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Applied and Environmental Microbiology, May 2000, p. 2057-2061, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mitotic Recombination and Genetic Changes in
Saccharomyces cerevisiae during Wine Fermentation
Sergi
Puig,1,2,*
Amparo
Querol,1
Eladio
Barrio,3 and
José
E.
Pérez-Ortín1,2
Departamento de Biotecnología,
Instituto de Agroquímica y Tecnología de Alimentos,
CSIC,1 and Departamento de
Bioquímica y Biología Molecular2
and Institut `Cavanilles' de Biodiversitat i Biologia
Evolutiva,3 Universitat de València,
Valencia, Spain
Received 12 July 1999/Accepted 23 February 2000
 |
ABSTRACT |
Natural strains of Saccharomyces cerevisiae are
prototrophic homothallic yeasts that sporulate poorly, are often
heterozygous, and may be aneuploid. This genomic constitution may
confer selective advantages in some environments. Different mechanisms
of recombination, such as meiosis or mitotic rearrangement of
chromosomes, have been proposed for wine strains. We studied the
stability of the URA3 locus of a URA3/ura3 wine
yeast in consecutive grape must fermentations. ura3/ura3
homozygotes were detected at a rate of 1 × 10
5 to
3 × 105 per generation, and mitotic rearrangements
for chromosomes VIII and XII appeared after 30 mitotic divisions. We
used the karyotype as a meiotic marker and determined that sporulation
was not involved in this process. Thus, we propose a hypothesis for the
genome changes in wine yeasts during vinification. This putative
mechanism involves mitotic recombination between homologous sequences
and does not necessarily imply meiosis.
| |
INTRODUCTION |
Saccharomyces cerevisiae
wine yeast strains have been selected for (i) their ability to quickly
and efficiently ferment grape musts with elevated sugar concentrations,
(ii) their resistance to high ethanol and sulfur dioxide
concentrations, and (iii) their survival during fermentation at
elevated temperatures (17). Thus, wine yeasts have unique
genetic and physiological characteristics that differentiate them from
other laboratory and industrial strains, such as baker's, brewer's,
and distiller's yeasts.
Natural yeasts are mostly prototrophic, homothallic, and heterozygous
(4, 15, 17). They sporulate poorly (3), although in the case of wine yeasts, between 0 and 75% of cells sporulate, depending on the ploidy of the strain (4). In wine yeasts, spore viability also varies greatly (0 to 98%) (4) and is
inversely correlated with heterozygosity (23). Wine yeasts
frequently are aneuploid, with disomies, trisomies, and, less
frequently, tetrasomies (3, 15). In some cases, these
strains are nearly diploid or triploid. This aneuploidy may confer
selective advantages by increasing the number of copies of beneficial
genes or by protecting the yeast against lethal or deleterious
mutations (3, 15). The electrophoretic karyotypes of wine
yeast strains differ in the number, size, and intensity of bands,
allowing the identification of every strain by its chromosome pattern
(37, 40). Wine strains do not have a stable and defined
karyotype, like flor yeasts (21), but their variability is
not as high as that reported for baker's yeasts (5, 10).
Chromosomal rearrangements have been described in wine yeast genomes
during vegetative growth, due to recombination between homologous
chromosomes (19) and to recombination between repeated or
paralogous sequences (24, 39). The maintenance of these polymorphisms in a population suggests that such exchanges might be the
result of an important adaptive mechanism of yeasts (1, 19).
Mortimer and coworkers (23) proposed a mechanism of
evolution for natural wine yeast strains, termed genome renewal. This hypothesis maintains that wine yeasts, which accumulate deleterious mutations as heterozygotes, can sporulate and, as homothallics, produce
completely homozygous diploids. Some of these new homozygotes would
replace the original heterozygote. However, sexual isolation in yeast
populations during wine production (34), the high level of
heterozygosity, and the low sporulation rates of wine yeasts (3,
4, 15) do not favor this hypothesis.
Our objective in this study was to test the genome renewal hypothesis
(23). We analyzed the formation of homozygotes from a
URA3/ura3 heterozygous wine strain during consecutive wine
fermentations. The chromosomal heteromorphism of this strain allowed us
to determine if the formation of the homozygotes occurred as a
consequence of sporulation. Chromosomal rearrangements during
vinifications also were studied. We hypothesize that the mechanism of
genome evolution for wine yeasts involves only mitotic recombinations.
| |
MATERIALS AND METHODS |
Strains and culture conditions.
We used the diploid,
homothallic S. cerevisiae wine yeast strain T73
(Spanish Type Culture Collection reference no. CECT1894) selected in
the region of Alicante, Spain (29), and commercialized by
Lallemand Inc. (Montreal, Quebec, Canada). A recombinant
T73 strain, named T73-6, was obtained by
transformation with an NdeI-StuI fragment of
plasmid pURA::KMX4, that contains the kan gene
conferring resistance to the antibiotic G418 (28).
T73-6 has one allele of the URA3 gene disrupted
by the insertion of the kanMX4 marker (38) and
the wild-type allele on the homologous chromosome. It is phenotypically
Ura+ and Kanr, and it will be either
Ura/Kanr or Ura+/Kans
if it becomes homozygous.
For laboratory cultures, yeast cells were grown at 30°C in YPD (1%
yeast extract, 2% bacteriological peptone, 2% glucose) or in SD
(0.67% yeast nitrogen base without amino acids [Difco Laboratories,
Detroit, Mich.], 2% glucose). For Ura screening,
107 cells were spread on a plate of 5-fluoro-orotic acid
(FOA) medium [SD without
(NH4)2SO4, 0.1% proline, 10 mg of
uracil per liter (22) containing 1 mg of FOA (Toronto
Research Chemicals, Ontario, Canada) per ml].
Escherichia coli DH5
was used for the construction of
plasmids. It was grown at 37°C in LBA medium (1% tryptone, 1% NaCl,
0.5% yeast extract, 50 mg of ampicillin per ml). Media were solidified
with 2%
agar.
DNA manipulations.
Standard protocols were followed
(33).
Yeast transformation protocol.
Wine yeast strain
T73 was transformed using lithium acetate to permeabilize
the cells (13), and transformants were selected by their
resistance to the antibiotic G418 sulfate (Geneticin; GIBCO-BRL,
Rockville, Md.) (28, 38).
Sporulation and tetrad analysis.
Sporulation was induced
(12), and asci were dissected with a micromanipulator
(35), as previously described.
Microvinification experiments.
Four consecutive
microvinifications with strain T73-6 were performed at
22°C, using 1 liter of red grape Bobal must (27). The
initial yeast inoculum was 2.5 × 105 cells/ml from
overnight cultures. At the end of each fermentation, wine was removed
and residual yeast cells were maintained for 2 weeks in the original
bottles at 22°C until fresh grape must, sterilized with dimethyl
dicarbonate (Velcorin; Bayer, Leverkusen, Germany), was added. Thus,
material from the previous fermentation was used as inoculum for the
next one. This procedure simulates the seasonal rebreeding that occurs
in wine cellars. During each microvinification, samples of cells were
spread on YPD and FOA plates, to determine the total number of viable
and Ura homozygous cells, respectively. We used reducing
sugar concentration to indicate fermentation progress.
Chromosomal DNA preparations and pulsed-field gel
electrophoresis.
Karyotypes were determined by the contour-clamped
homogeneous electric field electrophoresis (CHEF) technique with a
CHEF-DRIII apparatus (Bio-Rad Laboratories, Hercules, Calif.).
Chromosomal DNA was prepared in agarose plugs (7) and washed
three times in TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) at
50°C for 30 min and then twice in the same buffer at room temperature
for 30 min. Plugs were loaded into 1% agarose gels in 0.5× TBE buffer (44.5 mM Tris-borate, 1.25 mM EDTA [pH 8.0]); migration was at 14°C
and 6 V/cm for 13 h with 60 s between field changes, and then
9 h with 90 s between field changes.
Southern blot analysis.
The chromosomal DNA separated by
CHEF gel electrophoresis was transferred to nylon filters (Hybond-N;
Amersham-Pharmacia Biotech, Buckinghamshire, United Kingdom) as
suggested by the manufacturer. Karyotype filters were hybridized with
32P-labeled probes corresponding to rDNA (chromosome XII),
HSP42 (chromosome IV), CAR1 (chromosome XVI),
YML128w (chromosome XIII), URA3 (chromosome V),
CUP1 (chromosome VIII, right arm), and SNF6 (chromosome VIII, left arm) (33).
| |
RESULTS |
Characterization of T73 wine yeast strain.
Strain
T73 is approximately diploid, homothallic, and prototrophic
for most common requirements (data not shown). Sixty percent of
T73 cells sporulated, and spore viability was 70% (168 out of 240). Most of the tetrads analyzed had two or three viable spores.
The colony sizes (diameters) of the meiotic derivatives varied widely
(between one- and fourfold), suggesting that this strain is highly heterozygous.
The CHEF gel karyotype of T
73 has 14 different bands (Fig.
1), some of which have a lower intensity,
suggesting aneuploidy
or the presence of homologous chromosomes of
different sizes.
We used two tetrads, each with four viable spores, to
analyze
the karyotype following meiosis (Fig.
1). Small differences
were
detected for chromosomes XIII and I (data not shown). More
extensive
changes were observed for chromosomes XII, XVI, and VIII,
which
are represented by two bands of different sizes that segregate
2:2 in these tetrads (Fig.
2). To
demonstrate that chromosome
VIII was dimorphic, with the usual band of
580 kb and a second
of approximately 1,000 kb, we hybridized with
probes from both
arms of the chromosome with the same result. Thus, we
conclude
that T
73 has at least five pairs of heteromorphic
chromosomes.
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FIG. 1.
Electrophoretic karyotypes of wine yeast strain
T73 and two complete meiotic derivatives (2A to 2D; 3A to
3D). Putative chromosomes corresponding to every band according to the
pattern obtained for laboratory strain S288c are indicated.
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FIG. 2.
Hybridization of the karyotype of T73 and
its meiotic derivatives (Fig. 1) with probes from chromosomes (Chr.)
XII (rDNA; A), XVI (CAR1; B), and VIII (CUP1; C).
In the case of chromosome VIII, the same result was obtained by using
CUP1 (right arm) and SNF6 (left arm) probes (not
shown). Arrows indicate significant bands. Asterisk shows
cross-hybridization with an unidentified target.
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|
Genetic changes during consecutive wine fermentations.
Homozygous Ura cells were generated from the
URA3/ura3 heterozygote T73-6 during consecutive
microvinifications (Table 1). The
relative frequency of Ura cells increases with each
microvinification. A reduction in residual cells occurred between the
end of one microvinification and the beginning of the following (Table
1). This fact could be explained by the lower viability of
Ura cells than of Ura+ cells in these
conditions.
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TABLE 1.
Determination of the formation rate of Ura
strains during four consecutive microvinifications with
T73-6 straina
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|
We estimate that Ura
cells appear at a rate of 1 × 10
5 to 3 × 10
5 cells per generation
(Table
1). This result was lower in the
first fermentation. In our
calculations, we assume that the growth
rate (fitness) is the same for
both heterozygous and homozygous
cells in this medium. Preliminary
results from competition experiments
between these strains indicate
that Ura
cells have a lower fitness than the
heterozygotes. Thus, we underestimate
the rate of Ura
cell
formation.
Ura
cells could arise by different molecular mechanisms,
which would give genetically different strain patterns. We analyzed
the
URA3 loci of 48 Ura
strains (12 from each
microvinification) and found that all were
ura3::kanMX4/ura3::kanMX4 (Fig.
3; only one example shown). We
determined
the electrophoretic karyotypes of the 12 Ura
strains from
the last microvinification, apparently obtaining
the same pattern than
T
73-6 (Fig.
4A). However, two
of these strains
carried cryptic chromosome rearrangements that could
be detected
only when hybridized to chromosome specific probes (Fig.
5). These
changes involved chromosomes
VIII (strain 1) and XII (strain 11).
On the other hand, every strain
obtained by sporulation has a
different electrophoretic karyotype (Fig.
4B), due to segregation
of nonidentical sister chromosomes. From these
data, we conclude
that Ura
strains were not produced by
sporulation and subsequent mother-daughter
conjugation. The lack of
sporulation during vinification was confirmed
by the absence of spores
after staining with green malachite (frequency
of
10
5)
(
18). These data support the hypothesis that mitotic gene
conversion or recombination resulted in the Ura
strains,
but not that these strains arose by sporulation or mutation,
events
with frequencies between 10
8 and 10
9 in
S. cerevisiae (
20).
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FIG. 3.
(A) Southern analysis of the URA3 locus. DNA
from T73, T73-6, and one Ura
strain were digested with HindIII (H), and separated by
electrophoresis in a 1.2% agarose gel. The gel was transferred to a
nylon membrane and hybridized with a HindIII
URA3 probe of 1,170 bp. Three different bands can be
obtained: the wild-type locus produces a 1,170-bp band, and integration
of kanr in the URA3 locus produces
two bands of 1,640 and 920 bp (B).
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FIG. 4.
Electrophoretic karyotype of 12 Ura
strains that appeared during the consecutive fermentations (A) and 12 meiotic derivatives of T73-6 from four different tetrads
with three viable spores (B).
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FIG. 5.
Hybridization of 12 Ura strains karyotype
(Fig. 4A) with rDNA (Chr. [chromosome] XII) and CUP1 (Chr.
VIII) probes. Asterisks indicate cross-hybridization with undetermined
targets.
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DISCUSSION |
Chromosomal features of wine yeast T73.
S.
cerevisiae industrial yeasts commonly are aneuploid (3,
15). In wine yeasts, strains with approximately diploid DNA contents, such as T73, are well known (11, 15, 21,
24). This result does not imply that such strains are strictly
diploid. Indeed, preliminary results with the strain T73
suggest that chromosome IV may be aneuploid (J. V. Gimeno-Alcañiz and E. Matallana, personal communication). Other
wine strains are near diploid or triploid (3, 15). The
tolerance of wine yeasts to these DNA levels suggests that meiosis is
not a common occurrence in their life cycles (3).
Strain T
73 carries several homologous chromosomes of
different sizes. Thus, this strain possesses two chromosomes XII of
unequal
size, probably due to differences in the number of rDNA repeats
(Fig.
2A, lane 1), as has been demonstrated for other strains
(
9,
24,
25,
31,
32). T
73 also has two different-sized
homologues of chromosome VIII. The longer version of chromosome
VIII
has been observed in other wine strains (
6,
14,
19).
Goto-Yamamoto and coworkers (
14) have demonstrated
recombination
between chromosomes VIII and XVI located at the promoter
of
SSU1,
a gene coding for a plasma membrane protein. The
longer version
of chromosome VIII in T
73 could be explained
by the presence of
this reorganization. Rearrangements of chromosomes
XII and VIII
during vegetative growth also were observed (Fig.
5),
suggesting
that they may carry hot spots for mitotic crossing
over.
Mechanisms of genetic change in wine yeasts during
fermentation.
Mechanisms proposed for genomic evolution of wine
yeasts include (i) chromosomal length polymorphisms, (ii) aneuploidy,
and (iii) genome renewal in which meiosis is followed by diploidization and competition of the resulting completely homozygous strains (5,
6, 11, 19, 23).
The frequency of meiotic gene conversion for the
URA3 locus
is approximately 2% (
36), with mitotic gene conversion
being
3 to 4 orders of magnitude lower (
26). We estimate the
formation
of
ura3::kanMX4/ura3::kanMX4
homozygotes at a rate of 1 × 10
5 to 3 × 10
5 per generation during successive microvinifications,
but we have
no evidence for meiosis or sporulation. Therefore, we
interpret
these data to mean that mitotic gene conversion or mitotic
crossing
over is the most likely mechanism for their
formation.
We propose a process of gradual adaptation to vinification conditions,
as chromosomal rearrangements and aneuploidies acquired
following
numerous mitotic divisions are maintained vegetatively.
Mitotic
recombination at a frequency of 1 × 10
5 to 3 × 10
5, instead of sporulation, could eliminate the
deleterious mutations.
With a mechanism such as genome renewal, a
sporulation event leads
to complete homozygosity of homothallic
strains, and hence a loss
of polymorphisms and aneuploidies, which we
did not observe. This
reasoning does not mean that wine strains never
sporulate, but
it does suggest that sporulation is not significant with
respect
to their genome
evolution.
| |
ACKNOWLEDGMENTS |
We thank Tahía Benítez, Benjamín
Piña, Emilia Matallana, and Daniel Ramón for helpful
discussions and critical reading of the manuscript, and we thank P. Philippsen for providing kanMX plasmids.
This work was supported by grants ALI95-0566 and ALI98-1041 (to
J.E.P.-O.) from Comisión Interministerial de Ciencia y
Tecnología of the Spanish Government. S.P. was a recipient of
an FPI fellowship from the Ministerio de Educación y Cultura.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Biological Chemistry, University of Michigan Medical School, Medical Science I, 1301 Catherine Road, Ann Arbor, MI 48109-0606. Phone: (734)
764-7514. Fax: (734) 763-7799. E-mail:
spuig{at}neptune.biochem.med.umich.edu.
| |
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Applied and Environmental Microbiology, May 2000, p. 2057-2061, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
