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Appl Environ Microbiol, January 1998, p. 106-111, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vivo Characterization of
Dimethylsulfoniopropionate Lyase in the Fungus Fusarium
lateritium
Melissa K.
Bacic and
Duane C.
Yoch*
Department of Biological Sciences, University
of South Carolina, Columbia, South Carolina 29208
Received 5 June 1997/Accepted 22 August 1997
 |
ABSTRACT |
A fungus, Fusarium lateritium, with
dimethylsulfoniopropionate (DMSP) lyase activity was isolated from both
seawater and a salt marsh due to its ability to grow on DMSP (with the
evolution of dimethyl sulfide) as the sole source of carbon. This is
the first reported case of DMSP lyase activity in a fungus. Several other common fungal genera tested did not have DMSP lyase activity. DMSP was taken up more rapidly by F. lateritium than it was
utilized, leading to its intracellular accumulation. Inhibitor studies
with nystatin and cyanide indicated that DMSP uptake was an
energy-dependent process. The lyase was inducible by its substrate,
DMSP (Km, 1.2 mM), and by the substrate analogs
choline and glycine betaine. During induction, DMSP lyase activity
increased with time and then dropped rapidly. This loss of activity
could be prevented by spiking the culture with fresh DMSP or choline.
The Vmax for DMSP lyase was 34.7 mU · mg
of protein
1. The inhibitory effects of nystatin, and
p-chloromercuriphenylsulfonate on DMSP lyase activity
suggested that the enzyme is cytosolic. Because plants like
Spartina (a marsh grass) and marine algae contain high
concentrations of DMSP, we speculate that DMSP-utilizing fungi may be
involved in their decay.
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INTRODUCTION |
The role of marine fungi in
biodegradation is well documented; however the details involving
specific degradative processes have not been closely examined (15,
19, 24). Much of the research on marine fungi has focused on
taxonomy, or determining which fungal species is associated with a
particular host and whether the relationship is symbiotic, parasitic,
or saprophytic (19). Furthermore, considerable work has been
done on the mineralization (lignocellulolysis) of marine plant litter
by fungi (14, 27, 28) and its impact on the carbon and
nitrogen cycles (12). Fungi, possibly including marine
fungi, are believed to play a significant role in the global sulfur
cycle through mineralization and immobilization processes; however, the
specific enzymatic reactions involved in these processes are not well
characterized (23, 31). One aspect of the global sulfur
cycle to which marine ecosystems are major contributors is the
production of dimethyl sulfide (DMS) (2, 18). DMS arises
primarily from the enzymatic cleavage of dimethylsulfoniopropionate
(DMSP), which is produced as an osmoprotectant by salt marsh
cordgrasses (6) and by certain species of macroalgae
(3, 4, 16) and phytoplankton (1, 13, 17, 30). The
physiology and biochemistry of the DMSP-degrading enzyme, DMSP lyase,
has been studied in bacteria (9, 10, 21, 38), macroalgae
(3, 4, 11, 16, 28a, 33), and phytoplankton (17,
32). However, no data on fungal DMSP lyase have been reported,
although there was a brief mention of DMS emissions from added DMSP by
a penicillium (4a), and the potential contribution by fungi
in cleaving DMSP was suggested by Kiene (18). This paper
reports the discovery and in vivo characterization of DMSP lyase in
isolates of the fungus Fusarium lateritium. The potentially
significant role of this species, and others that may contain this
enzyme, in marine DMS emissions is discussed.
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MATERIALS AND METHODS |
Isolation of Fusarium spp.
Plates of a minimal
medium, which contained 1 mM DMSP as the sole source of carbon and
energy, were inoculated with either seawater or liquid from the surface
of a decaying jellyfish found on the beach near North Myrtle Beach,
S.C. Fungal hyphae, which appeared 5 days after inoculation, were
subcultured on this medium. Pure cultures were obtained by restreaking
onto separate plates containing 25 µg of chloramphenicol · ml1. Cultivation of fungal biomass for the experiments
described below was carried out in Vogel's medium (37)
modified by the addition of a trace elements solution (8).
This medium was further supplemented with 1.5% NaCl, 2 mg of
biotin · liter1 25 mg of chloramphenicol · liter1, and 2% sucrose.
DMSP lyase induction.
After 5 days of growth on sucrose
medium, Fusarium cultures were filtered onto a
0.45-µm-pore-size membrane filter (Gelman), washed with deionized
water, and resuspended in 50 mM sodium phosphate buffer (unless
otherwise indicated). Cell suspensions were incubated for 30 min prior
to experimentation so that ethanol, a sucrose fermentation product
(which has a peak on our gas chromatograph interfering with that of
DMS), could be metabolized or released to the air. DMSP lyase was
induced by adding 2 mM DMSP to this buffered cell suspension, which was
then incubated for 3 h at room temperature on a rotary shaker at
100 rpm. The same method was used to test acrylate and DMSP analogs as
potential inducers of DMSP lyase. The lyase-induced cells were
collected by filtration, rinsed, and resuspended in fresh buffer for
use in the various experiments described below.
DMSP lyase activity.
DMSP lyase activity in fungal
suspensions was determined by measuring the amount of DMS emitted from
1 mM DMSP. DMS was analyzed on a GC-8A gas chromatograph (Shimadzu,
Kyoto, Japan) equipped with a flame ionization detector and a Poropak R
column. The column temperature was maintained at 170°C, and the
carrier gas flow rate was 74 ml · min1. The amount
of DMS in the liquid phase was calculated from the measured amount of
gas in the headspace by using Henry's law constants (7).
Estimations of total protein were made by boiling hyphae in 2 M NaOH
for 5 min, centrifuging for 3 min, and adding Bradford reagent
(Bio-Rad) to an aliquot of the supernatant. Bovine serum albumin was
the standard.
Effect of pH and temperature on DMSP lyase activity.
The
optimum temperature for DMSP lyase activity was determined by
incubating induced cells at the temperatures indicated for 15 min
before adding the substrate and during the assay. The optimum pH was
determined by resuspending rinsed, induced cells in buffers ranging
from pH 4.0 to 10.0. After 15 min, 1 mM DMSP substrate was added and
the enzyme activity was measured.
Effect of inhibitors on DMSP lyase induction and activity.
Fungal cells were incubated with the inhibitor nystatin or
p-chloromercuriphenylsulfonate (p-CMBS) at the
concentrations indicated. To determine their effect on DMSP lyase
induction or turnover (activity), the inhibitors were incubated with
the cells 15 min before addition of the inducer or substrate. The data
shown are representative of at least two experiments.
DMSP uptake.
The rate of DMSP uptake by Fusarium
was measured by adding DMSP (250 µM) to fungal hyphae in 50 ml of
phosphate buffer. Aliquots of the fungus were removed and filtered
every 5 min after the addition of DMSP. The intracellular DMSP
concentration was determined by the addition of 1 ml of 4 M NaOH to the
filtered hyphae to cleave DMSP to DMS and acrylate. The effect of
inhibitors (cyanide, nystatin, and p-CMBS) on the rate of
DMSP uptake is presented as percent inhibition and was calculated as
follows: [(1 
rate of inhibitor-treated sample)/rate of sample
with DMSP only] × 100.
Chemicals.
DMSP was synthesized from DMS and acrylate by the
method of Chambers et al. (5). DMSP was standardized against
the pure commercial reagent obtained from Research Plus, Inc., Bayonne, N.J. Acrylate was obtained from Aldrich, Milwaukee, Wis. Nystatin was
obtained from Sigma Chemical Co., St. Louis, Mo.
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RESULTS AND DISCUSSION |
Characterization of the fungus.
Two fungal isolates growing on
minimal medium containing DMSP as the sole source of carbon were
purified and tested for the production of DMS. DMS emissions were
inhibited by nystatin but not chloramphenicol, indicating that a
fungus, not a bacterial contaminant, was responsible for DMSP lyase
activity. Comparative analysis of macroconidia (25)
indicated that both isolates were of the genus Fusarium
(Fig. 1). Comparison of fungal growth on various media and further analysis of the reproductive structures revealed that both isolates were the same genus and species, F. lateritium (12a).
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FIG. 1.
Scanning electron micrograph of F. lateritium
macroconidia. Fungal cultures were fixed in 2% glutaraldehyde and
postfixed in 1% OsO4. Magnification, ×1,200.
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Substrate utilization.
Yields of F. lateritium
grown in minimal medium with sucrose, DMSP, or acrylate as the sole
source of carbon were compared (Fig. 2).
After 8 days of growth, the fungal biomass was filtered, dried, and
weighed. Figure 2A compares the fungal biomass produced per mole of
added substrate, and Fig. 2B compares the fungal biomass produced per
gram of carbon. While the biomass yield per mole on sucrose was ca.
2.5-fold higher than on DMSP or acrylate, on a gram-of-carbon basis,
yields on acrylate were significantly higher (ca. 30%) than were
yields on DMSP or sucrose, which were approximately equal. Since methyl
carbon is ca. two-fifths of the total carbon, it is probable that the
lower growth yield (per gram of carbon) on DMSP was because the DMS
released could not be recaptured and utilized by this fungus.
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FIG. 2.
Growth of F. lateritium on various carbon
sources. (A) Fungal dry weight produced per mole of substrate; (B)
fungal dry weight per gram of carbon. Substrate concentrations were as
follows: sucrose, 2.5 mM; DMSP and acrylate, 5 mM each. The results
show the mean and standard deviation of three replicas.
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Fungal DMS production.
To determine if DMS emissions from
added DMSP occur in fungal metabolism generally, both
Penicillium spp. and Saccharomyces cerevisiae
were tested for DMSP lyase activity. Only F. lateritium had
this activity (Fig. 3). Several other
unidentified fungal species were also tested, but none evolved DMS from
DMSP.
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FIG. 3.
DMS emissions by various fungal genera. DMSP (1 mM) was
added to fungal cell suspensions containing approximately 40 mg of
biomass; the appearance of DMS in the gas phase was monitored by gas
chromatography.
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Induction of DMSP lyase.
As seen in Fig. 3, DMS was not
immediately evolved from F. lateritium upon addition of
DMSP, suggesting that DMSP lyase is an inducible enzyme. The optimum
DMSP concentration for lyase induction was 2.5 mM, although
concentrations as low as 0.25 mM would induce the enzyme (Fig.
4). In F. lateritium, this
enzyme was induced not only by its substrate DMSP
(Km, 1.2 mM) but also by the substrate analogs
choline, glycine betaine, and dimethyl glycine (Table
1). The selenium analog of DMSP,
dimethylselenoniopropionate, did not induce DMSP lyase. Furthermore,
neither proline, an osmoprotectant for many organisms (22),
nor acrylate, the product of the lyase reaction, was effective as an
inducer of DMSP lyase.
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FIG. 4.
Effect of DMSP concentration on DMSP lyase induction.
The kinetics of induction were monitored by assaying washed cells for
DMSP lyase activity after 3 h of induction.
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On examining the activity of DMSP lyase during induction in this
fungus, we observed that enzyme specific activity, as measured
by DMS
emissions from added DMSP, increased with time up to about
3 h and
then began to drop rapidly as the inducer was depleted
from the cell
suspension (Fig.
5, no addition). Since
this drop
in activity occurred even though fungal aliquots had been
filtered
and fresh DMSP had been added, the reason for the loss was
unrelated
to substrate availability. Furthermore, since the addition of
the reaction end product acrylate or DMS or lowering the pH had
no
effect on enzyme induction or activity (data not shown), this
loss of
activity was apparently not due to feedback inhibition.
However,
spiking the cell suspension with fresh DMSP or choline
(1 mM), but not
glycine betaine (1 mM), at 3 and 6 h resulted
in the fungal lyase
activity remaining constant (Fig.
5, bar graphs).
These molecules
either stabilized the lyase or induced the synthesis
of new enzyme.
Since glycine betaine induced the lyase but was
not effective in
maintaining lyase activity after spiking, it
appears that the molecules
which caused the lyase activity to
remain high did so by stabilizing
the enzyme rather than inducing
new enzyme. It is not uncommon for
enzymes to be stabilized by
their substrates occupying the active site
(
35), and these results
may be examples of such a
phenomenon.
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FIG. 5.
Effect of DMSP and various DMSP analogs on DMSP lyase
stability. DMSP (2 mM) was added to four identical fungal cell
suspensions at time zero. At the times indicated, an aliquot of cells
was removed, filtered, washed, and resuspended with 1 mM DMSP. The
cultures were amended after 3 and 6 h, as indicated by the arrows,
with 1 mM choline, glycine betaine, or DMSP. The line indicates the
course of an unamended culture. Bars show the level of activity after
the additions listed.
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DMSP storage.
During the induction process, the fungus took up
more DMSP than could be immediately used. After 3 h of induction,
1 ml of filtered, washed hyphae could contain between 0.35 and 1.0 µmol of DMSP per mg of cell protein. This stored DMSP could be
released by the addition of nystatin to the cell suspension (data not
shown).
Optimum conditions for DMSP lyase activity.
Although both
F. lateritium isolates came from the marine environment,
salinity variations in the growth or resuspension media had no effect
on DMSP lyase activity (data not shown). The temperature for optimal
DMS emissions was between 20 and 30°C (Fig.
6A). This compares with 37°C for the
DMSP lyase of aerobic marine bacteria (10). The lyase
activity was essentially independent of pH over the range measured (pH
4 to 10) (Fig. 6B), which may be considered unusual. However, since the
activity was measured in vivo, it may have been unaffected by the
external pH. DMSP lyases in bacteria were strongly dependent on
[H+], with maxima at approximately pH 8 (10).
The kinetics of DMSP utilization by DMSP lyase could not be determined
in these fungal isolates because they concentrated DMSP during the
induction period, and therefore the lyase did not respond to added DMSP
(data not shown).
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FIG. 6.
Effects of pH and temperature on DMSP lyase activity in
F. lateritium. Fungal cultures were induced with 1 mM DMSP;
the temperature and pH were adjusted for each aliquot of fungal
biomass. DMSP lyase rates were averaged from three separate experiments
and are shown with their standard deviations.
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DMSP uptake.
The kinetics of DMSP uptake by cells uninduced
for DMSP lyase (i.e., having no internal DMSP) are shown in Fig.
7. The intracellular pool of DMSP peaked
at ca. 80 min and then declined as the lyase that was induced during
this period began to turn over and deplete the pool. A steady-state
level of DMSP (1.5 to 2 µmol · mg of protein1)
was reached at about 3 h.
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FIG. 7.
DMSP uptake by F. lateritium as measured by
increases in intracellular DMSP concentrations with time. The inset
shows the effect of cyanide on DMSP uptake. The 0% inhibition of DMSP
uptake represents 1.62 µmol of DMS · mg of
protein1 after 40 min of uptake.
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Effect of inhibitors on DMSP metabolism.
The energy
requirement for the DMSP uptake process was seen by its strong
inhibition by the electron transport inhibitor cyanide (Fig. 7, inset).
To determine if DMS emission (DMSP lyase activity) and induction of
DMSP lyase were energy-requiring processes and to confirm that DMSP
uptake is energy dependent, the effects of nystatin on DMSP metabolism
were examined. Nystatin, which attacks fungal membrane sterols,
strongly inhibited DMSP uptake (Ki, ca. 2 × 107 M) and DMSP lyase induction
(Ki, ca. 8 × 108 M) when
added to cell suspensions (Fig. 8). While
it might be expected that DMSP uptake and turnover would be identical
in their sensitivity to nystatin, lyase activity (turnover) was less
sensitive to this inhibitor (Ki, ca.
106 M). This difference in sensitivities could be
explained by the fungus storing DMSP during the induction period. The
stored DMSP would be available to the enzyme (for turnover) and
therefore would be insensitive to low levels of added nystatin.
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FIG. 8.
Effect of nystatin concentrations on DMSP uptake and
induction and turnover of DMSP lyase. The 100% DMSP lyase activities
for induction and turnover were 0.06 and 0.03 µmol of DMS · min1 · mg of protein1, respectively.
DMSP uptake in the absence of inhibitor (0% was 1.6 µmol of DMS
· mg of protein1; all uptake measurements were done at
40 min.
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Addition to fungal suspensions of the thiol-binding reagent
p-CMBS, which cannot cross membrane barriers
(
36), helped determine
which of the proteins (or enzymes)
involved in DMSP metabolism
are exposed to the cell surface. Figure
9 shows that DMSP uptake
was inhibited
50% by ca. 0.3 mM
p-CMBS. This relatively low sensitivity
suggests that the DMSP uptake system, presumably located in the
cell
membrane, is only partially exposed to the thiol-binding
reagent. Since
induction of DMSP lyase also requires DMSP uptake,
the much stronger
inhibition of this process by
p-CMBS
(
Ki = 0.05
mM) suggests that this thiol reagent
may also be interfering with
other cellular processes. Finally, DMSP
lyase activity was inhibited
ca. 30% by 0.1 mM
p-CMBS, and
higher concentrations had no further
effect. This observation may be
explained by the fact, already
mentioned, that
F. lateritium
stores DMSP during the induction
period and therefore the rate of
enzyme turnover is affected by
the inhibition of DMSP uptake by
p-CMBS. These results suggest
that DMSP lyase in
F. lateritium is not exposed to the surface
and could possibly be
cytosolic. Furthermore, the fact that DMSP
lyase activity in this
fungus requires the active uptake of substrate
supports the cytosolic
location of this enzyme.
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FIG. 9.
Effect of p-CMBS concentrations on DMSP
uptake and induction and turnover of DMSP lyase. The 100% DMSP lyase
activities for induction and turnover were 0.037 and 0.031 µmol of
DMS · min1 · mg of protein1,
respectively. DMSP uptake was 1.2 µmol of DMS · mg of
protein1 in the absence of inhibitor (0% inhibition);
all uptake measurements were made at 40 min.
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In summary, we have definitively established that fungi are capable of
causing DMS emission from DMSP by the action of an
inducible DMSP
lyase. This activity in
F. lateritium requires
the uptake of
DMSP by an energy-dependent process. Potential sources
of DMSP for
F. lateritium are phytoplankton (
17) and
macroalgae
(
16) and salt marsh cordgrasses (
20,
29). The salt marsh
cordgrass
Spartina alterniflora
contains between 80 and 280 µmol
· g (dry weight) of
DMSP
1 (
6,
20,
29). Studies have shown an
increased rate of DMS
emission during senescence and decay of this
grass (
6,
34).
Furthermore, it has been shown that fungi are
the major mediators
of this decay (
26). Although the
significance of fungus-related
DMS emissions in the ocean and salt
marsh is not known, the high
concentrations of DMSP in marsh grasses
and marine algae lead
to the notion that DMSP-utilizing fungi may be
found in these
cordgrasses and could be involved in their senescence
and decay.
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ACKNOWLEDGMENTS |
We thank R. Hanlin and his technician, C. Rodriguez, University
of Georgia, Athens, Ga., for identifying the fungal isolates and Steven
Y. Newell for a critical reading of the manuscript.
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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.
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0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
