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Journal of Bacteriology, January 1999, p. 149-152, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
3-Nitroadipate, a Metabolic Intermediate for
Mineralization of 2,4-Dinitrophenol by a New Strain of a
Rhodococcus Species
Rafael
Blasco,1,*
Edward
Moore,2
Victor
Wray,2
Dietmar
Pieper,2
Kenneth
Timmis,2 and
Francisco
Castillo1
Departamento de Bioquímica y
Biología Molecular, Facultad de Ciencias, 14071 Córdoba,
Spain,1 and
GBF (National Research
Center for Biotechnology), D-38124 Braunschweig,
Germany2
Received 1 June 1998/Accepted 21 October 1998
 |
ABSTRACT |
The bacterial strain RB1 has been isolated by enrichment
cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and
energy source and characterized, on the basis of 16S rRNA gene sequence
comparison, as a Rhodococcus species closely related to
Rhodococcus opacus. Rhodococcus sp. strain RB1 degrades
2,4-dinitrophenol, releasing the two nitro groups from the compound as
nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in
two steps. First, the 2-nitro group is removed as nitrite, with the
production of an aliphatic nitro compound identified by 1H
nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its
nitro group as nitrite. Full nitrite assimilation upon reduction to
ammonia requires that an additional carbon source be supplied to the medium.
| |
INTRODUCTION |
Nitroaromatic compounds are common
pollutants of natural environments due to abusive industrial and
agricultural practices (7). These xenobiotic compounds are
produced by industries manufacturing explosives, herbicides,
pesticides, and dyes, and degradative pathways of these compounds are
widely distributed among bacteria (for a review, see reference
15). Among these microorganisms, some
Rhodococcus strains exhibit a high potential for the
degradation of polynitroaromatic compounds (4). The biological hydride transfer to the aromatic ring with the concomitant formation of a Meisenheimer complex was shown to occur during the
metabolism of picric acid by Rhodococcus erythropolis HL
24-2 (9). Further evidence for a reductive metabolic route
was given in the same paper, showing the accumulation of
2,4-dinitrophenol (2,4-DNP) from picric acid, and in an accompanying
communication reporting 4,6-dinitrohexanoate as the metabolite that
accumulated under anaerobic conditions (11). A similar
mechanism might account for the degradation of trinitrotoluene (TNT) by
a Pseudomonas sp. strain able to convert TNT into toluene;
the mineralization of the chemical was achieved by transferring the TOL
catabolic plasmid to the recipient strain (3).
In this work, we report the isolation and characterization of a
gram-positive bacterium, characterized as a new Rhodococcus strain on the basis of its 16S rRNA gene sequence, with a high biodegradative potential. As far as we know, there is, beside the
identification of 4,6-dinitrohexanoate (11), no evidence for
the metabolic fate of the putative Meisenheimer complex formed from
2,4-DNP. On the basis of our results and those cited above, we propose
a pathway for the mineralization of 2,4-DNP with 3-nitroadipate as a
key metabolic intermediate.
| |
MATERIALS AND METHODS |
Bacterial isolation, growth conditions, and biodegradative
potential.
Rhodococcus sp. strain RB1 was isolated by
aerobic enrichment cultivation of an activated sludge from a wastewater
plant in Alicante, Spain. The medium used was M9 mineral medium
(13) with 0.5 mM 2,4-DNP as the sole nitrogen and carbon
source. After enrichment (four reinoculation steps), samples of the
culture were plated on the same medium, which was solidified with 1.8% (wt/vol) Bacto Agar (Difco). One colony was purified and proved capable
of 2,4-DNP mineralization in liquid culture; it was used for further
studies. Where indicated, acetate and/or ammonium or nitrite were added
as the carbon and nitrogen sources.
Anaerobic conditions were obtained by completely filling screw-capped
bottles with the culture medium (10-ml volume). For strict
anaerobiosis, high-purity argon was bubbled through the cultures.
R. erythropolis HL 24-2 was kindly provided by H.-J.
Knackmuss (
10).
Taxonomic position and analysis of the 16S rRNA gene sequence of
Rhodococcus sp. strain RB1.
The phylogenetic position
of strain RB1 was estimated as described in detail previously
(6) with the evolutionarily conserved primary sequence and
secondary structure as the reference (5). Cluster analyses
were carried out with programs contained in the Phylogeny Inference
Package (PHYLIP), version 3.5c (J. Felsenstein, University of
Washington, Seattle).
Analytical determinations.
Bacterial growth was monitored
turbidimetrically at 600 nm. Nitrite, ammonium, and protein
concentrations were estimated as described previously (1).
Nitrophenol compounds and possible transformation products were
analyzed with a Beckman System Gold high-performance liquid
chromatograph (HPLC) equipped with SC columns (125 by 4.6 mm)
filled
with 5-mm Lichrospher 100 RP8 particles (Bischoff, Leonberg,
Federal
Republic of Germany). 2,4-DNP was quantified in an isocratic
elution
program of an aqueous solvent containing 35% (vol/vol)
methanol and
0.1% (vol/vol) H
3PO
4 in Milli-Q water (flow
rate,
1 ml/min). Usually, 20-ml samples were analyzed. For the
detection
of the Meisenheimer complex of picric acid, a gradient
elution
program using 10 mM phosphate buffer, pH 6.8 (solvent A), and
acetonitrile (solvent B) was performed as follows: a flow rate
of 1 ml/min consisting of 3 ml of gradient (0 to 45% solvent B),
followed
by a 5-ml smooth gradient (45 to 100% solvent B) and
1 ml at 100%
solvent B. The column effluent was simultaneously
monitored at 210 and
270 nm with a diode array detector, and the
scans between 200 and 600 nm of the significant peaks were stored
by the
system.
Extraction and analysis of metabolites.
The cells were
cultured with 50 mM acetate and 1 mM 2,4-DNP as the carbon and nitrogen
sources, respectively. Immediately after 2,4-DNP was exhausted
completely, the cells were removed by centrifugation and the
supernatant from a 1-liter culture was acidified with HCl to reach pH
2. The acidified supernatant was extracted twice with 100 ml of diethyl
ether, and the organic phases were pooled, dried over anhydrous
Na2SO4, and concentrated at 40°C under
vacuum. The solid concentrate was dissolved in 1 ml of methanol and
chromatographed though a semi-preparative HPLC column. The effluent was
monitored at 210 nm. Fractions (1 ml each) were collected, and those
corresponding to the main HPLC peak were pooled and dried at 40°C
under vacuum. The solid was dissolved in 1 ml of deuterated methanol
and stored at 
20°C for further analysis.
The purified sample was injected directly into a Carlo Erba/MEGA SERIES
gas chromatograph (GC) equipped with a 30-m DB1 capillary
column with
helium as the carrier gas. The temperature program
was 80°C for 2 min
and then a linear gradient of 10°C per min
up to 300°C. The GC was
directly coupled to a Kratos MS 50 mass
spectrometer (MS). For electron
ionization, a potential of 70
eV was used. Dynamic high-resolution MS
of the molecular ion and
the major fragment ions was performed with
perfluorokerosene as
an internal
reference.
High-resolution one- and two-dimensional correlated spectroscopy
1H nuclear magnetic resonance (NMR) spectra were recorded
on a
Bruker WM 400 NMR spectrometer locked to the major deuterium
resonance
of the solvent, CD
3OD. Chemical shifts are given
in parts per
million relative to tetramethylsilane and constants in
Hertz.
Chemicals.
All reagents were of the maximal purity
commercially available.
| |
RESULTS AND DISCUSSION |
(i) Isolation and characterization of the strain.
A bacterium,
strain RB1, capable of degrading 2,4-DNP was isolated from activated
sludge after incubation with this compound as the sole carbon,
nitrogen, and energy source. Amplification by PCR of the 16S rRNA gene
sequence and its comparison with reference 16S rRNA sequence data
(12, 17) indicated that strain RB1 clustered
phylogenetically with species of Rhodococcus rRNA group IV
(14), particularly with the sequence of Rhodococcus
opacus (DSM 43205T) (4 nucleotide differences). The
genus Rhodococcus is the subject of intense investigation
due to its implication in the degradation of a broad range of organic
pollutants (8, 18). An independent research group using a
similar approach to enrich cultures with 2,4-DNP as the target compound
also isolated two Rhodococcus strains (11).
The biodegradative potential of
Rhodococcus sp. strain RB1
was analyzed on gradient-agar plates and in liquid cultures. In
addition to 2,4-DNP (Fig.
1),
Rhodococcus sp. strain RB1 was also
able to use acetate,
benzoate, naphthalene, 3-hydroxybenzoate,
4-hydroxy-3-methoxycinnamate (ferulate), 3,4-dihydroxybenzoate
(protocatechuate), and 4-methoxybenzoate (
p-anisate) as the
sole
carbon source (2 mM ammonium as the nitrogen source). These
chemicals
were also used by the bacterium as the sole carbon source (5 mM
concentration) in liquid culture experiments with 2 mM ammonium
as
the nitrogen source.
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FIG. 1.
Time course of cell growth, 2,4-DNP uptake, and nitrite
release by Rhodococcus strain RB1 cultured with 2,4-DNP as
the sole nitrogen source. Cells were grown under aerobic conditions
with 50 mM sodium acetate and 0.5 mM 2,4-DNP.
A600 ( ), 2,4-DNP concentration ( ), and
nitrite concentration ( ) were measured at the indicated times.
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|
The bacterium used as nitrogen sources 2,4-DNP, ammonium, nitrate, and
nitrite, with 50 mM acetate as the carbon source. Mononitrophenols
were
not used by the bacterium as either a carbon or nitrogen
source. In
contrast, both 2-nitrophenol and 4-nitrophenol strongly
inhibited
2,4-DNP mineralization by
Rhodococcus sp. strain RB1
(data
not shown), even though
Rhodococcus sp. strain RB1 partially
transformed 4-nitrophenol into 4-nitrocatechol as a dead-end product
(data not
shown).
Rhodococcus sp. strain RB1 grew aerobically in liquid
mineral medium with 2,4-DNP as the nitrogen source at concentrations
of
up to 5 mM (50 mM acetate as the carbon source). The maximal
concentration of 2,4-DNP tolerated by the bacterium under the
same
conditions with 2,4-DNP as the sole carbon and nitrogen source
was 2 mM. No growth was observed with 2,4-DNP under anaerobic
conditions.
The degradation of 2,4-DNP as the sole carbon and nitrogen source in
liquid cultures was complete and took place with an almost
stoichiometric nitrite release (1 mol of 2,4-DNP to 1.65 mol of
nitrite). The nitrite released into the medium was not taken up
by the
bacterium unless acetate was added to the carbon-starved
cells (data
not shown). The increment in optical density at 600
nm
(
A600) observed in cultures with 1 mM 2,4-DNP
as the sole
carbon and nitrogen source was 0.18 ± 0.02. The
A600 in cultures
growing with 3 mM acetate as
the carbon source and 2 mM nitrite
as the nitrogen source was 0.2 ± 0.03, thus indicating that 2,4-DNP
was fully used as a carbon source
without major energy
losses.
The utilization of 2,4-DNP as a nitrogen source by
Rhodococcus sp. strain RB1 showed a diauxic growth curve
(Fig.
1). After
an initial adaptation period (cells precultured on
acetate and
nitrite), the first phase is characterized by a minute
growth
(doubling time, approximately 21 h) and a rapid
transformation
of 2,4-DNP with simultaneous release of nitrite (almost
1 mol
of nitrite per mol of 2,4-DNP consumed). During the second phase,
the strain grew more rapidly (doubling time, approximately 11
h)
and the nitrite was taken up. Nevertheless,
Rhodococcus sp.
strain RB1 used both nitro groups as the nitrogen source. This
conclusion was substantiated by comparing the
A600 obtained with
an excess of carbon source
(50 mM acetate) and that obtained with
limiting amounts of either
2,4-DNP or equivalent amounts of nitrite
as the nitrogen source
(
A600 = 1.6 ± 0.05 units/mol of nitrite
and
A600 = 3.15 ± 0.05 units/mol of
2,4-DNP). In order to further
confirm that both nitro groups were used
by
Rhodococcus sp. strain
RB1, experiments were performed in
the presence of ammonium as
an inhibitor of nitrite assimilation
(
2). As expected, ammonium
inhibited nitrite uptake. As a
matter in fact, in the presence
of a nonlimiting concentration of
ammonium (20 mM), up to 1 mM
nitrite accumulated in the medium from 0.5 mM 2, 4-DNP (data not
shown). When limiting ammonium concentrations (2 mM) were used
in cell cultures containing 0.5 mM 2,4-DNP and 50 mM
acetate,
up to 1 mM nitrite appeared in the medium, but it was taken up
again once ammonia was completely assimilated (Fig.
2). Ammonium
did not inhibit 2,4-DNP
uptake (Fig.
2) as it does in
Rhodobacter capsulatus
(
1).
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FIG. 2.
Time course of cell growth, 2,4-DNP uptake, and nitrite
release by Rhodococcus strain RB1 cultured with 2,4-DNP and
ammonium as nitrogen sources. Cells were grown under aerobic conditions
with 50 mM sodium acetate, 0.5 mM 2,4-DNP, and 2 mM ammonium chloride.
At the times indicated, A600 (), 2,4-DNP
concentration (), and nitrite concentration () were measured. The
arrow indicates the time at which the concentration of ammonium in the
medium was undetectable.
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|
(ii) Degradation pathway of 2,4-DNP by Rhodococcus sp.
strain RB1.
An initial reductive pathway has been demonstrated in
some gram-positive bacteria that degrade nitroaromatic compounds
(4). The transfer of a hydride to the aromatic ring of
picric acid and 2-chloro-4,6-DNP provokes the release of the 2-nitro or
2-chloro group with the concomitant formation of 2,4-DNP (9,
10). We have also observed the formation of 2,4-DNP from picric
acid in anaerobic resting-cell experiments with Rhodococcus
sp. strain RB1 (data not shown), thus suggesting a similar reaction. In
addition, Rhodococcus sp. strain RB1 cells induced with
2,4-DNP produced a brilliant red compound when transferred to medium
with picric acid (0.5 mM), indicating the putative formation of a
Meisenheimer complex. The formation of this compound from picric acid
was unambiguously demonstrated in R. erythropolis HL 24-2 (9), and therefore, we have used it as the reference strain.
The product formed by both R. erythropolis HL 24-2 and
Rhodococcus sp. strain RB1 from picric acid coeluted during
HPLC analysis and exhibited the same UV-visible light spectrum (data
not shown). As mentioned above, the hydrogenation of the aromatic ring
may result in the rearomatization and concomitant liberation of nitrite
or chloride (9, 10). Nevertheless, it is unlikely that this
mechanism (rearomatization) accounts for the degradation of 2,4-DNP by
Rhodococcus sp. strain RB1, since it should yield a
mononitrophenol derivative (probably 4-nitrophenol) which cannot be
mineralized by the bacterium. In spite of that, this mechanism cannot
be excluded in other bacteria, and in fact, 4-nitrophenol has been
detected very recently in small amounts during the degradation of
picric acid by Nocardioides simplex (16).
The ratio of nitrite released to 2,4-DNP consumed showed a
stoichiometry of 1/1 instead of 2/1 during the first growth phase
of
Rhodococcus strain RB1 with 2,4-DNP as the nitrogen source
(Fig.
1). As very little growth had occurred at the end of this
growth
phase, it can be assumed that the second nitrogen is bound
to an
intermediate. However, analysis of the supernatants of the
culture
medium from this phase by HPLC showed only minute peaks
absorbing at
210 nm. To amplify the signals, these supernatants
were extracted and
concentrated as described in Materials and
Methods. A complex mixture
of compounds absorbing at 210 nm was
detected by HPLC. The main
compound (approximately 70% in relative
units, at 5 min net retention
time) was purified by preparative
HPLC and showed a single absorption
maximum at 204 nm. The product
contained in this fraction was
identified as 3-nitroadipate dimethyl
ester according to
GC-high-resolution MS analysis (Table
1).
Nevertheless, we assume that a
partial dimethylation of the chemical
occurred due to the heating in
the presence of methanol during
the concentration step (see Materials
and Methods). This assumption
is based on the need for acidic
conditions (pH 2) for the full
extraction of the chemical from the
supernatant. In addition,
the
1H-NMR spectrum of the
extracted compound corresponded to that
of the free acid (Table
2) with a minute amount of the diester
(estimated to be around 2% according to the two singlet signals
at
3.71 and 3.73 ppm corresponding to the methyl groups), even
though, as
the free acid is not volatile, only the diester was
detected by GC-MS.
The 3-nitroadipate is obviously more oxidized
than the proposed initial
intermediate (Meisenheimer complex).
The nature of the oxidant is
unknown, but we suggest that it should
be O
2. The reason is
that, under anaerobic conditions,
R. erythropolis accumulates mainly 4,6-dinitrohexanoate from 2,4-DNP (
11) or
1,3,5-trinitropentane from picric acid (
9). The formation of
4,6-dinitrohexanoate may be achieved by the hydration of the double
bond of a putative dihydro-2,4-DNP intermediate, whereas the
dioxygenolytic
cleavage of the double bond would produce 3-nitroadipate
(Fig.
3).
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FIG. 3.
Proposed pathway for the degradation of 2,4-DNP by
Rhodococcus. Compounds in brackets are hypothetical
intermediates. The formation of 4,6-dinitrohexanoate has been described
previously (12). TAC, tricarboxylic acid cycle.
|
|
These results may be summarized in the degradation pathway depicted in
Fig.
3. We propose that 2,4-DNP mineralization by
Rhodococcus takes place by a metabolic pathway including
three different phases:
(i) reduction of the aromatic ring, as
previously demonstrated
(
10-12), by two successive hydride
transfers; (ii) aerobic
ortho ring fission, production of
3-nitroadipate, and concomitant release
of the
ortho nitro
group as nitrite (nevertheless, the reductive
formation of
4,6-dinitrohexanoate [
11] and subsequent oxidation
to
3-nitroadipate cannot be excluded); and (iii) further metabolism
of
3-nitroadipate with release of a second mole of
nitrite.
Experiments are in progress to determine if enzymes of the 3-oxoadipate
pathway, actually involved in benzoate metabolism
by
Rhodococcus strain RB1 (data not shown), are also involved
in the metabolism of 3-nitroadipate by this
bacterium.
| |
ACKNOWLEDGMENTS |
We thank the Spanish Dirección General de
Investigación Científica y Técnica (DGICYT grant
PB95 0554 CO2 02) and Plan Andaluz de Investigación for financial
support. Postdoctoral fellowships from the Ministerio de
Educación y Ciencia and European Environmental Research
Organization to R.B. and from Alexander von Humboldt to F.C. are also
gratefully acknowledged.
We also thank Rolf Wittich for critically reading the manuscript, R. Blanco for his help in HPLC analysis, and Manfred Nimtz for performing
GC-MS analysis. We also thank H.-J. Knackmuss for kindly supplying
R. erythropolis HL 24-2.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Bioquímica, Facultad de Ciencias, Avenida San Alberto Magno
s/n, 14071 Cordoba, Spain. Phone: 34 57218592. Fax: 34 57218606. E-mail: bb1blplr{at}uco.es.
| |
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Journal of Bacteriology, January 1999, p. 149-152, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
