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Applied and Environmental Microbiology, July 2000, p. 3010-3015, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Reactions Involved in the Lower Pathway for
Degradation of 4-Nitrotoluene by Mycobacterium Strain
HL 4-NT-1
Zhongqi
He
and
Jim C.
Spain*
Air Force Research Laboratory, Tyndall Air
Force Base, Florida 32403
Received 15 February 2000/Accepted 3 May 2000
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ABSTRACT |
In spite of the variety of initial reactions, the aerobic
biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the
degradation of nitroaromatic compounds revealed that some nitroaromatic
compounds are initially converted to 2-aminophenol rather than
dihydroxy intermediates by a number of microorganisms. The complete
pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been
elucidated previously. The pathway is parallel to the catechol
extradiol ring cleavage pathway, except that 2-aminophenol is the ring
cleavage substrate. Here we report the elucidation of the pathway of
2-amino-4-methylphenol (6-amino-m-cresol) metabolism during
the degradation of 4-nitrotoluene by Mycobacterium strain
HL 4-NT-1 and the comparison of the substrate specificities of the
relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate
that the 2-aminophenol ring cleavage pathway in strain JS45 is not
unique but is representative of the pathways of metabolism of other
o-aminophenolic compounds.
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INTRODUCTION |
Nitroaromatic compounds are
important industrial feedstocks due to the versatile chemistry of the
nitro group (22). Many nitroaromatic compounds are harmful,
and their release into the environment has caused concern. The
microbial degradation of nitroaromatic compounds is usually initiated
by an enzymatic attack on the nitro group (22). One of the
strategies involves partial reduction of the nitro group to a
hydroxylamino group. The hydroxylamino intermediates can be transformed
to catechols which enter the ring cleavage pathways of aerobic
degradation of aromatic compounds, as observed during degradation of
4-nitrobenzoate (6, 33), 3-nitrophenol (19), and
4-nitrotoluene (7, 26). Alternatively, the hydroxylamino
intermediates can be rearranged, by an intramolecular transfer of the
hydroxyl group (10; L. J. Nadeau, Z. He, and J. C. Spain, unpublished data), to ortho-aminophenols,
as observed during biodegradation of nitrobenzene (21, 24),
3- or 4-chloronitrobenzene (2, 16, 24), 3-nitrophenol
(28), 2-chloro-5-nitrophenol (29), 4-nitrotoluene
(30), and 2,4,6-trinitrotoluene (15) by various
microorganisms. The initial steps of each of the degradation pathways
have been elucidated; however, the lower pathway has been determined
only for the metabolism of 2-aminophenol during the degradation of
nitrobenzene by Pseudomonas pseudoalcaligenes JS45 (11,
14). The pathway is parallel to the catechol extradiol ring
cleavage pathway, except that 2-aminophenol is the ring cleavage substrate.
Mycobacterium strain HL 4-NT-1 is able to grow on
4-nitrotoluene as the sole source of nitrogen, carbon, and energy
(30). 4-Nitrotoluene is converted to 2-amino-4-methylphenol
(6-amino-m-cresol) via 4-hydroxylaminotoluene in reactions
catalyzed by a nitroreductase and an aminohydroxymutase. In reactions
analogous to those used by strain JS45, extracts from induced cells of
strain HL 4-NT-1 catalyzed the conversion of 2-amino-4-methylphenol to
a transient yellow intermediate (A385) which was
subsequently converted into another compound
(A270) identified as 5-methylpicolinic acid
(30). When NAD+ was included in the reaction
mixture, 5-methylpicolinic acid accumulated to a lesser extent and
ammonia was released (30). Based on the preliminary
observations and by analogy with the system in strain JS45, the authors
tentatively proposed that 2-amino-5-methylmuconic semialdehyde and
2-amino-5-methylmuconic acid were produced from 2-amino-4-methylphenol
(30). The goal of the present work was to rigorously
determine the steps in the lower pathway including the mechanism of
release of ammonia in Mycobacterium strain HL 4-NT-1 grown
on 4-nitrotoluene. We also compared the substrate specificities of the
relevant enzymes in strains JS45 and HL 4-NT-1 to provide a preliminary
evaluation of the relationship between the two pathways.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
Mycobacterium
strain HL 4-NT-1 was grown on nitrogen-free mineral medium with
succinate (10 mM) and 4-nitrotoluene (0.5 mM) as described previously
(30). Fully induced cells of strain HL 4-NT-1 were obtained
by incubation of the harvested cells in fresh medium with
4-nitrotoluene as the sole carbon source overnight. Uninduced cells
were obtained by substitution of NH4Cl for 4-nitrotoluene during growth. Cells were harvested by centrifugation and then broken
using a French press as described previously (30).
Enzyme assays.
The activity of the ring cleavage dioxygenase
(0.1 mg of protein/ml) in crude extracts was determined
spectrophotometrically by monitoring the rate of the disappearance of
the substrates (0.05 mM) in potassium phosphate buffer (50 mM, pH 8.0).
The semialdehyde dehydrogenase activity (0.084 mg of protein/ml) was
assayed by measurement of the decrease of A375
(
= 44,000 M
1) using 2-hydroxymuconic semialdehyde
(0.038 to 0.041 mM) as the substrate in the presence of 0.2 mM
NAD+ in potassium phosphate buffer (50 mM, pH 8.0)
(11). Dehydrogenation of amino-substituted semialdehydes was
assayed as reported previously by estimation of the increase in the
A326 which was contributed by the products of
dehydrogenation (9). The deaminase activity was determined
by monitoring the decrease of A326 of a reaction mixture containing enzyme preparations (0.016 to 0.063 mg of
protein/ml), substrate (2-aminomuconate, 0.088 mM, = 17,800 M1 [13], or 2-amino-5-methylmuconate,
0.098 mM, = 15,400 M1 [this work]), and 50 mM
Tris-HCl buffer, pH 7.5, as described previously (12, 13).
4-Oxalocrotonate decarboxylase activity was determined by the initial
rate of decrease in A236 caused by the
disappearance of 2-oxo-3-hexene-1,6-dioate (4-oxalocrotonate, 0.06 to
0.07 mM, = 7,200 M1) in the presence of 1 mM
MgSO4 and enzyme (0.035 mg of protein/ml) (8).
Protein was measured by the Coomassie Plus protein assay reagent from
Pierce (Rockford, Ill.) by using bovine serum albumin as a standard.
Ion-exchange chromatography.
A Hitrap Q column (5 ml;
Pharmacia) was equilibrated with potassium phosphate buffer (10 mM, pH
7.0), loaded with 12.5 ml of crude extract from strain HL 4-NT-1, and
washed with 25 ml of the buffer. Proteins were eluted with a 48-ml
linear gradient from 0 to 0.5 M NaCl in buffer at a flow rate of 0.8 ml · min1. Fractions (2 ml) were collected and
assayed for enzyme activities and protein concentration.
Preparation of 2-amino-5-methylmuconate.
The muconate was
prepared from 4-methyl-2-aminophenol by the combined action of
2-aminophenol 1,6-dioxygenase of strain JS45 (5) and the
partially purified 2-amino-5-methylmuconic semialdehyde dehydrogenase
obtained by the ion-exchange chromatography described above. The molar
extinction coefficient of 2-amino-5-methylmuconate at 326 nm was
determined after the reaction went to completion in the presence of
excess dehydrogenase as indicated by the absence of accumulation of
ring cleavage intermediates (A382). The
accumulation of NADH was eliminated by the addition of pyruvate (0.5 mM) and lactate dehydrogenase (5 U per ml). The product was purified by ion-exchange chromatography as described previously for 2-aminomuconate (13).
Biochemical reagents and chemicals.
Purified 2-aminomuconate
semialdehyde dehydrogenase and 2-aminomuconate deaminase from strain
JS45 were obtained as described previously (9, 12).
2-Aminophenol 1,6-dioxygenase was obtained from an Escherichia
coli clone containing the structural genes encoding the enzyme
from strain JS45 (5). The 4-oxalocrotonate decarboxylase
from strain JS45 was partially purified as described previously
(11). 2-Hydroxy-5-methylmuconic semialdehyde was prepared
from 4-methylcatechol by the catechol 2,3-dioxygenase in an E. coli clone containing the appropriate gene from
Comamonas sp. strain JS765 (23). 2-Aminomuconate
was enzymatically synthesized as previously described (13).
2-Hydroxymuconic semialdehyde and 2-oxo-3-hexene-1,6-dioate
(4-oxalocrotonate) were prepared as reported previously (9,
14). All other chemicals were from Sigma (St. Louis, Mo.) or
Aldrich (Milwaukee, Wis.).
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RESULTS |
Ring cleavage dioxygenase and semialdehyde dehydrogenase activities
in crude extracts.
As observed previously (30), the
crude extracts of strain HL 4-NT-1 catalyzed the oxidation of
2-amino-4-methylphenol and 2-aminophenol, but not 4-methylcatechol
(Table 1). In contrast to the previous
report (30), we found that catechol was also a substrate of
the dioxygenase, but the activity ceased after a half-minute,
indicating that catechol was a suicide substrate of the
2-amino-4-methylphenol 1,6-dioxygenase, as it is for the 2-aminophenol
1,6-dioxygenase from strain JS45 (5).
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TABLE 1.
Comparison of the relevant enzyme activities in
Mycobacterium strain HL 4-NT-1 and P. pseudoalcaligenes JS45
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The
A375 of 2-hydroxymuconic semialdehyde
decreased when the crude extracts of strain HL 4-NT-1 were incubated
with the substrate
in the presence of NAD
+, but no decrease
in the absorbance was observed in the absence
of NAD
+. The
observation indicated that strain HL 4-NT-1 contains a semialdehyde
dehydrogenase but not a semialdehyde hydrolase. The result suggested
that 2-amino-5-methylmuconic semialdehyde, the ring fission product
from 2-amino-4-methylphenol, is dehydrogenated to
2-amino-5-methylmuconate,
as proposed previously (
30), and
by the same mechanism as in
the metabolism of 2-aminophenol in strain
JS45 (
14). Incubation
of 2-amino-4-methylphenol with the
crude extracts in the presence
of NAD
+, however, did not
lead to a spectral change characteristic of
the proposed
2-amino-5-methylmuconate, probably due to a high
activity of the
subsequent deaminase
enzyme.
Partial purification of 2-amino-5-methylmuconic semialdehyde
dehydrogenase and production of 2-amino-5-methylmuconate.
To
investigate the individual enzymes, the cell extracts of strain HL
4-NT-1 were loaded on a Hitrap-Q anion-exchange column and partially
purified enzymes were obtained as described in Materials and Methods.
The 2-amino-4-methylphenol 1,6-dioxygenase was not detected
spectrophotometrically in any fractions, probably due to the lability
of the dioxygenase. 2-Hydroxymuconate semialdehyde was used for routine
assay of the activity of the semialdehyde dehydrogenase because
2-amino-5-methylmuconic semialdehyde is unstable and spontaneously
converts to 5-methylpicolinate (17, 30). The dehydrogenase
activity eluted at 0.35 M NaCl (Fig. 1).
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FIG. 1.
Hitrap-Q chromatography of crude extracts of strain HL
4-NT-1. Diagonal line, NaCl elution gradient from 0 to 0.4 M; dotted
line, protein concentration;  , semialdehyde dehydrogenase;  ,
2-amino-5-methylmuconate deaminase;  , 4-oxalocrotonate
decarboxylase.
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The activity of the semialdehyde dehydrogenase from strain HL 4-NT-1
was optimal at pH 8, whereas 2-aminomuconic semialdehyde
dehydrogenase
from strain JS45 had an optimal activity at pH 7.3
(
9). Both
enzymes were able to act on the hydroxy analogs (Table
1). The
2-aminophenol 1,6-dioxygenase (
5) was used to produce
amino-substituted semialdehydes. The incubation of
2-amino-4-methylphenol
with the combination of the partially purified
semialdehyde dehydrogenase
and the 2-aminophenol 1,6-dioxygenase
produced a previously unreported
metabolite with an absorbance maximum
at 326 nm (Fig.
2). When
the purified
2-aminomuconic semialdehyde dehydrogenase (
9)
from strain
JS45 was substituted for the dehydrogenase fraction
from strain HL
4-NT-1, identical results were obtained. When the
purified
2-aminomuconate deaminase (
12) from strain JS45 acted
on the
product, stoichiometric release (90%) of ammonia was observed.
Based
on the catalytic mechanisms of the dioxygenase and dehydrogenase
in
strain JS45 (
9,
14) and the stoichiometry of ammonia
release,
the product seems to be 2-amino-5-methylmuconate. The spectrum
of 2-amino-5-methylmuconate was similar to that of 2-aminomuconate,
but
the molar extinction coefficient of the former was determined
to be
15.4 ± 0.2 mM
1 cm
1 (average of three
determinations), whereas that of the latter
is 17.8 mM
1
cm
1 (
13).
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FIG. 2.
Enzymatic formation of 2-amino-5-methylmuconate by the
action of partially purified 2-amino-5-methylmuconic semialdehyde
dehydrogenase (fraction 17). 2-Amino-5-methylmuconic semialdehyde
(A382) was instantly produced from
2-amino-4-methylphenol (0.05 mM) by the 2-aminophenol 1,6-dioxygenase
of strain JS45 (0.31 mg of protein per ml). The reaction mixture also
contained potassium phosphate (50 mM, pH 8.0), fraction 17 (0.084 mg of
protein per ml), and 0.2 mM NAD+. Recordings were taken at
0.5-min intervals. The A326 increased during the
course of the reaction.
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Deamination of 2-amino-5-methylmuconate.
When
2-amino-5-methylmuconate was incubated with crude extracts of HL
4-NT-1, the A326 decreased with time. The enzyme
activity eluted from the Hitrap-Q anion-exchange column at 0.21 M NaCl (Fig. 1). When 2-amino-5-methylmuconate (113 µmol in 1 ml) was incubated with the active fraction, stoichiometric release of ammonia
(112 µmol) was observed. This result indicated that the enzyme was
5-methyl-2-aminomuconate deaminase. During the deamination of
2-amino-5-methylmuconate, a new absorbance maximum appeared at 297 nm
along with an increase of absorbance at about 237 nm (Fig.
3A). The enzyme also catalyzed the
deamination of 2-aminomuconate with a higher relative activity (Table
1). During deamination of 2-aminomuconate by the HL 4-NT-1 deaminase,
the absorbance changed directly from 326 to 236 nm (the putative oxo
isomer, 2-oxo-3-hexene-1,6-dioate) without a significant
A297 (the putative enol isomer,
2-hydroxymuconate) (Fig. 3B). The spectral change was similar to that
catalyzed by the purified 2-aminomuconate deaminase from strain JS45
(12). Control experiments indicated that the product from
5-methyl-2-aminomuconate in reactions catalyzed by purified JS45
deaminase also had an absorbance maximum at 297 nm (data not shown).
The relative contributions of the enol and oxo isomers of
5-methyl-2-oxo-3-hexene-1,6-dioate to the absorbance spectra have not
been determined rigorously. Based on analogy with previous work,
the reaction catalyzed by the HL 4-NT-1 deaminase seems to be the
same as that catalyzed by the JS45 deaminase. That is, the product of
deamination of 5-methyl-2-aminomuconate seems to be the oxo isomer
5-methyl-2-oxo-3-hexene-1,6-dioate, not the enol isomer
5-methyl-2-hydroxymuconate.
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FIG. 3.
Spectral changes during metabolism of
2-amino-5-methylmuconate (A) and 2-aminomuconate (B). Buffer was
Tris-HCl (50 mM, pH 7.5). The amount of partially purified deaminase
(fraction 10) from strain HL 4-NT-1 in the reaction mixture (1 ml) for
panels A and B was 0.063 and 0.016 mg of protein, respectively. The
intervals between scans are 4 min for panel A and 2 min for panel B.
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The decarboxylase-hydratase fraction.
The protein fraction
that eluted at 0.31 M NaCl from the Hitrap-Q anion-exchange column
catalyzed the decarboxylation of 2-oxo-3-hexene-1,6-dioate in the
presence of MgSO4, as monitored by the decrease of the A237 (8). There was no activity in
the absence of MgSO4. During the decarboxylation, a
transient absorbance peak at 265 nm was observed (Fig.
4), indicating that the hydratase
coeluted with the decarboxylase, a common observation for the two
enzymes (8, 11, 18). It was not possible to determine
accurately the specific activity of decarboxylation of
5-methyl-2-oxohexene-1,6-dioate because no authentic substrate was
available. The decarboxylases from both bacteria showed less than 10%
of the relative activity on the 5-methyl analog (Table 1). The
existence of the decarboxylase and hydratase in strain HL 4-NT-1
suggested that the later steps in the pathway of the metabolism of
4-methyl-2-aminophenol were similar to the corresponding steps in
catechol extradiol pathways.
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FIG. 4.
Spectral changes during metabolism of
2-oxo-3-hexene-1,6-dioate. The substrate (A236)
was preequilibrated with its enol form (2-hydroxymuconate,
A296) in Tris-HCl buffer (50 mM, pH 7.0).
Fraction 14 was used for the source of the decarboxylase (0.035 mg of
protein per ml). Reactions were initiated by the addition of
MgSO4 (1 mM final concentration). Recordings were taken at
0.5-min intervals. The transient increase of absorbance around 265 nm
indicates that the 2-oxo-4-pentenoate hydratase coexisted in the
fraction.
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DISCUSSION |
Microorganisms exhibit extensive metabolic diversity that enables
them to degrade a variety of aromatic compounds. In general, pathways
for the aerobic degradation of aromatic compounds can be considered to
consist of two parts: an upper pathway and a lower pathway (25,
32). Since the catechol extradiol pathway (Fig.
5) was reported for the first time in the
1960s (3, 4, 27), it has been found to be one of the most
common lower pathways involved in degradation of both naturally
occurring and man-made aromatic compounds (1, 22, 25, 32).
The ortho-aminophenol intermediates produced during
degradation of some nitroaromatic compounds do not have the two
adjacent hydroxyl groups on the aromatic ring that were previously
thought to be necessary for metabolism through the extradiol ring
cleavage pathway. The pathway for the metabolism of 2-aminophenol
during the degradation of nitrobenzene by strain JS45 is parallel to
the catechol extradiol pathway (Fig. 5) (11, 14). Results
presented here suggest that the pathway is not confined to the
degradation of nitrobenzene and might be common among bacteria that
degrade nitroaromatic compounds or the corresponding aminophenols.
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FIG. 5.
General catechol extradiol ring cleavage pathway
(pathway 1) and general o-aminophenol extradiol-like ring
cleavage pathway (pathway 2). In pathway 1, R can be hydrogen or a
methyl, hydroxyl, carboxyl, chloro, or other group; in pathway 2, R has
been shown to be a hydrogen (11) and methyl group (this
work). I, ring cleavage dioxygenase; II, aldehyde dehydrogenase; III,
tautomerase; III', deaminase; IV, decarboxylase; V, hydratase; VI,
aldolase; VII, hydrolase. Vertical arrows represent spontaneous
conversion. When A is (4-methyl)catechol, B is
2-hydroxy-(5-methyl)muconic semialdehyde, C is
2-hydroxy-(5-methyl)muconate, D is
(5-methyl)-2-oxo-3-hexene-1,6-dioate, E is 2-oxo-4-pen(or hex)enoate,
and F is 2-oxo-4-hydroxyvalerate(or hexanoate). When A' is
2-amino-(4-methyl)phenol, B' and C' are the corresponding amino analogs
of B and C.
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Our results indicate that both 2-aminophenol and 2-amino-4-methylphenol
are degraded through the o-aminophenol cleavage pathways in
either strain JS45 or HL 4-NT-1 even though the specific activities of
the enzymes were not the same in the two strains. Catechol 2,3-dioxygenase similarly catalyzes cleavage of 4-methylcatechol (1, 3). ortho-Aminophenolic compounds can be
considered the analogs of catechols in which one of the hydroxyl groups
is replaced by an amino group. The amino and hydroxyl groups are comparable in size and electronegativity, and both are electron donating by resonance effects. The amino group is basic, and the hydroxyl group shows similar but weaker basicity (20).
However, 2-aminophenol is a very poor substrate for catechol
2,3-dioxygenase; it is oxidized at only about 0.01% of the rate of
catechol (5). On the other hand, catechol was a suicide
substrate of the two o-aminophenol 1,6-dioxygenases from
strains JS45 and HL 4-NT-1. Therefore, considering the fact that there
is only a single hydroxyl group in o-aminophenols, it seems
more appropriate to categorize the o-aminophenol
dioxygenases as extradiol-like ring cleavage dioxygenases. Because the
two pathways are similar but not identical, it is better to name the
o-aminophenol ring cleavage route an extradiol-like pathway.
In recent years, nitrobenzene, 3-chloronitrobenzene,
4-chloronitrobenzene, 3-nitrophenol, 2-chloro-5-nitrophenol,
4-nitrotoluene, and probably 2,4,6-trinitrotoluene have been
reported to be transformed to the corresponding
o-aminophenols via mutase-catalyzed rearrangements in
various microorganisms (22). The proliferation of reports suggests that o-aminophenolic compounds may be central
intermediates in the biodegradation of a variety of nitroaromatic
compounds. It will be useful to elucidate the pathways used during the
degradation of other nitroaromatic compounds via substituted
o-aminophenol intermediates. As the catechol pathway is the
archetype of extradiol cleavage pathways for degradation of various
aromatic compounds, the 2-aminophenol pathway might serve as an
archetypal extradiol-like ring cleavage pathway for degradation of
relevant nitroaromatic compounds. Biochemical characterization and
sequence analysis of 2-aminophenol 1,6-dioxygenases from strain JS45
and another bacterium, Pseudomonas sp. strain AP-3, show
that the enzymes share some features with extradiol dioxygenases, but
the amino acid sequences are only distantly related (5, 17,
31). 2-Aminomuconic semialdehyde dehydrogenase from P. pseudoalcaligenes JS45 is most similar to its counterparts in the
catechol pathway in both biochemical properties and amino acid
sequences (9). However, the substrates for 2-aminomuconate
deaminase and 4-oxalocrotonate tautomerase are not interchangeable,
even though the two reactions seem superficially similar. The
N-terminal amino acid sequence of the deaminase from JS45 does not
match that of the tautomerase (12). Further comparative
investigation of the enzymology and molecular biology of the two
pathways will be required to explain the biochemistry of the relevant
reactions and the evolution of the extradiol-like pathway.
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ACKNOWLEDGMENTS |
The work was supported in part by the U.S. Air Force Office of
Scientific Research and the Strategic Environmental Defense Research
Program. This work was also supported in part by an appointment to the
research Participation Program at the U.S. Air Force Research Laboratory, Tyndall Air Force Base, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and Tyndall Air Force Base.
We thank Hiltrud Lenke for providing strain HL 4-NT-1, Becky Parales
for providing the E. coli clone which contains the catechol 2,3-dioxygenase gene from JS765, and Shirley Nishino for reviewing the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: AFRL/MLQR, 139 Barnes Dr., Suite 2, Tyndall Air Force Base, FL 32403. Phone: (850) 283-6058. Fax: (850) 283-6090. E-mail:
jim.spain{at}tyndall.af.mil.
Present address: USDA-ARS, New England Plant, Soil, and Water Lab,
University of Maine, Orono, ME 04469.
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Applied and Environmental Microbiology, July 2000, p. 3010-3015, Vol. 66, No. 7
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Abstract
Introduction
