ELSEVIER
FEMS Microbiology
Ecology
19 (1996) 239-247
Metabolism of methylated osmolytes by aerobic bacteria from
Mono Lake, a moderately hypersaline, alkaline environment
Mara R. Diaz
Dir,ision
of Marine
and Atmospheric
Chemisty.
Rosenstiel
Miami,
*, Barrie
F. Taylor
School of Marine
FL 33149-1098.
Received 20 May 1995; revised 19 January
and Atmospheric
Science. University
of Miami.
USA
1996: accepted 25 January
1996
Abstract
Three strains of aerobic bacteria were isolated from water and sediment samples of Mono Lake, a moderately hypersaline
(90 ppt), alkaline (pH 9.7) lake in California. The organisms, Gram-negative rods, grew fastest at about pH 9.7 with no
growth or much slower growth at pH 7.0. All three isolates grew on glycine betaine (GB) and respirometric experiments
indicated that catabolism was by sequential demethylation with dimethyl glycine and sarcosine as intermediates. Two of the
isolates also grew on dimethylsulfoniopropionate
(DMSP), one with cleavage of the DMSP to yield dimethyl sulfide (DMS)
and acrylate, and the other by demethylation with 3-methiolpropionate
(MMPA) as an intermediate and the production of
methanethiol from MMPA. The methylated osmolytes supported growth at salinities similar to those in Mono Lake, but, at
higher salinities. catabolism was suppressed and GB and DMSP functioned as osmolytes. GB and DMSP probably originate
from cyanobacteria and/or phytoplankton
in Mono Lake and this report is the first indication of both the DMS and
demethylation/methanethiol-producing
pathways for DMSP degradation in a nonmarine environment.
Keywords;
Compatible
Dimethylsulfoniopropionate
solutes; Mono Lake
(DMSP):
3-Mercaptopropionate
1. Introduction
The methylated compounds, glycine betaine (GB)
and dimethylsulfoniopropionate
(DMSP), are osmolytes that are synthesized by macroalgae, phytoplankton, and higher plants [I-3]. GB and DMSP
have been detected in some marine invertebrates
[4-61 where they probably originated by trophic
transfer of the plant osmolytes. GB is also synthesized by some invertebrates [7].
” Corresponding
author.
Tel.:
+
I (305) 361 4941; Fax: + I
(305) 361 4600.
016%6496/96/$15.00
P/I
SOl68-6496(96)00014-!
0 1996 Federation
of European
Microbiological
(MPA):
3-Methiolpropionate
(MMPA);
Glycine
betaine
(GB);
GB and DMSP, serve as osmolytes
in bacteria,
and as carbon and energy substrates for methylotrophic and other heterotrophic organisms [8,9].
Besides its potential use as a growth substrate and/or
osmolyte,
DMSP is an important
precursor
of
dimethyl sulfide (DMS). It has been reported DMS
accounts for 90% of the total natural sulfur emissions from marine environments
[IO]. DMS, after
photochemical
oxidation
in the atmosphere
to
methane sulfonic acid and sulfuric acid, enhances
cloud formation that may lead to global cooling [ Ill.
The aerobic catabolism of GB proceeds with sequential N-demethylations
to glycine [ 121. The aerobit degradation of DMSP is more varied; one route
Societies. All rights reserved
yielded DMS and acrylate [13] and other pathways
involved
demethylation
to 3-methiolpropionate
(MMPA) followed either by another demethylation
yielding 3-mercaptopropionate
(MPA), or by demethiolation to give CH,SH [ 14,151.
Mono Lake, California, is a meromictic lake located in the base of the Sierra Nevada mountains. Its
waters are alkaline (pH 9.7) and moderately hypersaline (90 ppt). The predominant cation is Na+ and
the principal anions are carbonate, bicarbonate, sulfate, borate and chloride [16l. Because of the production of methylated osmolytes in saline environments.
we examined the function of GB and DMSP as
osmolytes and as sources of carbon and energy for
several bacterial isolates from Mono Lake.
2. Materials
and methods
2. I. Isolatio?l and growth of bacteria
Inocula of Mono Lake water and sediment were
added to filter-sterilized (0.2 pm pores) Mono Lake
water which was supplemented with 5 mM NH,Cl,
0.37 mM KHzPO,, SL4 trace metal solution (I ml
1~ ’ ) [ 171 and 5 mM of GB, DMSP or MMPA as the
source of carbon and energy. The enrichments were
transferred to a synthetic medium containing:
1.5 M
NaCl; 10 mM Na,SO,:
2.5 mM MgCl?; 6 mM
KNO,; 5 mM NH,Cl; 0.37 mM KH,PO,;
0.05 M
(cyclohexylamino)-2-hydroxy1-propane-sulfonic
acid (CAPSO) and SL4 trace metals (1 ml 1-l > [ 171.
GB, DMSP, MMPA, acrylate or propionate served
as carbon sources at a final concentration of 5 mM.
Incubations
were in the dark at room temperature
(about 25°C). Pure cultures were derived from single
colonies by streaking the bacteria onto medium solidified with 1.5% (w/v) Bacto Agar. The water
column bacterial strains, ML-G and ML-D, were
selectively
isolated from GB and DMSP enrichments, respectively. The bacterium, MM-P, was isolated from the sediments and selectively enriched
with MMPA. The cultures were incubated in a rotory
shaker at 25°C. Growth in liquid media was determined by turbidity, either visually or with a KlettSummerson calorimeter. Protein was determined by
the bicinchonic acid assay [ 181.
2.2. Optimum pH for growth
To determine the optimum pH of the isolates, the
synthetic medium described above was buffered with
bis(tris(hydroxy-methylaminol-propane
(BTP) in the
range pH 7 to pH 9, or with 2 amino-2-methyll-propanol (AMP) and/or CAPS0 when the pH ranged
from pH 9 to pH IO. 3-(Cyclohexylamino)1-propanesulfonic (CAPS) was used at pH 11. GB (5 mM)
served as the carbon source and growth was monitored by turbidity with a Klett calorimeter.
2.3. .Effects of GB and DMSP 011 bacterial groM,th
To determine the effect of GB and DMSP on
bacterial growth in relation to salinity tolerance, the
strains grown on 10 mM propionate were inoculated
into media containing 5 mM propionate and different
NaCl concentrations (0.5 M-4.0 Ml with or without
GB or DMSP at a final concentration
of 0.1 mM.
The reported osmolality values for NaCl solutions of
0.5. 1.5. 2.5, 3, 3.5 and 4 M NaCl, are 0.919. 2.84,
5.02, 6.25, 7.59 and 9 OS kg-‘, respectively [19].
2.-i. Respiration
experiments
Cells were harvested by centrifugation (10,000 X
g, 10 mitt, 4°C) and resuspended in 0.05 M CAPS0
buffer (pH 9.7) containing
1.5 M NaCl, 0.05 M
MgSO,. and 0.01 M KCl. The washed cells were
kept overnight at 4°C to diminish the endogenous
rates from approximately
200 nmol 0, min-’
per
mg of protein to approximately
10 nmol 0: min-’
per mg of protein. Respiration rates were measured
in a 5 ml reaction volume at 30°C with a Clark-type
oxygen electrode. The salinity ranged from 0.5 M to
3.5 M NaCl. Once endogenous
rates were determined, substrates were added at final concentrations
of 400 PM. To determine if Na’ or Kf were
required for the oxidation of GB, DMSP, and MMPA,
the cells were resuspended in the presence or absence of 1.5 M NaCl and/or
1.5 M KCl. All the
experiments were replicated at least once.
2.5. Accumulation
of DMSP at different salinities
To determine whether the cells could take up and
accumulate
DMSP under different salinity condi-
M.R. Dia;
B.F. Taylor/
FEMS Microbiology
tions, cells were grown in media containing 5 mM
propionate with different salinity levels (0.5 M-3.0
M NaCI) and 0.1 mM DMSP. At the end of the
exponential phase. cells were harvested by centrifugation. washed and resuspended
in the synthetic
medium; duplicate samples were analyzed for DMSP
content. Intracellular DMSP concentrations
were calculated using estimates of cell volumes [20]. The
experiments were replicated at least once.
2.6. l$ect
lation
Ecology
241
19 (1996) 239-247
2.8. Chemicals
Chemicals were obtained from Sigma (St. Louis,
MO) or Aldrich (Milwaukee. WI). DMSP was purchased from Research Plus (Bayonne, NJ) and was
also synthesized from acrylate and DMS [2 11. MMPA
was obtained from its methyl ester by alkaline hydrolysis. Methanethiol was prepared by reduction of the
dimethyl-disulfide
(DMDS) by 0.5 mM tributylphosphine 1221.
qf GB and propionate in DMSP accurnu3. Results
To determine whether GB or propionate affected
the intracellular accumulation of DMSP. 0.1 mM GB
or 0.05 mM propionate was added to 0.5 ml of
ML-G cells suspension up-shocked with 3 M NaCl
(final concentration)
and containing 0.1 mM DMSP.
Cells were then incubated for different time intervals
in the presence or absence of GB or propionate and
the DMSP content of the cells and of the supematant
was determined.
2.7. Analytical
methods
To measure the production of DMS and MSH.
either 0.25 ml or 0.5 ml of cell suspensions were
placed in 13 ml serum bottles. At time zero, 100 ~1
or 50 ~1 of 4 mM DMSP was injected and the head
space (100 ~1 samples) was analyzed for DMS or
MSH.
DMS and MSH were analyzed with a Shimadzu
gas chromatograph
with a flame ionization detector
and Carbopak BHT- 100 column (Supelco Bellefonte,
PA, USA). The column temperature was 100°C and
nitrogen was the carrier gas at a flow rate of 60 ml
min-‘. Retention times for DMS and MSH were 1.2
and 0.6 min. respectively. Detection limits for DMS
and MSH were 1 nmol 100 ~1~ ‘. Standards for
DMSP and MSH were prepared by consecutive dilutions from 4 mM stock solutions.
DMSP was quantified by the indirect method of
alkaline decomposition
[3]. The sample (0.5 ml of
washed cell suspension or supematant)
was transferred to a 13 ml serum vial and treated with 1 ml of
5 N NaOH. After 1.5 h, 100 ~1 of the gas phase was
withdrawn, injected in the gas chromatograph,
and
DMSP determined as DMS.
3. I. Characterization
of strains
The isolates ML-G, ML-D and MM-P were
Gram-negative.
non-spore-forming
rods. The average
sizes of ML-D and ML-G were 1.7 pm X 0.4 pm,
and 0.3 pm X 0.2 pm, respectively. Strains ML-G
and ML-D were motile; MM-P was non-motile. Of
the three strains, only ML-D fermented sucrose.
Growth substrate utilization by the strains is described in Table 1.
ML-G, and ML-D strains when grown on GB,
Table 1
Substrate
utilization
by Mono Lake strains
Substrate
Choline
Glycine betaine
N.N-Dimethylglycine
Sarcosine a
Propionate
Glucose
DMSP
Acrylate
3-Methiolpropionate
(MMPA)
Acetate
Trimethylamine (TMA)
Dimethylamine (DMA)
Monomethylamine
(MMA)
Methanol
Glycine
Bacterial strain
ML-G
ML-D
MM-P
+
+
+
+
+
+
_
_
+
+
+
+
+
+
+
+
_
+
+
+
+
+
+
+
+
+
+
_
NT
_
_
_
_
NT
_
_
_
_
_
NT
_
-
The cells were grown at 1.5 M NaCl on different substrates at a
final concentration of 5 mM. + , growth; - . no growth: NT, not
tested.
a All strains showed a weak growth on sarcosine.
242
M.R. Dia:.
B.F. Tqlor/
FEMS Microbiology
Table 2
Compounds oxidized by strains ML-G, ML-D, and MM-P grown
at I .5 M NaCl on 5 mM GB. 5 mM DMSP. 5 mM MMPA. 5 mM
propionate (P), 5 mM acrylate (A)
Net oxygen uptake rate
(nmol min- ’ per mg of protein)
Strains:
Substrate:
ML-G
GB
ML-G
P
ML-D
GB
ML-D
DMSP
ML-D
A
MM-P
MMPA
GB
DMG a
Sarcosine
Glycine
DMSP
Acetate
Propionate
Glucose
MMPA
Acrylate
230
237
13
7
-
118
145
_
86
110
_ h
_
56
42
_
_
92
79
_
_
159
114
_
_
35
58
ND“
ND
ND
-
41
20
22
ND
_
7
80
27
_
I5
42
42
208
77
ND
97
56
’ Dimethylglycine.
h Signifies rate not above endogenous
’ Not determined.
(no substrate)
rate.
mainly oxidized GB and N,N-dimethylglycine.
No
significant oxidation rates were recorded for other
intermediate products of GB degradation, i.e., sarcosine and glycine (Table 2). With other growth substrates. GB and NNdimethylglycine
were still
rapidly oxidized, but some oxidation of other substrates was also observed. Acetate oxidation rates
were similar to those of GB and N,N-dimethylglycine when ML-D strain was grown on acrylate.
Strain MM-P, grown on MMPA. showed rapid oxidation, not only of GB and N.N-dimethylglycine.
but
also of MMPA, acetate, acrylate and propionate.
None of the strains oxidized any of the substrates
in 0.5 M NaCl, although endogenous rates were high
(ML-G: 292 nmol mm’
per mg of protein. ML-D:
138 nmol mini’ per mg of protein. and MM-P: 229
nmol min- ’ per mg of protein). At higher salinities,
the endogenous rates were markedly decreased (9 to
20 nmol min- ’ per mg of protein). All three strains
had optimum oxidation rates at NaCl concentrations
between 1.O to 2.0 M with drastic decrease in rate at
NaCl concentration of 3.0 M or greater (Table 3). At
3.0 M NaCl, GB oxidation rates in ML-G and ML-D
cells were reduced by 74% and 87%, whereas MMPA
oxidation rates were reduced by 45% in strain MM-P.
The oxidation rates of GB. DMSP and MMPA
did not appear to have an absolute dependence on
Ecology
t 1996)
I9
239-247
Table 3
Effect of NaCl on GB, DMSP. and MMPA oxidation
rates
Net oxygen uptake rate
(nmol min-’ per mg of protein)
Strains:
Substrate li:
Salts:
0.5
1.0
1.5
2.0
2.5
3.0
3.5
M
M
M
M
M
M
M
ML-D
GB
ML-G
GB
ML-D
DMSP
MM-P
MMPA
_ 0
_
_
_
244
218
I54
60
32
I4
X6
81
59
37
22
9
35
41
26
ND
IO
_
ND’
97
93
ND
53
ND
Cells were grown at 1.5 M NaCl on 5 mM GB, 5 mM DMSP and
5 mM MMPA. Cells were harvested. washed and resuspended in
0.05 M Capso (pH 9.7) containing 0.05 M MgSO.,. 0.01 M KCI.
various concentrations of N&I, and substrate at 0.3 mM and 1.5
M NaCl and substrate at 0.4 mM final concentration.
’ The same substrate was used for growth and for oxidation.
h Rate not above endogenous rate (no substrate rate).
’ Not determined.
Na+ since substrate oxidation occurred when KC1
was substituted for NaCl (Table 4). These experiments were not definitive since reagent-grade
KCI
did contain low traces of Naf (0.002%). sufficient to
yield a final concentration
of about 0.03 mM Na+.
The GB oxidation rate for ML-G was 60% less with
KC1 than with NaCl: oxidation rates for strain MM-P
were higher in the presence of KCl.
Table 4
Oxidation of GB and DMSP by ML-G and ML-D in the presence
or absence of NaCl
Net uptake oxygen rate
(nmol min- I per mg of protein)
Strains:
Substrate:
ML-G
GB
ML-D
GB
ML-G
DMSP
MM-P
MMPA
I .5 M NaCl
I .5 M KCI
I.5 M KCI
+ 0.05 M NaCl
I .5 M KC1
+l.OMNaCl
217
86
81
4.5
11
10
36
97
I78
145
11
23
115
*2
70
-3
Cells were grown at 1.5 M NaCl on 5 mM GB, 5 mM DMSP and
5 mM MMPA. harvested, washed and resuspended in 0.05 M
Capso buffer (pH 9.7) containing NaCl or KCI and 0.4 mM
substrate.
’ Rate not above endogenous rate.
M.R. Dia:.
B.F. Taylor/
FEM.5 Microbiology
The pH optima of the isolated strains were pH
8-pH 10 (ML-G), pH 9-pH 10 (ML-D), and pH
9-pH 9.7 (MM-P). Strain ML-G was the only strain
able to grow at pH 7, at a rate of 0.14 hh’. Calculated specific growth rates for ML-G, ML-D and
MM-P at pH 9 were 0.29 hh’, 0.13 hh’, and 0.17
h- ‘, respectively. Growth at pH 11 using 0.08 M
CAPS as buffer occurred at rates as low as 0.050
h- ’ and 0.048 hh ’ for ML-G and ML-D, respectively. However, this is not a suitable buffer since its
buffering capacity was lost after two days. even at
concentrations
as high as 0.08 M. No growth occurred when 2-amino-2-methyl1-propanol was used
as buffer.
3.2. &@ect of osmolytes
Ecology
243
19 (19961239-247
mj;C
OH bacterial growth
Strain ML-G is a moderately halophilic bacterium
that grows on propionate at NaCl levels between 0.5
M and 2.5 M in the absence of added osmolytes
(Fig. 1). However, GB or DMSP in the growth
B
Fig. 2. Effect of added osmolytes
on the growth of strain ML-D:
(A)0.1mM GB: (B) 0.1 mM DMSP. Growth was determined by
turbidity.
T
Fig. 1. Effect of added osmolytes
.-,e
(h:
on the growth of strain ML-G:
(A)0.1mM GB: (B)0.1mM DMSP. Cells were grown on 5 mM
propionate. Growth was determined
was 5 mM propionate.
by turbidity.
Growth substrate
Growth substrate
was 5 mM propionate.
medium permitted growth in the presence of 3.0 M
NaCl. Strains ML-D and MM-P were more tolerant
of higher salinities, since they grew in the absence of
osmolytes at salinities between 0.5 M and 3 M (Fig.
2, Fig. 3). Growth of ML-D and MM-P at 3.5 M
NaCl was observed only in the presence of GB (Fig.
2A, Fig. 3). None of the strains grew at 4 M NaCl.
even in the presence of potential osmolytes. In general, additions of GB or DMSP decreased the lag
phases for strains growing at high salinities. ML-D
showed no difference in growth rate at salinities
between 0.5 M and 2.5 M NaCl in the presence of
the osmolytes. A similar response was observed for
ML-G, when grown in the presence of DMSP (Fig.
1B). However at 2.5 M NaCl the growth rate of
ML-G increased by 45% in the presence of GB (Fig.
IA). The effect was more pronounced
for ML-D
grown at 3 M NaCl, where the growth rate in the
presence of GB was 70% more than in its absence
(Fig. 2A). GB addition also improved the growth of
MM-P, especially at high salinities (Fig. 3). For
example, at 3.0 M NaCl, the lag phase was de-
M.R. Dia:.
B.F. Taylor/
FEMS Microhiolo,q~
Ecology
19 f 19915) 23Y-247
(20 nmol mini’ per mg of protein), by ML-D cells
grown on DMSP in a medium containing
1.5 M
NaCl. When ML-D was grown at salinities between
0.5 M NaCl and 1.5 M NaCl, most of the DMSP
added was completely catabolized at the beginning
of the stationary phase. DMSP did not accumulate in
the cells at 0.5 M NaCI. but did accumulate at higher
salinities (Table 5). In contrast, strain ML-G. which
Fig. 3. Effect of GB on the growth of strain MM-P. Growth was
determined by turbidity. Cells were grown on 5 mM propionate.
+
creased and the growth rate was 78% higher in the
presence of GB than in its absence.
3.3. Metabolic fate of DMSP ad
MMPA
Cells of strain MM-P, when grown on MMPA,
produced MSH from MMPA at a rate of 5 nmol
mini’
per mg of protein. When the cells were
grown on DMSP and either DMSP or MMPA was
added, the production rates of MSH were significantly lower (0.8 nmol min-’ per mg of protein and
1.3 nmol min-’ per mg of protein, respectively).
There was a rapid production of DMS from DMSP
Table 5
Effect of NaCl concentration
ML-D and ML-G
NaCl (M)
on DMSP incorporation
DMSP
ML-G
ML-D
0.5
1.5
2.5
3.0
in cells of
I-DMSP
Pellet
(nmol per
mg of protein)
(mM)
-2
_
28
59
250
II
23
96
Pellet
(nmol per
mS of protein)
I-DMSP
fmM)
600
700
800
1000
226
269
308
385
Cells were grown in propionate
(5 mM) to the end of the
exponential phase in the synthetic medium containing 0.5 to 3.0
M NaCl and 0.1 mM DMSP. At the end of the exponential phase
the cells were harvested, washed and resuspended in the synthetic
medium without propionate. DMSP in the resuspended pellet was
determined and the intracellular DMSP (I-DMSP) was calculated
[171.
A Not detectable.
Fig. 3. The effect of GB and propionate on the accumulation of
DMSP by strain ML-G. The cells were grown on 5 mM propionate and up-shocked by addition of 3 M NaCl (final concentration). (A) Cells treated with 0. I mM DMSP. (B) Cells treated with
0.1 mM DMSP and 0.1 mM GB. (C) cells treated with 0.1 mM
DMSP and 0.05 mM propionate. Data represent mean of duplicate
for each treatment.
did not grow on (Table 1) or oxidize (Table 2)
DMSP, accumulated this compound at all salinities
tested. DMSP accumulation was related to osmolarity; higher concentrations were accumulated at higher
NaCl concentrations.
At 3 M NaCl, the calculated
intracellular
DMSP accumulation
by ML-D and
ML-G were 96 mM and 385 mM, respectively.
ML-G cells. up-shocked by additions of 3 M
NaCl (final concentration) in the presence of 0.1 mM
DMSP (control) accumulated up to 76 mM DMSP
(Fig. 4A). In the presence of 0.1 mM GB, intracellular DMSP accumulation
was greatly decreased (Fig.
4B). However. additions of 0.05 mM propionate did
not have a significant effect on DMSP accumulation;
the DMSP accumulation level was similar to that of
the control (Fig. 4C). The DMSP uptake rates for
samples treated with GB. propionate and control
were 0.36 nmol mini’ per mg of protein. 0.73 nmol
mini’ per mg of protein. and 0.52 nmol min-’ per
mg of protein, respectively.
4. Discussion
Catabolism of GB by some aerobic bacteria has
been described [23-251. GB is progressively demethylated by Rhi:obium
meliloti through dimethylglycine and sarcosine to glycine with the formation
of I -carbon fragments [24]. ML-D, ML-G. and MM-P
strains appear to degrade GB and N,N-dimethylglycine
by successive
demethylations;
further
catabolism to glycine did not occur. This observation
was supported by growth and oxidation experiments.
Further experiments involving the detection of GB
metabolites need to be done to support the above
observation.
The studied isolates were highly specific for demethylation
of GB and N,N-dimethylglycine. However, when ML-D was grown on DMSP,
oxidation of additional
substrates (including
nonmethylated compounds) was possible (Table 2). The
oxidation of GB and N,N-dimethylglycine
by all
three strains irrespective of growth substrate suggests
that the demethylating
enzyme(s) are constitutive.
On the other hand, absence of growth on TMA,
DMA and MMA showed the inability of those strains
to use as substrate the intermediate
products of
anaerobic degradation.
Catabolism of GB by the three strains and DMSP
by ML-D occurred by two different mechanisms. GB
was degraded
by successive
N demethylations.
whereas DMSP was enzymatically
cleaved (DMSP
lyase) yielding DMS at a rate similar to that reported
for a marine bacterium [23].
Strain MM-P was induced to MMPA metabolism
when grown on MMPA, but catabolism of DMSP
(via the dem e th y 1,dt’ion-demethiolation
pathway) with
the concomitant
production of MSH, was possible
only when it was grown on DMSP. The inability of
MM-P to metabolize DMSP when grown on MMPA,
suggests that this bacterium needs separate enzymes
to metabolize DMSP and MMPA. The enzyme system responsible for demethylation-demethiolation
of
DMSP was induced by DMSP. When grown on
MMPA. demethiolation,
but not demethylation.
occurred. Studies done with a marine bacterium isolated from MMPA enrichments
also demonstrated
separate enzymatic
systems in the catabolism
of
DMSP and MMPA [ 151.
Addition of osmolytes to the growth medium
relieves or restores the growth of bacteria inhibited
by excessive salinity [26,27]. Exogenous additions of
GB and DMSP enhanced the growth of bacteria at
moderate salinities and allowed a higher maximal
salinity for growth. The utilization
of DMSP as
osmoprotectant
has been reported in Klebsiella
pneumoniae [28] and in a marine strain [23]. As an
osmoprotectant.
GB has been reported
in Escherichia coli [29], R. meliloti [24] and Salmorlella
tphimurium
[30]. Even though DMSP decreased the
lag phases at high salinities, GB was a more effective osmolyte than DMSP. There was more growth
in the presence of GB at high salinities. GB. but not
DMSP, permitted growth of strain ML-D at 3.5 M
NaCl. The presence of three methyl groups in the
structure of GB versus the two methyl groups in
DMSP may be pertinent to the effectiveness of GB
over DMSP. Sequential
methylations
of glycine
derivatives have been reported to increase the osmotolerance of barley leaf malate dehydrogenase
[31].
For example, GB was a more effective osmolyte than
N.N-dimethylglycine.
According to that study, the
shielding of the cationic charge on the nitrogen
increased with increase in the number of methyl
groups. Apparently
this increases effectiveness
in
osmoprotection
by allowing the osmoprotectant
to
remain as a zwitterion at physiological pH.
In ML-G, DMSP was accumulated intracellularly
and functioned as an osmolyte under high osmotic
stress conditions.
The highest intracellular
DMSP
concentration (385 mM) for ML-G strain was at 3 M
NaCI. Although ML-G was unable to grow on DMSP.
this strain possessed a DMSP transport system that
was active and resulted in DMSP accumulation (226
mM) at NaCl concentrations as low as 0.5 M. Analogously, the presence of a GB transport system in
cells unable to use GB as a growth substrate has
been reported for E. coli [32]. in which GB was
actively transported
and could reach intracellular
concentration
as high as 246 mM when grown at
0.65 M NaCl.
Sudden additions of 3 M NaCl (final concentration) stimulated the uptake of DMSP (Fig. 4). Since
the uptake was not rapid the DMSP transport is
probably inducible. There was no significant impact
of propionate (0.05 mM) on DMSP uptake. suggesting that storage products functioned in generating the
electrochemical
proton gradient thought to be the
main driving force in bacterial active transport systems [33]. Inhibition of DMSP uptake by the ML-G
strain in the presence of GB suggests competition for
these substrates (Fig. 4). Structural similarities between the substrates might explain the observed inhibition. Indeed. GB uptake in the sulfur bacterium.
Ectothior-llodo.~pi~~
hcrloch1ori.s was strongly inhibited by proline betaine and other GB analogues [33]
indicating that the uptake showed structural specificity for carboxyl and methyl groups. The DMSP
uptake system might have structural specificities
similar to those for GB and this topic deserves
further investigation.
The Mono Lake strains used GB as a carbon
and/or
energy source as well as osmoprotectant.
The ability to use GB as a substrate under non-stress
and stress conditions might have a dual advantage
for survival under natural conditions where GB is
present. Although no report in the literature specifically documents the presence of GB or DMSP in
Mono Lake, cyanobacteria as well as some halophilic
phototrophic
bacteria do produce and accumulate
significant amounts of GB in many hypersaline environments [34] and such phytoplankton
communities
as well as benthic communities have been described
for the Mono Lake ecosystem
[ 16.35,36]. The
cyanobacterium.
O.scillcrtotk.
and the phototrophic
bacterium. Ectothio~llodospirn.
may be examples of
DMSP and GB producers in Mono Lake. Since GB
and DMSP were successfully used either as carbon
sources or osmolytes this could imply fundamental
roles for both compounds in halotolerance
and as
carbon and energy sources for Mono Lake organisms. DMSP and GB are important substrates for a
wide variety of microorganisms.
Their utilization as
substrate and/or osmolyte will depend on the environmental conditions. their availability in the natural
environments.
and the presence of uptake systems.
Furthermore, this study strongly supports the possibility that alternate routes for DMSP metabolism
occur not only in marine environments,
but in a
moderately extreme environment such as Mono Lake.
Acknowledgements
We thank Drs. Ed Carpenter, H.S. Levinson and
P.T. Visscher for careful reviews and perceptive
questions which helped to improve the manuscript.
Dr. R. Oremland provided water samples and sediments from Mono Lake, CA. Financial assistance
was provided by the National Science Foundation
grant OCE 9012 IS7 and the W. Burghardt Turner
Fellowship (to Mara Diaz).
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