Download Enzymes responsible for chlorate reduction by Pseudomonas sp

FEMS Microbiology Letters 247 (2005) 153–159
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Enzymes responsible for chlorate reduction by Pseudomonas sp.
are different from those used for perchlorate reduction by Azospira sp.
Lisa M. Steinberg a, John J. Trimble b, Bruce E. Logan
a
a,*
Department of Civil and Environmental Engineering, 212 Sackett Building, The Pennsylvania State University, University Park, PA 16802, USA
b
Department of Biology, Saint Francis University, Loretto, PA 15940, USA
Received 2 February 2005; received in revised form 29 April 2005; accepted 1 May 2005
First published online 17 May 2005
Edited by S. Silver
Abstract
Pseudomonas sp. PDA is an unusual bacterium due to its ability to respire using chlorate under aerobic conditions. The chlorate
reductase produced by PDA was shown to be intrinsically different from the enzyme responsible for chlorate and perchlorate [(per)chlorate] reduction produced by Azospira sp. KJ based on subunit composition and other enzyme properties. The perchlorate reductase
from strain KJ appeared to have two subunits (100 and 40 kDa) while the chlorate reductase from PDA had three subunits (60, 48,
and 27 kDa). N-terminal amino acid sequencing of the 100 kDa protein from strain KJ showed a 77% similarity with the perchlorate
reductase alpha subunit from another perchlorate-respiring bacterium, Dechloromonas agitata, while the N-terminus amino acid
sequence of the 60 kDa protein from strain PDA did not show a similarity to previously isolated chlorate or perchlorate reductases.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Biodegradation; Chlorate reductase; Electron acceptor; Perchlorate reductase; Remediation; Respiration
1. Introduction
The US Environmental Protection Agency (EPA)
estimates that the drinking water of 15 million people
is affected by perchlorate contamination [1]. Perchlorate
reducing bacteria have been isolated from a variety of
natural environments, and are widely dispersed among
a-, b-, c-, and e-subclasses of the Proteobacteria [2–4].
Members of two genera of the b-subclass of Proteobacteria, Dechloromonas and Azospira, are thought to represent the dominant perchlorate-reducing bacteria in
the environment [5,6]. All bacteria capable of perchlorate respiration can respire using chlorate, but the reverse is not necessarily true. Bacteria capable of
*
Corresponding author. Tel.: +1 814 863 7908; fax: +1 814 863 7304.
E-mail address: [email protected] (B.E. Logan).
chlorate, but not perchlorate, respiration include members of Proteus, Pseudomonas, and Rhodobacter [2–4].
Oxygen is known to prevent chlorate reduction by all
chlorate and perchlorate [(per)chlorate] reducing strains
except for Pseudomonas sp. PDA [7].
Perchlorate and chlorate are sequentially reduced
according to the same sequence of intermediates, via
ClO
4 ! ClO3 ! ClO2 ! Cl þ O2 [8]. Chlorite dismutase catalyzes the disproportionation of chlorite to chloride and oxygen, and the oxygen that is produced is
consumed for cell respiration and does not accumulate
in solution [9]. In perchlorate-respiring bacteria a single
enzyme is used for both chlorate and perchlorate reduction, but few enzymes have been isolated. Perchlorate
reductases isolated from strains GR-1 [10] and perc1ace
[11], and chlorate reductases from Ideonella dechloratans
[12] and Pseudomonas chloritidismutans strain AW-1 [13]
0378-1097/$22.00 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2005.05.003
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L.M. Steinberg et al. / FEMS Microbiology Letters 247 (2005) 153–159
all show some similarity to the selenate reductase of
Thauera selenatis and to other reductases, dehydrogenases, and heme proteins based on sequencing data.
However, these enzymes differ in their physical and biochemical properties, suggesting that a wide variety of enzymes may catalyze chlorate and perchlorate reduction.
Pseudomonas sp. PDA is a relatively novel chloraterespiring bacterium due to its ability to completely degrade chlorate in the presence of dissolved oxygen [7].
While other chlorate respiring bacteria are known to
constitutively express chlorite dismutase, no other bacterium is known to constitutively express both chlorite dismutase and chlorate reductase. In order to further
examine this unique microorganism, we used native gel
electrophoresis to isolate the chlorate reductase from
strain PDA and the perchlorate reductase from strain
KJ. In addition, partially purified enzymes from the
periplasmic space of these two strains were used to compare enzyme properties. Chlorite reductase was not
examined here as it has already been well characterized
[2,9,14–16].
ally contained sucrose (0.75 M). Protease inhibitor cocktail (2.5 ml), lysozyme (100 mg), and EDTA (10 mM
final concentration; all from Sigma) were added to each
tube, and the tubes were shaken vigorously by hand for
2 to 3 s. Samples were incubated on ice in an anaerobic
chamber for 2 h. All reagents except the protease inhibitor cocktail, EDTA, and lysozyme, were prepared and
stored under anoxic conditions.
Spheroplasts from tube A (observed using phase
microscopy) were collected by centrifugation (2500g,
20 min) with the supernatant retained for use as the
‘‘periplasmic fraction’’. Spheroplasts were suspended in
buffer (50 ml) with 10% (w/v) glycerol, and broken by
sonification (on ice) for 1 min. Insoluble material was
collected by centrifugation (40,000g, 10 min), with the
supernatant collected as the ‘‘cytoplasmic fraction’’.
Broken cells from tube B were treated in the same manner, with the supernatant collected as the ‘‘whole cell lysate’’. The insoluble material was resuspended in buffer
(50 ml) with glycerol (10% w/v) and Triton-X 100 (1% w/
v) as the ‘‘membrane fraction’’.
2.2. Enzyme activity
2. Materials and methods
2.1. Bacterial strains and cell preparation
Azospira sp. KJ (formerly Dechlorosoma sp. KJ) was
isolated from an acetate-fed, perchlorate-degrading
packed bed bioreactor and has been used in drinking
water bioremediation tests [7,16–18]. KJ is a Gram-negative, facultatively anaerobic rod capable of coupling
the oxidation of lactate or acetate to the complete reduction of nitrate, chlorate, or perchlorate. Pseudomonas
strain PDA was isolated from primary digester sludge
[17] and can grow on lactate or acetate using oxygen
or chlorate, but not nitrate or perchlorate, although
some nitrate reduction has been observed in cell-free
suspensions [7]. Strain KJ is a member of the b-subclass
of Proteobacteria, while PDA falls within the c-subclass
(see Fig. 2 in [6]).
Cells were prepared as whole cell lysate, periplasm,
cytoplasm, and membrane fractions, and each fraction
was tested for chlorate and perchlorate reduction. Cells
were grown in basal medium [7] with NH4H2PO4 (6.1
mM). Strain KJ was grown using perchlorate (1.2 g/l)
or chlorate (1.3 g/l) while strain PDA was grown using
only chlorate (1.3 g/l). Cultures were grown with stirring
in an anaerobic glove box at 30 C in 1 l capped media
bottles, and harvested at an optical density (600 nm) of
0.7–0.8 (typically within 2–3 days).
Cell suspensions were split into two 500 ml portions,
centrifuged (10,000g, 5 min), and the pellets washed
twice with buffer (Tris–HCl, 50 mM, pH 8.0; 100 ml).
Both pellets (tubes A and B) were resuspended in 100
ml of buffer and 10% (w/v) glycerol, but tube B addition-
Chlorate and perchlorate reductase activities (triplicate samples) were determined for each fraction using
reduced methyl viologen assays [10]. Each 1 ml reaction
mixture contained 0.5 mM methyl viologen, 0.35 mM
sodium dithionite, and 10 to 100 ll of enzyme in MOPS
buffer (50 mM, pH 8.0), which yielded a starting absorbance of approximately 1.0. The reaction was initiated
by addition of the substrate (10 mM; chlorate or perchlorate), and activity quantified based on a linear decrease in absorbance at 600 nm (PalmSpec, Ocean
Optics, Inc.; OOIBASE32 software) for 1 to 2 min in
an anaerobic chamber with the rate corrected based on
biotic controls (no substrate; duplicate samples). Abiotic
controls containing no enzyme were also performed. A
cytoplasmic marker, soluble malate dehydrogenase,
was used to determine the extent of spheroplast rupture
according to a standard assay [19]. Specific activity was
defined as Units of activity per mass of protein (U/mg)
based on the oxidation of 1 mmol of methyl viologen
per min (600 nm). A previously determined millimolar
extinction coefficient of 13 for methyl viologen at 600
nm was used to relate absorbance to concentration
[20]. Protein concentrations were measured using the
Bradford assay reagent (Sigma).
Enzyme activities of periplasmic fractions were measured at set pH values of 6 to 6.5 using MES (2-(N-Morpholino) ethanesulfonic acid, pKa 6.1), 7 to 8 using
MOPS (3-(N-Morpholino) propanesulfonic acid, pKa
of 7.2), or 8.5 to 10 using CHES (2-(N-Cyclohexylamino) ethanesulfonic acid, pKa of 9.3) (all from Sigma).
Buffers were prepared at 50 mM in deionized water,
adjusted to a final pH using 1 M HCl or NaOH, and
L.M. Steinberg et al. / FEMS Microbiology Letters 247 (2005) 153–159
degassed by stirring the solution for two days in an
anaerobic chamber prior to use.
2.3. Isolation and N-terminal sequencing of perchlorate
reductase enzymes
Enzymes were separated by ammonium sulfate precipitation in an anaerobic chamber. Ammonium sulfate
(saturated solution of 4.1 M in MOPS buffer; final concentration 20%, 40%, 55%, 67%, and 76% saturation)
was dripped into the periplasmic fraction of samples
with slow stirring for 10 min. The precipitate was collected by centrifugation (25,000g, 10 min) and the proteins were re-solubilized in 5 ml of MOPS buffer and
analyzed for chlorate and perchlorate reductase activities using reduced methyl viologen assays. The protein
fraction with the greatest enzyme activity was passed
through an ultrafiltration membrane (Nanosep 30K
membrane, Pall Corporation) and resuspended in 0.5
ml of 50 mM Tris–HCl buffer with glycerol (10% w/v),
and mixed with 0.5 ml of Tris-Gly Native Sample Loading Buffer (NuPage, Invitrogen Life Technologies). Proteins were separated using agarose gels (0.8%; 15 cm
wide by 10 cm long) by native gel electrophoresis
(Sub-Gel GT Wide Mini submarine gel rig, Bio-Rad
Laboratories) at 50 V (5 V/cm) for 4 h in 1· TAE buffer
at a pH of 9.1 (strain PDA) or 7.2 (strain KJ).
The location of chlorate and perchlorate reductase
activities in the gels were determined using methyl viologen. Gels were placed in a tray and stained blue by
reducing methyl viologen (2 mg/ml) with sodium dithionite (2 mg/ml) in 50 mM MOPS buffer. Sodium chlorate
(5 ml, 1 M) was poured over the top of the gel, with the
enzyme location indicated by a clear band following oxidation of methyl viologen. In tests with proteins from
strain KJ, the gel was rinsed twice with 50 ml of buffer,
and repeated using sodium perchlorate (5 ml, 0.1 M) to
confirm that the locations were the same for perchlorate
and chlorate. The center of the clear band in both cases
was extracted from the gel and dialyzed in 5 ml of 1·
TAE buffer (pH 9.1 for PDA and pH 7.2 for KJ) using
a high-retention cellulose membrane (Sigma). The membrane was placed across the width of the electrophoresis
chamber, and protein electroeluted from the agarose gel
into the buffer in the dialysis membrane at a constant
potential of 5 V/cm for 1 h. The buffer was removed
from the dialysis membrane and the extracted protein
was concentrated using an ultrafiltration membrane
(Nanosep 30 K, Pall Corporation). Specific activity
was determined at each step in the purification process.
Protein fractions were further analyzed using SDS–
PAGE analysis in a NuPage 4–12% Bis-Tris gel (Novex
XCell Mini-Cell vertical gel rig, Invitrogen Life Technologies) at 15 V/cm for 2 h along with wide-range
molecular weight markers (Sigma). Proteins were transferred to a PVDF membrane using a Western blot
155
apparatus (XCell II Blot Module, Invitrogen Life
Technologies). Proteins were stained by immersing the
membrane in 100% methanol for a few seconds, rinsing
with deionized water for 5 min (with shaking), immersion for 30 s in Coomassie Blue (0.1% in 40% methanol
with 1% acetic acid), and rinsing 4 to 5 times in 50%
methanol. The membrane was washed several times with
deionized water over the course of 12 h to remove any
remaining SDS. The stained proteins were cut from
the PVDF membrane and sequenced by the Macromolecular Core Facility at the Penn State College of
Medicine.
2.4. Effect of oxygen on enzyme activity
The effect of oxygen on enzyme activity was examined using 100 ll periplasmic fractions (collected in 1.5
ml microcentrifuge tubes, on ice) that had been passed
through an ultrafiltration membrane (Nanosep 30K) in
an anaerobic chamber. Samples were exposed to air in
a refrigerator (on ice), and activity periodically tested
(in duplicate) using the reduced methyl viologen assay
after first reducing the oxygen in the sample with dithiothreitol (100 ll, 30 mM) in MOPS buffer. Enzyme stability during the experiment was verified using an anoxic
control kept in the anaerobic chamber. The time needed
to saturate the samples with dissolved oxygen was 40
min (FOXY fiber optic probe, Ocean Optics).
3. Results
3.1. Isolation and analysis of the enzyme from strain KJ
Perchlorate and chlorate reductase activity was identified for strain KJ to be greatest in the periplasm fraction, based on analysis of enzyme activities in whole
cell lysate and three different cell fractions (Table 1).
Malate dehydrogenase assays showed that spheroplasts
from KJ grown on chlorate or perchlorate were 86%
and 91% intact, respectively. Enzyme activity was larger
in the periplasm fraction than in the cytoplasm fraction
for chlorate and perchlorate, independent of whether
cells were originally grown on chlorate or perchlorate.
Based on these results, the periplasm fraction was selected for further analysis.
The analysis of enzyme activity from the proteins prepared by precipitation using the periplasm fraction
showed that perchlorate reductases from strain KJ could
be isolated in the 40% to 55% saturated ammonium sulfate fraction. This protein fraction was selected for further purification using native gel electrophoresis. At a
pH of 9.1 the perchlorate reductase of strain KJ was observed to migrate towards the cathode. The separation
was therefore performed in reverse, and at a pH of 7.2
perchlorate reductase activity was found to be localized
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L.M. Steinberg et al. / FEMS Microbiology Letters 247 (2005) 153–159
Table 1
Activity with chlorate and perchlorate in fractions obtained from strain KJ grown with chlorate, KJ grown with perchlorate, and PDA grown with
chlorate
Substrate for activity assay
Cell fraction
Activitya(Units/mg protein)
KJ grown with ClO
3
KJ grown with ClO
4
PDA grown with ClO
3
ClO
3
ClO
3
ClO
3
ClO
3
Membrane
Cytoplasm
Periplasm
Whole cell lysate
0.11 ± 0.04
0.47 ± 0.19
0.60 ± 0.13
0.38 ± 0.10
0.07 ± 0.02
0.46 ± 0.19
0.80 ± 0.44
0.45 ± 0.23
5.86 ± 0.02
18.96 ± 1.36
40.48 ± 2.59
43.31 ± 1.44
ClO
4
ClO
4
ClO
4
ClO
4
Membrane
Cytoplasm
Periplasm
Whole cell lysate
0.02 ± 0.02
0.01 ± 0.01
0.72 ± 0.01
0.31 ± 0.01
0.00 ± 0.02
0.00 ± 0.01
0.06 ± 0.01
0.000 ± 0.00
0.15 ± 0.11
0.30 ± 0.16
0.60 ± 0.05
0.51 ± 0.00
a
One Unit equals the oxidation of 1 mmol of methyl viologen per minute.
SDS–PAGE analysis of proteins obtained from the
gel indicated the presence of two bands at 100 and 40
kDa (Fig. 2(a)). Sequencing of the first 15 amino acids
of the N-terminus of the 100 kDa band (SATDISGAFEYSGGE) indicated a 77% similarity to the alpha subunit of a perchlorate reductase obtained from
Dechloromonas agitata strain CKB [21].
3.2. Isolation and analysis of the enzyme from strain PDA
Fig. 1. The purification of reductase enzymes from strains KJ and
PDA with native gel electrophoresis: (a) the chlorate reductase of
strain PDA, (b) the perchlorate reductase of strain KJ. The entire gel
width of the gel was utilized, but only a strip of the gel is shown.
Chlorate reductase activity in strain PDA was primarily associated with the periplasm fraction (Table
1), consistent with results found with strain KJ. Malate
dehydrogenase assays showed that spheroplasts prepared using PDA were 67% intact. As expected, there
was little enzyme activity relative to chlorate with perchlorate using these different protein fractions from
Table 2
Specific activity (U/mg protein) of enzymes from strains KJ and PDA
with chlorate or perchlorate at each step of the enzyme purification
Whole cells
Periplasm
Ammonium
sulfate precipitation
Gel-purified
Strain KJ,
ClO
3
Strain KJ,
ClO
4
Strain PDA,
ClO
3
1.0 ± 0.1
2.4 ± 0.5
3.8 ± 0.7
0.57 ± 0.03
0.86 ± 0.13
1.0 ± 0.2
27 ± 3
36 ± 1
48 ± 1
0
6.0 ± 0.1
0.13 ± 0.12
in a band that migrated 5 cm from the well towards the
cathode (Fig. 1(a)). Clear bands formed within 10 min
after adding chlorate or perchlorate to the gel. Unfortunately, gel electrophoretic separation reduced enzyme
activity. As shown in Table 2, enzyme activity normalized to the protein concentration increased in the periplasm and ammonium sulfate-precipitated fractions
relative to the whole cell fraction. Enzyme activity was
reduced by an order of magnitude, however, following
preparation of this fraction using gel electrophoresis.
Fig. 2. Western blot to PVDF membrane of an SDS–PAGE gel of the
isolated reductase enzymes from KJ and PDA: (a) the perchlorate
reductase of strain KJ showing the presence of two subunits, (b) and
the chlorate reductase of strain PDA showing the presence of three
subunits. The entire gel width of the gel was utilized, but only a strip of
the gel is shown.
L.M. Steinberg et al. / FEMS Microbiology Letters 247 (2005) 153–159
strain PDA, consistent with previous reports that strain
PDA is unable to respire using perchlorate [7].
The characteristics of the chlorate reductase obtained
from strain PDA were substantially different from those
from strain KJ. Chlorate reductase activity of strain
PDA was found to be associated with the precipitate
from the 55–67% saturated ammonium sulfate fraction.
When the protein fraction from strain PDA was purified
by gel electrophoresis, the reductase was isolated at a
pH of 9.1, with the protein band appearing 12 cm from
the well towards the anode (Fig. 1(b)). A clear band
indicating the presence of chlorate reductase appeared
within 2 min after adding sodium chlorate to the gel.
However, overall activity of the chlorate reductase was
reduced by an order of magnitude after electrophoresis,
a reduction similar to that found with proteins from
strain KJ (Table 2).
SDS–PAGE analysis of the chlorate reductase from
strain PDA indicated three bands with molecular
weights of 60, 48, and 27 kDa (Fig. 2(b)). The first 15
amino acids of the N-terminus of the 60 kDa band
(SATDISGAFEYSGGE) did not show similarity to
any other previously isolated chlorate or perchlorate
reductase [10–13]. The closest match was a 73% similarity to the chaperonin GroEL proteins from Pseudomonas aeruginosa and Azotobacter vinelandii. These
proteins serve as molecular chaperones for proteins expressed under conditions of environmental stress,
including heat and osmotic stress, and the use of nonoptimal growth components. While it is possible that a
protein other than the chlorate reductase was identified
by sequencing, the similarity in the purification steps
using strains KJ and PDA suggests that the three bands
obtained here are indicative of a substantially different
chlorate reductase than has been previously identified.
Further work is needed to confirm these findings.
3.3. Enzyme activity as a function of pH
Chlorate reductase activity using the periplasm fraction from strain PDA showed a maximum at a pH of
8.5 (Fig. 3). Enzyme activities using the periplasm fraction from strain KJ showed no apparent peak in activity
as a function of pH with either chlorate or perchlorate.
3.4. Effect of oxygen on enzyme activity
Chlorate reductase activity of the periplasmic fraction from strain KJ was reduced by 50% after 76 h, while
perchlorate reductase activity was reduced by 70% after
76 h (data not shown). Chlorate reductase activity with
the periplasmic fraction from strain PDA was still 50%
of the original rate even after 15 days. No change in
activity was seen in any of the anoxic controls kept on
ice in the anaerobic glove box, suggesting the decline
in activity was due to exposure to oxygen.
157
100
10
1
0.1
0.01
6.0
KJ grown with ClO3KJ grown with ClO4PDA grown with ClO37.0
8.0
9.0
10.0
Fig. 3. (Per)chlorate reductase activity at various pH values of the
periplasmic fraction of cells of strain KJ grown with chlorate or
perchlorate, and the periplasmic fraction of cells strain PDA grown
with chlorate.
4. Discussion
Chlorate and perchlorate reductase activity was localized within the periplasmic fractions for strains KJ and
PDA, indicating these enzymes are soluble and not
membrane-bound. The location of these enzymes appears consistent with several other (per)chlorate respiring strains. Perchlorate and chlorate reductase
enzymes isolated from strains GR-1 [10], perc1ace
[11,26], and from I. dechloratans [12] were also found
to be soluble and located in the periplasm. In contrast,
nitrate reductase A, which possesses chlorate reductase
activity, and chlorate reductase C from Proteus mirabilis
are bound to the cytoplasmic membrane [22]. Chlorate
reductase from P. chloritidismutans is localized in the
cytoplasm of the cell [13].
The perchlorate reductase from strain KJ shows similarity with previously isolated perchlorate reductase enzymes. The two subunits of the perchlorate reductase
with molecular weights of 100 and 40 kDa are similar
in size to those of a perchlorate reductase from strain
GR-1, which possessed subunits 95 and 40 kDa in size
[10]. The perchlorate reductase of strain KJ had a basic
isoelectric point (pH > 9.1) and demonstrated greater
activity with chlorate than perchlorate, consistent with
findings for the perchlorate reductase from strain GR1. Sequencing of the 100 kDa protein revealed a 77%
similarity to an internal sequence of the perchlorate
reductase from D. agitata strain CKB [21]. The main
limitation of our isolation procedure was a loss of enzyme activity following separation by electrophoresis.
Perchlorate reductase activity of the protein fraction
markedly declined following electrophoretic separation
in the native gel, likely due to inactivation of the
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L.M. Steinberg et al. / FEMS Microbiology Letters 247 (2005) 153–159
enzyme. As a result, the final purified protein fraction
had a much lower level of specific activity than the perchlorate reductase of strain GR-1 [10] or the perchlorate
reductase of perc1ace [11].
The chlorate reductase from strain PDA had properties different from enzymes previously identified from
chlorate or perchlorate respiring bacteria. The optimum
pH for chlorate reductase activity in periplasmic extracts
(pH 8.5) was higher than that reported for chlorate
reductase from P. chloritidismutans (7.5) [13] or perchlorate reductase from perc1ace (7.5–8.0) [11]. Chlorate
reductase activity was higher than activity reported for
GR-1 [10] or perc1ace [11], although it was less than reported for P. chloritidismutans [13]. Sequencing data for
the 60 kDa band from strain PDA showed no resemblance to other reductases or to dehydrogenases; the
closest similarity (73%) was to chaperonin GroEL proteins from Pseudomonas and Azotobacter. This finding
is unusual in that all sequenced perchlorate and chlorate
reductase enzymes to date have shown similarity to
other reductases, dehydrogenases, and heme proteins
[10–13]. This lack of a similarity to other data could
be due to a GroEL motif at the N-terminus, so further
analysis of this 60 kDa protein (and the other two subunits) is needed. However, the combined characteristics
in terms of optimum pH, isoelectric point, and activity
in the presence of dissolved oxygen all support the unique nature of this enzyme compared to characteristics
reported for previously isolated chlorate reductases.
Strain PDA is the only known chlorate reducer that
expresses chlorate reductase and chlorite dismutase under aerobic conditions [7]. It was observed here that
chlorate reductase activity was only reduced by about
50% even after 15 days. In contrast, extracts from strain
KJ lost 50% of the chlorate reductase activity and 70%
of the perchlorate reductase after only 3 days. Kengen
et al. [10] suggested a half-life of a perchlorate reductase
from strain GR-1 of 2 to 3 days. Wolterink et al. [13]
stated only that cell extracts of the chlorate-reducing
strain P. chloritidismutans lost chlorate reductase activity when stored under air, but they also did not provide
supporting data. Song and Logan [23] found using
whole cells that perchlorate reduction by strain KJ
was lost after 12 h although the cells used in tests were
still viable. The stability of chlorate reductase activity
in strain PDA under aerobic conditions, combined with
a known constitutive activity of this enzyme in cells
grown aerobically, support the unique nature of chlorate reduction by strain PDA.
The purification method used here to isolate perchlorate and chlorate reductases has not been previously reported for (per)chlorate respiring bacteria. However, a
similar methyl viologen-based assay was used by others
to locate nitrate reductase enzymes in a native gel, but
the enzymes were not subsequently extracted from the
gel [24,25]. While our method may provide insight into
the unique nature of the respiratory enzymes used by
strain PDA, the loss of activity after the electrophoresis
limits confirmation of the protein compared to other
separation techniques. Thus, while our findings regarding the sequences obtained from the 60 kDa protein
should be considered preliminary, these and other findings provide a basis for undertaking additional studies
to examine the unique nature of oxygen-tolerant chlorate reductase enzymes.
Acknowledgement
This research was supported by National Science
Foundation Grant BES-0001900.
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