracellular low-molecular-mass substance, termed TPT-DF, which is constitutively produced in the growth of strain CNR15.

FIG. 2.

Transformation of TPT to DPT by resting-cell suspensions and cell-free culture supernatant of strain CNR15. DPT concentrations were determined by post-column HPLC analysis. The resting cells from culture medium growth with TPT (▴) and without TPT (⧫) and the cell-free culture supernatants from culture media grown with TPT (■) and without TPT (●) were prepared as described in Materials and Methods, and they were incubated in 20 mM potassium phosphate buffer (pH 7.2) at 28°C. TPT was added to yield a final concentration of 130 μM TPT. TPT-grown resting cells (optical density of 5 at 600 nm) and the culture supernatant contained 135 and 13 μM TPT in the reaction mixture, respectively, prior to the addition of TPT. The culture supernatant prepared from TPT-grown culture also contained 17.5 μM DPT in the reaction mixture. The results are the means of three independent experiments.

Characterization of TPT-DF.

TPT-DF from strain CNR15 was partially purified and concentrated by the C₁₈ solid-phase extraction. The solid-phase extract of the culture supernatant was a yellow color with an absorption maximum at 398 nm in 20 mM potassium phosphate buffer (pH 7.2). TPT-DF was stable under acid (pH 1.0 for 72 h) and alkaline (pH 10 for 72 h) conditions, and the residual activities were 93 and 97%, respectively. The optimum pH range for the TPT-DF activity was pH 7.5 to 8, and the relative activities were reduced to approximately 40 to 50% at pHs 5.5 and 9.5. TPT-DF was also highly stable during heat treatment, and no loss in TPT-DF activity was observed at 100°C for 10 min. In addition, the rate of TPT degradation was dependent on the temperature. The reactions performed at 50 and 80°C showed 7.3- and 13-fold-higher activities, respectively, than that at 30°C, although no TPT degradation occurred at 80°C without TPT-DF. The chemical degradations of TPT have been summarized by Bock (6): hydrolysis by boiling or strong acids or bases; oxidation and reduction by strong oxidation or reducing agents; and photolysis by UV radiation or sunlight (3). These reactions quickly led to the dephenylation of TPT and produced the corresponding organotins and benzene. In contrast, the TPT degradation reaction by TPT-DF occurred under mild conditions in the dark. Thus, TPT-DF is a new catalyst capable of accelerating TPT degradation.

Stoichiometric amounts of DPT were released during the TPT degradation at an initial rate of 0.88 nmol min⁻¹ mg of substance⁻¹ (Fig. 3). The apparent K_m value for TPT was 59.8 μM. Although the apparent initial concentration of TPT for the half-maximal rate of DPT production by the strain CNR15 culture was twofold higher than the apparent K_m value of TPT-DF, the result may be illustrated by the decrease in TPT concentration in the culture medium with adsorption by the cells or debris.

FIG. 3.

TPT degradation by the solid-phase extract prepared from non-TPT-grown culture of strain CNR15. The solid-phase extract was prepared as described in Materials and Methods, and the reaction was performed in 10 mM potassium phosphate buffer (pH 7.2) containing 0.5 mg of extract/ml and 130 μM TPT at 30°C. TPT (▴) and DPT (●) concentrations were determined by post-column HPLC analysis. The values are the means of three independent experiments.

All metabolites produced by TPT dephenylation using TPT-DF were detected by an HPLC-connected photodiode array detector (215 to 650 nm). The HPLC condition was modified from the condition of the post-column HPLC analysis to separate the peaks of TPT and benzene, which is one of the putative metabolites. The detection limits of TPT, DPT, and benzene were 1.5, 2.0, and 9.5 μM, respectively, when 50 μl of the sample solution was injected. The HPLC analysis for the 5-h incubation of 1.0-mg/ml TPT-DF and 257 μM TPT in 300 μl of reaction mixture revealed that there was degradation of TPT (to 85.3 μM, 17.0 min) and production of two new metabolites. These metabolites were identified as DPT (141 μM, 8.5 min) and benzene (91.8 μM, 13.8 min) by comparison with the authentic samples. This was further confirmed in three different chromatographic conditions with coelution of their standards (see Materials and Methods). The amount of released benzene was not stoichiometric; only 60% of the theoretical ratio was recovered, probably due to its high volatility. No other changes in the HPLC chromatograms were confirmed during the incubation of the reaction mixture. These results suggest that TPT-DF directly catalyzes the dephenylation of TPT to produce DPT and benzene.

The molecular mass of TPT-DF was estimated to be about 1,000 Da by gel filtration chromatography (Fig. 4). The elution peaks were simultaneously detected at 214 and 398 nm, since the applied sample (the solid-phase extraction of the culture supernatant) was yellow (λ_max = 398 nm, pH 7.2). Two main peaks (14.3 and 15.2 min) were observed at 214 nm, and the active fraction of TPT-DF was consistent with the peak (15.2 min) detected at 398 nm (Fig. 4). In the further purification step of TPT-DF, we also found that the two yellow active peaks (F-I and F-II) exhibiting the TPT-DF activity were separated by Resource S cation-exchange chromatography. Interestingly, the spectra of these substances were consistent with that of a pyoverdine produced by P. fluorescens at pHs 7.2 and 5.5 (32, 38) (Fig. 5). Furthermore, the activities of TPT-DF using 0.43 mg of F-I/ml and 0.47 mg of F-II/ml were completely inhibited in the reaction mixtures (containing 20 mM MOPS [pH 7.5] instead of 10 mM potassium phosphate buffer [pH 7.2]) preincubated for 5 min with 1 mM FeCl₃ (data not shown). These results suggest that the most likely substance functioning as TPT-DF may be pyoverdine, which is a yellow-green chromopeptide siderophore with a molecular mass of 1,000 to 1,500 Da (1), or its analogous compound.

FIG. 4.

The gel filtration chromatography profile of the solid-phase extract from strain CNR15 and molecular mass determination of TPT-DF. Thirty microliters of the solid-phase extract (5.0 mg/ml) was injected into the HPLC system as described in Materials and Methods and was detected at 214 nm (——) and 398 nm (–––). The fractions showing TPT-DF are indicated by the hatched area. The peptide standards (—–—) were detected at 214 nm; 1, Gastrin I (M_r = 2,126); 2, Substance P (M_r = 1,348); 3, glycine hexamer (M_r = 360); 4, glycine trimer (M_r = 189). Inset, the calibration curve obtained with the peptide standards.

FIG. 5.

Absorption spectra of strain CNR15 TPT-DFs F-I (A) and F-II (B). Both TPT-DFs (50 μg/ml) were measured in 20 mM MES buffer (pH 5.5) and 20 mM potassium phosphate buffer (pH 7.2). The pH values are indicated beside the spectra.

Pyoverdine is also the major exogenous siderophore that is characteristically produced by fluorescent pseudomonads in a medium similar to the SG medium under iron-deficient conditions (30). For a given fluorescent pseudomonad strain, it is known that various forms of pyoverdine, differing only in the acyl substituent bound to the amino group on C3 of the chromophore, are produced (2, 27). These reports may illustrate our result that various forms of TPT-DF are produced by P. chlororaphis CNR15 (Fig. 5). Metal chelation (especially of Fe³⁺) by pyoverdine involves a stable octahedral complexation with the catecholate group of the chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline and two bidenate groups derived from two hydroxyaminoacyl residues of the peptide moiety, such as δ-N-hydroxyornithyl or β-hydroxyaspartyl (2). Recently, Xiao and Kisaalita have found that pyoverdines bind and oxidize Fe²⁺ to Fe³⁺ (38). The Sn-C bond can be polarized in either direction, so it can be attacked by nucleophilic and electrophilic reagents (9). Interestingly, Martin and Walton have reported that the tin-phenyl cleavage of DPT occurs with the coordination and nucleophilic attack of a chelating agent, such as 8-hydroxy quinoline in dimethyl sulfoxide (29). These reports, along with our results, may also support the possibility that the microbial chelators, such as pyoverdine or its analogs, play a role as the potent catalysts that accelerate the cleavage of the Sn-C bond to stabilize complexation concomitant with TPT chelation. DPT produced by TPT degradation may immediately form a soluble complex with pyoverdine that is present in the culture supernatant (Fig. 2); however, further studies are required to reveal whether the TPT-DF is pyoverdine or not.

TPT degradation by other fluorescent pseudomonad.

In order to elucidate whether other fluorescent pseudomonads exhibit the TPT degradation activity, the fluorescent pseudomonads producing the well-characterized pyoverdines (18), P. chlororaphis ATCC 9446, P. fluorescens ATCC 13525, P. putida ATCC 12633 and P. aeruginosa ATCC 15692, were cultured and assayed (Fig. 6). During growth on SG medium supplemented with 130 μM TPT, a change in the color of the medium to yellow and a significant DPT production were observed in the P. chlororaphis ATCC 9446 and P. aeruginosa ATCC 15692 cultures (Fig. 6). P. aeruginosa ATCC 15692 also exhibited the activity when the culture was incubated at 37°C, although strain CNR15 had no activity. We found that the DPT levels in the cultures gradually decreased similarly to those of strain CNR15 (Fig. 1 and 6). These results suggest that further degradation of DPT occurs; however, the identification of the metabolite remains unknown. P. fluorescens ATCC 13525 and P. putida ATCC 12633 exhibited little degradation activity, although the cell growth was not inhibited by the initial concentration of 130 μM TPT.

FIG. 6.

Biodegradation of TPT by ATCC strains of fluorescent pseudomonads in SG medium supplemented with TPT. The concentrations of DPT produced by TPT (130 μM) degradation were determined by post-column HPLC analysis. Similar results were obtained in three independent experiments. Symbols: ●, P. chlororaphis ATCC 9446; ▴, P. fluorescens ATCC 13525; ⧫, P. putida ATCC 12633; ■, P. aeruginosa ATCC 15692.

All solid-phase extracts prepared from non-TPT-grown cultures of these bacteria showed absorption maxima at 398 nm in 20 mM potassium phosphate buffer (pH 7.2) and at approximately 380 nm in 20 mM MES buffer (pH 5.5) except for P. putida ATCC 12633. These extracts, except for P. putida, also exhibited TPT degradation activities (expressed as nanomoles minute⁻¹ milligram of substance⁻¹) of 0.25 (P. chlororaphis ATCC 9446), 0.11 (P. aeruginosa ATCC 15692), and 0.12 (P. fluorescens ATCC 13525), suggesting the production of TPT-DF by these fluorescent pseudomonads. This result is likely to contradict our suggestion that TPT-DF may be pyoverdine or its analog, since the extract from P. putida did not exhibit TPT-DF activity. However, it should be noted that the pyoverdines produced by P. aeruginosa ATCC 15692, P. fluorescens ATCC 13525, and P. chlororaphis ATCC 9446 that exhibit TPT-DF activity have a closely related structure and cross-reactivity to the iron transport system, whereas P. putida ATCC 12633 produces a strain-specific pyoverdine (10, 18, 27). Small differences in the nature and locations of amino acids and chelating bidenate groups in the peptide chain determining the strain specificity for the pyoverdines may have an influence on the coordination or dephenylation of TPT.

This study has demonstrated that the low-molecular-mass substance (TPT-DF) secreted into the culture medium by P. chlororaphis CNR15 catalyzes the stoichiometric dephenylation to DPT from TPT. Similar results have also been observed with some ATCC strains of a fluorescent pseudomonad. The organotin degradation by extracellular substances seems to be reasonable for microorganisms, since some organotin-resistant microorganisms accumulate organotin in the cell envelope by a non-energy-requiring process (5, 33, 40). Barug has also suggested the possibility of bis (tributyltin) oxide degradation with the reactants of P. aeruginosa ATCC 13388 (4). However, it should be noted that TPT-DF is produced in the culture medium with or without TPT and seems to have the physiological function of a metal chelator (probably siderophore) rather than a catalyst to degrade TPT. These observations indicate that TPT degradation with TPT-DF by a fluorescence pseudomonad should be a cometabolite reaction, although it remains unknown whether these bacteria are capable of utilizing the benzene concomitantly produced with the TPT degradation. The mineralization of radiolabeled TPT to CO₂ in soil and sediment samples (3, 22) suggests that unknown mechanisms play an important role in the microbial degradations of TPT. In that case, DPT and benzene accumulation by a fluorescent pseudomonad could contribute to codegradation with bacteria in the natural population.