FEMS Microbiology Letters 180 (1999) 325^330
Salt stress a¡ects sterol biosynthesis in the halophilic black yeast
Hortaea werneckii
U. Petrovic­ a , N. Gunde-Cimerman b , A. Plemenitas­
a;
*
a
b
Institute of Biochemistry, Medical Faculty, Vrazov trg 2, 1000 Ljubljana, Slovenia
Department of Biology, Biotechnical Faculty, Vec­na pot 111, 1000 Ljubljana, Slovenia
Received 9 August 1999; received in revised form 21 September 1999; accepted 23 September 1999
Abstract
Hortaea werneckii is a black yeast recently isolated from salterns in Slovenia. Some of the adaptations of halophilic
microorganisms to increased salinity and osmolarity of the environment are alterations in membrane properties. By
modulating the fluidity, sterols play an important role as a component of eukaryotic biological membranes. We studied the
regulation of sterol biosynthesis in H. werneckii through the activity and amount of 3-hydroxy-3-methylglutaryl coenzyme A
reductase (HMG R), a key regulatory enzyme in the biosynthesis of sterols. We found some differences in the characteristics of
HMG R and in its regulation by different environmental salinities in H. werneckii when compared to the mesophilic baker's
yeast, Saccharomyces cerevisiae. Our results suggest that halophilic black yeast regulates sterol biosynthesis through HMG R in
a different way than mesophiles, which might be a consequence of the different ecophysiology of halophilic black yeasts. From
this perspective, H. werneckii is an interesting novel model organism for studies on salt stress-responsive proteins as well as on
sterol biosynthesis in eukaryotes. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science
B.V. All rights reserved.
Keywords : Halophilic black yeast; Sterol biosynthesis; Hortaea werneckii ; Salt stress; 3-Hydroxy-3-methylglutaryl coenzyme A reductase
1. Introduction
Recently, several types of black yeasts have been
isolated from salterns and some of them were characterized as adaptively halophilic or extremophilic
fungi [1,2]. The dominant species among black yeasts
was Hortaea werneckii, isolated for the ¢rst time
from salterns in Slovenia in 1995 [1]. Based on pop-
* Corresponding author. Tel.: +386 (61) 312 357; Fax:
+386 (61) 132 00 16; E-mail: [email protected]
ulation dynamics [1] it appears that salterns are the
natural habitat of H. werneckii. Salterns are an extreme environment because of the variations in the
concentration of di¡erent ions, especially NaCl. Species able to grow actively in such environments must
be adapted to rapid changes in the environment and
these adaptations should also be detectable in biochemical studies.
Some of these adaptations to increased salinity
and consequently osmolarity of their environment
are mediated through changes in the membrane
properties [3,4]. Pinocytotic vesiculation of plasma
membranes had been observed earlier under stress
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
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conditions in the halotolerant fungus Aspergillus repens. Many of the hyphal cells under stress also exhibited numerous intravacuolar membranes [5]. In
eukaryotes, sterols function as a component of biological membranes by modulating their properties,
especially their £uidity. Changes in sterol contents
have been observed in halophilic black yeasts in
our laboratory [6]. We assumed that halophilic fungi
might regulate the sterol content of their membranes
in a di¡erent manner than their mesophilic counterparts. The key regulatory enzyme in sterol biosynthesis is 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG R) [7,8] which is present in Archaea
and presumably all eukaryotes [9]. The enzyme has a
highly conserved carboxy-terminal domain which is
responsible for the conversion of hydroxymethyl-glutaryl coenzyme A to mevalonate. The activity of
HMG R is tightly regulated in all organisms in a
variety of ways, including end-product-mediated
degradation and cross-regulation in which HMG R
activity is regulated by processes independent of the
mevalonate pathway [9].
Papers on the puri¢cation and characterization of
HMG R in halophilic bacteria have been published
[10^12], but no data exist on HMG R in halophilic
or halotolerant eukaryotes. In this study we present
data on the basic characteristics of HMG R in H.
werneckii in comparison to that of a mesophilic
yeast, Saccharomyces cerevisiae. The e¡ect of di¡erent salinities on both in vitro activities and amounts
of HMG R are also presented.
2. Materials and methods
2.1. Strains and growth conditions
H. werneckii (Ascomycota, Dothideales) from the
culture collection of the Slovenian National Institute
of Chemistry (MZKI), strain B-736 isolated from
salterns near Sec­ovlje, Slovenia [1] was used in this
study. The reference mesophilic strain was S. cerevisiae MZKI K-86. Cells were grown in supplemented
synthetic de¢ned medium (Bio-101): 1.6 g yeast nitrogen base, 0.8 g complete supplement mixture, 5 g
(NH4 )2 SO4 , 20 g glucose per liter of deionized water,
pH adjusted to 7.0, with di¡erent NaCl concentrations, and were harvested in the mid-exponential
phase. H. werneckii was grown on a rotary shaker
at 27³C at 180 rpm and S. cerevisiae at 30³C and 200
rpm. For determining the e¡ect of lovastatin, cells
were grown for 3 h before harvesting in the presence
of 1 mM lovastatin and after harvesting were washed
thoroughly with 50 mM Tris bu¡er.
2.2. Measurements of HMG R in vitro activity
HMG R in vitro activity was assayed as in [13]
with some alterations. Harvested cells were washed
with 50 mM Tris bu¡er, pelleted, frozen in liquid
nitrogen and mechanically disintegrated (Mikro-Dismembrator II, B. Braun). Powdered frozen lysates
were then resuspended in the activity measurement
bu¡er: 50 mM Tris bu¡er with pH 5^10, 0.5% Triton X-100 and various concentrations of NaCl and
glycerol (see Table 1 and Fig. 1 for details), and were
then incubated on ice for 1 h. Protease inhibitor
cocktail (50 Wl ml31 , Sigma) was added to each sample. Samples were centrifuged at 8000Ug for 15 min
at 4³C and the supernatants were used for the measurement of the enzyme activity. Proteins were determined by the Lowry method. Aliquots of samples
containing at least 500 Wg of protein were used for
the HMG R activity assay and 25 Wl of NADPH
regeneration system (50 mM Tris bu¡er, pH 7.5,
with 0.4 mg dithiothreitol, 0.4 mg NADP‡ , 4 Wl
14% glucose 6-phosphate and 1 Wl glucose 6-phosphate dehydrogenase (100 U ml31 , Sigma)) was
added to each aliquot. [14 C]3-Hydroxy-3-methylglutaryl coenzyme A (2.5 nmol) was added to each sample at zero time to start the reaction. The reaction
was terminated with 10 Wl of concentrated HCl at
di¡erent times (Table 1), and 2.5 nmol of
[3 H]mevalonate was added as a reference standard
and incubated for an additional 15 min to convert
the mevalonate into mevalonolactone. The samples
were then analyzed on precoated Silica Gel 60 TLC
plates (Merck) with a benzene/acetone 50:50 (v/v)
solvent system. The mevalonolactone-containing
area was scratched o¡ the plates into scintillation
vials and dissolved in 1 ml of ethanol and 3 ml of
scintillation counting liquid (0.5% 2,5-diphenyloxazol and 0.01% 1,4-bis-2/4-methyl-5-phenyloxazol
benzene in toluene). The radioactivity was measured
by a scintillation counter using the dual-count (14 C/
3
H) mode and the absolute values for HMG R in
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327
vitro activities expressed as (pmol produced mevalonate) min31 (mg total protein)31 were calculated.
2.3. Immunoprecipitation and immunoblot of
HMG R
Cell lysates, obtained as described in Section 2.2,
were resuspended in IP bu¡er (15 mM
NaH2 PO4 +NaOH, pH 7.5, 150 mM NaCl, 2% Triton X-100, 0.1% SDS and 0.5% Na-deoxycholate)
and 50 Wl ml31 of the protease inhibitor cocktail
(Sigma) was added. The samples were incubated
for 1 h on ice to solubilize the HMG R from the
membranes and then centrifuged at 16 000Ug for
30 min at 4³C. Aliquots of samples containing 1 mg
of protein were used for further manipulations. Rabbit IgG (10 mg ml31 ) was added in a concentration
of 1 Wl per 50 Wl of sample for non-speci¢c binding
and samples were incubated for 6 h at 4³C. After the
incubation, protein A-Sepharose was used to remove
the IgG-protein complexes with incubation for 2 h at
4³C while being shaken. Antibodies for the catalytic
domain of HMG R were then used for immunoprecipitation of HMG R using 2 Wl ml31 of antibodies
at 17 (mg protein) ml31 . The samples were incubated
overnight at 4³C. Protein A-Sepharose was added,
incubated for an additional 2 h and pelleted. Protein
A pellets were washed several times with washing
bu¡ers: twice with 0.15 M NaCl in 0.1 M Tris
bu¡er, pH 8.0; twice with 0.5 M LiCl in 0.1 M
Tris bu¡er, pH 8.0; once with 0.15 M NaCl, 1%
Triton X-100 and 1% Na-deoxycholate in 0.05 M
Tris bu¡er, pH 7.4; once with 0.15 M NaCl in
0.05 M Tris bu¡er, pH 7.4; and ¢nally with PBS
bu¡er. The washed samples were then resuspended
in electrophoresis sample bu¡er (62.5 mM Tris-HCl
bu¡er, 8 M urea, 1.5% SDS, 0.1 M dithiothreitol, 5%
glycerol and 0.002% bromophenol blue, pH 6.8) and
the proteins separated by SDS-PAGE electrophoresis
(Bio-Rad MiniProtean Cell, 10% gel). The proteins
were transferred to a PVDF membrane by electro-
Fig. 1. In vitro HMG R activities in H. werneckii: (a) pH dependence, (b) e¡ect of NaCl content (%) in the bu¡er. Shown
are mean values of at least three independent experiments.
transfer (Bio-Rad MiniProtean Cell). Immunoblotting was performed with the same anti-HMG R antibodies as for immunoprecipitation at a dilution of
1:6000 using the ECL detection system (Amersham).
Table 1
Optimal conditions for in vitro HMG R activity in H. werneckii and S. cerevisiae
Species
pH
Concentration of NaCl
Concentration of glycerol
Temperature
Time of reaction
H. werneckii
S. cerevisiae
9
7
0.5%
0%
20%
0%
25³C
37³C
25 min
15 min
In both cases the bu¡er was 50 mM Tris with 0.5% Triton X-100. For details see Section 2.
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Fig. 2. E¡ect of NaCl concentration in the growth medium on
the absolute in vitro HMG R activities in S. cerevisiae (a) and
H. werneckii (b). Shown are mean values of at least three independent experiments.
3. Results and discussion
3.1. Characteristics of H. werneckii HMG R in vitro
Since to our knowledge this is the ¢rst study on
sterol biosynthesis in halophilic or halotolerant fungi, we ¢rst determined the optimal conditions in
which the HMG R was active in vitro. Because it
is known for some proteins of the halophilic Archaea
in general [14] and for HMG R in particular [10,12]
that the presence of ions in their surroundings is
necessary for their native tertiary structure and activity, we optimized the conditions for in vitro HMG
R activity for the presence of di¡erent concentrations of NaCl, pure and non-re¢ned from salterns,
KCl, Mg2‡ , and glycerol. The pH, time and incubation temperature of the reactions were also optimized. Sensitivity of the enzyme to lovastatin, a
known competitive inhibitor of HMG R [15,16],
was tested as well. In all experiments, S. cerevisiae
was used as a reference mesophilic strain. The optimal conditions of HMG R in vitro activity for H.
werneckii in comparison to S. cerevisiae are summarized in Table 1.
Calculated absolute values (Figs. 1 and 2) for in
vitro HMG R activities ranged from 150 to 350 pmol
min31 (mg protein)31 in S. cerevisiae and from 10 to
40 pmol min31 (mg protein)31 in H. werneckii. The
lower activity in H. werneckii may be partly due to a
longer generation period of the halophilic cells, but it
could also demonstrate that halophilic HMG R may
be regulated in a di¡erent manner than that of S.
cerevisiae, since generation times of H. werneckii
were not that much longer than those of S. cerevisiae
(data not shown). Lovastatin at a concentration of
1 mM decreased the HMG R activity in H. werneckii
to approximately one third of that in untreated cells.
Glycerol, which is known to be an osmoprotectant in
some osmotolerant microorganisms [17], had the
most positive e¡ect on H. werneckii in vitro HMG
R activity at a concentration of 20% (Table 1), while
it decreased in vitro activity in S. cerevisiae at any
concentration. The pH optimum for the H. werneckii
HMG R activity was pH 9 (Table 1, Fig. 1a),
although H. werneckii had been isolated from its
habitat at a close to neutral pH [1]. These data together with di¡erences in optimal temperatures and
times of reaction (Table 1) point to the di¡erent
nature and/or regulatory mechanisms of HMG R
in halophiles. Sensitivity of the enzyme for lovastatin
on the other hand suggests that the catalytic domain
of the enzyme should be similar to other species.
Although H. werneckii can tolerate up to 30%
NaCl in the medium, 0.5% NaCl turned out to be
optimal for HMG R in vitro activity (Table 1, Fig.
1b). The same was observed for other species of
black yeasts isolated from salterns and studied in
our laboratory (data not shown). Addition of KCl
and Mg2‡ at any concentration lowered this activity
in both H. werneckii and S. cerevisiae (data not
shown). We observed that pure, analytical grade
NaCl was better than non-re¢ned saltern salt which
contained traces of Mg2‡ , K‡ and SO23
4 ions. These
results show that the salt requirements for the in
vitro measurements of the H. werneckii HMG R
are very di¡erent from those in Halobacterium sali-
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329
Fig. 3. Degradation of HMG R in S. cerevisiae and H. werneckii grown in media with di¡erent salinities, analyzed by Western blot.
Shown are speci¢c bands for HMG R and its degradation product with a putative size of 60 kDa.
narum and Haloferax volcanii HMG Rs which were
only active in the presence of at least 3 M KCl
[10,12].
3.2. E¡ect of salinity on HMG R in vivo
Since the growth characteristics of H. werneckii in
the presence of di¡erent salt concentrations are very
di¡erent from those of mesophilic yeasts and fungi
[1,2] and since H. werneckii HMG R is sensitive to
ionic strength and the nature of the salt in its environment, we tested how salinity a¡ects H. werneckii
in comparison to S. cerevisiae. Fig. 2 shows HMG R
activities from H. werneckii cells and S. cerevisiae
grown in the presence of di¡erent NaCl concentrations. For S. cerevisiae 0% NaCl in the medium represents the optimal growth condition while 3^5%
NaCl represent an increased salt stress. Growth of
S. cerevisiae at higher NaCl concentrations is diminished. For H. werneckii 17% NaCl is considered optimal salinity for growth [2]. We chose 0% NaCl as a
hyposaline and 25% NaCl as a hypersaline environment. Di¡erent pro¢les of HMG R activities were
observed for the two species: the in vitro activity
of S. cerevisiae HMG R increased with increased
salinity of the medium (Fig. 2a) which suggests
that at least one of the two isozymes [9] responds
to salt stress with increased activity. In S. cerevisiae
one of the HMG R isozymes, Hmg2p, behaves like a
stress protein that is speci¢cally produced in oxygendepletion stress and degraded when the stress is removed [9]. In contrast, H. werneckii cells grown in
medium with 17% NaCl had the lowest in vitro
HMG R activity, while cells from both higher and
lower NaCl concentrations showed increased HMG
R activities (Fig. 2b). We may speculate that 17%
NaCl is the optimal salinity for H. werneckii growth,
and that the increase of the HMG R activity is a
response to both hyperosmotic stress with 25%
NaCl and hypoosmotic stress without NaCl. Indeed,
ecological studies of H. werneckii showed a major
increase in the population when the NaCl concentration exceeded 15% [1]. It is known that in some
plants and bacteria HMG R isozymes respond to
various types of stresses; moreover, this e¡ect has
even been detected in mammals and is known as
cross-regulation [9].
The degradation pattern of HMG R was analyzed
for S. cerevisiae and H. werneckii cells grown in optimal growth conditions and under stressed conditions. In S. cerevisiae we observed an increase in
the amount of whole HMG R correlated with the
increase of NaCl in the growth medium (Fig. 3). It
could be argued that the S. cerevisiae Hmg2p protein, known to be a protein responsive to changes in
oxygen concentrations [9], also reacts to salt stress
with an increase in both its activity and amount.
However, H. werneckii reacts to increased salinity
in a di¡erent manner: cells grown in 17% NaCl
had a smaller amount of whole HMG R than those
grown in 25% or 0% NaCl (Fig. 3). Along with the
bands representing the whole enzymes, di¡erences in
the degradation patterns were also observed: a fragment with a putative size of 60 kDa accumulated in
cells grown in optimal conditions (Fig. 3). On the
basis of the decreased HMG R activity (Fig. 2) we
assume that it is a product of HMG R degradation
and is catalytically inactive. These results suggest
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that salt stress induces changes in HMG R activity
through the regulated degradation of the enzyme.
We speculate that 17% NaCl is optimal salinity for
H. werneckii and that an increase in both the amount
(Fig. 3) and activity (Fig. 2) of HMG R is the response to both hyper- and hypo-osmotic stress. Thus
these experiments con¢rmed our hypothesis that at
least one of the H. werneckii HMG R isozymes responds to salt stress. However, the mechanism of
this regulation remains to be resolved.
The results presented here show that an extremophilic eukaryote, the halophilic black yeast H. werneckii, regulates sterol biosynthesis, one of the essential metabolic pathways of all eukaryotes, in a quite
di¡erent way than S. cerevisiae, a generally accepted
eukaryotic model organism. The characteristics of
the HMG R are also di¡erent from those of archaeal
HMG Rs [10^12]. H. werneckii is thus an interesting
model organism for studies on salt stress-responsive
proteins and on sterol biosynthesis. However, for the
determination of general adaptations to the extremely high salinities that H. werneckii can tolerate,
further studies of various cellular compartments are
necessary.
Acknowledgements
We would like to thank John A. Watson for antiHMG R antibodies and for useful comments on the
manuscript. We would also like to thank all our
colleagues for their useful suggestions and especially
Milena Marus­ic­ for her technical assistance. This
work was supported by a grant from the Ministry
of Science and Technology of the Republic of Slovenia.
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