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Applied and Environmental Microbiology, August 2000, p. 3492-3498, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Degradation of Triphenyltin by a Fluorescent
Pseudomonad
Hiroyuki
Inoue,1,*
Osamu
Takimura,1
Hiroyuki
Fuse,1
Katsuji
Murakami,1
Kazuo
Kamimura,2 and
Yukiho
Yamaoka1
Marine Biological Technology Section, Chugoku
National Industrial Research Institute, Hiroshima
737-0197,1 and Department of
Biological Function and Genetic Resources Science, Faculty of
Agriculture, Okayama University, Okayama
700-8530,2 Japan
Received 22 February 2000/Accepted 6 June 2000
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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.
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INTRODUCTION |
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.
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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
Km 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 (Mr = 2,126), Substance P (Mr = 1,348), glycine
hexamer (Mr = 360), and glycine trimer
(Mr = 189).
The TPT-DF fractions from the gel filtration chromatography were
concentrated by using Sep-Pack C
18 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 C
18 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).
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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 (
ex)
and emission wavelengths (em) were 410 and 506 nm,
respectively, which correspond to values between the TPT-fisetin complex (ex = 405 nm, em = 510 nm) and DPT-fisetin complex (ex = 417 nm,
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
(ex = 410 nm) for the on-flow scanning measurement.
Strain CNR15 accumulated 16 µM DPT when cultured for 72 h in the
screening medium.
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FIG. 1.
TPT biodegradation by P. chlororaphis CNR15
in SG medium supplemented with TPT. (A) Chromatograms of post-column
HPLC analysis (ex = 410 nm, 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.
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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,
-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
(
V1/2max = 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.
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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.
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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
Km 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
Km 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.
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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.
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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 (
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 FeCl
3
(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.
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|
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 (Mr = 2,126); 2, Substance P
(Mr = 1,348); 3, glycine hexamer
(Mr = 360); 4, glycine trimer
(Mr = 189). Inset, the calibration curve
obtained with the peptide standards.
|
|
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|
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 Fe
3+) 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
-
N-hydroxyornithyl or
-hydroxyaspartyl
(
2).
Recently, Xiao and Kisaalita have found that pyoverdines
bind and
oxidize Fe
2+ to Fe
3+ (
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.
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|
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
CO
2 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.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Marine
Biological Technology Section, Chugoku National Industrial Research
Institute, 2-2-2, Hiro-Suehiro, Kure, Hiroshima 737-0197, Japan. Phone:
81-823-72-1935. Fax: 81-823-73-3284. E-mail:
inoue{at}cniri.go.jp.
| |
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Top
Abstract
Introduction
