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Previous Article | Next Article 
Journal of Bacteriology, May 2001, p. 3251-3255, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3251-3255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cloning and Expression of Two Genes Coding for
Sodium Pumps in the Salt-Tolerant Yeast Debaryomyces
hansenii
Anabel
Almagro,1,2,
Catarina
Prista,3
Begoña
Benito,2
Maria C.
Loureiro-Dias,3 and
José
Ramos1,*
Departamento de Microbiología,
Escuela Técnica Superior de Ingenieros Agrónomos, E-14071
Córdoba,1 and Departamento de
Biotecnología, Escuela Técnica Superior de Ingenieros
Agrónomos, Universidad Politécnica de Madrid, E-28040,
Madrid,2 Spain, and Departamento de
Botânica e Engenharia Biológica, Instituto Superior de
Agronomia, 1349-017 Lisbon, Portugal3
Received 10 November 2000/Accepted 27 February 2001
 |
ABSTRACT |
Two genes encoding Na+-ATPases from Debaryomyces
hansenii were cloned and sequenced. The genes, designated
ENA1 from D. hansenii (DhENA1) and
DhENA2, exhibited high homology with the corresponding genes from Schwanniomyces occidentalis. DhENA1 was
expressed in the presence of high Na+ concentrations, while
the expression of DhENA2 also required high pH. A mutant of
Saccharomyces cerevisiae lacking the Na+ efflux
systems and sensitive to Na+, when transformed with
DhENA1 or DhENA2, recovered Na+
tolerance and also the ability to extrude Na+.
| |
TEXT |
Two strategies for adapting to the
presence of high salt have-been identified in different organisms. The
best-known mechanism is to exclude sodium from the cytoplasm and to
accumulate high concentrations of compatible solutes to avoid water
loss (excluder organisms), while some organisms use and accumulate high
Na+ concentrations without becoming intoxicated (includer
organisms) (26).
Saccharomyces cerevisiae is an excluder yeast which keeps
cytoplasmic sodium at low levels by extruding the cation out of the
cell or driving it into the vacuole. Nha1 and Ena1-4 mediate sodium
efflux processes. Nha1 works as a Na+
(K+)/H+ antiporter (4, 23), and
Ena proteins are P-type ATPases (5, 9, 11, 28). Deletion
of the corresponding genes renders the cells sensitive to sodium,
specially in the case of ENA1 (11). In
addition, Nhx1 drives sodium into the vacuole in exchange with
H+ (17). Schwanniomyces
occidentalis and Schizosaccharomyces pombe are also
excluder yeasts. While two ENA genes have been identified in
S. occidentalis (3), in the case of the fission
yeast, which is more sensitive to external sodium, only a
Na+/H+ antiporter (Sod2) has been found
(10, 13). Finally, in Zygosaccharomyces rouxii,
an osmotolerant yeast, there is evidence of the existence of both, two
antiporters (ZSOD2 and ZSOD22) (12,
19) and one ATPase (ZrENA) (unpublished data)
(GenBank TM/EMBL accession number D78567).
Debaryomyces hansenii can be isolated from salty
environments (14, 32). More than 30 years ago, it was
reported that among several marine yeasts, D. hansenii was
the least affected by high concentrations of NaCl (21,
27). D. hansenii accumulates high amounts of
Na+, and in this yeast, Na+ is not more toxic
than K+ (18, 24, 30). More recently it was
shown that Na+ improves the performance of D. hansenii under different stress conditions (1).
Therefore, we have proposed that D. hansenii behaves as a
Na+ includer yeast and that it may be considered a
halophilic yeast, in particular at low K+ concentrations.
In any case, a role for glycerol, as a compatible solute, must be
reserved (2, 20), since a glycerol/Na+
symporter with homeosmotic function in this yeast has been described (15, 16). In addition, biochemical work has shown the
existence of sodium efflux processes that may be involved in sodium
tolerance (for a review, see reference 26). There is no
specific molecular information on the processes involved in such good
performance in the presence of salt and in sodium fluxes in D. hansenii. This information would be essential in the understanding
of the molecular basis of halotolerance and halophilism in yeasts.
Effect of pH on the Na+ efflux in D. hansenii.
The existence of Na+ efflux processes
in D. hansenii has been previously reported (22, 24,
30). The mechanisms described may involve
Na+-ATPases or Na+/H+ antiporters.
While antiporters can be strongly affected by pH, the activity of
Na+-ATPases is constant over a wide range of pH. As a first
approach to investigate the nature of the system(s) involved in
Na+ extrusion, we measured the effect of pH on this
process. D. hansenii (strain PYCC 2968) was grown in YNB
medium (Difco), and cells were harvested at the early exponential
growth phase. Cells were then loaded with 200 mM NaCl over 3 h,
resulting in an increase in Na+ content from 77 to 290 nmol/mg (dry weight) of cells. Cells were resuspended in assay buffer:
Tris-citrate (10 mM Tris brought to pH 3.5 with citric acid), MES [10
mM MES (2-morpholinoethanesulfonic acid) brought to pH 5.5 with
Ca(OH)2], or TAPS {10 mM TAPS
[N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid]
brought to pH 7.5 with Ca(OH)2}. Tris buffer was
supplemented with CaCl2 (1 mM), and all buffers also
contained 0.1 mM MgCl2 and 2% (wt/vol) glucose. The efflux
process was studied as described previously (24), and
results indicate that it was not significantly affected by pH in the
range from 3.5 to 7.5 (data not shown). This was an indication that
under the conditions tested, a Na+/H+ antiport
was not the main mechanism involved. This idea is supported by the
observation that D. hansenii can grow at high pH (7.8) and
high Na+ concentration (1.5 M) (1). Under
these conditions, the activity of a Na+ ATPase should be
required in order to extrude the cation in the absence of favorable
Na+ or pH gradients.
Cloning and sequence analysis of ENA genes in D. hansenii.
Taking into account the previous result and the
similarity of the kinetics of Na+ extrusion in D. hansenii and in S. cerevisiae (24), we
investigated the existence of ENA-type genes in D. hansenii by a PCR approach. Manipulation of nucleic acids was
performed by standard protocols (29) or by following the
manufacturer's instructions. We used degenerated primers designed from
conserved sequences of Na+- and Ca2+-ATPase
genes (6). Using two of these primers corresponding to the
conserved amino acids, CSDK (as the sense primer) and DDNFASSI (as the
antisense primer), we amplified from genomic DNA of D. hansenii two fragments of 1.0 and 1.2 kb. In order to obtain the complete sequence of the cDNAs, we extended the sequences up to the 5'
end by reverse transcription-PCR using a 5'3'RACE kit (Boehringer) and
to the 3' end by reverse transcription-PCR using first an anchored
Not-dT18 commercial primer for reverse transcription and then an
antisense anchor primer and a sense specific primer for the PCR
amplification of each cDNA. Full-length cDNAs of 3,309 and 3,219 bp,
respectively, were obtained. Analysis of their translated sequences
showed 75 and 73% identity with SoEna1 and SoEna2, respectively. Figure 1 shows the phylogenetic
relationships between the different fungal Na+-ATPases. It
is interesting that cta3Sp, a putative calcium transporter, appears to
be closely related to the Ena proteins.
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FIG. 1.
Phylogenetic tree of Na+- and
Ca2+-ATPases from fungi. Alignment of the sequences was
performed with the CLUSTALX program (31) using the core
sequences starting at the beginning of the f and ending at the end of
the j conserved regions (28). Accession numbers are as
follows: ScENA1, X67136; ZrENA, D78567;
cta3Sp, P22189; NcENA1, AJ243520;
DhENA2, AF263248; SoENA2, AF030861;
DhENA1, AF247561; SoENA1, AF030860.
|
|
On the basis of sequence homologies, the corresponding genes were
designated ENA1 of D. hansenii
(DhENA1) and DhENA2. Southern blot analyses
revealed that the probes prepared for DhENA1and DhENA2 were specific and hybridized only with their own
genes and with total DNA from D. hansenii.
Regulation of transcription of DhENA1 and
DhENA2.
In order to perform Northern blot analyses,
cells were grown in YPD medium (1% [wt/vol] yeast extract, 2%
[wt/vol] peptone, and 2% [wt/vol] glucose) (pH 5.8) at 28°C and
harvested at the early exponential phase. Total RNA was prepared from
D. hansenii cells incubated for 2 h under different
conditions (Fig. 2) which were chosen by
taking into account previous data. The concentration of 0.5 M NaCl
improves the performance of D. hansenii (1). KCl and sorbitol were used at concentrations equivalent to that of
NaCl. The selected concentration of LiCl was lower (100 mM) because
Li+ is toxic for D. hansenii (24).
Finally, a high pH was selected because it regulates the expression of
ENA genes in other yeasts (3). Total RNA
purification and hybridizations were performed as described previously
(3).
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FIG. 2.
Northern blot analyses of DhENA1 and
DhENA2 transcripts in D. hansenii cells grown
under the conditions indicated at the top of the figure. Total RNA was
fractionated, transferred to a nylon membrane, and probed. Filters were
stripped and stained with methylene blue as a loading control
(bottom).
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|
Northern blot analyses showed that DhENA1 and
DhENA2 were differentially expressed. While the expression
of DhENA1 was mainly dependent on the presence of high
Na+ concentrations, high expression of DhENA2
required high Na+ concentrations together with high pH.
This is not surprising, since these two factors are present in natural
habitats of D. hansenii, such as marine water.
K+ and Li+ triggered poor expression of both
genes. When the cells were incubated with 15% (wt/vol) sorbitol, the
expression of the genes was also poor, even at pH 7.5 (data not shown).
This observation eliminated the hypothesis that osmotic factors were
involved in the regulation of expression. Similar results were obtained
when the cells were grown under the same conditions, instead of having been incubated for 2 h (not shown).
Functional expression of DhENA1 and DhENA2
in S. cerevisiae.
DhENA1 and
DhENA2 were inserted in pYPGE15 after the PGK1 promoter
(8). A strain of S. cerevisiae (B31)
(4) lacking all Na+ efflux systems was
transformed with the plasmid without any insert (strain AD0) or with
the plasmid containing the DhENA1 or DhENA2 gene.
Growth of several transformants was assessed in plates with 0.5 M NaCl,
in which the performance of strains carrying DhENA1 and
DhENA2 genes was similar. From these, two were selected to study heterologous expression in more detail. The two transformants were named AD1, carrying DhENA1, and AD2, carrying
DhENA2. Salt tolerance was studied in these strains. Growth
experiments were carried out in solid medium (YPD) with
Na+, K+, or Li+ (Fig.
3A). For comparison, growth of S. cerevisiae (wild type and AD0 strain) and of D. hansenii is also shown. Strain AD0 did not grow in the presence of
the cations tested. The strains carrying any of the genes were able to
grow in plates with 1 M NaCl after 60 h of incubation. It is worth
mentioning that Na+ tolerance in both strains was at the
same level as in the wild strain of S. cerevisiae but never
reached the level in D. hansenii, which grew well in the
presence of 2 M NaCl (data not shown). The tolerance to Li+
was only slightly increased by DhENA2 but was clearly
improved by DhENA1, suggesting a selective role of both
genes in the process of tolerance to cations. As in the case of
Na+, the presence of any of the ENA genes
increased the capacity to grow at high K+ concentrations
and with the same level of tolerance as the wild strain of S. cerevisiae. In order to compare the efficiency of Ena1 from
D. hansenii and from S. cerevisiae, strain B31
was also transformed with ScENA1 under the control of the
same promoter. Tolerance tests showed that this strain behaved like AD1
(Fig. 3A).
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FIG. 3.
Alkali cation tolerance and efflux in strains of
S. cerevisiae transformed with DhENA1 and
DhENA2. (A) Tolerance to alkali cations was tested in solid
YPD in the S. cerevisiae B31 null mutant (ena
1-4 nha1) transformed with
DhENA1 (AD1) or with DhENA2 (AD2). The same
strain containing the plasmid without any insert (AD0) or with
ScENA1 and a D. hansenii strain were used as
controls. Plates were inoculated with 10-µl drops of cultures at the
middle of the exponential phase, and growth was scored after 60 h.
The efflux of Na+ (B) or Li+ (C) was evaluated
in AD1 ( ), AD2 ( ), and AD0 ( ). For determination of
Na+ and Li+ efflux rates, cells were grown in
YNB medium and loaded with the cations by incubation in the same medium
plus 200 mM NaCl or 100 mM LiCl for 3 h. Cells were harvested and
resuspended in the assay buffer (pH 5.5) supplemented with 50 mM KCl to
trigger the efflux process. Samples were taken at regular intervals,
filtered, and treated as previously described (24, 25).
The experiments were repeated at least three times. The standard errors
were 20% lower than the corresponding mean values.
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|
In addition, we performed growth experiments in liquid media containing
NaCl or LiCl. The results confirmed that expression of
DhENA1 or DhENA2 improved growth in the presence
of either or both cations. The doubling time of strains AD1 and AD2 in
the presence of 0.5 M NaCl were 3.4 and 4.3 h, respectively,
significantly lower than the value obtained with AD0 (6.9 h). In the
presence of Li+, growth was significantly improved in the
case of AD1 strain and only very slightly in AD2 (the doubling times of
strains AD0, AD1, and AD2 in the presence of 0.05 M LiCl were 8.5, 4.1, and 7.0 h, respectively). All these results suggest that the
increase in Na+ and Li+ tolerance could be due
to an increase in the process of Na+ and Li+
efflux in cells carrying the DhENA genes. In order to
confirm this hypothesis, we studied Na+ and Li+
efflux in Na+-loaded (Na+in, 205 to
221 nmol/mg [dry weight] of cells) or Li+-loaded
(Li+in, 52 to 59 nmol/mg [dry weight] of
cells) cells of the three strains. The results indicate that the
proteins were located at the plasma membrane (Fig. 3B and C).
Interestingly, the efflux of Li+ in strain AD2 was very
poor, a result that fits with the fact that DhENA2 improved
Li+ tolerance only to a small extent. It is worth
mentioning that Na+ efflux was clearly higher in the strain
expressing DhENA2 than in the strain expressing
DhENA1, although this fact was not linked to a higher
tolerance to Na+. It has been reported that a certain
amount of the protein could be located in some intracellular
organelles, increasing tolerance without important changes in the
efflux process (5). It is worth noting that the different
ENA genes previously described for other yeasts confer
Na+ and Li+ tolerance, although Wieland et al.
(33) have proposed a specific role in Li+
tolerance in the case of ScENA2 (also named
PMR2B). In our case, DhENA1 and DhENA2
conferred Na+ and K+ tolerance, but while
DhENA1 improved Li+ tolerance, the effect of
DhENA2 in this process was almost negligible. Two results
indicate that this is the case: very little complementation was
conferred by DhENA2 when Li+ was the toxic
cation in the growth medium, and no important Li+ efflux
was detected in S. cerevisiae cells carrying
DhENA2.
We have cloned, sequenced, and expressed two genes coding for sodium
efflux systems in D. hansenii. To our knowledge, this is the
first report on genes specifically involved in salt tolerance in a
halophilic yeast. The existence of several Na+ transporter
genes has been previously described for the osmotolerant yeast Z. rouxii. This yeast is considered to be highly sugar tolerant, but
different strains exhibit dramatically different responses to NaCl
(reference 7 and references therein). Four different results support the idea that DhENA1 and DhENA2
are P-type ATPases involved in Na+ extrusion: (i) homology
with sequences of previously reported ENA genes from
S. cerevisiae, S. occidentalis, or Z. rouxii;
(ii) complementation of salt tolerance of a mutant of S. cerevisiae lacking the Na+ efflux systems; (iii)
complementation of the sodium efflux defect in the same mutant; and
(iv) the observation that the extrusion of sodium is pH independent in
D. hansenii.
A question remains: what is the physiological relevance of a
Na+ efflux system in a yeast which is not normally
intoxicated by the cation? We propose that Ena proteins play an
important role in maintaining balanced levels of intracellular cations,
ensuring the ionic homeostasis of the cell. Therefore, these pumps
would transport alkali cations out of the cells, not because they are specifically toxic for D. hansenii but because excessive
Na+ (or K+) accumulation would cause osmotic problems.
Our research on the mechanisms involved in salt tolerance in D. hansenii will continue. We are looking for other genes involved in
this process. The heterologous expression of DhENA genes in a sensitive mutant of S. cerevisiae lacking the
Na+ extrusion systems recovered salt tolerance in this
mutant up to the level of the wild strain but still far from the
tolerance level of D. hansenii. Salt tolerance is a complex
challenge for which nature has developed a number of strategies. Some
have been identified in a physiological perspective. We expect to
identify other genes and find clues to establish the relative
importance of each mechanism in overall salt tolerance.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge A. Rodríguez-Navarro and B. Garciadeblas for stimulating discussion of the manuscript and technical assistance.
This research was partially supported by grants BIO4-CT97-2210
(Commission of the European Communities) and PB98-1036 (M.E.C.) to
J.R. C.P. was the recipient of grant PRAXIS XXI BD/9089/96.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Departamento de
Microbiología, Escuela Técnica Superior de Ingenieros
Agronomos, E-14071 Cordoba, Spain. Phone: 34 957 218521. Fax: 34 957 218563. E-mail: mi1raruj{at}uco.es.
Present address: Institute of Molecular Biology, Academia Sinica,
Taipei 11529, Taiwan.
| |
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Journal of Bacteriology, May 2001, p. 3251-3255, Vol. 183, No. 10
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.10.3251-3255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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