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Applied and Environmental Microbiology, April 2001, p. 1412-1417, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1412-1417.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Long-Range 1H-15N
Heteronuclear Shift Correlation at Natural Abundance: a Tool To Study
Benzothiazole Biodegradation by Two Rhodococcus
Strains
Pascale
Besse,1
Bruno
Combourieu,1
Gaëlle
Boyse,1
Martine
Sancelme,1
Heleen
De
Wever,2 and
Anne-Marie
Delort1,*
Laboratoire de Synthèse et Etude de
Systèmes à Intérêt Biologique, UMR 6504 du
CNRS, Université Blaise Pascal, 63177 Aubière Cedex,
France,1 and Laboratory for Soil
Fertility and Soil Biology, Katholieke Universiteit Leuven, 3001 Heverlee, Belgium2
Received 24 October 2000/Accepted 8 January 2001
 |
ABSTRACT |
The biodegradation of benzothiazole and 2-hydroxybenzothiazole by
two strains of Rhodococcus was monitored by reversed phase high-pressure liquid chromatography and by 1H nuclear
magnetic resonance (NMR). Both xenobiotics were biotransformed into a
hydroxylated derivative of 2-hydroxybenzothiazole by these two strains.
The chemical structure of this metabolite was determined by a new NMR
methodology: long-range 1H-15N heteronuclear
shift correlation without any previous 15N enrichment of
the compound. This powerful NMR tool allowed us to assign the
metabolite structure to 2,6-dihydroxybenzothiazole.
| |
INTRODUCTION |
Benzothiazoles (BTs) are a family of
xenobiotics widely used in a variety of industrial products and
processes, such as pharmaceuticals, herbicides, fungicides, slimicides
in the paper and pulp industry, antialgal agents, and, mainly,
catalysts in the vulcanization process in rubber. BTs have been
detected not only in industrial wastewater but also in various
environmental compartments (15, 27) and are of concern for
aquatic environment due to their limited biodegradability and potential
toxicity (18). To date, only a few bacterial isolates have
been shown to degrade some BTs as pure culture. These all belong to the
Rhodococcus genus (8, 9, 17). However, the
biodegradative pathways in these bacteria remain poorly known. De Wever
et al. (8, 9) studied the biodegradation of BT and
2-hydroxybenzothiazole (OBT) by two Rhodococcus isolates
(Rhodococcus erythropolis and Rhodococcus rhodochrous) by using high-pressure liquid chromatography (HPLC) with a UV detector and mass spectrometry. A common intermediate was
detected and identified as dihydroxybenzothiazole (diOHBT), but its
chemical structure could not be determined precisely (8). This prompted us to develop a new methodology in order to determine the
structure of this metabolite, with our final goal being to use this
tool more generally to study other benzothiazole derivatives.
Nuclear magnetic resonance (NMR) spectrometry is a powerful tool used
to determine chemical structures and has been used, for example, to
study the biodegradation of xenobiotics by microorganisms (7). However, most of the studies rely on specific
enrichment of the compounds (e.g., 2H, 13C,
15N, and 19F). To overcome the difficulty of
labeled-compound synthesis and also to increase the sensitivity of
detection of metabolites, in situ 1H NMR has been recently
developed that allows the study of molecules at natural abundance
directly on the incubation medium without any previous purification
step. This technique was successfully applied to study microbial
metabolism (3) and, more specifically, to elucidate
biodegradative pathways (2, 4-6, 16, 26). In this study
we used 1H NMR to study the biodegradative pathway of BT
and OBT by R. erythropolis and R. rhodochrous.
Though this approach led to the characterization of some structural
elements, it was not sufficient to assign unambiguously the position of
the hydroxyl group on the benzene ring of the metabolite. To go
further, we had to use long-range 1H-15N
heteronuclear shift correlation by using GHMBC (gradient heteronuclear multiple-bond correlation) experiments at natural abundance. Although a
large number of natural compounds and also pharmaceuticals and organic
pollutants contain N atoms, only a few studies have been reported thus
for that take advantage of the valuable information contained in
1H-15N scalar couplings. This can be easily
explained by the difficulty of detecting the 15N nucleus
because its sensitivity is very low due to a low natural abundance
(0.37%) and a low gyromagnetic ratio. HMBC techniques (inverse-detection methods) have been used to measure long-range 1H-13C heteronuclear shift correlations
(1) for the last 15 years but could not be applied to
1H-15N correlations at natural abundance until
the 1995s, when spectrometers were equipped with gradients. Gradients
greatly improved the sensitivity of detection for various reasons: (i)
they suppress t1 noise and so improve the
signal-to-noise ratio, (ii) they eliminate time-consuming phase cycling
as a result of coherence pathways selection, and (iii) they can be used
for solvent suppression. Other technical improvements were reached by
using specific probes or new NMR sequences. More details are available
in a recent and very interesting review (24) about the
application of this technique to the determination of natural product
structure, particularly of alkaloids. One- and two-dimensional
inverse 1H-15N experiments have been used to
detect 15N-enriched metabolites, mainly amino acids in
mammalian (21, 28), insect (11, 12), and
plant (25) cells. To our knowledge, this approach has
never been applied in the field of biodegradation of xenobiotics by
microorganisms. The present study is thus the first example of the
determination of the structure of a metabolite issued from xenobiotic
biodegradation using long-range 1H-15N
heteronuclear shift correlation at natural abundance.
| |
MATERIALS AND METHODS |
Chemicals.
BT and OBT were purchased from Aldrich.
Tetradeuterated sodium trimethylsilylpropionate (TSPd4) was
purchased from Eurisotop (Saint Aubin, France).
Growth conditions.
R. erythropolis and R. rhodochrous were grown in 100-ml portions of Trypticase soy broth
(bioMérieux, Marcy l'Etoile, France) in 500-ml Erlenmeyer flasks
incubated at 30°C at 200 rpm. The cells were harvested after 20 h of culture.
Incubation with xenobiotic compounds.
Cells were centrifuged
at 9,000 × g for 15 min at 5°C. The pellet was
washed twice with Knapp buffer (K2HPO4, 1 g/liter; KH2PO4, 1 g/liter; FeCl3 4 mg/liter; MgSO4 · 7H2O, 40 mg/liter [pH
6.7]) and finally resuspended in this buffer (5 g of wet cells in 100 ml of buffer). The resting cells were incubated with 3 mM BT or OBT in
500-ml Erlenmeyer flasks at 30°C under agitation (200 rpm). The
negative controls consisted of preparations incubated under the same
conditions without a substrate or cells. Samples (1 ml) were taken
every 30 min directly in the culture medium. They were centrifuged at
12,000 × g for 5 min and prepared for HPLC analysis or
for 1H NMR analysis.
HPLC analyses.
HPLC analyses were performed using a Waters
600E chromatograph fitted with a reversed-phase column (Interchrom
Nucléosil C18; 5 µm, 250 by 4.6 mm; Interchim) at
room temperature. The mobile phase was acetonitrile-water (20/80, by
volume), with a flow rate of 1 ml/min. Detection was performed with a
Waters 486 UV detector set at 295 nm.
1H-NMR spectroscopy.
The preparation of NMR
samples and the methods for performing a spectrum on a Bruker Avance
300 spectrometer at 298 K using 5-mm-diameter tubes have already been
described (4), as was the method for quantification of the metabolites.
1H-15N GHMBC experiments.
The GHMBC
experiments were performed on a Bruker Avance 300 spectrometer at 298 (±0.2) K. A 5-mm triple-tuned
1H-13C-15N probe equipped with a
z-gradient coil was used. 1H and 15N 90°
pulse lengths were 7.5 and 27 µs, respectively. Spectral widths were
adjusted in both dimensions to encompass all 1H and
15N signals. Delay to allow
nJNH correlations was set to 80 or
140 ms. The responses of 32 scans for each of 128 t1 increments were acquired. Zero-filling and
phase-shifted sine window function in t1 and
sine-squared window function in t2 were applied
prior to two-dimensional Fourier transformation. A recycle delay of
1.5 s was used.
Ammonia and sulfate measurements.
Weatherburn's method
(30) was used to assay for ammonia. To 100 µl of the
sample was added successively 500 µl of solution A (phenol, 1 g;
sodium nitroprusside [Na2(Fe(CN)5NO),
2H2O], 5 mg; distilled water, 100 ml) and 500 µl of
solution B (NaOH, 0.5 g; sodium hypochlorite, 0.8 ml; distilled
water, 100 ml). The mixture was stirred vigorously and put for 20 min
in a water bath at 37°C. A blue coloration appeared, and the
absorbance was measured at 625 nm. A standard curve was plotted using
standard solutions of ammonium sulfate in Knapp buffer.
Sulfate was measured according to the method of Mainprize et al.
(23). To 950 µl of the sample was added 50 µl of
"developer solution" containing NaCl (7.5 g), ethanol (10 ml)
concentrated HCl (3 ml), glycerol (5 ml), and distilled water (30 ml).
BaCl2 (5 mg) was then added, and the solution mixed
thoroughly for 1 min. The turbidity of the BaSO4
suspension, stabilized in the developer solution, was measured at 540 nm. A standard curve was plotted using standard solutions of ammonium
sulfate in Knapp buffer.
Isolation of the unknown metabolite.
For the isolation of
the unknown metabolite, a resting-cell experiment with R. erythropolis was performed as described above but on a larger
scale. R. erythropolis (45 g) was incubated in 900 ml of
Knapp buffer with 3 mM OBT (nine Erlenmeyer flasks). The biodegradation
kinetics was monitored by HPLC in order to stop the experiment at the
maximum concentration in metabolite. After 2 h of incubation
(metabolite concentration of 0.5 mM), the reaction mixture was
centrifuged, and the supernatant was extracted with ethyl acetate for
24 h. The organic layer was dried on MgSO4,
concentrated under vacuum, and purified over a silica gel column
(eluent, ethyl acetate-chloroform [30/70]). The pure metabolite was
obtained as a pale yellow solid (melting point of 219 to 221°C). In
order to determine the structure of this compound, it was analyzed by
mass spectroscopy (Electronic Impact) on a Hewlett-Packard MS 5989B spectrometer.
| |
RESULTS AND DISCUSSION |
In order to confirm the biodegradative pathways of BT and OBT by
R. rhodochrous and R. erythropolis that were
described previously (8) and to demonstrate the formation
of other metabolites that were not detected by HPLC with a
monochromatic UV detector, new assays of biodegradation were performed
using 1H NMR spectroscopy. The biodegradation conditions
were slightly modified; in particular, the xenobiotic concentration was
increased (3 mM instead of 1 mM). The samples were analyzed in parallel by inverse-phase HPLC and by 1H NMR as previously described
(4).
Biodegradation of BT and OBT by R. erythropolis.
The biodegradation of BT (3 mM) by resting cells of R. erythropolis was monitored by both analytical methods used, i.e.,
HPLC and 1H NMR. After 30 min of incubation, a first
metabolite was detected. Two 1H NMR spectra recorded after
0 and 1.25 h of incubation are shown in Fig.
1. By studying the 1H NMR
spectra obtained at different times, we could observe the signals of BT
(7.57 [t], 7.65 [t], 8.14 [2xd], and 9.28 [s]) decreasing and
new signals appearing in the aromatic region, i.e., two triplets at
7.24 and 7.38 ppm and two doublets at 7.29 and 7.58 ppm. They were
assigned to OBT by adding the commercially available compound to the
sample. This was also confirmed by HPLC (coelution of the OBT
standard). After BT exhaustion, another metabolite appeared on the HPLC
chromatogram. Its retention time (7 min) was shorter than that of OBT
(25 min), indicating a higher polarity of this compound. Using
1H NMR, we could observe the disappearance of OBT signals.
However, no signal corresponding to the new metabolite observed by HPLC could be detected due to the lower sensitivity of NMR.
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FIG. 1.
1H NMR spectra of the samples taken after 0 and 1.25 h of incubation of BT (3 mM) with resting cells of
R. erythropolis.
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|
The use of standard curves for the identified compounds in HPLC and the
addition of a reference in the sample (TSPd
4 solution
of
known concentration in D
2O) allowed the quantification of
each
compound. (For the metabolite, the quantitative curves, for both
methods, were plotted only after its isolation and the determination
of
its chemical structure.) Figure
2 shows
the time courses of
the concentration of BT and its metabolites with
R. erythropolis,
as analyzed by HPLC and
1H NMR.
The results obtained by both techniques are very similar.
Both methods
are quantitative.
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FIG. 2.
Time courses of the concentration of BT ( ), OBT
( ), and the unknown metabolite ( ) during the biodegradation of BT
(3 mM) by resting cells of R. erythropolis. A comparison of
the results obtained by HPLC (left) and by 1H NMR (right)
is shown. Similar results were obtained in three independent
experiments.
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|
With this strain, BT was quantitatively transformed into OBT, which was
then converted to an unknown, more-polar metabolite.
This one, whose
highest concentration reached 0.6 mM after 2 h
30 min, was then
degraded in its turn within 6 h of
incubation.
The degradation of OBT (3 mM) by
R. erythropolis was also
studied by both analytical methods: HPLC and
1H NMR (data
not shown). OBT was completely degraded within 5 h.
The sole
metabolite, observed only by HPLC, presented the same
retention time (7 min) as the compound formed during the biodegradation
of BT.
Coinjection of samples issued from both of these biodegradations
showed
only one peak on the HPLC chromatogram whatever the elution
conditions.
The maximum concentration in metabolite (0.54 mM)
was obtained after
3 h of
incubation.
Since the metabolite has completely disappeared in its turn within
6 h, we wanted to know if mineralization had occurred.
Preliminary
assays to determine the production of ammonia and
sulfate were carried
out using spectrophotometric means according
to the methods of
Weatherburn (
30) and Mainprize et al. (
23),
respectively. The stoichiometry for the degradation of OBT and
ammonia
and sulpfate production obtained was 1/0.6/0.4 (theoretical
stoichiometry = 1/1/1). A very similar ratio was obtained during
BT degradation. The values obtained are lower than those expected
for a
complete mineralization, but this is generally the case
in such
experiments (
23), in particular because nitrogen and
sulfur can be either assimilated by the cells or discharged in
a
different form than NH
4+ and
SO
42
(
10).
The BT and OBT degradation pathways are closely related and go through
the same unknown, more-polar metabolite. In both cases,
the medium
turned
black.
Biodegradation of BT and OBT by R. rhodochrous.
The kinetics of BT and OBT biodegradation observed with R. rhodochrous showed a different behavior for this strain. No OBT was detected during the BT biodegradation whatever analytical method
was used. Only the unknown metabolite was observed. Some hypotheses can
be proposed: either the life span of OBT in this case is too short (its
rate of biodegradation being very high), its concentration remains too
low to be detected even by HPLC, or R. rhodochrous follows a
different pathway, converting BT directly into the unknown metabolite.
Several experiments of HPLC coinjection of different samples obtained
with both strains and/or with both xenobiotics (BT and OBT) gave
evidence that the metabolite observed was always the same one.
The time courses of BT, OBT, and their metabolite concentrations
measured by HPLC are presented in Fig.
3.
The biodegradative
rates of BT and OBT with
R. rhodochrous
were much slower than
those observed with the other strain: the
complete disappearance
of the xenobiotic was observed after 8 h of
incubation for BT
(1.5 h for
R. erythropolis) and after
10 h for OBT (5 h for
R. erythropolis). Moreover, with
this strain the metabolite concentration
remained very low throughout
the biodegradation (<0.2 mM).
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FIG. 3.
Biodegradation of 3 mM BT (left) or 3 mM OBT (right) by
R. rhodochrous. Symbols: , BT; , OBT; , unknown
metabolite.
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|
The stoichiometry for the degradation of OBT and ammonia and sulfate
production obtained was 1/0.3/0.3 (theoretical stoichiometry
= 1/1/1). A very similar ratio was obtained during BT degradation.
As
observed previously with
R. erythropolis, the values
obtained
are lower than those expected for a complete
mineralization.
Determination of the chemical structure of the unknown
metabolite.
In order to determine the chemical structure of the
unknown metabolite obtained during the BT and OBT degradation, a
quantitative assay was carried out with R. erythropolis,
this strain giving the highest concentration in metabolite. R. erythropolis (45 g) was incubated in 900 ml of Knapp buffer with 3 mM OBT. The biodegradation kinetics was monitored by HPLC in order to
stop the experiment at the maximum concentration in metabolite (0.5 mM
after 2 h of incubation). Extraction of the supernatant with ethyl
acetate, followed by purification over a silica gel column, yielded the pure metabolite as a pale yellow solid.
We checked that the isolated product corresponded to the unknown
metabolite observed previously by coelution with a sample
taken during
the degradation of OBT and also by thin-layer chromatography.
The
purified product was first analyzed by
1H NMR in
CD
3OD. Only three signals are visible in the aromatic
region, each one corresponding to an equivalent number of protons
(Fig.
4). So a substituent was present on the
aromatic ring. By
analyzing the coupling constants, we could deduce
that the substituent
was either at position 5 or position 6 (Fig.
5). Indeed, the doublet
at 6.97 ppm
presented a coupling constant of 8.5 Hz (
3J), meaning that
this proton was coupled with another proton away
from three bonds. The
same coupling constant was observed on the
signal at 6.74 ppm (dd).
This proton was then not only coupled
with the doublet at 6.97 ppm but
also coupled with the proton
at 6.89 ppm, with a small coupling
constant (
4J = 2.6 Hz) corresponding to a four-bond
coupling. All of this
information showed that the substituent was
either at position
5 or position 6. However, the assignment of protons
4 and 7 was
not possible because of the difficulty of knowing the
electronic
effects of the nitrogen and the sulfur atoms on these
protons.
Neither a semiempirical approach nor ab initio calculations
are
precise enough to determine nuclear shielding constant within
0.5 ppm (
14).
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FIG. 4.
1H-15N GHMBC spectra of OBT
metabolite recorded in CD3OD; the experiments were set up
for assumed 3J (delay = 80 ms) (a) or 4J
(delay = 140 ms) (b) long-range couplings.
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Another method to get information on the metabolite structure is mass
spectrometry (Fig.
5). The mass spectrum of the isolated
compound
presented a peak with a mass/charge ratio of 167, differing
from the
molecular weight of OBT by 16. Thus, the metabolite formed
during the
biodegradation of BT and OBT is a hydroxylated derivative
of the
substrate
OBT.
To assign unambiguously the position of the hydroxyl group on the
benzene ring of the metabolite, long-range
1H-
15N heteronuclear shift correlation and,
more precisely GHMBC were
used. Among the new sequencing methods
available recently (
24),
the choice of the GHMBC
experiment (
20) was guided by the ease
in setting up this
kind of experiment and by its high distribution
on most commercial
spectrometers. In the scheme of the pulse program
(for details, see
reference
24) a variable delay is included
between the two
first 90°
15N pulses. This evolution period allows the
system to be converted
into zero and double quantum coherence and is
proportional to
1/(2 ×
nJ
15N-
1H).
Thus, this delay must be optimized in order to select
2J,
3J, or
4J couplings.
2J and
3J are generally in the range of 5 to 10 Hz, while only few
precise
data are available for
4J. We can only estimate
that such values are in the range of 0.5
to 4 Hz (
24).
Another point of interest is that the desired
coupling constant can be
overestimated (until 30%) without important
loss of information.
Usually, the
4J
NH are difficult to observe in
nonaromatic systems since the
corresponding coupling could be very
weak. In our case, the delay
to allow
nJ
NH correlations was set to 80 and
140 ms in order to select assumed
3J
NH (6.2 Hz)
and
4J
NH (3.5 Hz),
respectively.
As chemical shift is concerned, since there is not a single chemical
shift referencing, we used CH
3NO
2 as an
external reference
(0 ppm) and more precisely enriched urea (for
practical convenience)
calibrated at
306 ppm from nitromethane
(
13). The chemical
shifts are reported negative upfield
from CH
3NO
2 resonance.
Figure
4a represents the
1H-
15N GHMBC recorded
with an 80-ms evolution period. On this spectrum, only one correlation
can be
observed with the doublet at 6.97 ppm. Thus, this proton can be
assigned to H4, since no other
3J can be observed in this
compound. In Fig.
4b, the delay fixed
at 140 ms (while other conditions
remain the same) allows the
observation of a
4J coupling
between nitrogen and the doublet of doublets resonating
at 6.73 ppm.
This proton can be assigned to H5 because of its
8.5-Hz
(
3J) coupling constant with H4. No doubt exists concerning
the attribution
of the substituent in position 6. So the unknown
metabolite is
2,6-dihydroxybenzothiazole (diOBT), corresponding to a
hydroxylation
in position 6 of OBT (Fig.
5).
The same sequence of experiments (Fig.
5) was carried out starting from
a quantitative assay with
R. erythropolis and led
to an
identical structure for the metabolite, observed during
the
biodegradation of BT and OBT with this
strain.
The case of structural determination solved in these experiments using
1H-
15N GHMBC is quite ideal for two reasons:
(i)
4J
NH could be observed easily (the coupling
constant seems to be
relatively important) without looking for several
delays and (ii)
no
2J couplings exist in this compound,
which avoids confusion between
two- and three-bond correlations
(indeed,
2J and
3J are usually of the same
order).
Even if it would be the case, some solutions have been developed
recently. Concerning the observation of
4J
NH,
ACCORD-HMBC (
29) and IMPEACH-MBC (
19) offer
the capability
of surveying broad ranges of potential long-range
couplings in
a single experiment. The second point mentioned above
could be
circumvented by the use of the
2J,
3J-HMBC experiment (
22), which
is capable of differentiating
two-bond from three-bond couplings.
However, this sequence was
checked only for
1H-
13C
correlations.
One last point is the development of cold metal or superconducting coil
NMR probes, which allow the detection of smaller quantities
with an
increased signal-to-noise ratio. This technical aspect
will certainly
be one of the most important improvements of the
15N
detection method in the
future.
In conclusion, the complementarity of a classical analytical method
such as HPLC equipped with a UV detector, which has a
great
sensitivity, and
1H NMR carried out directly on the culture
medium without any purification
allowed us to confirm structural
information previously obtained
by De Wever et al. (
8).
With
R. erythropolis, BT is first oxidized
into OBT, which
is further transformed into diOBT (Fig.
6); the
same metabolite is obtained when
OBT is incubated with the cells.
With
R. rhodochrous, the
biodegradative pathways of BT and OBT
are very similar, except that OBT
was not detected during the
BT degradation. The determination of the
exact chemical structure
of the metabolite formed was possible by using
long-range
1H-
15N heteronuclear shift
correlation at natural abundance. The hydroxyl
group was shown to be at
the C6 position on the benzene ring (Fig.
6). diOBT is a common
intermediate to OBT and BT degradation by
both strains of
Rhodococcus. This metabolite was then degraded
quite
rapidly. This result was confirmed by direct incubation
of diOBT (1.7 mM) with resting cells of
R. rhodochrous. It was
completely
degraded within 4 h of incubation, and no other metabolite
was
detected. However, further experiments are still needed in
order to
understand which mechanism underlies this initial oxidation.
The major interest of our work is to show that long-range
1H-
15N heteronuclear shift correlation can be
used to study metabolites
without any previous
15N
enrichment of the starting xenobiotic (at natural abundance).
Xenobiotics that contain N nuclei are numerous, so this tool could
be
developed in the future in the environmental
field.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Synthèse et Etude de Systèmes à Intérêt
Biologique, UMR 6504 du CNRS, Université Blaise Pascal, 63177 Aubière Cedex, France. Phone: 33-4-73-40-77-14. Fax:
33-4-73-40-77-17. E-mail:
amdelort{at}chimtp.univ-bpclermont.fr.
| |
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Applied and Environmental Microbiology, April 2001, p. 1412-1417, Vol. 67, No. 4
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.4.1412-1417.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
