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Applied and Environmental Microbiology, September 2000, p. 4119-4123, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Hydrogen Peroxide Sensitivity of
Catechol-2,3-Dioxygenase: a Cautionary Note on Use of
xylE Reporter Fusions under Aerobic Conditions
Daniel J.
Hassett,1,*
Urs A.
Ochsner,2
Stephanie L.
Groce,3
Kislay
Parvatiyar,1
Ju-Fang
Ma,1 and
John D.
Lipscomb3
Department of Molecular Genetics,
Biochemistry and Microbiology, University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267-05241;
Department of Microbiology and Immunology, University of
Colorado Health Sciences, Denver, Colorado
802622; and Department of Biochemistry,
Molecular Biology and Biophysics, University of Minnesota,
Minneapolis, Minnesota 55455-03263
Received 16 March 2000/Accepted 15 June 2000
 |
ABSTRACT |
Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida,
encoded by the xylE gene, was found to be sensitive to
hydrogen peroxide (H2O2) when used as a
reporter in gene fusion constructs. Exposure of Pseudomonas
aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding 
-galactosidase)
or -xylE fusion plasmids to H2O2
stimulated -galactosidase activity, while there was little or no
detectable C23O activity in these strains. More than 95% of C23O
activity was lost after a 5-min exposure to equimolar H2O2, while a 10,000-fold excess was required
for similar inhibition of -galactosidase. Electron paramagnetic
resonance spectra of the nitrosyl complexes of C23O showed that
H2O2 nearly stoichiometrically oxidized the
essential active-site ferrous ion, thus accounting for the loss of
activity. Our results suggest using caution in interpreting data
derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.
| |
TEXT |
The use of gene reporter cassettes,
including those encoding -galactosidase (lacZ),
luciferase (luxAB), and catechol-2,3-dioxygenase (C23O)
(xylE), has been a tremendous benefit to scientists studying gene regulation. The lacZ gene is the most widely used
reporter in bacteria, while others that include luxAB,
gfp (green fluorescent protein), phoA (alkaline
phosphatase), and xylE fusions are used less often. The use
of lacZ fusions in bacteria has proven to be the best tool
with which to accurately assess promoter activity under conditions of
oxidative stress. For example, in Escherichia coli,
lacZ fusions were used to show that the transcriptional activity of oxidant-regulated genes including sodA (encoding
Mn-superoxide dismutase), katE (encoding
catalase/hydroperoxidase II), and katG (encoding
catalase/hydroperoxidase I), among others, is markedly increased upon
exposure to either O2- or
H2O2-generating agents (16, 29, 34).
Unlike the reliable lacZ reporters, other gene fusions have
some drawbacks. For example, the use of the Vibrio harveyi
luxAB genes (encoding luciferase) in transcriptional gene fusions
was cautioned against, because it generates
O2
(12). Fusions linked to
phoA are limited to proteins expressed in the periplasm
(21), while those linked to gfp are only
semiquantitative (24). The use of xylE fusion
constructs does not appear to have any of these disadvantages
(32).
The product of the xylE gene of Pseudomonas
putida, C23O, is an important component in the degradation
pathways of toluene and xylenes and catalyzes the dioxygenolytic
cleavage of the aromatic ring (8, 11, 18, 25).
Interestingly, it has been observed that optimal toluene degradation
occurs under reduced oxygen tension with some C23O enzymes
(17). This suggests a sensitivity to oxygen or one of its
reduced forms, despite the fact that C23O requires oxygen for activity.
Oxygen sensitivity may be derived from the fact that each of four C23O
subunits contains an essential iron atom which must be in the Fe(II)
oxidation state for activity in extradiol-type aromatic dioxygenases
(3, 4, 25). Oxidation of this iron would inactivate the
enzyme. Indeed, early studies showed that C23O is inactivated by low
levels of the oxidizing reagent H2O2 in vitro
(25). Our later studies of other Fe(II)-containing dioxygenases showed that H2O2 treatment
resulted in enzyme inactivation and the appearance of electron
paramagnetic resonance (EPR) signals attributable to ferric ion in most
but not all cases (3, 23, 37). Several other iron-containing
enzymes including the antioxidants iron superoxide dismutase and
catalase are also sensitive to elevated levels of
H2O2 (7, 9). In this study, we
describe the sensitivity of C23O to H2O2
measured both in vivo using isogenic catalase mutants (Table
1) of the aerobic gram-negative bacterium
Pseudomonas aeruginosa and in vitro using purified enzyme.
We suggest caution in interpreting data obtained using xylE
reporter fusions under aerobic conditions.
Sensitivity of C23O but not -galactosidase to
H2O2 in vivo using P. aeruginosa
catalase gene lacZ and xylE fusions.
The
katA gene of P. aeruginosa encodes the major,
constitutively expressed catalase, KatA (13, 20). In
contrast, KatB activity is only detected upon exposure of bacteria to
H2O2 or the redox-cycling agent paraquat
(6). As shown in Fig. 1A, XylE
reporter activity in the katA mutant, where catalase
activity is virtually undetectable (20), was reduced 93%.
The KatA-XylE reporter activity in the katA katB double
mutant was reduced ~70%. Since H2O2 is
required to activate the katB gene (6), it is not
surprising that KatB reporter activity was very low or undetectable. In
fact, KatB-LacZ reporter activity was reduced ~5,000-fold relative to
KatA-LacZ reporter activity (Fig. 1B).
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FIG. 1.
Quantification of catalase gene reporter activity in
wild-type and catalase mutant bacteria. P. aeruginosa PAO1
katA, katB, and katA katB strains
(13) harboring plasmids containing katA- or
katB-lacZ or -xylE transcriptional fusions were
grown aerobically for 24 h in L broth containing carbenicillin
(0.4 mg/ml) for plasmid maintenance. Bacteria were washed twice in
either ice-cold 50 mM potassium phosphate buffer (pH 7.5) (for C23O
assays) or Z buffer (for -galactosidase assays) (22)
containing 39 mM 2-mercaptoethanol and were sonicated in an ice water
bath for 10 s with a Heat-Systems, Inc. (Farmington, N.Y.), model
W-225 sonicator at setting 5. Cell extracts were assayed for C23O and
-galactosidase activities as previously described (19,
28). The results are expressed as the means ± standard
errors of the means of three replicates. (A) Lane 1, PAO1/pkatA::xylE; lane 2, PAO1/pkatB::xylE; lane 3, katA mutant/pkatA::xylE;
lane 4, katA
mutant/pkatB::xylE; lane 5, katB mutant/pkatA::xylE;
lane 6, katB
mutant/pkatB::xylE; lane 7, katA
katB mutant/pkatA::xylE; lane 8, katA katB
mutant/pkatB::xylE. (B) Lanes are
identical to those in panel A except that the fusions are to
lacZ. The values in panel A are all statistically
significantly different from one another (P < 0.01,
Student's t test). The differences between lanes 1, 3, 5, and 7 of panel B are not statistically significant.
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Treatment with H2O2 stimulates catalase
gene lacZ reporter activity but inhibits xylE
reporter activity in catalase-deficient strains.
To test the
hypothesis that H2O2 increases
katA::lacZ and
katB::lacZ but not
katA::xylE or
katB::xylE activity, aerobic,
mid-logarithmic-phase organisms were exposed to a sublethal 1 mM dose
of H2O2 for 1 h, a condition which
markedly increases KatB activity and, to a far lesser extent, KatA
activity (6). Figure 2A shows
that KatA-LacZ activity was only slightly increased in wild-type
bacteria but was increased 1.4-fold following
H2O2 treatment in the katA katB
mutant. KatA-XylE activity was also increased upon exposure to
H2O2 in wild-type bacteria but was dramatically
inhibited in the control and H2O2-treated
katA katB strains (Fig. 2B). In contrast to KatA activity,
KatB activity is stimulated upon exposure to H2O2 (6). This is reflected in the
KatB-LacZ results shown in Fig. 2B. Wild-type and katA katB
mutant KatB-LacZ activity increased 10.7- and 35-fold upon exposure to
H2O2. In contrast, KatB-XylE activity in the
H2O2-treated katA katB mutant was
reduced ~15-fold and was not detectable in control bacteria.
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FIG. 2.
H2O2-mediated activation of
katA and katB gene transcription: demonstration
of increased -galactosidase activity but not C23O activity in
catalase-deficient bacteria. Bacteria were grown aerobically to
mid-exponential phase (optical density at 600 nm = 0.6) in L broth
and were allowed to grow an additional hour in the presence (lanes 2 and 4) or absence (lane 1 and 3) of 1 mM H2O2.
Cell extracts were then assayed for -galactosidase (A and C) and
C23O (B and D) activity. The results are expressed as the percentage of
reporter activity in uninduced wild-type bacteria (n = 3). Lane 1, PAO1; lane 2, PAO1 + H2O2; lane 3, katA katB; lane 4, katA katB + H2O2. In panel A,
values in lanes 1, 2, and 4 are not statistically significant, yet that
in lane 3 is significantly reduced (P < 0.05). All
remaining values are statistically significantly different at
P values of < 0.01.
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Sensitivity of purified C23O but not of -galactosidase to
H2O2.
The in vivo results suggest that
H2O2 is much more damaging to C23O than to
-galactosidase. To compare the sensitivity of C23O and
-galactosidase to H2O2, aliquots of the
purified enzymes were incubated at room temperature with increasing
concentrations of H2O2. As shown in Fig.
3A, the C23O activity loss was directly correlated with the nearly stoichiometric titration of the active-site Fe(II) in the sample with H2O2. Most of the
activity was lost after a 1:2 titration, and there was no detectable
C23O activity after the enzyme was incubated with a 20-fold excess of
H2O2. In contrast, 100 mM
H2O2 (a 10,000-fold excess) was required for ~50% inhibition of -galactosidase activity (Fig. 3B).
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FIG. 3.
Relative sensitivity of purified P. putida
C23O (A) and -galactosidase (B) to H2O2.
C23O was isolated from P. putida mt-2 cells maintained on
m-toluate and grown in 10-liter batch cultures on benzoate
as the sole carbon source from 1-liter liquid starter cultures
containing 1:2 m-toluate-benzoate as carbon sources. The
total culture time on benzoate was 12 h, and the extradiol
catechol oxidation activity was approximately 105 U/100 g
(wet weight) of cells. The purification was as previously described
(4), except that 50 mM morpholinepropanesulfonic acid (MOPS)
buffer (pH 7.0) was used in place of potassium phosphate buffer in all
steps. Samples with a specific activity greater than 150 U/mg were
pooled, concentrated, and stored at  80°C in small aliquots until
use. Purified P. putida C23O (10 U) and E. coli
-galactosidase (10 U; Sigma) were incubated with increasing
concentrations of H2O2 at room temperature for
5 min. C23O and -galactosidase activities were then assayed as
previously described (19, 28) and expressed as the
percentage of the control without H2O2. The
bars indicate the means + standard errors of the means of three
replicates. Concentrations of H2O2 added
follow. (A) Lane 1, control; lane 2, 1 µM; lane 3, 10 µM; lane 4, 100 µM. (B) Lane 1, control; lane 2, 1 mM; lane 3, 10 mM; lane 4, 100 mM; lane 5, 200 mM; lane 6, 300 mM; lane 7, 400 mM; lane 8, 500 mM.
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EPR analysis of H2O2-treated C23O.
The
effect of H2O2 on the active-site iron of C23O
can be directly evaluated by EPR spectroscopy. Our past studies have
shown that complexing of the enzyme with NO converts an EPR-silent
metal center into an EPR-active species with two of the three
resonances symmetrically distributed around g values of 4 (3, 4). This is the characteristic spectrum of a spin (S)
3/2 species that is formed by transferring one electron from the iron
to the NO and antiferromagnetic coupling between the resulting species
(5). This species is easily distinguished from that
resulting from oxidation of the Fe(II) to Fe(III), which yields an S
5/2 EPR spectrum with g values near 4.3. As shown in Fig.
4, untreated C23O yielded an intense S
3/2 EPR spectrum from the active-site Fe(II)-NO complex. Quantitation
of the spectra showed that all of the iron in the sample formed a
complex. After a 30-min incubation with a twofold excess of
H2O2 on ice, a dramatic decrease in the S 3/2
spectrum from Fe(II)-NO occurred, accompanied by a 96% (±3%) loss of
activity and the appearance of a broad signal from Fe(III) (g = 4.3). Quantitation of the S 3/2 signal showed that
87% (±5%) of the Fe(II) was oxidized. Incubation of an identical
sample with a 200-fold excess of H2O2 resulted
in a complete loss of the S 3/2 signal and activity as well as a
further increase in the S 5/2 signal from Fe(III). These results show
directly for the first time that the origin of the loss of activity of
C23O following H2O2 treatment is oxidation of
the active-site iron in accord with previous indirect experiments
(25). Many studies have indicated that the mechanism of
extradiol dioxygenases requires Fe(II), and thus its oxidation would
inhibit the enzyme (2, 33). Because the Fe(III) EPR signal
of the inactivated C23O is very broad, it is likely either that the
iron is lost from the enzyme following oxidation or that the
environment of the iron becomes highly disordered.
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FIG. 4.
EPR spectra of the Fe-nitrosyl complex of purified
P. putida C23O show that loss of activity correlates with
oxidation of the active-site Fe(II). Purified P. putida C23O
(160 µM) was incubated on ice with 0 µM ( ), 300 µM
(....), or 30 mM (- - -)
H2O2 for 30 min at pH 7. Then 7.5 U of bovine
liver catalase was added to destroy any residual
H2O2, and the enzyme activity was determined
(this concentration of catalase cannot be detected by EPR). The samples
were then transferred by a gastight syringe to an EPR tube under argon.
Nitric oxide (NO) was added by slowly bubbling the gas through the
sample under argon flow. Trace oxygen was removed from the argon gas by
passage over a BASF copper catalyst at 160°C. Samples were flushed
with argon after NO addition to remove excess NO from the headspace.
The samples were frozen by slow immersion in liquid N2, and
the EPR spectra were recorded. The enzyme activity of the EPR samples
after the measurement was determined by first thawing them under argon.
The samples were transferred by a gastight syringe to a serum-stoppered
vial under argon. Subsequently, the NO was removed by cycles of
evacuation and flushing with Ar. Activity measurements of the samples
were approximately unchanged from those determined before exposure to
NO. The loss of signal in the 4-g region is proportional to
the oxidation of Fe(II) to Fe(III) in the sample. At least three
slightly different S 3/2 species are present with resonance pairs at
g values of 4.18 and 3.82, 4.11 and 3.91, and 4.02 and 3.98. Multiple species are usually seen for nitrosyl complexes of Fe(II)
dioxygenases. EPR measurement conditions using a Bruker E-500
spectrometer equipped with an Oxford ESR-910 liquid helium cryostat
follow: temperature, 2 K; modulation amplitude, 10 G; modulation
frequency, 100 kHz; microwave power, 200 µW; and microwave frequency,
9.63 GHz. Data were digitally recorded and analyzed as previously
reported (10). Spin quantitations were performed by single
or double integration of the first derivative spectra (1)
using an Fe(II)-NO-EDTA complex as a standard. EPR spectra of S 3/2 and
S 5/2 complexes were analyzed as previously reported (23,
37). Iron content was quantified by atomic absorption.
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Conclusions.
In this study, we demonstrate that C23O is very
sensitive to approximately stoichiometric levels of
H2O2 both when it is present as a gene fusion
product in vivo and as the purified enzyme in vitro. Arguably, the
attractiveness of the xylE reporter system in monitoring
gene-promoter activity is based upon its rapid spectrophotometric assay
that is zero-order with respect to its substrate catechol. However,
this study raises concern over the interpretation of data obtained
under highly aerobic conditions. Recently we have cloned,
overexpressed, and characterized another extradiol dioxygenase similar
to C23O that catalyzes the cleavage of 3,4-dihydroxyphenyl acetic acid
(homoprotocatechuate) between the 2- and 3-ring carbons to yield a
yellow product analogous to the product of the C23O-catalyzed reaction
(23, 35). Homoprotocatechuate-2,3-dioxygenase (H23O, encoded
by hpcd) is unique among Fe(II)-containing dioxygenases in
that it exhibits very low sensitivity to H2O2.
Moreover, a homologous H23O containing Mn(II) rather than Fe(II) has
also been cloned and characterized and was also found to be insensitive to H2O2 (27, 36). Thus, gene fusions
to either of these enzymes could offer an alternative to C23O when
experiments require highly aerobic and/or oxidative stress conditions.
Such experiments are in progress.
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ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant AI-45041 and
Cystic Fibrosis grant HASSET97PO to D.J.H. and NIGMS grant 24689 to
J.D.L.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Genetics, Biochemistry and Microbiology, University of
Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH
45267-0524. Phone: (513) 558-1154. E-mail:
Daniel.Hassett{at}UC.Edu.
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Applied and Environmental Microbiology, September 2000, p. 4119-4123, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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