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Applied and Environmental Microbiology, October 1998, p. 3591-3598, Vol. 64, No. 10
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effects of Temperature, Salinity, and Medium
Composition on Compatible Solute Accumulation by
Thermococcus spp.
Pedro
Lamosa,1
Lígia O.
Martins,1
Milton
S.
Da Costa,2 and
Helena
Santos1,*
Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, 2780 Oeiras,1 and
Departamento de
Bioquímica, Universidade de Coimbra, 3000 Coimbra,2 Portugal
Received 29 December 1997/Accepted 23 July 1998
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ABSTRACT |
The effects of salinity and growth temperature on the accumulation
of intracellular organic solutes were examined by nuclear magnetic
resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and
Thermococcus zilligii (strain AN1). In addition, the
effects of growth stage and composition of the medium were studied in
T. litoralis. A novel compound identified as
-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly
discovered compound, T. litoralis accumulated
mannosylglycerate, aspartate, 
-glutamate,
di-myo-inositol-1,1'(3,3')-phosphate, hydroxyproline, and
trehalose. The hydroxyproline and -galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the
other Thermococcus strains examined.
Di-myo-inositol-1,1'(3,3')-phosphate, aspartate, and
mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated -glutamate. The only nonmarine species studied, T. zilligii,
accumulated very low levels of -glutamate and aspartate. The levels
of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in
response to salt stress, while
di-myo-inositol-1,1'(3,3')-phosphate was the major
intracellular solute at supraoptimal growth temperatures. The phase of
growth had a strong influence on the types and levels of compatible
solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while
di-myo-inositol-1,1'(3,3')-phosphate was the predominant
organic solute during the stationary phase of growth. This work
revealed an unexpected ability of T. litoralis to scavenge
suitable components from the medium and to use them as compatible
solutes.
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INTRODUCTION |
Most microorganisms subjected to
water stress accumulate organic solutes to control their internal water
activity, maintain the appropriate cell volume and turgor pressure, and
protect intracellular macromolecules (14). The compatible
solutes, also called osmolytes, include sugars, amino acids and their
derivatives, polyols and their derivatives, betaines, and ectoines
(11, 12, 14). The term compatible solute is generally used
for low-molecular-weight organic compounds that accumulate to high
intracellular levels under osmotic stress and that are compatible with
the metabolism of the cell. Compatible solutes can be synthesized de
novo or, if present in the medium, can be taken up by the organisms.
The latter strategy is preferred when appropriate substances are
present in the environment or in the growth medium (14).
Glycine betaine, for example, is a very common compatible solute in
mesophilic bacteria and archaea that is not synthesized by most
microorganisms but is taken up from the medium and used for
osmoadaptation (11, 14).
Recent investigations of water and temperature stress in thermophilic
and hyperthermophilic microorganisms led to the identification of
several new compatible solutes, some of which (mannosylglycerate [MG], di-glycerol-phosphate, and -glutamate) accumulate primarily in response to salt stress. Others, such as
di-myo-inositol-1,1'(3,3')-phosphate (DIP) and
cyclic-2,3-bisphosphoglycerate, accumulate primarily in response to
growth at supraoptimal temperatures (9, 15, 20-22, 26, 31).
A growing body of evidence also suggests that some solutes may
stabilize macromolecules facing potential thermal denaturation; for
example, DIP, cyclic-2,3-bisphosphoglycerate, and MG have been shown to
stabilize enzymes (15, 27, 31). These results suggest that
these solutes play an important role in protecting cellular components
in vivo that may account, in part, for the thermophilic nature of the
organisms. Other solutes, such as
di-myo-inositol-1,3'-phosphate and
2-O--di-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate, whose levels increase in response to high temperatures in some hyperthermophilic organisms, may also have similar properties (21,
22). Therefore, in this paper, the term compatible solute is used
for major intracellular organic solutes that accumulate in response to
osmotic or temperature stress.
Recently, we examined several thermophiles and hyperthermophiles for
the accumulation of organic solutes (20-22, 26); in particular, we observed that Pyrococcus furiosus accumulates
only the following two compatible solutes: MG, whose concentration increases as the salinity of the medium increases; and DIP, whose concentration increases sharply at supraoptimum growth temperatures (20). Moreover, the same solutes were also observed in
Pyrococcus woesei (31). Given the close
phylogenetic relationship between the genera Pyrococcus and
Thermococcus (30), we decided to compare the
compatible solutes and strategies of thermoadaptation and osmoadaptation in species of these two genera. In this work, we examined the effects of salinity and growth temperature on the intracellular solute pools of four Thermococcus species. A
new solute, -galactopyranosyl-5-hydroxylysine (GalHL), was
identified in Thermococcus litoralis. Furthermore, we found
that the composition of the medium had a major effect on the type of
organic solutes that accumulated during salt stress in this species and
that the proportions of several solutes changed significantly with the growth stage.
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MATERIALS AND METHODS |
Strains and culture conditions.
T. litoralis DSM
5474T, Thermococcus celer DSM 2470T,
Thermococcus stetteri DSM 5262T, and
Thermococcus sp. strain AN1T (= DSM
2770T), recently classified as Thermococcus
zilligii (30), were obtained from the Deutsche Sammlung
von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.
Unless indicated otherwise, T. litoralis, T. celer, and T. stetteri were grown in Bacto Marine Broth
(Difco). For growth of T. celer and T. stetteri
this medium was supplemented with sodium sulfide (final concentration,
2 g · liter
1). T. zilligii was grown in
the medium described by Lanzotti et al. (18) supplemented
with S0.
Cultures of T. litoralis were routinely grown in a 2-liter
fermentation vessel with continuous gassing with pure N2
and stirring at 100 rpm. T. stetteri was grown under the
same conditions except that the gas mixture contained N2
and CO2 (80:20). T. zilligii and T. celer were grown in static 2-liter flasks that were flushed with
N2 for 1 h before the medium was autoclaved. Cell
growth was monitored by measuring turbidity at 600 nm. Most
experimental cultures were grown until the late exponential phase,
harvested by centrifugation (8000 × g, 30°C, 10 min), and washed once with a solution lacking organic nutrients but
otherwise identical to the medium in which the cells were grown. The
effect of salinity on the levels of intracellular solutes was examined
by changing the NaCl concentration while the same concentrations of the
other medium components were maintained. The effect of growth phase on
the accumulation of solutes was examined in T. litoralis by growing the cultures in a 5-liter fermentation vessel with medium containing 4% NaCl. Samples (500 ml) were collected at appropriate culture times and treated as described above.
The effects of organic components on the accumulation of compatible
solutes by T. litoralis were examined by replacing peptone (5.0 g · liter1) with Casitone (5.0 g · liter1) or tryptone (5.0 g · liter1)
or by replacing yeast extract (1.0 g · liter1)
with maltose (2.0 g · liter1) in a medium
containing 3% NaCl and having the same basal salts composition as
Bacto Marine Broth.
Extraction of intracellular solutes, cell protein determination,
partial purification and hydrolysis of -galactosyl-hydroxylysine,
and quantification of organic solutes.
Cells were extracted twice
with boiling 80% ethanol by the method of Reed et al. (28),
modified as previously described by Martins and Santos (20).
Freeze-dried extracts were dissolved in D2O for nuclear
magnetic resonance (NMR) analysis. Quantification was performed by
1H NMR by using formate as an internal concentration
standard. Glutamate content was determined by the enzymatic assay of
Lund (19), and hydroxyproline was determined by using a
Pico-Tag amino acid analysis system (Waters, Milford, Mass.).
The protein content of cells was determined by the Bradford assay
(
6) after treatment of the cells with 1 M NaOH (100°C,
10 min) and neutralization with 1 M HCl.
The novel compound GalHL was purified by ion-exchange chromatography on
an SP-Sepharose C-25 (Pharmacia, Uppsala, Sweden)
column (19 by 2.6 cm). The extract was eluted with a sodium phosphate
buffer (pH 7.0)
gradient ranging from 5 mM to 1 M. Fractions were
eluted and examined
by NMR for the presence of the compound. Fractions
containing the
compound were pooled and freeze-dried. The residue
was dissolved in a
small volume of water, salt was removed by
passage through a Sephadex
G-25 column (type PD-10; Pharmacia),
and the eluted sample was
concentrated by freeze-drying. The residue
was dissolved in
D
2O for NMR analysis. Part of the sample was
hydrolyzed
with 3.0 M HCl at 100°C for 3 h under an N
2
atmosphere
in a sealed ampoule to liberate the sugar moiety from the
amino
acid.
NMR spectroscopy.
All spectra were obtained with a Bruker
model AMX500 spectrometer. 13C NMR spectra were obtained at
125.77 MHz by using a 5-mm carbon selective probe head. Typically,
spectra were acquired with a repetition delay of 1.5 s and a pulse
width of 7 µs corresponding to a 90° flip angle. Proton decoupling
was applied during the acquisition time only by using the wide-band
alternating-phase low-power technique for zero-residue splitting
sequence. Chemical shifts were referenced to the resonance of external
methanol at 49.3 ppm.
1H NMR spectra were acquired with water presaturation, a
6-µs pulse width corresponding to a 60° flip angle, and a
repetition
delay of 15 s. Chemical shifts were determined relative
to 3-(trimethylsilyl)propanesulfonic
acid (sodium salt). Formate was
added as an internal concentration
standard. Two-dimensional spectra
were obtained by using standard
Bruker pulse programs. Phase-sensitive
nuclear Overhauser effect
spectroscopy (NOESY), proton-homonuclear
shift correlation spectroscopy
(COSY), and total-correlation
spectroscopy (TOCSY) were performed
by collecting 4,096 (
t2) × 512 (
t1) data
points; in
1H-
13C heteronuclear multiple
quantum coherence (HMQC) spectra (
3),
a delay of 3.5 ms was
used for evolution of
1J
CH. The heteronuclear
multiple bond connectivity (HMBC) spectrum
was obtained by collecting
4,096 (
t2) × 256 (
t1)
data points;
a delay of 73.5 ms was used for evolution of long-range
couplings.
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RESULTS |
Identification of organic solutes in Thermococcus
spp.
The 13C-NMR spectra of ethanol extracts of the
thermococci grown in Bacto Marine Broth contained several sets of
resonances that were assigned to DIP, MG, trehalose, -glutamate,
hydroxyproline, and aspartate (Fig. 1).
Resonances were assigned by comparison with the carbon chemical shift
values reported previously (7, 26, 31). The identities of
the compounds were confirmed by 1H NMR, and assignment of
the resonances due to DIP was further confirmed by spiking a T. litoralis extract with an aliquot of a P. woesei
extract, in which DIP was initially identified (31). Initially, the remaining resonances in the 13C- NMR
spectrum of T. litoralis (176.0, 102.5, 75.5, 75.5, 72.8, 71.2, 68.9, 61.4, 55.0, 43.4, 28.4, and 27.1 ppm) could not be assigned
to a known compound, but identification was significantly facilitated
by partial purification of the extract. The resonances at 176.0 and
102.5 ppm were assigned to a carboxylic group and an anomeric CH group,
respectively. The HMQC spectrum showed that the latter resonance was
connected to a proton resonance at 4.49 ppm (Fig.
2). The resonances at 27.1, 28.4, 43.4, and 61.4 ppm were due to methylene groups, while the remaining
resonances were due to methyne groups. The final structure of the
molecule was established by using the results of a set of COSY, NOESY,
TOCSY, and HMBC experiments. The COSY and TOCSY spectra showed that the anomeric carbon at 102.5 ppm belonged to a network of a six-carbon monosaccharide (resonances at 102.5, 75.5, 72.8, 71.2, 68.9, and 61.4 ppm). The HMBC experiments showed that there was a connection between
the anomeric carbon of the hexose and the methyne group of the nonsugar
moiety at 76.5 ppm. Further analysis of the COSY and TOCSY spectra led
to identification of this moiety as 5-hydroxylysine. The hexose and the
amino acid fragments were firmly identified as galactose and
5-hydroxylysine after hydrolysis of the intact compound and spiking of
the hydrolysate with pure galactose and 5-hydroxylysine. The carbon
chemical shift values of the original compound indicated that the
galactose moiety was in the pyranose configuration rather than the
furanose configuration (5). The presence of connectivities
between H1 and H2 and between H3
and H5 but not between H1 and H4 in
the NOESY spectra led to the conclusion that galactose was in the
-pyranosyl configuration. This conclusion was supported by the low
chemical shift value for galactose H1 (4.49 ppm) and the
magnitude of the coupling constants (3JH1,H2
=7.7 Hz, 1JC1,H1 =160 Hz), which unambiguously
indicated that the configuration was a -pyranosyl configuration
(2, 4, 23). There was also a clear connectivity between
H1 of galactose and H5 of 5-hydroxylysine in
the NOESY spectrum, which showed that the location of the linkage was
between C-1 of galactose and the hydroxyl group of the amino acid. The
position of this linkage was also confirmed by the HMBC spectrum. On
the basis of these results, the unknown compound was identified as
GalHL (Fig. 2, inset). The corresponding carbon and proton chemical
shift values are shown in Table 1.
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FIG. 1.
Proton decoupled 13C-NMR spectrum of an
ethanol extract of T. litoralis grown in Bacto Marine Broth
containing 4% NaCl at 85°C. Resonances due to aspartate (peaks a),
MG (peaks b), DIP (peaks d), glutamate (peaks g), GalHL (peaks h), and
trehalose (peaks t) are indicated.
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FIG. 2.
13C-1H correlation spectrum
through one bond coupling (heteronuclear multiple quantum correlation)
of the novel compound GalHL. (Inset) Schematic representation of
GalHL.
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Effects of temperature and salt stress on the accumulation of
compatible solutes by Thermococcus spp.
The optimum
growth temperatures of T. litoralis, T. celer,
T. stetteri, and T. zilligii are 85, 88, 88, and
75°C, respectively. All of these organisms except T. zilligii, which is not halophilic, have optimum salinities for
growth of about 2.0 to 2.5% NaCl (17, 24, 25, 37). Under
optimal growth conditions in Bacto Marine Broth, the total solute pools
of these organisms were small; the highest levels of organic solutes
were found in T. celer, in which they did not exceed 0.6 µmol · mg of protein1 (Table
2). As the growth temperature or the
salinity of the medium was increased above the optimal level for
growth, the total solute pool also increased due to differential
accumulation of some solutes (Table 2, and Fig.
3 and 4).
There was, for example, a positive correlation in T. litoralis between growth in medium containing higher
concentrations of NaCl and pronounced increases in the levels of
aspartate, MG, and (to a lesser extent) GalHL. On the other hand, the
accumulation of compatible solutes was less pronounced when the
temperature was raised above the optimum temperature for growth.
However, DIP became the major solute during growth at supraoptimum
temperatures.
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FIG. 3.
Effect of the growth temperature, at the optimum
salinity, on the accumulation of the following compatible solutes by
T. litoralis (A), T. celer (B), and T. stetteri (C): DIP ( ), MG ( ),
glutamate
( ),
aspartate ( ), trehalose( ), GalHL
( ), and
hydroxyproline ( ).
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FIG. 4.
Effect of the NaCl concentration of the medium, at the
optimum growth temperature, on the accumulation of the following
compatible solutes by T. litoralis (A), T. celer
(B), and T. stetteri (C): DIP (), MG
(), glutamate
(),
aspartate (), trehalose (), GalHL
(),
and hydroxyproline ().
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Trehalose, GalHL, and hydroxyproline were not detected in
T. celer or
T. stetteri, which at elevated salinities
accumulated
significant levels of MG and aspartate. The levels of MG
also
increased at supraoptimum temperatures, but the most pronounced
alterations of the organic solute pools were due to increases
in the
DIP concentrations from about 0.4 to 1.2 µmol · mg of
protein
1 in
T. celer and from 0.1 to 0.4 µmol · mg of protein
1 in
T. stetteri.
T. zilligii was the only nonhalophilic and nonhalotolerant
species examined in this study, and, unlike the other species of
the
genus
Thermococcus,
T. zilligii had very low
intracellular
solute concentrations (Table
2). No solutes other than
aspartate
and glutamate were detected in this organism, and no clear
correlation
between the levels of these solutes and a high growth
temperature
was evident.
Effect of the growth phase on the accumulation of solutes in
T. litoralis.
Alterations in the levels of the organic
solutes during the growth cycle of T. litoralis were
determined in Bacto Marine Broth containing 4% NaCl at 85°C (Fig.
5). During the exponential phase of
growth, aspartate was the most abundant solute, but trehalose, MG, DIP,
glutamate, and GalHL were also detected in moderate amounts, while
hydroxyproline was present at very low levels. At the onset of the
stationary phase, increases in the concentrations of DIP and GalHL were
observed concomitant with slight decreases in the concentrations of all
other solutes except glutamate, whose level decreased notably. The
total solute pool decreased significantly after prolonged incubation of
the culture; only DIP, MG, and GalHL were detected, and DIP was the
predominant organic solute during this stage of growth.
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FIG. 5.
Correlation between growth phase and accumulation of
organic solutes by T. litoralis. The culture was grown in
medium containing 4.0% NaCl at 85°C ( ). The intracellular
concentrations of the following solutes were determined at different
phases of growth: DIP (), MG (),
glutamate
(),
aspartate (), trehalose (), GalHL
(),
and hydroxyproline ().
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Dramatic alterations in the relative amounts of the compatible solutes
with the growth stage were observed in cells grown
at 85°C in Bacto
Marine Broth containing 3% NaCl when peptone
was replaced by tryptone.
In particular, the levels of MG changed
drastically; the concentrations
of MG ranged from 0.37 µmol ·
mg of protein
1
when cells were harvested during the mid-exponential phase to
vestigial
during the early stationary phase. On the other hand,
the levels of DIP
remained almost constant. These results show
that it is critical to
control the cell growth stage if meaningful
compatible solute
accumulation data are going to be obtained.
Effect of the growth medium on the accumulation of organic solutes
in T. litoralis.
Due to the uniqueness of GalLH and the
rarity of hydroxyproline in prokaryotes, we though that it was
important to ascertain whether these solutes were synthesized de novo
or taken up from medium containing peptone or yeast extract. For this
reason, T. litoralis was grown in media that were identical
to Bacto Marine Broth except that they contained Casitone or tryptone
instead of peptone and in media in which maltose replaced yeast
extract. T. litoralis accumulated DIP, MG, trehalose, and
glutamate in medium containing yeast extract plus Casitone or
tryptone, but, unlike growth in medium containing peptone,
aspartate, GalHL, and hydroxyproline were not detected (Fig.
6). In these media there were also
pronounced increases in the levels of DIP and MG compared to the levels
accumulated by the organism in peptone-containing medium. Substitution
of yeast extract for maltose in medium containing peptone, Casitone, or
tryptone led to an absence of trehalose.
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FIG. 6.
Effect of medium composition on the accumulation of
organic solutes by T. litoralis grown at 85°C with 3.0%
NaCl. The media contained peptone plus yeast extract (bar A), peptone
plus maltose (bar B), Casitone plus yeast extract (bar C), Casitone
plus maltose (bar D), tryptone plus yeast extract (bar E), and tryptone
plus maltose (bar F). The intracellular concentrations of the following
solutes were determined: DIP (), MG (),
glutamate
(),
aspartate (), trehalose (), GalHL
(),
and hydroxyproline ().
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DISCUSSION |
The results presented in this paper show that T. celer,
T. stetteri, and T. litoralis accumulate
different solutes in response to two different types of stress. The
levels of MG, aspartate, and GalHL increased in response to salt
stress, while the levels of DIP responded primarily to temperature
stress. In T. litoralis, however, the effect of the salinity
of the medium on the accumulation of compatible solutes was much more
pronounced than the effect of the growth temperature. The reverse was
true in T. celer; in this organism the growth temperature
had a clear effect on the accumulation of compatible solutes, primarily
the levels of DIP, while there were only slight increases in the
concentrations of solutes as the salinity of the medium was increased
(Fig. 3 and 4). In P. furiosus, on the other hand, both
temperature and salinity had very pronounced effects on the
accumulation of compatible solutes (20). Accumulation of MG
and DIP in the species of the genus Thermococcus was
expected because these organisms are closely related to
Pyrococcus species, in which these solutes are also encountered (20). However, we did not expect to find that
aspartate was a major compatible solute in the halophilic
Thermococcus species grown in peptone-containing
medium, while glutamate played a lesser role in the osmoadaptation of
these organisms. Glutamate has been shown to be a common compatible
solute during low-level osmoadaptation in many bacteria and archaea
(11, 12, 14), while aspartate is generally found at very low
intracellular concentrations and contributes only slightly to the
compatible solute pool (34). Aspartate accumulates to
higher levels in Methanococcus thermolithotrophicus and Methanosarcina thermophila, but there is only a slight
positive correlation between the level of aspartate and salinity and
this amino acid is not thought to behave like a compatible solute
(29, 33). However, under salt stress conditions aspartate is
an important compatible solute of halophilic thermococci grown in
medium containing peptone, in which it reaches levels comparable to
those of MG.
The newly identified solute GalHL was detected only in peptone-derived
T. litoralis cells, in which it accumulated in response to
increasing levels of salinity. Aside from choline and acetylcholine, which are taken up from the medium by Lactobacillus
plantarum (16), no other known compatible solute has a
net positive charge (12, 14). However, two neutral lysine
derivatives are known to serve as compatible solutes;
N
-acetyl--lysine accumulates in several
methanogens, such as Methanosarcina thermophila and
Methanogenium cariaci (33), and
N-acetyl-lysine behaves like an osmolyte in a
few moderately halophilic bacteria (13). It is also
important to note the resemblance between GalHL and the
-glucosyl--galactosyl-5-hydroxylysine moiety found in mammalian
collagen. In fact, GalHL is probably derived from collagen, since this
protein is present in peptone (13a); it may be formed from
the diglycosyl-hydroxylysine component after removal of the terminal
glucose residue by the microorganisms or during the production of
peptone. It should also be noted that hydroxyproline is a major amino
acid in collagen and that low levels of this amino acid also accumulate
in T. litoralis. Accumulation of hydroxyproline was detected
previously in Listeria monocytogenes under water stress
conditions in peptone-containing media or when hydroxyproline or
prolyl-hydroxyproline was added to the medium (1). The
observation that GalHL and hydroxyproline were detected only in
T. litoralis cells grown in peptone-containing media leads us to believe that these solutes are derived from peptone. It is,
however, difficult to explain why aspartate was not accumulated from
Casitone or tryptone by this organism, since this amino acid is present
in these casein-based preparations. It is also not known why GalHL and
hydroxyproline were not detected in the other Thermococcus
species or in the closely related species P. furiosus grown
in media that also contain peptone (22).
The intracellular levels of compatible solutes in the thermococci never
balanced the levels of osmotically active substances in the medium.
This indicates that inorganic ions (namely, K+ and
Cl) may significantly contribute to the osmolyte pools of
these organisms. Large contributions by inorganic ions to the osmolyte pools have been found in some methanogens and have been inferred for
other hyperthermophilic archaea (9, 20, 21), and such contributions may also occur in the thermococci. However, it should be
pointed out that the values obtained were calculated by assuming that
the cell volume was independent of the salinity of the medium, which is
unlikely.
Trehalose is a common disaccharide in bacteria and archaea and may play
a role in osmotic adaptation in several organisms (11, 14).
Yeast extract, in which the trehalose level is about 11% (wt/wt)
(36), is a common source of this disaccharide for many
organisms. Our results show that trehalose accumulated in T. litoralis only when yeast extract was added to the medium; therefore, we concluded that this disaccharide was not synthesized de
novo under the conditions examined in this study. Rather, trehalose was
taken up from yeast extract by the recently described high-affinity maltose-trehalose transport system, which was shown to be induced by
trehalose (36). Our results reinforce the importance of
solute uptake by organisms under water stress conditions and extend
previous results showing that unusual compatible solutes, such as
carnitine, dimethylsulfoniopropionate, 3-morpholino-1-propanesulfonic
acid (MOPS), and arsenobetaine (10, 14, 16, 32) are taken
up.
This work revealed the an unexpected ability of T. litoralis
to scavenge suitable components from the medium and to use them as
compatible solutes, thus bypassing de novo synthesis and saving energy.
It is noteworthy that neither the other Thermococcus species examined nor the closely related Pyrococcus species were
able to derive such a variety of solutes from the medium.
The differential accumulation of osmolytes observed during the growth
cycle of T. litoralis corroborates previous results obtained
with hyperthermophilic archaea, mesophilic bacteria, and yeasts
(8, 20, 32, 35). For example, it is often observed that the
total solute pool decreases during the stationary phase, although no
explanation for this observation has been given. Perhaps some
compatible solutes replace others that accumulate during the
exponential phase because they are not as easily lost to the external
environment. Some compatible solutes that accumulate during the
exponential phase of growth may also be preferentially consumed when
the concentrations of nutrients in the medium decrease below critical
levels.
Unlike the other organisms examined here, T. zilligii
AN1T was isolated from a low-salinity continental hot
spring and does not tolerate the levels of NaCl required by the other
species of the genus Thermococcus for growth (17,
30). This organism does not accumulate appreciable levels of
compatible solutes at the optimum growth temperature, nor does it
accumulate compatible solutes as the growth temperature is raised.
Other nonhalophilic thermophilic and hyperthermophilic organisms, such
as Thermotoga thermarum, Pyrobaculum islandicum,
and Fervidobacterium islandicum, unlike slightly halophilic
members of the same genera, do not accumulate compatible solutes at the
optimum growth temperature (21, 22). The observation that
only halophilic strains accumulate compatible solutes at supraoptimum
growth temperatures suggests that the role played by these solutes may
be something other than a role in thermoadaptation. However, it is
possible that solutes such as DIP, cyclic-2,3-bisphosphoglycerate, and
(to some extent) MG may play a role as thermoprotectants primarily when
two stresses (namely, water stress and temperature stress) are applied
simultaneously. In fact, it is difficult to explain the consistent
accumulation of specific solutes by most hyperthermophilic organisms at
supraoptimal temperatures unless a role in thermoprotection is
envisioned. Intrinsic properties of cell components are very important
for growth at high temperatures, but with the present state of
knowledge we cannot discount the contribution of extrinsic factors,
such as the thermostabilizing attributes of some compatible solutes, to
the growth of microorganisms at high temperatures.
| |
ACKNOWLEDGMENTS |
This work was supported by grant BIO4-CT96-0488 from the European
Community Biotech Programme (Extremophiles as Cell Factories) and by
Praxis XXI and FEDER Programme grants PRAXIS 2/2.1/BIO/20/94 (to H.S.
and M.S.D.) and PRAXIS/2/2.1/BIO/1109/95 (to H.S.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Tecnologia Química e Biológica, Apartado 127, 2780 Oeiras, Portugal. Phone: 351 1 4469828. Fax: 351 1 4428766. E-mail:
santos{at}itqb.unl.pt.
| |
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Abstract
Introduction
