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Applied and Environmental Microbiology, July 2001, p. 3134-3139, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3134-3139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Nuclear Magnetic Resonance Analysis of
[1-13C]Dimethylsulfoniopropionate (DMSP) and
[1-13C]Acrylate Metabolism by a DMSP Lyase-Producing
Marine Isolate of the 
-Subclass of
Proteobacteria
John H.
Ansede,1,
Perry J.
Pellechia,2 and
Duane C.
Yoch1,*
Department of Biological
Sciences1 and Department of Chemistry
and Biochemistry,2 University of South
Carolina, Columbia, South Carolina 29208
Received 15 March 2001/Accepted 8 May 2001
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ABSTRACT |
The prominence of the 
-subclass of Proteobacteria in
the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of
dimethylsulfoniopropionate (DMSP) metabolism in a representative
isolate of this phylotype, strain LFR. [1-13C]DMSP was
synthesized, and its metabolism and that of its cleavage product,
[1-13C]acrylate, were studied using nuclear magnetic
resonance (NMR) spectroscopy. [1-13C]DMSP additions
resulted in the intracellular accumulation and then disappearance of
both [1-13C]DMSP and
[1-13C]
-hydroxypropionate
([1-13C]-HP), a degradation product. Acrylate, the
immediate product of DMSP cleavage, apparently did not accumulate to
high enough levels to be detected, suggesting that it was rapidly
-hydroxylated upon formation. When [1-13C]acrylate was
added to cell suspensions of strain LFR it was metabolized to
[1-13C]-HP extracellularly, where it first accumulated
and was then taken up in the cytosol where it subsequently disappeared,
indicating that it was directly decarboxylated. These results were
interpreted to mean that DMSP was taken up and metabolized by an
intracellular DMSP lyase and acrylase, while added acrylate was
-hydroxylated on (or near) the cell surface to -HP, which
accumulated briefly and was then taken up by cells. Growth on acrylate
(versus that on glucose) stimulated the rate of acrylate metabolism
eightfold, indicating that it acted as an inducer of acrylase activity.
DMSP, acrylate, and -HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the
pathway of DMSP and acrylate metabolism in the -proteobacterium, strain LFR.
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INTRODUCTION |
Dimethylsulfoniopropionate (DMSP) is
an abundant sulfonium compound in marine environments (1, 30,
33), where it appears to function as a compatible solute for
osmoregulation in marine algae and phytoplankton (10, 11,
34). DMSP is released into the water column or sediment pore
water by autolytic processes related to algal senescence or zooplankton
predation and by leaching from zooplankton fecal pellets (8, 23,
27, 31). The enzymatic cleavage of DMSP by DMSP lyase in some
microbial species results in the production of dimethylsulfide (DMS)
and acrylate (5, 7, 9, 18, 22), while other species
demethylate it to methyl-3-mercaptopropionate and mercaptopropionate
(21, 22, 32). The positive correlation between chlorophyll
a and bacterial numbers in DMSP-producing phytoplankton
blooms (4, 13, 29, 35), where intracellular DMSP can range
from 0.01 to >100 mM (17), suggests that DMSP and
acrylate could be an important carbon source for bacterioplankton. The
accumulation of 1 to 7 mM acrylate in the colony mucus of
Phaeocystis spp. (28) is another source that would be
readily available at the time of senescence to microbes in the
vicinity. The products of DMSP metabolism are all substrates for
various marine bacteria (3, 16, 19, 20, 28a, 32, 37).
Understanding the biochemistry of DMSP metabolism has resulted from
work with anoxic sediments, anaerobic isolates (20, 36,
38) and, more recently, aerobic marine (3, 12, 24, 40) and freshwater (D. C. Yoch, R. N. Hardee, R. Friedman, and N. Kulkarni, submitted for publication) isolates. The
most detailed analysis of DMSP metabolism has been on the salt marsh
isolate, Alcaligenes faecalis strain M3A. Its metabolism of
DMSP to acrylate and DMS and of acrylate to -hydroxypropionate
(-HP)
(HOCH2CH2CO2
) all
occurs on the cell surface, with only the -HP being transported into
the cytoplasm, where it serves as an energy source and inducer of DMSP
lyase and acrylase (3). While there is no evidence that
the -subclass of Proteobacteria
(-Proteobacteria; like A. faecalis) are major
players in DMS production (2), this process is common
among Roseobacter isolates, an abundant subclass of the
-Proteobacteria in marine surface waters (12, 13, 14). Since DMSP metabolism is not well understood in this
prominent group of DMS producers, it was the goal of this work to
analyze this process in an isolate from this group; strain LFR, which has a cytosolic DMSP lyase (26), was chosen.
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MATERIALS AND METHODS |
Growth and preparation of cell suspensions.
Strain LFR was
grown in 50-ml batch cultures of seawater-based f/2 medium
(15) supplemented with either glucose or acrylate (5 mM)
as the sole carbon and energy source. Cultures were incubated on a
rotary shaker (140 rpm) for 24 h at 30°C, harvested by
centrifugation (11,000 × g for 10 min), and
resuspended in half the volume of filter-sterilized seawater, and 10-ml
aliquots were placed in 36-ml glass serum bottles. Cultures were
allowed to equilibrate for 30 min at 100 rpm prior to the addition of
DMSP or acrylate. To monitor the metabolism of DMSP to acrylate and the
disappearance of the latter from the medium, cells were removed at
various time intervals and centrifuged in an Eppendorf Microfuge for 1 min, and the supernatant was analyzed by high-performance liquid
chromatography as described previously (3). DMSP,
acrylate, and -HP were tested as inducers of DMSP lyase in strain
LFR by adding various concentrations of these putative inducers to
glucose-grown cell suspensions. Aliquots were removed at 2-h intervals,
microcentrifuged for 30 s, and resuspended in an equal volume of
seawater to which the putative inducer was added. DMSP metabolism was
monitored by gas chromatography, and the rate of DMS production by the
cells removed at each time point was assumed to be proportional to the extent of DMSP lyase induction. Data presented below in Results are
representative of at least two experiments.
NMR analysis of [1-13C]DMSP and
[1-13C]acrylate metabolites.
[1-13C]DMSP was synthesized as described previously
(6) using [1-13C]acrylate in place of
unlabeled acrylate. Nuclear magnetic resonance (NMR) analysis
determined the [1-13C]DMSP preparation to be >95% pure,
and the concentrations of the reactants (DMS and acrylate) in the
product were below detection limits. To have a basis for identifying
labeled DMSP metabolites in the cells, the gradient-enhanced
1H 13C heteronuclear multiplequantum coherence
(1H{13C} HMQC) spectrum of the
[1-13C]DMSP standard was analyzed. It showed one
13C resonance at 177 ppm which had two 1H
correlations at 3.38 and 2.65 ppm, which were indicative of two
methylene groups adjacent to the 1-13C-labeled carboxyl
group of DMSP (data not shown).
To determine the pathway of [1-13C]DMSP and
[1-13C]acrylate metabolism by using NMR analysis, cells
were grown for 24 h in 250 ml of f/2 medium supplemented with
acrylate (final A600 
0.32), harvested
by centrifugation, resuspended in 12 ml of filter-sterilized seawater,
and placed in a 64-ml glass serum bottle. The cells were incubated at
140 rpm for 30 min prior to the addition of 5 mM
[1-13C]DMSP or [1-13C]acrylate. After the
addition of 13C-labeled DMSP or acrylate, the metabolites
were identified by removing 1-ml aliquots of the cell suspensions at
0.5-h (for DMSP) or 5-min (for acrylate) time intervals and
centrifuging them in an Eppendorf Microfuge for 2 min. The supernatant
(the extracellular medium) was separated from the cell pellet and
immediately frozen until needed for NMR analysis. The cell pellet was
washed once with seawater, and the intracellular constituents were
extracted in 1 ml of 100% ethanol by gentle agitation overnight at
room temperature on a rotary shaker. The mixture was then centrifuged for 2 min to remove all nonsoluble debris. The ethanol extract was
dried in a rotary evaporator for 2 h and resuspended in 1 ml of
deuterium oxide for NMR analysis. NMR data on
[1-13C]DMSP, [1-13C]acrylate, and their
metabolic product(s) were collected on a Varian Unity Inova 500 NMR
spectrometer as described previously (3).
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RESULTS |
DMSP and acrylate metabolism.
The metabolism of DMSP and
acrylate by cell suspensions of strain LFR grown in f/2 medium
supplemented with either glucose or acrylate is presented in Fig. 1A
and B, respectively. DMSP metabolism was
determined by measuring DMS production, while acrylate metabolism was
measured by following its disappearance from the extracellular medium.
The addition of DMSP (500 µM) to suspensions of either glucose- or
acrylate-grown cell suspensions resulted in a low level of DMS
production followed by a rapid increase in this rate. The DMSP added at
time zero served as the substrate for DMSP lyase, as evidenced by DMS
released in the gas phase; however, acrylate could not be detected in
the extracellular medium of cells grown in either glucose or acrylate,
suggesting that acrylate was sequestered inside the cell.
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FIG. 1.
DMSP and acrylate metabolism in cell suspensions of
strain LFR cultured on either glucose (A) or acrylate (B). Substrates
and products were measured in either the extracellular medium or gas
phase following the addition of DMSP or acrylate (500 µM) to the cell
suspension. The cells had an A600 of ca. 0.5 and
protein concentration of 0.12 mg · ml1. Symbols:
 , DMS production from added DMSP;  , extracellular acrylate
resulting from DMSP cleavage;  , uptake (or metabolism) of added
acrylate.
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When acrylate was added to a parallel set of glucose- and
acrylate-grown cell suspensions it was consumed without a lag; however,
the rate of disappearance differed dramatically. In glucose-grown
cells
(Fig.
1A), the low initial rate of acrylate disappearance
from the
extracellular medium during the first several hours was
followed by a
threefold increase in this rate. In the presence
of the protein
synthesis inhibitor gentamicin, the rate of acrylate
disappearance in
glucose-grown cells remained low (data not shown),
indicating that this
organism had a low constitutive level of
the acrylate-degrading (or
transporting) enzyme. In suspensions
of acrylate-grown cells, the rate
of disappearance of added acrylate
was ca. eight times faster than that
for glucose-grown cells,
resulting in a depletion of the acrylate from
the extracellular
medium within 1 h. These results confirm that
acrylate metabolism,
like that of DMSP, is an inducible process in
strain
LFR.
[1-13C]DMSP metabolism.
1H and
13C NMR analyses were used to determine the fate of
[1-13C]DMSP added to concentrated cell suspensions of
strain LFR. The NMR characteristics of [1-13C]DMSP are
given in Materials and Methods. To identify the metabolite(s) produced
as a result of DMSP catalysis, [1-13C]DMSP was added to a
concentrated suspension of acrylate-grown cells. The intracellular and
extracellular metabolites were both analyzed by NMR at half-hour
intervals. 1H spectra from gradient-enhanced
1H{13C} HMQC analyses of the cytosolic
extracts prepared at various time points following the addition of
[1-13C]DMSP are presented in Fig.
2. Four resonances, centered at 3.71, 3.38, 2.65, and 2.35 ppm, were seen in the cytosol at the first time
point. To confirm the identity of the molecules represented by these
signals, gradient-enhanced 1H{13C} HMQC
spectrum of the 0.5-h sample was collected (Fig.
3). The proton chemical shifts at 3.38 and 2.65 ppm correlated to a 13C chemical shift of 177 ppm,
identical to that of [1-13C]DMSP. The second molecule in
the cytosol had proton chemical shifts at 3.71 and 2.35 ppm, indicative
of the two methylene groups adjacent to a 13C-labeled
carboxyl group of [1-13C]-HP (3). Both of
the 13C-labeled molecules began to decrease in
concentration after the first half hour, and by 3 h, only a small
amount of the [1-13C]DMSP could be detected in the
cytosol. Conspicuously absent in the cytosol was the appearance of an
acrylate signal following the addition of labeled-DMSP.
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FIG. 2.
1H NMR analysis of intracellular
[1-13C]DMSP and its metabolite(s) following the addition
of 5 mM [1-13C]DMSP to a cell suspension of strain LFR
(acrylate-grown). Samples were taken at the indicated time intervals
and prepared as described in Materials and Methods. The NMR spectra
were acquired by using the same conditions and number of scans for each
sample so that the peak intensity would represent an approximate
concentration of each metabolite(s) relative to each sample.
1H resonances centered at 3.38 and 2.65 ppm and 3.71 and
2.35 ppm were identified as those belonging to
[1-13C]DMSP and [1-13C]-HP,
respectively.
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FIG. 3.
The gradient-enhanced
1H{13C} HMQC spectrum of the cell extract
taken at 0.5 h after the addition of 5 mM [1-13C]DMSP to
a concentrated cell suspension of strain LFR. NMR parameters were
exactly the same as those reported elsewhere (3).
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The extracellular medium analyzed at the first time point (0.5 h) after
labeled DMSP was added showed two sets of resonances.
One set, centered
at 3.38 and 2.65 ppm, was from [1-
13C]DMSP, and it
disappeared with time (data not shown). The second
set of resonances,
having proton chemical shifts centered at 3.71
and 2.35 ppm, was
recognized as
-HP. The
-HP was observed only
at this first time
point, after which it disappeared from the
extracellular medium (data
not
shown).
[1-13C]acrylate metabolism.
To determine why
[1-13C]acrylate was not detected as a result of
[1-13C]DMSP cleavage in strain LFR,
[1-13C]acrylate was added to cell suspensions and both
the extracellular medium and the ethanol-extracted cell pellets
(cytosol) were analyzed by NMR
spectroscopy. Both the extracellular (Fig. 4) and cytosolic (Fig.
5) metabolites were examined at 5-min
intervals over a 30-min time course. The extracellular sample indicated
that at the first time point (5 min), proton resonances due to both
acrylate (6.05, 5.90, and 5.55 ppm, which correlate with a
13C resonance at 175 ppm) and -HP (resonances at 3.64 and 2.35 ppm) were observed (Fig. 4). The decrease in the intensity of the acrylate proton resonances over time corresponded to an increase in
the intensity of the -HP resonances. After the -HP concentration reached a maximum in the extracellular medium, it decreased, suggesting that it was taken up by the cells (data not shown).
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FIG. 4.
1H NMR analysis of the extracellular medium
following the addition of 5 mM [1-13C]acrylate to a
concentrated cell suspension. Samples were taken at the various time
intervals indicated; the cells were removed by centrifugation and the
supernatant was assayed directly. The NMR spectra were acquired using
the same conditions and number of scans for each sample so that the
peak intensity would represent an approximate concentration of each
metabolite relative to each sample. 1H resonances at 5.55, 5.90, and 6.05 ppm and at 2.35 and 3.64 ppm were identified as those
belonging to that of acrylate and -HP, respectively.
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FIG. 5.
Gradient-enhanced 1H{13C}
HMQC spectrum of the cell extract (cytosol) taken at 10 min after the
exposure of cell suspension to [1-13C]acrylate. NMR
parameters are as reported previously (3). (Inset)
Cytosolic concentration of -HP following the addition of
[1-13C]acrylate, as measured by the intensity of the
-C protons (3.71 ppm) of the 180-ppm 13C-labeled
component from HMQC data (i.e., -HP).
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Next, it was determined if any of the [1-
13C]acrylate
added to cell suspensions, or products derived from its metabolism,
could
be found in the cytosol of strain LFR. The
1HNMR
spectrum of the ethanol extracts showed many overlapping
peaks which
could not be interpreted; however, the gradient-enhanced
1H{
13C} HMQC spectrum showed one resonance
at 180 ppm (Fig.
5). This
resonance was assumed to be an acrylate
metabolite due to its
high intensity, and it resembled
-HP. Cytosol
samples of strain
LFR prepared over the time course showed an increase
in this 180-ppm
signal, which reached its maximum at 20 min and
thereafter declined
(Fig.
5, inset). The intracellular intermediate was
confirmed
as being
-HP by adding this compound to the cytosolic
sample
and showing that the intensity of the resonance centered at 180
ppm increased dramatically. Because this cytosolic signal disappeared
with time and no other resonances appeared, it indicated the
13C-labeled carboxyl group of
-HP was subsequently
decarboxylated.
In addition to the resonances attributed to
-HP, several other peaks
were observed in the gradient-enhanced
1H{
13C} HMQC spectrum between 65 and 100 ppm in the
13C dimension and between 5.2 and 3.2 ppm in the
1H dimension as seen in Fig.
5. Since these peaks did not
increase
in intensity before or after the addition of
[1-
13C]acrylate, they are not products of either
[1-
13C]acrylate or [1-
13C]
-HP
metabolism. This indicated that strain LFR produced a metabolite(s)
that was stored at high enough concentrations within the cell
cytoplasm
to be detected by NMR spectroscopy, suggesting that
it may function as
a compatible solute. No attempt was made to
identify this molecule(s);
however, there was a resemblance to
[
13C]sucrose chemical
shifts observed in extracts of
Chromatium salexigens (
39). It is noteworthy that none of the
[1-
14C]acrylate added to the cell suspensions could be
detected in
the
cytosol.
DMSP lyase induction.
Both DMSP and acrylate are known to
induce DMSP lyase activity in strain LFR (24). With the
discovery that -HP was the product of both DMSP and acrylate
metabolism by strain LFR, we reexamined the inducer profile for DMSP
lyase and found that -HP was an even better inducer than DMSP and
acrylate. The effectiveness of DMSP and its metabolites as putative
inducers of DMSP lyase is proportional to the initial rate of DMS
production from cells preincubated with the putative inducer and washed
before DMSP is added. Induction rates for -HP, DMSP, acrylate, and
the constitutive activity were 0.35, 0.23, 0.2, and 0.09 µmol
· ml of culture1, respectively. The inducers were
tested over a range of 100 to 1,000 µM and each was maximally
effective at approximately 500 µM.
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DISCUSSION |
Studies of DMSP and acrylate metabolism in natural marine samples
yield results that are the sum of the activities of the diverse
population of microbes carrying out these processes (1, 18, 23,
25). Such results can be difficult to interpret, and they
therefore provide a rationale for our focus on the pathway of DMSP
cleavage of individual - and 
-Proteobacteria isolates (3, 40). As those two isolates, A. faecalis and
Pseudomonas doudoroffii, showed considerable variability in
this process, there was no way to know a priori how the
-Proteobacteria, now recognized as probably the most
prominent group of DMS producers in the marine environment
(12-14), would metabolize DMSP. This report describes the
mechanism of DMSP and acrylate metabolism in one culturable isolate of
-Proteobacteria, strain LFR.
While strain LFR was similar to A. faecalis in metabolizing
DMSP to acrylate and then to -HP, the details relating to location of the relevant enzymes and uptake of intermediates differed. The facts
that DMSP was not taken up by A. faecalis and that the product of DMSP lyase was seen only outside the cell both suggested that the lyase was an extracellular enzyme in that organism.
Conversely, strain LFR took up DMSP before it was cleaved, suggesting
an intracellular lyase (24), which was confirmed here with
13C-labeled DMSP (Fig. 2). Acrylase (the
acrylate-hydroxylating enzyme) in A. faecalis was
extracellular (3), while the NMR results provided
convincing evidence for both an intracellular and extracellular
acrylase in strain LFR. Specifically, the intracellular 13C-labeled DMSP and -HP detected following
[1-13C]DMSP addition indicated an intracellular acrylase,
even though the acrylate intermediate could not be detected. The
13C-labeled -HP detected extracellularly following
[1-13C]acrylate addition indicated an external location.
The term "extracellular enzyme" is loosely interpreted here to mean
an enzyme whose product(s) was released to the solution outside the
cell. No claim can be made as to whether the enzyme is periplasmic or
bound to cytoplasmic or outer membranes. Our observations, in fact,
have not ruled out the possibility of a single transmembrane acrylase
that could hydroxylate both extracellular and cytosolic acrylate.
A couple of apparent anomalies in the results need to be discussed. The
first is the absence of detectable acrylate in the cytosol following
the addition and cleavage of DMSP. This observation is rationalized by
suggesting that strain LFR has a very active acrylase such that the
acrylate pool does not accumulate to levels that can be detected by
NMR. The second anomaly is the observation of extracellular -HP
following the addition of DMSP to cell suspensions. This might be
explained if strain LFR had an extracellular DMSP lyase, but this is
contradicted by the kinetic data of Ledyard and Dacey
(24). They clearly showed that DMSP uptake into the cytosol preceded its cleavage to form DMS, thus proving that the DMSP
lyase resides in the cytosol. A likely, though unproven, explanation
for the brief appearance of -HP outside the cells is that leakage
occurred during the cell processing procedure. The fact that
extracellular -HP was not detected at later time points coincided
with its decrease in the cytosol (Fig. 2), suggesting that -HP
leakage stopped when the cytosolic levels decreased.
The progressive disappearance of the [1-13C]-HP signal
from the cytosol following addition of [1-13C]acrylate
(Fig. 5, inset) indicates that -HP is decarboxylated to some unknown
compound X by strain LFR. If a metabolite of -HP was decarboxylated,
a new set of NMR signals would have appeared, and they did not, meaning
that -HP itself was decarboxylated. The model for DMSP metabolism of
this -proteobacterial isolate is putative but best fits the
experimental data presented here (Fig.
6). It remains to be determined if other
members of the -Proteobacteria metabolize DMSP and
acrylate by a similar pathway.
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FIG. 6.
A model comparing the metabolism of DMSP and acrylate in
strain LFR and A. faecalis M3A. Abbreviations: DL, DMSP
lyase; Ac*, constitutive levels of acrylase; Ac, inducible acrylase;
BP, uptake/binding proteins; acrylate*, intracellular acrylate (assumed
to be present, but not actually observed, presumably due to low pool
size).
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ACKNOWLEDGMENTS |
This work was supported in part by grants from the S.C. Sea Grant
Consortium (R-MX-8) and from DOE/SCUREF.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of South Carolina, Columbia, SC 29208. Phone: (803) 777-2322. Fax: (803) 777-4002. E-mail:
yoch{at}biol.sc.edu.
Present address: University of Maryland Center for Vaccine
Development, Baltimore, MD 21201-1509.
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Applied and Environmental Microbiology, July 2001, p. 3134-3139, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3134-3139.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
