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Applied and Environmental Microbiology, October 2000, p. 4421-4426, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
MET17 and Hydrogen Sulfide Formation in
Saccharomyces cerevisiae
Apostolos
Spiropoulos and
Linda F.
Bisson*
Department of Viticulture and Enology,
University of California, Davis, Davis, California 95616-8749
Received 11 April 2000/Accepted 9 August 2000
 |
ABSTRACT |
Commercial isolates of Saccharomyces cerevisiae differ
in the production of hydrogen sulfide (H2S) during
fermentation, which has been attributed to variation in the ability to
incorporate reduced sulfur into organic compounds. We transformed two
commercial strains (UCD522 and UCD713) with a plasmid overexpressing
the MET17 gene, which encodes the bifunctional
O-acetylserine/O-acetylhomoserine sulfhydrylase
(OAS/OAH SHLase), to test the hypothesis that the level of activity of
this enzyme limits reduced sulfur incorporation, leading to
H2S release. Overexpression of MET17 resulted
in a 10- to 70-fold increase in OAS/OAH SHLase activity in UCD522 but had no impact on the level of H2S produced. In contrast,
OAS/OAH SHLase activity was not as highly expressed in transformants of UCD713 (0.5- to 10-fold) but resulted in greatly reduced
H2S formation. Overexpression of OAS/OAH SHLase activity
was greater in UCD713 when grown under low-nitrogen conditions, but the
impact on reduction of H2S was greater under high-nitrogen
conditions. Thus, there was not a good correlation between the level of
enzyme activity and H2S production. We measured cellular
levels of cysteine to determine the impact of overexpression of OAS/OAH
SHLase activity on sulfur incorporation. While Met17p activity was not
correlated with increased cysteine production, conditions that led to
elevated cytoplasmic levels of cysteine also reduced H2S
formation. Our data do not support the simple hypothesis that variation
in OAS/OAH SHLase activity is correlated with H2S
production and release.
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INTRODUCTION |
Hydrogen sulfide (H2S)
is an undesirable by-product of alcoholic fermentation by yeast. Very
low levels of H2S can be detected due to its low aroma
threshold (11), so trace amounts can have a profound effect
on final product quality. Subtle changes in yeast metabolic behavior
appear to have a major impact on the presence or absence of this
spoilage character. It is highly desirable, therefore, to have yeast
strains available for wine production that will not produce and release
H2S. Lowering the amount of H2S produced could
be achieved by supplementation of grape juice with one or more organic
sources of sulfur: methionine, cysteine, glutathione, or the
intermediate homocysteine, as transsulfuration pathways exist, thereby
negating the need for sulfate reduction (7, 34). However,
these sulfur-containing compounds can be metabolized to other volatile
sulfur compounds by Saccharomyces and other yeasts and
bacteria present in fermenting juice (8), producing
characters (feces or rotten seafood) as objectionable as or more
objectionable than that of H2S (rotten egg).
Several environmental factors have been implicated in the appearance of
hydrogen sulfide: high residual levels of elemental sulfur (26,
27), presence of sulfur dioxide (13, 17, 31), presence
of organic compounds containing sulfur (1, 19), pantothenate
deficiency (35, 36), high threonine content (37), high methionine or cysteine content relative to other amino acids (1, 19, 37), and nitrogen limitation (12, 13,
37). Volatile-sulfur-compound production varies widely among
commercial strains and natural isolates in response to these
environmental conditions (1, 9, 12, 15, 37). This variation
suggests that differences in internal levels of enzymatic activities or their regulation have a profound effect on the appearance of hydrogen sulfide. If the basis of this genetic variation can be identified, then
it may be possible to construct commercial strains with reduced sulfur production.
The MET17 gene (also called MET15 or
MET25) encodes a sulfhydrylase (SHLase) capable of using
either O-acetylserine (OAS) or O-acetylhomoserine
(OAH) as the substrate in vitro and is the last step of the sulfate
reduction pathway. met17 mutants are methionine auxotrophs,
and no OAS/OAH SHLase activity is detectable (22). Only
O-acetylhomoserine appears to be the substrate in vivo, with
O-acetylserine serving instead as an inducer of the sulfate
reduction pathway (22, 23, 25), in contrast to the situation
in bacteria (18). A mutational analysis indicated that the
transsulfuration pathway is the only pathway by which cysteine is
synthesized in Saccharomyces (7) and that the
direct conversion of O-acetylserine to cysteine does not
occur. The MET17 gene, therefore, encodes the only enzymatic
activity responsible for incorporation of reduced sulfide in this yeast.
The levels of OAS/OAH SHLase can vary 10-fold in different strains of
Saccharomyces cerevisiae (22). One hypothesis is
that low levels of reduced sulfur incorporation result in
"leakiness" of reduced sulfur and subsequent release of
H2S (16, 19). Thus, strains that are high
producers of H2S should have relatively low levels of
OAS/OAH SHLase activity. In support of this hypothesis, Omura et al.
(21) reported that H2S formation was reduced in a brewing yeast strain overexpressing the MET17 gene, which
encodes OAS/OAH SHLase.
Our objective in this study was to determine if low levels of OAS/OAH
SHLase activity accounted for increased H2S production in
two commercial yeast strains: one, UCD522, which produces high levels
of H2S under commercial conditions, and another, UCD713, which is a low-to-moderate-level producer of this compound. Additional objectives of this study were (i) to overexpress the MET17
gene in two commercial strains of S. cerevisiae to further
test the hypothesis that increased levels of expression would increase efficiency of incorporation of sulfur and reduce H2S
formation and (ii) to study the consequences of the overproduction of
OAS/OAH SHLase during alcoholic fermentation simulating wine production conditions.
| |
MATERIALS AND METHODS |
Plasmids, DNA manipulations, and transformation methods.
Restriction and modification enzymes were used according to the
manufacturer's instructions. The control vector pEG25C was constructed
by restriction digestion of the pEG25 (21) multicopy 2µm
vector with BamHI (Gibco-BRL, Gaithersburg, Md.) in order to
remove the MET17 insert, cutting the appropriate band of DNA from a 0.7% agarose gel, cleaning the DNA (QIAquick gel extraction kit; QIAGEN, Valencia, Calif.), and subsequent ligation of the DNA
using T4 DNA ligase (Gibco-BRL). The resulting control vector lacked
the MET17 coding region but retained the sequence
corresponding to the glyceraldehyde-3-phosphate dehydrogenase gene
promoter used to overexpress MET17 (21). S. cerevisiae was transformed using the lithium acetate method
(28), and Escherichia coli was transformed using
the method described by Inoue et al. (14). E. coli INV
F' (Invitrogen, Carlsbad, Calif.) was used for plasmid preparations. Luria-Bertani medium with ampicillin was used for selection of transformed E. coli cells.
Yeast strains and culture conditions.
Two S. cerevisiae industrial wine yeast isolates were used from our
culture collection (Department of Viticulture and Enology, University
of California, Davis): UCD522 (Montrachet) and UCD713 (French White)
(Universal Foods, Milwaukee, Wis.). These commonly used commercial
strains are likely not true diploids and contain extra copies of some
chromosomes (2).
Yeast strains were maintained and grown on yeast
extract-peptone-dextrose medium with 2% glucose (YPD) (29).
The same medium (YPD) with geneticin (G418, 30 ppm) was used for
transformant selection and maintenance. Four transformants were
constructed: UCD522 transformed with pEG25
(UCD522pMET17) and with the vector pEG25C
(UCD522pVector) and UCD713 transformed with pEG25
(UCD713pMET17) and pEG25C (UCD713pVector).
Fermentation media and conditions.
In the fermentation
experiments, the synthetic grape juice medium "Minimal Must Medium"
(MMM) (12), modified from the original recipe described by
Spiropoulos et al. (30), was used. The two nitrogen levels
were generated by using 0.8 g of L-arginine/liter and
1 g of ammonium phosphate/liter for media containing 433 mg of N
equivalents/liter and 0.2 g of L-arginine/liter and
0.5 g of ammonium phosphate/liter for media containing 208 mg of N
equivalents/liter (30).
Fermentations were initiated at a density of 10
6 cells/ml
by inoculation with stationary-phase cells from a culture pregrown
in
MMM starter medium (
30). Fermentations were conducted in
500-ml Erlenmeyer flasks containing 300 ml of medium. Each flask
was
connected to a hydrogen sulfide trap (
30). The flasks were
incubated by shaking (120 rpm) on a rotary shaker at room temperature
(23 to 28°C). Fermentations were monitored by using weight loss
as an
estimate of CO
2 production (
30). Completion of
fermentation
(absence of reducing sugars) was confirmed by using the
Clinitest
(Bayer, Elkhart, Ind.). Fermentations with higher nitrogen
levels
were performed in triplicate, while fermentations with lower
nitrogen
levels were performed in duplicate. All the fermentations
reached
dryness (less than 0.5% residual sugar), except
UCD713p
MET17 (2%
residual sugar). Values presented
represent the averages of the
replicate samples. Levels of
H
2S in replicates run simultaneously
differed by less than
5%. In replicate experiments not run at
the same time, H
2S
values typically varied by 20% or less. This
elevated variation
probably was due to differences in room temperature
in experiments not
conducted at the same time. In the few cases
where the variation was
greater than 20%, the trend of peaks and
valleys was the
same.
Analytical methods.
H2S was measured using the
cadmium trap assay as previously described (30). Protein
extracts were prepared using the method of Brzywczy and Paszewski
(4). OAS SHLase activity was measured using the method of
Paszewski and Grabski (24). One unit of activity was defined
as the amount of enzyme producing 1 nmol of cysteine per mg of protein
per min. Cysteine was extracted from yeast cells by the method of
Tezuka et al. (32) and was measured by the method of
Gaitonde (10). The protein level was estimated by the method
of Bradford with bovine serum albumin as the standard (3).
Plasmid loss was monitored during the entire course of fermentation in
MMM by plating samples on YPD alone and YPD plus 30 mg of G418/liter.
| |
RESULTS |
Characterization of H2S formation in commercial wine
strains of Saccharomyces.
The formation of H2S
by two commercial wine strains of Saccharomyces was
evaluated during fermentation in synthetic grape juice media to
determine if our assay conditions would yield variation in
H2S production similar to that observed for these strains
under commercial conditions. UCD522 produced significantly higher
amounts of sulfide than did UCD713 under nutrient-sufficient conditions (Fig. 1), consistent with reports for
these strains during wine production.
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FIG. 1.
Pattern of production of H2S in UCD522 (A)
and UCD713 (B) at two concentrations of nitrogen. The levels of
H2S represent the total amount accumulated in the cadmium
trap for that interval. For example, the point at 43 h represents
the amount accumulated between 21 and 43 h. Open symbols, high
nitrogen; solid symbols, low nitrogen.
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|
Nitrogen limitation affects the production of H
2S during
alcoholic fermentation (
12,
13,
37). Most strains release
increased
H
2S when nitrogen is limited. We monitored
sulfide production
during an alcoholic fermentation of synthetic grape
juice media
but with approximately half (208 mg/liter) of the original
nitrogen
(433 mg/liter) (Fig.
1). Sulfide formation increased
significantly
for both isolates, but overall UCD522 produced almost
twice as
much total H
2S as UCD713 (Table
1). While these two wine isolates
display
distinctly different behavior in the basal level of production
of
H
2S, both react similarly to a reduction in medium nitrogen
levels and increase the amount of H
2S produced.
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TABLE 1.
Hydrogen sulfide production under low- and high-nitrogen
conditions in transformed and untransformed strains
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|
The H
2S produced by yeast strains is driven from the
fermentation medium into the trap by the CO
2 produced from
the catabolism
of sugar. Differences in the fermentation rate may
therefore impact
the detection of H
2S. The nitrogen levels
used were chosen because
there were no significant differences in
fermentation behavior
(Fig.
2A), growth
rate (Fig.
2B), or maximal fermentation rate
(Table
1) for these two
strains under these conditions. Both
displayed a slightly higher
maximal fermentation rate under low-nitrogen
conditions. Thus, the two
commercial strains displayed differences
in H
2S formation
not associated with differences in growth or
fermentation performance.
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FIG. 2.
Fermentation (A) and growth (B) of UCD522 and UCD713 in
low-nitrogen (solid symbols) and high-nitrogen (open symbols) media.
 ,  , UCD522;  ,  , UCD713.
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|
Comparison of OAS/OAH SHLase activity and H2S
formation.
To determine if differences in the production of
H2S were associated with differences in rates of reduced
sulfur incorporation, we measured OAS/OAH SHLase activity in both
strains, as well as in strains that were transformed with the vector
encoding G418 resistance. Under high-nitrogen conditions across the
time course of fermentation, activity values ranged from 2.2 to 12 U
for UCD522 and from 2.5 to 13 U for UCD713. The two strains showed a
similar pattern of expression of activity, with activity peaking within the first 24 to 48 h and then gradually declining to a low value late in fermentation (Fig. 3A) (data
shown for strains carrying the vector only). At most time points, the
activity value for UCD713 is generally 30 to 50% lower than for
UCD522.
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FIG. 3.
H2S and OAS/OAH SHLase activity in
transformed strains of UCD522 (, ) and UCD713 (, ). Solid
symbols, H2S production; open symbols, OAS/OAH SHLase
activity. (A) High nitrogen, strains transformed with vector; (B) low
nitrogen, strains transformed with vector; (C) high nitrogen, strains
transformed with pMET17; (D) low nitrogen, strains
transformed with pMET17. OAS/OAH SHLase activity is measured
in arbitrary units.
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|
Under low-nitrogen conditions, activity in UCD522 similarly ranged from
3 to 13 U, with the peak in activity occurring around
92 h into
the fermentation (Fig.
3B). OAS/OAH SHLase activity
was reduced in
UCD713 under nitrogen-limiting conditions and ranged
from 1.7 to 7.5 U. Except for very late time points, activity
was two- to threefold higher
in UCD522 than in
UCD713.
Hydrogen sulfide production also was evaluated for the time course of
fermentation (Fig.
3). Under high-nitrogen conditions,
UCD522 produced
higher levels of H
2S throughout the time course
of
fermentation than did UCD713. Both strains showed the same
pattern of
peaks and valleys of H
2S release. Thus, while enzymatic
activity did not vary significantly under these conditions for
these
two strains, UCD522 consistently produced greater levels
of reduced
sulfur. Note that the enzymatic levels represent activity
in a cell
extract made from a sample taken at that point in time,
while the
H
2S levels represent the amount accumulated in the trap
over the time period indicated. As enzymatic activity decreased
in
UCD522, there was a tendency for H
2S levels to rise. If the
change in enzymatic activity between time points was plotted versus
the
level of H
2S produced, an
R2 value
of 0.83 was obtained for UCD522, while that for UCD713
was only 0.018. Changes in activity were therefore somewhat correlated
with changes in
H
2S production in one strain, UCD522, but not
in the
other.
Both strains produced higher levels of H
2S under
low-nitrogen conditions. The total amount of H
2S formed for
UCD522 doubled
with a 50% decrease in nitrogen content (Table
1).
Total H
2S
production increased over fourfold for UCD713.
Thus UCD713 displays
a much stronger response in increasing
H
2S formation under nitrogen
limitation. The peak of
H
2S production occurred earlier in UCD522
than in UCD713
(Fig.
3B). H
2S formation did not correlate well
with
changes in OAS/OAH SHLase activity in either UCD522 or UCD713
under
low-nitrogen conditions (
R2 values of 0.12 or
0.50, respectively). OAS/OAH SHLase activity
differed in the two
strains, as did H
2S formation, but there was
not a good
correlation between changes in the level of activity
of this enzyme and
release of H
2S, with the exception of UCD522
under
high-nitrogen
conditions.
Effect of overexpression of MET17.
We transformed both
strains with a multicopy plasmid carrying the MET17 gene,
leading to overproduction of OAS/OAH SHLase, and evaluated Met17p
enzymatic activity and H2S production. In UCD522 under
high-nitrogen conditions, the presence of the MET17 gene on
the plasmid resulted in a 10- to 70-fold increase in Met17p activity
(Fig. 3C). However, total H2S production was not reduced but instead was slightly increased (Table 1). Met17p activity displayed
a similar increase under low-nitrogen conditions (Fig. 3D).
H2S production did not decrease and also displayed a very slight but reproducible elevation in the strain carrying the
MET17 gene, compared to the strain without plasmid or with
the vector.
Transformation of UCD713 with
MET17 increased OAS/OAH SHLase
activity and decreased H
2S formation at both nitrogen
concentrations
(Fig.
3C and D). The increase in OAS/OAH SHLase activity
over
basal level was about 10-fold under low-nitrogen conditions but
was only slightly higher than the levels found in the control
strain
under high-nitrogen conditions. H
2S production under
high-nitrogen
conditions was much less than under low-nitrogen
conditions in
spite of the much higher levels of OAS/OAH SHLase
activity in
the latter case. At both nitrogen levels,
H
2S production in UCD713p
MET17 was far below
that of UCD522 and of UCD713 with or without the
vector.
The fermentation and growth profiles for the transformed strains at
both nitrogen concentrations also were evaluated (Fig.
4). Neither the control vector nor the
MET17 plasmid had any dramatic
impact on growth or
fermentation in UCD522. The presence of the
control vector did not
impact UCD713. Overexpression of the
MET17 gene decreased
the fermentation rate and growth in UCD713 under
high-nitrogen
conditions. This observation suggests that high
levels of expression of
OAS/OAH SHLase may be deleterious in certain
genetic backgrounds.
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FIG. 4.
Fermentation (A) and growth (B) of
pMET17-(solid symbols) and vector-transformed (open symbols)
strains of UCD522 and UCD713. Testing was performed on UCD522 at
high-nitrogen (, ) and low-nitrogen ( ,  ) concentrations and
on UCD713 at high-nitrogen (, ) and low-nitrogen ( ,  )
concentrations.
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The differences in levels of enzymatic activity of the two strains and
of UCD713 at different nitrogen concentrations might
be explained by
differences in the stability of the
MET17 plasmid
in these
two genetic backgrounds. Plasmid loss was not detected
in either
transformant (data not shown). This stability suggests
that differences
in the level of overexpression between the two
strains may reflect
differences in plasmid copy number or in the
transcriptional or
posttranscriptional regulation of the
protein.
Analysis of cysteine levels.
To determine if increased Met17p
activity leads to increased incorporation of reduced sulfate, we
measured the cellular pool levels of cysteine. Cytoplasmic cysteine
levels were higher in strains grown under high-nitrogen conditions than
in strains grown under low-nitrogen conditions (Fig.
5). All strains showed a similar pattern
for internal cysteine levels. Levels were high early in the time
course, peaking by 48 h. Pool levels then decreased but remained
at a higher level under high-nitrogen conditions than under
low-nitrogen conditions. Cysteine levels tended to increase late in
fermentation in the case of high-nitrogen conditions but remained low
in the nitrogen-limiting state. Overexpression of OAS/OAH SHLase
activity in UCD522 had little or no effect on cysteine levels and, in
some cases, led to an apparent decrease in the internal concentration
of the amino acid. Overexpression of OAS/OAH SHLase resulted in
slightly higher internal levels of cysteine in UCD713, especially under
high-nitrogen conditions, where the decrease in H2S
formation was greatest. Thus, there is a relationship between high pool
levels of cysteine and low levels of H2S release. High
cysteine levels may indicate more efficient incorporation of reduced
sulfur. Alternately, high cysteine levels may alter the level of
activity or expression of the enzymes involved in sulfate reduction, as
has been proposed (22).
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FIG. 5.
Cysteine levels in transformed strains of UCD522 and
UCD713 under high-nitrogen (open symbols) or low-nitrogen (solid
symbols) conditions. Testing was performed on vector-transformed UCD522
(, ) and pMET17-transformed UCD522 (, ) and on
vector-transformed UCD713 (, ) and pMET17-transformed
UCD713 (, ).
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| |
DISCUSSION |
The discovery of variation in the levels of OAS/OAH SHLase
activity led us to the hypothesis that differences in the activity of
this enzyme might explain the differences in hydrogen sulfide formation
observed with different yeast genetic backgrounds. Low enzymatic
activity was predicted to be correlated with reduced efficiency of
incorporation of reduced sulfur and increased release of
H2S. Our analysis of commercial strains known to produce
high and low levels of H2S does not support this
hypothesis. OAS/OAH SHLase activity was generally higher in UCD522, the
strain that produced more H2S, than in UCD713 under all
conditions. Overexpression of the MET17 gene did not inhibit
H2S formation in UCD522 but did reduce volatile sulfur
production in UCD713. Thus, enzyme activity appears to be limiting only
in the strain that produced low levels of H2S and cannot
explain the difference between the two strains.
The lack of an effect of manipulation of OAS/OAH SHLase activity on
H2S production in UCD522 suggests that if decreased
incorporation of reduced sulfur is responsible for H2S
release, then it is due to other factors such as the limiting
substrate, OAH. If OAH is limiting, then increasing enzymatic activity
will have no impact on incorporation. The fact that H2S
production in UCD522 is high at both nitrogen concentrations compared
to that in UCD713 may reflect generally lower pool levels of the
substrate rather than of enzymatic activity. This situation is also
consistent with there being little to no change in the cytoplasmic
levels of cysteine in this strain. Cysteine is synthesized via the
transsulfuration pathway, involving conversion of homocysteine to
cystathionine and then to cysteine (7). Low levels of OAH
would prevent formation of cysteine. We predict that H2S
formation would be greater under low-nitrogen conditions, when pool
levels of OAH would be even further reduced.
The presence of the MET17 gene on a multicopy plasmid led to
a slight increase in H2S formation in UCD522 that was
reproduced in all experiments under both nitrogen conditions. This
result suggests that increased activity of OAS/OAH SHLase decreases
efficiency of incorporation. Increasing activity, while not affecting
cysteine concentration, may lead to an increase in the level of
methionine and S-adenosylmethionine, both of which are
thought to repress the expression of genes encoding enzymes upstream in
the sulfate reduction pathway (5, 6, 20, 33). However, if
the pathway were repressed, we would expect a reduction in the
production of H2S, not an increase. Methionine also
represses expression of the MET2 gene, encoding
homoserine-O-transacetylase, which would lead to
reduced levels of OAH (5, 6, 20, 33). If the reduction in
OAH levels were greater than the decrease in activity of the sulfate
reduction pathway, greater H2S release would occur. This
hypothesis also is consistent with reduced levels of OAH being
primarily responsible for increased release of H2S in
UCD522 in general. It is also possible that our knowledge of the
regulation of sulfate reduction in Saccharomyces is
incomplete and that other regulatory mechanisms also impact
reduced sulfur incorporation. For example, if the sulfate
reduction sequence existed as a multienzyme complex, then
overexpression of MET17 could interfere with formation of
the complex by titering other components, thus reducing the efficiency
of incorporation. Other speculative models are equally plausible.
Overexpression of MET17 dramatically reduced H2S
production in UCD713 under both low- and high-nitrogen conditions. This
result suggests that the activity of this enzyme may indeed be limiting for sulfide incorporation in this strain. The increase in enzymatic activity was greater under low-nitrogen conditions, but the production of H2S was greater in these fermentations. It is likely
that under nitrogen-limiting conditions, OAH levels also are affected,
reducing the level of the substrate, which would negate the effect of
elevated activity and account for an increase in H2S
production. However, the increase in H2S production does
not approach the values observed for the control strains not carrying
multiple copies of the MET17 gene, suggesting that in this
strain, enzymatic activity may be truly limiting for incorporation.
Our work suggests that the factors resulting in inefficient reduced
sulfur incorporation in these two strains are fundamentally different.
In the case of UCD522, OAS/OAH SHLase activity is not limiting and the
data are consistent with pool levels of OAH influencing H2S
production and release. In contrast, in UCD713, enzymatic activity does
appear to be limiting for reduced sulfur incorporation. This study
underscores the importance of a thorough understanding of the nuances
of variation in pathway regulation in different strains, as a prelude
to the development of strategies for the generation of commercial
strains with improved physiological characteristics.
| |
ACKNOWLEDGMENTS |
This research was supported by grants from the American Vineyard
Foundation and the California Competitive Grant Program for Research in
Viticulture and Enology. A. Spiropoulos was supported by scholarships
from the American Society of Enology and Viticulture, the Wine
Spectator, and the Jastro Shields Graduate Research Awards.
We thank K. Spiropoulou (Department of Food Science and Technology,
Agricultural University of Athens, Athens, Greece) for suggestions and
technical assistance during this study and F. Omura for generously
providing plasmid pEG25. We also thank the reviewers of the manuscript
for numerous excellent suggestions for improving the clarity of the presentation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Viticulture and Enology, University of California, Davis, Davis, CA
95616-8749. Phone: (530) 752-3835. Fax: (530) 752-0382. E-mail:
lfbisson{at}ucdavis.edu.
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Abstract
Introduction
