From the E. C. Slater Institute, University of
Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands, the § Kluyver
Laboratory of Biotechnology, Delft University of Technology,
Julianalaan 67, 2628 BC Delft, The Netherlands,
¶ Gist-brocades B. V., PO Box 1, 2600 MA Delft, The Netherlands, and the ** Department of
Chemical Engineering, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands
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ABSTRACT |
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The kinetics of glucose transport and the
transcription of all 20 members of the HXT hexose
transporter gene family were studied in relation to the steady state
in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h Carrier-mediated hexose transport across the plasma membrane is an
essential step in the utilization of hexoses by Saccharomyces cerevisiae (1). Based on measurements of zero-trans
influx of radioactive glucose into washed cells, two kinetically
distinguishable transport systems have been described: a low affinity
system (Km Genetic studies and analysis of the S. cerevisiae genome
have implicated the HXT family of homologous genes in
encoding the hexose transporters expressed by this yeast. The 20 members of this family include HXT1-HXT17,
GAL2 (encoding the galactose transporter), and
SNF3 and RGT2 (encoding putative sensors of high
and low glucose concentrations, respectively) (4). The glucose
transport kinetics of some individual HXT gene products have
been analyzed in genetically modified yeast strains; it was found that
a strain with null mutations in HXT1-HXT7 (the
hxt null strain) was not able to grow on various concentrations of glucose (5). When these seven HXT genes
were expressed individually as single chromosomal copies in the
hxt null strain, they were all able to confer growth on
glucose or fructose medium with the exception of HXT5.
However, the growth properties of the single-HXT strains
differed on media with different glucose concentrations, suggesting
that the genes are regulated differently in response to glucose or that
they encode transporters with different affinities for glucose
(5).
Thus far, regulation of individual HXT genes or subsets of
the HXT gene family has been studied by various means,
including immunodetection, mRNA analysis, and expression of
HXT promoter-lacZ fusions (6-11). These studies
indicate that HXT1 is induced by high glucose
concentrations, whereas HXT2, HXT4, and HXT6/7
are induced at low glucose concentrations and HXT3 is
induced by glucose irrespective of concentration. The presence of
HXT promoters on multicopy plasmids has been shown to affect
expression of other HXT genes (10, 12), indicating that
regulation of HXT genes is preferably studied in a wild-type context.
With few exceptions (13, 14), published studies on glucose transport
kinetics and HXT gene expression in S. cerevisiae are based on batch cultivation in shake flasks. In such cultures, rapid
changes occur in substrate availability (e.g. from sugar excess to sugar depletion), metabolite concentrations (e.g.
ethanol accumulation), pH, and oxygen tension. Moreover, the substrate affinity of glucose transport changes during batch growth on glucose (3). In contrast to batch cultivation, chemostat cultivation allows for
studies on effects of specific environmental parameters because each
parameter can be varied independently (15). In particular, the specific
growth rate µ (defined as the relative biomass increase in time) can
be easily manipulated, because it is numerically equal to the dilution
rate (D) in steady state chemostat cultures. By selecting
appropriate nutrient limitation regimes and specific growth rates,
chemostat cultivation permits studies on glucose transport in cells
grown under steady state conditions, both under sugar limitation and in
the presence of excess sugar (16).
The aim of the present study was to investigate the regulation of
hexose transport in S. cerevisiae under a wide range of defined growth conditions. Glucose transport kinetics were determined for cells grown at a fixed specific growth rate under different nutrient limitations and for cells from aerobic glucose-limited chemostat cultures grown at various specific growth rates.
Transcription of all 20 HXT genes was studied by Northern
analysis of RNA isolated from these cultures. This approach enabled a
comparison of the expression of the individual HXT genes
with the glucose transport kinetics of the cultures, taking into
account the kinetic characteristics of the transporter proteins
inferred from earlier studies with single-HXT strains
(17).
Yeast Strain and Maintenance--
The haploid, prototrophic
S. cerevisiae strain CEN.PK113-7D
(MATa, MAL2-8c
SUC2) was kindly provided by Dr. P. Kötter (Frankfurt,
Germany). Precultures were grown to stationary phase in shake-flask
cultures on mineral medium (18), adjusted to pH 6.0 and containing 2% (w/v) glucose. After adding glycerol (30% v/v), 2-ml aliquots were
stored in sterile vials at Chemostat Cultivation--
Chemostat cultures were run in
laboratory fermenters (Applikon) at a stirrer speed of 800 rpm and at
30 °C. The working volume was kept at 1.0 liter by a peristaltic
effluent pump coupled to an electrical level sensor (19). The exact
working volume was measured after each experiment. The pH was kept at
5.0 ± 0.1 by an ADI 1030 biocontroller (Applikon), via automatic
addition of 2 M KOH. Aerobic cultures were flushed with air
at a flow rate of 0.5 liter·min
Carbon recoveries were 91.3% for the aerobic glucose-limited chemostat
with D = 0.38 h Media for Chemostat Cultivation--
For aerobic carbon-limited
growth, a defined mineral medium containing vitamins (18) was used. The
concentration of hexoses in the reservoir media was 7.5 g·liter Gas Analysis--
The exhaust gas was cooled in a condenser
(2 °C) and dried with a Perma-Pure dryer (type PD-625-12P).
O2 and CO2 concentrations were determined with
a Servomex type 1100A analyzer and a Beckman model 864 infrared
detector, respectively. Measurements of exhaust gas flow rate and
calculation of specific rates of CO2 production and
O2 consumption were performed as described previously (22, 23).
Determination of Culture Dry Weight and Protein Content--
The
dry weight of chemostat biomass was determined by filtration of culture
samples (10 ml) over preweighed nitrocellulose filters (0.45 µm pore
size; Gelman). After removal of medium, filters were washed with
demineralized water, dried in a Sharp R-4700 microwave oven (20 min at
360 W output), and weighed. Duplicate determinations differed by less
than 1%. The protein content of chemostat biomass was estimated by a
modified biuret method (21) using bovine serum albumin (fatty
acid-free, Sigma) as a standard.
Substrate and Metabolite Analyses--
Cell suspensions were
rapidly (within 3 s) transferred from the culture into liquid
nitrogen. The frozen suspension was thawed on ice, with gentle shaking
to keep the cell suspension at 0 °C. Subsequently, cells were
removed by centrifugation at 4 °C (2 min at 11,600 × g). Ammonium, acetate, glucose, and fructose were determined
with Roche Molecular Biochemicals kits 1112732, 148261, 716251, and
139106 respectively. Ethanol was determined enzymatically (24).
Galactose was determined spectrophotometrically at 340 nm after
addition of Batch Cultivation--
Cells were grown in batch on a rotary
shaker at 200 rpm and 30 °C in a medium containing 2% (w/v)
glucose, 0.67% (w/v) yeast nitrogen base, and 0.1 M
potassium phthalate at pH 5.0. Growth was monitored by measuring the
optical density at 600 nm. Yeast cells were grown to exponential phase
(A600 0.5), to the diauxic shift
(i.e. before growth resumed on ethanol), or until 5 h
after the diauxic shift (i.e. growth on ethanol).
In Vitro Hexose Transport Assays--
Cells were harvested by
centrifugation at 4 °C (5 min at 4000 × g), washed
three times in ice-cold 0.1 M potassium phthalate buffer
(pH 5.0), and resuspended in 0.1 M potassium phthalate buffer (pH 5.0) to a concentration of approximately 4 g·liter Statistical Analysis of Transport Data--
Kinetic parameters
of hexose transport were calculated using a computer program written
especially for this purpose with the Matlab software package (27). The
program used the Levenburg-Marquardt nonlinear fitting procedure (28),
with the average of the influx rates at each substrate concentration as
initial weighting factors. The fitting was repeated four times using
the results of the previous fit as new weighting factors according to
the method of iteratively reweighted least squares (28). The models
used for the fitting procedure were one- or two-component
Michaelis-Menten kinetics. The results of a one-component fit were
checked for significant lack of fit by an F-test that compared the lack
of fit to the random error in the data. This test was accompanied by
visual inspection of residual plots. If significant lack of fit was
found with a one-component model, the data were fitted to a
two-component Michaelis-Menten model. Confidence intervals for the
estimated kinetic constants were calculated by Monte Carlo analysis
using the size of the proportional random error. A detailed description of the statistical analyses will be presented
elsewhere.1
RNA Isolation--
Culture samples (approximately 7 mg dry
weight) were centrifuged at room temperature (30 s at 14,000 × g). RNA extraction from the pellets was performed by a
modified acid phenol procedure (29). Briefly, cells were resuspended in
400 µl of AE buffer (50 mM sodium acetate, 10 mM EDTA, pH 5.0), 40 µl of 10% SDS, and 400 µl of
AE-saturated phenol/chloroform/isoamyl alcohol (25:24:1 vol:vol:vol) by
vortexing for 30 s. The samples were incubated at 65 °C for 5 min and then stored at Oligonucleotide Probes--
This study required oligonucleotide
probes specific to each of the 20 members of the HXT hexose
transporter gene family. These were designed by visual inspection of
the aligned protein and open reading frame sequences and by computer
analysis of pairwise and multiple sequence alignments of highly similar
open reading frames and their 5' and 3' flanking sequences. Some of the
HXT open reading frames are very similar; for example, the
open reading frames of HXT6 and HXT7 are 99.825%
identical, and they differ by only three nucleotides. However, regions
3' of these open reading frames that are quite dissimilar in sequence
are transcribed,2 and
gene-specific probes were designed that hybridize to these regions as
necessary. The analysis used the sequence of the complete S. cerevisiae genome at the Saccharomyces Genome Data Base
(http://genome-www.stanford.edu/Saccharomyces/), and the Wisconsin
Package software (Wisconsin Package, version 9.0, Genetics Computer
Group). The probes (Table I) were
designed such that they were similar in length and percentage of
guanine and cytosine bases (with optima of 24 nucleotides and 42% GC), so that they would have similar melting temperatures. The resulting oligonucleotide sequences were compared with the sequence of the yeast
genome using the BLAST algorithm to ensure that they were unique.
Northern Analysis--
5-µg quantities of total RNA, or the
poly(A)+ RNA from 150 µg of total RNA, were separated by
electrophoresis in 1% agarose formaldehyde gels (30), transferred
under vacuum to nylon membranes (Bio-Rad), and cross-linked to the
membranes by ultraviolet irradiation. 5 pmol of oligonucleotide
(Isogen) were labeled with T4 polynucleotide kinase (Roche Molecular
Biochemicals) and 25 µCi of [ Physiology of S. cerevisiae CEN.PK113-7D in Steady State Chemostat
Cultures--
In aerobic glucose-limited chemostat cultures of
S. cerevisiae CEN.PK113-7D grown at various dilution rates,
the glucose consumption and product formation profiles were essentially
as described previously (32). At dilution rates below 0.30 h
At D = 0.30 h
Other nutrient limitations were investigated in chemostat cultures
grown at a fixed dilution rate of 0.10 h
Glucose-excess conditions were obtained by growing cultures with
ammonium limitation on glucose. This resulted in increased glucose
consumption rates and significant production of ethanol (Table II).
Ammonium-limited cells had a reduced protein content, probably due to
glycogen accumulation (33).
Kinetics of Glucose Transport--
The apparent glucose transport
kinetics of washed and aerated cells sampled from the chemostat
cultures were assessed by measuring the zero-trans influx of
14C-glucose during 5 s. A statistical test was
developed for the kinetic data to discriminate between a single kinetic
component for glucose transport and two kinetic components.
Cells grown at low dilution rates in aerobic glucose-limited chemostats
generally displayed single-component high affinity glucose transport
kinetics with a Km of approximately 1 mM
(Table III). The cultures grown at
D = 0.1 h
The galactose-limited and anaerobic glucose-limited cultures
(D = 0.1 h Comparison between in Situ Glucose Consumption and Zero-trans
Influx Kinetics--
Glucose transport rates in the chemostat cultures
were calculated using the transport kinetic parameters of the cultured
cells estimated from the zero-trans influx measurements and
the residual glucose concentrations of the cultures. These rates were
compared with the in situ glucose consumption rates
calculated from the dilution rates of the chemostats and the
concentrations of biomass and glucose in the cultures (see Fig.
6A). In the aerobic glucose-limited chemostat cultures grown
at lower dilution rates, the calculated transport rates exceeded the
in situ glucose consumption rates. Conversely, at high
dilution rates, the calculated transport rates were lower than the
in situ consumption rates; at dilution rates of 0.35 and
0.38 h
The calculated glucose transport rates for the nitrogen-limited
cultures (D = 0.1 h Transcription of HXT Genes in Aerobic Glucose-limited Chemostat
Cultures--
To investigate whether the changes in hexose transport
kinetics as a function of growth conditions could be correlated with the transcription of individual HXT genes, mRNA levels
of the 20 HXT genes were determined in cells sampled from
each of the steady state chemostat cultures. Transcript levels of
HXT1-HXT7 and GAL2 were high enough
under some conditions to be detected in total RNA samples (Figs.
2 and 4). The signals from
SNF3 and RGT2 mRNA were too weak to detect
them satisfactorily due to the background that resulted from
nonspecific hybridization of the probes to ribosomal RNA. However,
these mRNAs were readily detected in samples of
poly(A)+ RNA (Figs. 3 and 5).
No signal was detectable for HXT8-HXT17 in total RNA
samples, but very low transcript levels of some of these HXT
genes were detected on poly(A)+ RNA blots (data not
shown).
In the aerobic glucose-limited chemostat cultures grown at dilution
rates from 0.05 to 0.30 h
Additional information was obtained by determining the levels of
HXT mRNAs in a glucose-grown batch culture. In
exponentially growing cells (extracellular glucose concentration,
approximately 100 mM), HXT1, HXT3, and
HXT4 were highly expressed, whereas transcripts from other
HXT genes were not detectable (Fig. 2, lane 11).
Conversely, in cells grown to glucose exhaustion, mRNAs of
HXT1, HXT3, and HXT4 were undetectable, and those
of HXT5, HXT6, and HXT7 were abundantly expressed
(Fig. 2, lane 12). Five h after glucose exhaustion, when the
culture was growing on ethanol, the level of HXT5 mRNA had increased, whereas the levels of HXT6 and
HXT7 had decreased (Fig. 2, lane 13).
HXT2 mRNA was not detectable in any phase of batch
culture tested.
Transcription of HXT Genes in Chemostat Cultures Grown under
Different Nutrient Limitations--
In anaerobic glucose-limited
chemostats (D = 0.10 h
GAL2 mRNA, which was not detectable under any of the
other growth conditions tested, was abundant in aerobic
galactose-limited chemostat cultures. HXT5 was also
transcribed in galactose-limited cultures, albeit at low levels (Fig.
4, lanes 1 and 2). In the fructose-limited
chemostats, which had an extracellular fructose concentration of 0.61 mM, low levels of HXT5 and high levels of HXT2 and HXT7 mRNA were detected (Fig. 4,
lanes 7 and 8). Although the feed medium of
ethanol-limited cultures did not contain any hexoses, mRNAs of
HXT2, HXT5 and HXT7 were detectable at low to moderate levels (Fig. 4, lanes 3 and 4). This
might be related to the low concentration of glucose that was found in
the residual media of the ethanol-limited chemostats.
In the anaerobic nitrogen-limited chemostats, which had a high
extracellular glucose concentration of 154 mM, HXT1,
HXT2, HXT3, and HXT4 were expressed to various levels
(Fig. 4, lanes 5 and 6). In the aerobic
nitrogen-limited chemostats, which had a lower extracellular glucose
concentration of 3 mM, HXT6 and HXT7
mRNAs were also detectable (lanes 9 and 10).
The SNF3 and RGT2 genes were both expressed at
relatively high levels in the aerobic galactose-, fructose-, and
nitrogen-limited chemostats (Fig. 5,
lanes 1, 2, 5, 6, 9, and 10). SNF3
expression was depressed slightly in the ethanol-limited chemostats
(lanes 3 and 4) and significantly in the
anaerobic nitrogen-limited chemostats (lanes 5 and
6). RGT2 expression was depressed significantly
in the ethanol-limited chemostats and elevated slightly in the
anaerobic nitrogen-limited chemostats.
Kinetics of Hexose Transport in Chemostat Cultures--
The
accuracy of the kinetic parameter estimates was dependent on whether
one or two kinetic components were detected. This was partly due to the
observed proportionality between transport rate and experimental error.
When two components were present, estimation of the low affinity
kinetic parameters was predominantly based on those substrate
concentrations at which the transport rates, and hence the experimental
errors, were highest. Another factor influencing parameter estimation
is that each apparent kinetic component may in reality represent the
contribution of multiple, co-expressed transporters with substrate
affinities that are not sufficiently different to be resolved
analytically. Despite these inherent problems in analyzing glucose
transport kinetics in S. cerevisiae, some distinct
differences in transport kinetics as a function of growth conditions
were observed.
Single-component high affinity glucose transport kinetics were observed
for steady state S. cerevisiae cultures with low
extracellular glucose (<0.6 mM), with the exception of the
aerobic glucose-limited chemostats at D = 0.1 and 0.2 h
Glucose transport was assayed by measuring zero-trans influx
over short (5 s) intervals. Calculation of kinetic parameters from
these experiments was based on Michaelis-Menten kinetics and rested on
the assumption that the intracellular glucose concentration was zero
during the uptake assays. This requires the presence of sufficient
hexokinase activity and ATP. If this criterion is also met in the
chemostat cultures, then their in situ glucose consumption
rates should equal the glucose transport rates calculated from the
transport kinetics and the residual glucose concentration. Any
discrepancies would indicate that Michaelis-Menten kinetics do not
adequately describe glucose transport in either the off-line transport
assays or the steady state chemostat cultures (37, 38).
At lower dilution rates, the calculated transport rates exceeded the
in situ glucose consumption rates in the aerobic
glucose-limited chemostats (Fig.
6A). This suggests that an
inhibitor affected glucose uptake in the growing chemostat cultures.
The most likely inhibitor is intracellular glucose: a previous study
showed that intracellular glucose could account for a 50% difference
between measured zero-trans influx rates and observed
glucose consumption rates (38).
At higher dilution rates, the calculated transport rates were lower
than the in situ glucose consumption rates (Fig.
6A). This suggests that positive effectors influenced the
glucose transport step in the chemostat cultures, or, alternatively,
that the zero-trans influx assays systematically
underestimated the in situ transport capacity. It has
previously been observed that zero-trans glucose influx is
lower than the glucose consumption rate when a low affinity component
of glucose transport is prominent (38), as is the case for some of
these cultures.
Among the chemostats with various nutrient limitations, only the
anaerobic glucose-limited cultures had a calculated glucose transport
rate that was lower than the in situ glucose consumption rate. Eadie-Hofstee plots of glucose transport kinetics of these cultures displayed convex curves (not shown). This behavior has also
been found for an S. cerevisiae strain lacking glucose
kinase activity and may therefore reflect an accumulation of
intracellular glucose (37).
Transcriptional Regulation and Transport Phenotype of HXT
Genes--
Transcription of HXT genes in steady state
chemostats and batch cultures was strongly influenced by the carbon
source, its extracellular concentration, and the specific growth rate.
The observed regulatory patterns for the individual HXT
genes are discussed briefly below.
HXT1--
In the aerobic glucose-limited chemostat cultures,
HXT1 transcript levels were positively correlated with the
extracellular glucose concentration; significant HXT1
mRNA was detected at extracellular glucose concentrations of 0.65 mM and above. The transcript levels increased with
increasing extracellular glucose, but not to the level observed in the
exponentially growing batch-cultured cells at >100 mM
glucose. In nitrogen-limited cultures, HXT1 mRNA was more abundant under aerobic conditions (extracellular glucose, 3 mM) than under anaerobic conditions (extracellular glucose, 154 mM). Apparently HXT1 transcription is not
exclusively regulated by extracellular glucose concentrations but is
also affected by, for instance, oxygen or nitrogen availability. An
earlier study with batch cultures showed induction of HXT1
transcription by high glucose concentrations (>100 mM) and
repression at low glucose concentrations (<25 mM) (6).
Meijer (39) found that HXT1 mRNA levels in aerobic
nitrogen-limited chemostats were essentially constant at extracellular
glucose concentrations between 1 and 72 mM. This is
consistent with our observation that HXT1 transcription is
already induced by moderate glucose concentrations.
Transcription of HXT1 was consistently associated with
expression of low affinity glucose transport, in agreement with the previous observation that HXT1 confers low affinity glucose
transport on an hxt null strain (17).
HXT2--
This gene was transcribed at high levels in cultures
with low extracellular glucose concentrations. In the respiratory
glucose-limited chemostat cultures, the extracellular glucose
concentration did not appear to be correlated with the dilution rate.
Nevertheless, transcription of HXT2 reached a maximum at
D = 0.2 h
HXT2 expression is associated with the presence of high
affinity glucose transport; in particular, it is low in the anaerobic nitrogen-limited chemostats, the high affinity component of which has a
relatively high Km of 4.7 mM. This is
consistent with the previous observation that HXT2 can
confer relatively high substrate affinity to an hxt null
strain (17). In that study, the HXT2-only strain also
displayed lower affinity kinetics or biphasic kinetics; our results
cannot verify this behavior, but are not inconsistent with it. In
particular, such behavior could explain the strong dual-component
kinetics of the fructose-limited chemostats. It is possible that the
affinity of Hxt2 is modulated by, for example, posttranslational
modification or the lipid composition of the plasma membrane in
response to the nutritional status of the culture.
HXT3--
The pattern of HXT3 transcription was
virtually identical with that of HXT1. Specifically,
HXT3 was not expressed at low glucose concentrations,
neither in the aerobic glucose-limited chemostats nor in batch culture
on glucose. This seems to contradict an earlier study on batch-grown
S. cerevisiae in which HXT3 expression was found
to be independent of the glucose concentration (6). A comparison of
HXT1 and HXT3 mRNA levels in exponentially
growing batch cultures or aerobic versus anaerobic
nitrogen-limited chemostats indicates that, in our experiments, the
susceptibility of HXT3 to glucose induction is the same as
or even slightly greater than HXT1. In line with our
results, DeRisi et al. (11) found elevated HXT3
expression in cells early in batch culture, at approximately 100 mM glucose.
HXT3 transcription was observed in cells with a substantial
low affinity transport component, consistent with the conclusion based
on expression in an hxt null strain (17) that
HXT3 encodes a low affinity hexose transporter.
HXT4--
Similar to HXT1 and HXT3,
HXT4 was induced by elevated glucose concentrations. This
conclusion is evident in the batch culture (compare the samples from
exponential stage (Fig. 2, lane 11) with those from
glucose-depleted and ethanol-consuming stages (Fig. 2, lanes
12 and 13)) and in the nitrogen-limited chemostats. It
is also the trend in the dilution rate series, despite the erratic
behavior of HXT4 expression (see below). Contradictions are
found in the descriptions of HXT4 regulation in previous
studies. Using fusions of the HXT4 promoter to
lacZ on multicopy plasmids as a reporter, it was found that
the HXT4 gene is strongly glucose-repressed (6, 10) and is
co-regulated with HXT2 (6). DeRisi et al. (11)
detected HXT4 mRNA late in fermentative growth and upon glucose exhaustion. In contrast, Ciriacy and Reifenberger (41) cite
unpublished data that show HXT4 expression is significant on
both low and high glucose. Our results indicated that HXT4 is induced by high glucose and co-regulated with HXT1 and
HXT3, and in batch culture, its mRNA is scarce upon
glucose exhaustion. Possible reasons for these discrepancies include
strain differences (we have found considerable differences in the DNA
sequences upstream of the different HXT4 genes in the
GenBankTM data base; data not shown), lack of specificity
of the probes previously used to detect HXT4 mRNA (for
example, the open reading frames of HXT4 and HXT7
are 82% identical) or to artifacts associated with the use of fusions
of HXT promoters to lacZ on multicopy plasmids as
reporters of HXT gene expression (10, 12).
The respirofermentative glucose-limited cultures and the
nitrogen-limited cultures that displayed a low affinity glucose
transport component expressed HXT4. The
Km previously reported for an hxt null
strain expressing HXT4, approximately 9.5 mM
(17), falls outside the ranges of Km values measured
in the HXT4-expressing cultures in this study. However, in a
multicomponent glucose transport system, it is difficult to distinguish
a transporter with an intermediate affinity for glucose.
HXT5--
Previous studies on regulation of HXT genes
in batch cultures have not identified conditions in which the
HXT5 gene is highly expressed. Furthermore, overexpression
of HXT5 confers only a weakly positive growth phenotype to
the hxt null strain (42). DeRisi et al. (11)
found HXT5 expression in batch culture upon glucose
depletion. We found that HXT5 was abundantly transcribed in
cultures without glucose in the feed medium; thus, it was expressed in
fructose-, galactose-, and ethanol-limited chemostats. In
glucose-limited cultures, HXT5 mRNA was only detected at
the lowest dilution rates tested, which yielded low extracellular
glucose concentrations and low specific rates of glucose consumption.
HXT5 mRNA was also abundant in batch-cultured cells
after glucose exhaustion. These results are consistent with previous
observations, because even the low glucose concentrations that were
used to assay growth (5) would repress HXT5 and prevent it
from conferring growth on glucose. Two sequences that match the
consensus binding site of the HAP2/3/4/5 transcriptional activator
complex occur in the HXT5 promoter region (data not shown).
The HAP complex regulates some genes positively in the absence of
glucose (for a review, see Ref. 43), which is the pattern of expression
that is observed for HXT5. This suggests that
HXT5 is under HAP complex regulation and is expressed in
glucose-deprived cells to ensure that they are able to utilize the
sugar rapidly when it becomes available.
No kinetic data have been published for hexose transport by Hxt5. In
the current study, HXT5 expression was not unequivocally associated with either high or low affinity transport. We are currently
examining the kinetics of glucose transport displayed by strains
expressing only HXT5.
HXT6 and HXT7-HXT7 (and, in aerobic
glucose-limited chemostats, HXT6) was expressed at high
levels in cultures with low extracellular glucose, and at lower levels
in chemostats with higher extracellular glucose (up to 19 mM). At even higher glucose concentrations (e.g. in the anaerobic nitrogen-limited chemostats or exponentially growing
batch-cultured cells) HXT6 and HXT7 transcripts
were not detected. Expression of HXT7 increased with
increasing growth rate up to D = 0.3 h
HXT6 and HXT7 expression has also been detected
during batch culture on glycerol/ethanol and galactose media (7, 42); these findings are in accord with our observation that HXT7
was expressed in the ethanol-limited chemostats. However, we did not detect HXT6 or HXT7 in the galactose-limited
chemostats. Perhaps in batch culture, the cells had consumed enough
galactose (and produced enough ethanol) to allow a higher level of
expression than was observed in the steady state cultures.
HXT7 (and in some cases HXT6) was highly
expressed in most of the cultures that displayed a strong high affinity
kinetic component. Its expression was lower in cultures with a
diminished high affinity transport capacity (namely the
respirofermentative glucose-limited cultures and the ethanol-limited
chemostats). These results are in clear agreement with the conclusion
of Reifenberger et al. (17) that HXT6 and
HXT7 are high affinity hexose transporters. In that study,
the transport capacity of cells expressing HXT6 was
considerably lower than that of cells expressing HXT7, which is in broad agreement with the expression pattern we observed.
GAL2--
GAL2 transcription was only observed in the
galactose-limited chemostat cultures. This result was expected, because
GAL2 is repressed by glucose and induced by galactose (44,
45). Gal2 is able to transport glucose as well as galactose (7, 17). Previous studies have found both high and low affinity transport kinetics associated with Gal2 expression (7, 17, 46). In the present
study, cells expressing the GAL2 gene displayed a single,
high affinity component of glucose transport. It is possible that under
galactose-limited conditions, the Gal2 transporter is only present in a
high affinity state.
SNF3 and RGT2--
The SNF3 and RGT2 genes
have been proposed to encode glucose sensors, rather than transporters
of metabolically significant quantities of glucose (35, 47), and to
transduce a signal that leads to differential expression of some
HXT genes according to the glucose concentration in the
medium. It has previously been reported that SNF3 expression
is repressed by glucose and that RGT2 expression is
constitutive (35, 48, 49). We observed that the patterns of
SNF3 and RGT2 transcription were virtually identical; exceptions were the ethanol-limited chemostats, in which
RGT2 expression was low, and the anaerobic nitrogen-limited chemostats, in which RGT2 expression was high and
SNF3 expression was low.
Snf3 has been reported to repress HXT6 and/or
HXT7 on glucose (7) and to induce HXT2 on low
glucose (35), and Rgt2 has been reported to induce HXT1 on
high glucose (35). However, in the present study SNF3 was
co-expressed with HXT7 under conditions of low extracellular
glucose and fructose and with HXT6 in some of the aerobic
glucose-limited chemostats. Similarly, SNF3 was expressed
under conditions of low extracellular glucose in the absence of
significant HXT2 expression (e.g. the anaerobic
glucose-limited chemostats). RGT2 expression was high in the
aerobic glucose-limited chemostats at lower dilution rates, and
declined at higher dilution rates, which is the opposite pattern of
that observed for HXT1 expression. These results suggest
that differences in the expression of the target genes of the
SNF3- and RGT2-dependent pathways are due to posttranscriptional control of the Snf3 and Rgt2 sensors or that
other regulatory systems can predominate over the Snf3 and Rgt2 signals
in these growth conditions.
Reproducibility of HXT4 and HXT6 Transcription in Chemostat
Cultures--
Transcription of HXT4 and HXT6 in
the dilution rate series did not follow a consistent trend, in contrast
to the other HXT mRNAs analyzed. For example, they were
much more abundant at D = 0.15 h Conclusions--
This study is the first comprehensive survey of
the expression of hexose transporter genes and glucose transport
kinetics in wild-type S. cerevisiae. The results confirm
some previous descriptions of their expression patterns and kinetic
properties. They point to the potential importance of HXT5
as a source of latent glucose transport capacity in cells grown on
non-glucose media. They display some discrepancies with previous
findings on the expression of HXT2, HXT3, and
HXT4 and of SNF3 and RGT2. Furthermore, they reveal significant differences in the regulation of
HXT6 and HXT7.
1 under various
nutrient-limited conditions (anaerobically glucose- or nitrogen-limited
or aerobically glucose-, galactose-, fructose-, ethanol-, or
nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h
1 in aerobic glucose-limited cultures. Transcription of
HXT1-HXT7 was correlated with the extracellular glucose
concentration in the cultures. Transcription of GAL2,
encoding the galactose transporter, was only detected in
galactose-limited cultures. SNF3 and RGT2, two
members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8-HXT17 transcripts were
detected at very low levels. A consistent relationship was observed
between the expression of individual HXT genes and the
glucose transport kinetics determined from zero-trans
influx of 14C-glucose during 5 s. This relationship
was in broad agreement with the transport kinetics of Hxt1-Hxt7 and
Gal2 deduced in previous studies on single-HXT strains. At
lower dilution rates the glucose transport capacity estimated from
zero-trans influx experiments and the residual glucose
concentration exceeded the measured in situ glucose
consumption rate. At high dilution rates, however, the estimated
glucose transport capacity was too low to account for the in
situ glucose consumption rate.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10-50 mM) expressed by
cells growing in media with a high glucose concentration, and a high
affinity system (Km
1-3 mM) expressed by cells in low glucose media (2, 3). Cells growing in low
glucose media often display both a high affinity component and a low
affinity component (2).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. These frozen stocks were used to
inoculate precultures for chemostat cultivation.
1 using a Brooks 5876 mass flow controller. The dissolved oxygen concentration was monitored
with an O2 electrode (model 34 100 3002; Ingold) and
remained above 60% of air saturation. For anaerobic cultivation,
fermenters were equipped with Norprene tubing to avoid O2
diffusion and flushed with nitrogen gas at a flow rate of 0.5 liter·min
1. Chemostats were started at a dilution rate
(D, equal to specific growth rate in steady state cultures)
of 0.10 h
1. Steady states at other dilution rates were
achieved by changing the dilution rate in 0.05 h
1
increments. At D values of 0.20 h
1 and above,
these increments were reduced to 0.025 h
1. After each
increase of D, cultures were allowed to establish a new
steady state (defined as the situation in which at least five volume
changes had passed after the last change in growth conditions, and in
which the biomass concentration and specific rates of CO2
production and O2 consumption had remained constant (<2%
variation) for at least two volume changes). Culture purity was
routinely monitored by phase-contrast microscopy and by plating on YPD
medium (1% yeast extract, 2% peptone, 2% glucose, 2% agar) (20).
1, 93.4 ± 1.6% for
the aerobic nitrogen-limited chemostats, 93.5 ± 0.8% for the
anaerobic glucose-limited chemostats, and >96% for all other
chemostats. Carbon recoveries were calculated assuming a carbon content
of dry biomass of 48%. Calculations of carbon recovery did not take
into account the minor loss of ethanol in the off-gas that occurred at
high dilution rates due to evaporation (18) and the dilution of
cultures as a result of KOH titration.
1, and the ethanol concentration was 6.75 g·liter
1. For anaerobic cultivation, media were
supplemented with the anaerobic growth factors ergosterol and Tween 80 (10 and 420 mg·liter
1, respectively) (21) and the
glucose concentration in the medium was increased to 25 g·liter
1. For aerobic nitrogen-limited growth on
glucose, the (NH4)2SO4 concentration in the mineral medium was reduced to 1.0 g·liter
1, and the glucose concentration was increased
to 25 g·liter
1. Similarly, for anaerobic
nitrogen-limited growth on glucose, the
(NH4)2SO4 concentration was reduced
to 0.25 g·liter
1, and the glucose concentration was
increased to 37.5 g·liter
1. In nitrogen-limited media,
the reduced concentration of
(NH4)2SO4 was compensated for by
addition of equimolar amounts of K2SO4 .
-galactose dehydrogenase to an assay mixture containing
100 mM Tris-HCl (pH 7.6), 20 mM NAD and sample.
Glycerol and pyruvate in culture supernatants were determined by high
pressure liquid chromatography using an HPX-87H Aminex ion exchange
column (300 × 7.8 mm, Bio-Rad) at 60 °C. The column was eluted
with 5 mM H2SO4 at a flow rate of
0.6 ml·min
1. Pyruvate was detected by a Waters 441 UV
meter at 214 nm, coupled to a Waters 741 data module. An ERC-7515A
refractive-index detector (ERMA), coupled to a Hewlett-Packard type
3390A integrator, detected glycerol.
1 protein. Cells were kept on ice until further
use. Zero-trans influx of glucose and fructose was
determined at 30 °C according to Walsh et al. (3).
Briefly, cells were aerated for 2 min before beginning the assay. Fifty
µl of cells were mixed with 12.5 µl of 5-fold concentrated glucose
or fructose labeled with D-[U-14C]glucose or
D-[U-14C]fructose (Amersham Pharmacia
Biotech) respectively and incubated for approximately 5 s
(accurately timed). Uptake was quenched by transfer of 50 µl of the
resulting mixture to 10 ml of quench buffer (0.1 M
potassium phosphate (pH 6.5)/0.5 M D-glucose,
5 °C), and the cells were rapidly harvested by vacuum filtration onto a glass fiber filter (GF/C, Whatman). The filters were washed twice with quench buffer and transferred rapidly to scintillation vials
containing 10 ml of Emulsifier Scintillator Plus (Packard). Disintegrations per min were determined by scintillation counting. Triplicate determinations were performed at each of 10 glucose or
fructose concentrations (from 250 mM (6 MBq·mol
1) to 0.25 mM (1400 MBq·mol
1)) for each batch of cells. The concentration
of the nonradioactive hexose used in the assays was determined as
described (25). The protein concentration of the cell suspensions was
determined by the method of Lowry et al. (26) using bovine
serum albumin (fatty acid-free, Sigma) as a standard. The protein and
glucose concentrations were measured on COBAS-BIO and COBAS-FARA
automatic analyzers (Roche).
80 °C. After thawing, the samples were
centrifuged at room temperature for 15 min at 16,000 × g. The supernatant was extracted with AE-saturated
phenol/chloroform/isoamyl alcohol and centrifuged at room temperature
for 4 min at 16,000 × g. The supernatant was combined
with 40 µl of 3 M sodium acetate and 1 ml 100% ethanol
and stored at
20 °C. The precipitated RNAs were recovered by
centrifugation at room temperature for 15 min at 16,000 × g, resuspended in formamide, and quantitated as described (30). Poly(A)+ RNA was exhaustively purified from 150-µg
samples of total RNA using oligo(dT) Dynabeads (Dynal) according to the
manufacturer's instructions.
Oligonucleotide probes used to detect mRNAs of the members of the
S. cerevisiae HXT gene family
-32P]ATP (Amersham
Pharmacia Biotech) and purified with the QIAquick nucleotide removal
kit (Qiagen). The specific activities of the probes were routinely
1.75-2.5 µCi/pmol. Prehybridization was carried out for 1 h at
45 °C in 5 ml of 6× SSC, 0.1% SDS, 5× Denhardt's solution, and
100 µg·ml
1 sheared, denatured salmon sperm DNA.
Hybridization was carried out by addition of the oligonucleotide probe
and further incubation for 4 h. The blots were washed twice for 5 min at room temperature in 6× SSC, 0.1% SDS, twice for 5 min at
45 °C in 6× SSC, 0.1% SDS, and once for 5 min at 45 °C in 2×
SSC, 0.1% SDS. A final wash of 5 min at 45 °C in 1× SSC, 0.1% SDS
was performed if required. Autoradiograms were developed after exposure
of Kodak X-Omat AR film to the blots in the presence of an intensifying
screen at
70 °C. Exposure times were overnight for total RNA blots
and 48 h for poly(A)+ RNA blots. Four identical blots
of total RNA samples were used for each experiment; after
autoradiography, the probes were stripped from the blots by treatment
in 0.1× SSC, 0.1% SDS at 75 °C three times for 30 min prior to
rehybridization with other probes. A 1.1-kilobase pair
BamHI/NcoI fragment of the S. cerevisiae
PDA1 gene (encoding the
subunit of pyruvate dehydrogenase) was
used to control for variation in sample loading (31).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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1, growth was completely respiratory (Fig.
1B). Over this range of
dilution rates, the specific rate of glucose consumption increased linearly with the dilution rate; however, the extracellular glucose concentration did not change significantly with changes in the dilution
rate (Fig. 1C). This peculiar phenomenon was observed earlier with a different S. cerevisiae strain (13).
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Fig. 1.
Growth of S. cerevisiae
CEN.PK113-7D in aerobic glucose-limited chemostat cultures at
various dilution rates. A, biomass yield
(Yxs, g of biomass·(g of
glucose) 1) and specific rate of ethanol production
(qethanol, mmol·(g of
biomass)
1·h
1). B, specific
rates of O2 consumption (qO2, mmol·(g of
biomass)
1·h
1) and CO2
production (qCO2, mmol·(g of
biomass)
1·h
1). C, specific
rate of glucose consumption (qglucose, mmol·(g of
biomass)
1·h
1) and the extracellular
glucose concentration (mmol·liter
1) in culture
supernatants.
1, the specific
O2 consumption rate of the cultures reached a maximum (Fig.
1B). At higher dilution rates, respiration and alcoholic
fermentation (including some pyruvate, acetate and glycerol production)
occurred simultaneously (Fig. 1, A and B). The
strong increase of the glucose consumption rate at high growth rates,
which results from the lower energetic efficiency of alcoholic fermentation, was accompanied by an increase of the extracellular glucose concentration in the cultures and a reduced biomass yield (Fig.
1, A and C).
1. All aerobic
hexose-limited cultures were completely respiratory at this dilution
rate, as is evident from the absence of fermentation products in
culture supernatants, a respiratory quotient close to 1, and a high
biomass yield of 0.5 g of biomass·(g of glucose)
1
(Table II). The anaerobic glucose-limited
cultures were completely fermentative, with specific rates of glucose
consumption that were 5-fold higher than the aerobic cultures (Table
II). Aerobic ethanol-limited cultures served as a reference situation
in which no net hexose uptake occurred during growth (Table II).
Remarkably, significant concentrations of glucose were detected in
supernatants of the ethanol-limited (110 µM),
galactose-limited (140 µM), and fructose-limited (750 µM) cultures. Glucose concentrations in the reservoir
media were 10, 10, and 100 µM, respectively, indicating that the glucose present in these cultures was a result of yeast metabolism.
Biomass yield (Yxs; g of biomass · (g of
substrate)1), residual substrate concentration, protein
content (g of protein · (g of dry biomass)
1), and
specific rates (q) of oxygen consumption, carbon dioxide production,
glucose consumption, and ethanol production in steady-state chemostat
cultures of S. cerevisiae CEN.PK113-7D (D, 0.10 h
1) under different growth limitations. Data are presented as
average ± S.D. of two independent steady-state chemostat
cultures.
1 and D = 0.2 h
1 also displayed a low affinity component, with a
Km of about 13 mM. At dilution rates
above 0.3 h
1, two components were consistently detected:
a high affinity component with a Km similar to that
observed at the lower dilution rates, and a low affinity component with
an average Km of 35 mM. Given the
uncertainties of the estimates, the overall Vmax
of glucose transport showed relatively little variation at the
different dilution rates and ranged from 578 to 797 nmol·min
1·mg
1.
Maximum velocity (nmol·min1·mg
protein
1) and Michaelis constant (mM) of
glucose transport by cells grown in aerobic glucose-limited chemostats
at various dilution rates and in batch culture on glucose
1) displayed single-component
high affinity glucose transport. All of the other cultures examined at
this dilution rate displayed both high and low affinity kinetics (Table
IV). The high affinity Km values of the ethanol- and anaerobic
nitrogen-limited cultures were significantly higher than those of the
high affinity components found in other cultures. The overall
Vmax values were relatively low for the ethanol-
and anaerobic glucose-limited chemostats and relatively high for the
fructose-limited chemostats.
Maximum velocity (nmol·min1·mg
protein
1) and Michaelis constant (mM) of
glucose transport by cells grown in duplicate chemostats with various
nutrient limitations at a dilution rate of 0.1 h
1
1 the calculated rates were only 49 and 72% of the
in situ consumption rates, respectively.
1) exceeded their
in situ glucose consumption rates. Similarly, the calculated
fructose transport rates exceeded the in situ fructose consumption rates for the fructose-limited chemostats. However, in the
anaerobic glucose-limited cultures, the calculated rate of glucose
transport was 23% lower than the in situ glucose
consumption rate (see Fig. 6B).
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Fig. 2.
Expression of HXT genes in
aerobic glucose-limited chemostat cultures at various dilution rates
and glucose-fed batch cultures at various growth stages. Blot
analysis of total RNA samples was performed with oligonucleotide probes
specific for the genes HXT1-HXT7. A DNA probe
for the PDA1 gene was used to monitor transcript levels of
this constitutively expressed gene to control for variation in sample
loading. The dilution rates were 0.05 (lane 1), 0.1 (lane 2), 0.15 (lane 3), 0.2 (lane 4),
0.25 (lane 5), 0.28 (lane 6), 0.3 (lane
7), 0.33 (lane 8), 0.36 (lane 9), and 0.38 h 1 (lane 10); the stages of batch cultivation
on glucose were exponential growth (lane 11), diauxic shift
(glucose exhaustion) (lane 12), and growth on ethanol (5 h
after glucose exhaustion) (lane 13).
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Fig. 3.
Expression of SNF3 and
RGT2 in aerobic glucose-limited chemostat cultures at
various dilution rates. Blot analysis of poly(A)+ RNA
samples was performed with oligonucleotide probes specific for the
SNF3 and RGT2 genes. The dilution rates were 0.05 (lane 1), 0.1 (lane 2), 0.15 (lane 3),
0.2 (lane 4), 0.25 (lane 5), 0.28 (lane
6), 0.3 (lane 7), 0.33 (lane 8), 0.36 (lane 9), and 0.38 h 1 (lane 10).
The PDA1 probe was as described for Fig. 2.
1, the extracellular glucose
concentration remained below 0.2 mM and did not change
significantly with dilution rate (Fig. 1C). Some remarkable
patterns in the HXT transcript levels could be observed in
these cultures (Fig. 2). Transcription of HXT1 and HXT3 was low throughout the lower range of dilution rates
but then increased sharply above D = 0.30 h
1, a pattern that seems to follow the changes in
extracellular glucose concentration. In contrast to this,
HXT7 appeared to be transcribed to a considerable extent at
all growth rates, but somewhat less at the highest growth rates. A
similar pattern was observed for HXT2. HXT5 mRNA was
detected only in slowly growing cells. Transcript levels of
HXT4 and HXT6 were erratic throughout the range
of dilution rates. The SNF3 and RGT2 genes were
both maximally transcribed at the lowest dilution rates; their
expression levels declined gradually with increasing dilution rates,
but transcription of both genes was readily detected even at
D = 0.38 h
1 (Fig. 3).
1), which exhibited
an extracellular glucose concentration approximately 2.5-fold higher
than aerobic glucose-limited chemostats grown at the same dilution
rate, HXT7 was abundantly expressed, and HXT2 and
HXT6 were expressed at low levels (Fig.
4, lanes 11 and 12).
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Fig. 4.
Expression of HXT genes in
chemostat cultures with various growth limitations at a dilution rate
of 0.1 h 1. Total RNA was
extracted from cells harvested from duplicate chemostats maintained at
steady state with various growth limitations. Blot analysis of total
RNA samples was performed with oligonucleotide probes specific for the
genes HXT1-HXT7 and GAL2. The steady state
culture conditions were aerobic galactose limitation (Gal)
(lanes 1 and 2), ethanol limitation
(EtOH) (lanes 3 and 4), anaerobic
nitrogen limitation (Nitr Anaer) (lanes 5 and
6), aerobic fructose limitation (Fru)
(lanes 7 and 8), aerobic nitrogen limitation
(Nitr Aer) (lanes 9 and 10), and
anaerobic glucose limitation (Glu Anaer) (lanes
11 and 12). The PDA1 probe was as described
for Fig. 2.
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Fig. 5.
Expression of SNF3 and
RGT2 in chemostat cultures with various growth
limitations at a dilution rate of 0.1 h 1. Poly(A)+ RNA
was extracted from cells harvested from duplicate chemostats maintained
at steady state with various growth limitations. Blot analysis of the
RNA samples was performed with oligonucleotide probes specific for the
SNF3 and RGT2 genes. The steady state culture
conditions were aerobic galactose limitation (Gal)
(lanes 1 and 2), ethanol limitation
(EtOH) (lanes 3 and 4), anaerobic
nitrogen limitation (Nitr Anaer) (lanes 5 and
6), aerobic fructose limitation (Fru)
(lanes 7 and 8), aerobic nitrogen limitation
(Nitr Aer) (lanes 9 and 10), and
anaerobic glucose limitation (Glu Anaer) (lanes
11 and 12).
DISCUSSION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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1. Two transport components were found for the latter
cultures, for cultures with higher extracellular hexose levels
(0.6-154 mM), and for the cultures with ethanol in the
feed medium. These results are consistent with previous observations
that high affinity glucose transport is not detected in
glucose-repressed (i.e. high extracellular glucose) cultures
(3, 34). They support the proposal that S. cerevisiae is
able to sense the extracellular glucose concentration and express
transport systems with a substrate affinity that is appropriate to the
available concentration of glucose (35, 36). The presence of two
kinetic components in the ethanol-limited cultures, each with a low
Vmax, suggests that both high and low affinity
transporters are expressed at a basal level when no glucose is
furnished to the culture.
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Fig. 6.
Relationship between in situ
hexose consumption rates and calculated hexose transport rates in
hexose-limited chemostats. The rates of hexose transport were
calculated from the zero-trans influx kinetics and the
residual hexose concentrations. The calculated hexose transport rate is
expressed as a percentage of the in situ hexose consumption
rate for aerobic glucose limitation at various dilution rates
(A) and for various nutrient limitations at a fixed dilution
rate of 0.1 h 1 (B). Glu, glucose;
aer, aerobic; anaer, anaerobic; nitr,
nitrogen; fruc, fructose.
1. Expression was also high in
the fructose-limited chemostats and, to a lesser extent, in the aerobic
nitrogen-limited chemostats. Reduced transcript levels were observed in
respirofermentative glucose-limited cultures grown at higher dilution
rates, in which the extracellular glucose concentration increased up to
19 mM. At the even higher glucose concentration of the
anaerobic nitrogen-limited cultures, HXT2 mRNA levels
were very low. The levels were also low in the ethanol- and aerobic
galactose-limited chemostats and, surprisingly, in the anaerobic
glucose-limited chemostats. HXT2 expression was undetectable
in any phase examined of batch culture on glucose, in agreement with
previous observations (8). Earlier observations that HXT2
expression is high only when batch-cultivated cells are shifted from
high to low glucose media led to the proposal that expression requires
both a low glucose concentration and exponentially growing cells (8,
40). Our results are consistent with this model, but they point to an
effect of aerobiosis as well.
1.
In some previous studies using batch cultures, expression of HXT6 and HXT7 was repressed by high glucose and
induced by low glucose (7, 42), in agreement with the findings
presented here. Others have reported that HXT6 and
HXT7 expression increased early in batch growth, when the
glucose concentration was still approximately 100 mM
(11).
1 than at
0.1 or 0.2 h
1 and were much less abundant at
D = 0.3 h
1 than at 0.28 or 0.33 h
1. We have examined HXT1, HXT4,
HXT6, and HXT7 mRNA levels in samples from three
different aerobic glucose-limited chemostats at D = 0.1 h
1 and from two such chemostats at D = 0.2 h
1 (including the samples shown in Fig. 2,
lanes 2 and 4) and found that the mRNA levels
of HXT4 and HXT6 were particularly variable (data
not shown). In one of the D = 0.1 h
1
samples and in the other D = 0.2 h
1
sample, HXT4 and HXT6 mRNA levels were
higher, consistent with the levels seen in Fig. 2, lanes 1, 3, and 5. There are two plausible explanations for
these results. The cultures with aberrant expression of HXT4
and HXT6 may not have attained the same steady state as the
other cultures with respect to expression of these genes, despite being
in a steady state with respect to measured physiological parameters and
expression of the other HXT genes. Alternatively, a
heritable change may have occurred to a cis- or
trans-acting factor that influences the regulation of
HXT4 and HXT6, resulting in their anomalous
expression in these samples. Nutrient-limited chemostats impose a
strong selective pressure for changes that adapt the cells for more
efficient capture of the limiting nutrient (see e.g. Refs.
50-53). HXT6 has been observed previously to be the subject
of genetic modifications during growth in glucose-limited chemostats
(54). In that case, the result was duplications and rearrangements of
the tightly linked HXT6 and HXT7 loci, resulting in increased levels of HXT6 mRNA. Surprisingly, the
erratic behavior of HXT6 that we observed was not
accompanied by similar behavior by HXT7. This suggests that
these two genes are subject to different regulatory influences despite
being tightly linked and virtually identical in the sequences of their
open reading frames.
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FOOTNOTES |
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* This work was financially supported by the Foundation for Chemical Research of the Netherlands Organization for Scientific Research, the Associaton of Biotechnology Centers in the Netherlands, Gist-brocades B. V., and the European Community (Framework IV projects BIO4-CT95-0132 and BIO4-CT95-0107 of the BIOTECH program).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Current address: Lead Discovery Unit/ADG, Organon, P. O. Box
20, 5340 BH Oss, The Netherlands.
To whom correspondence should be addressed: Dept. of Molecular
Cell Physiology, Faculty of Biology, Free University of Amsterdam, de
Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. Tel.:
31-20-4447235; Fax: 31-20-4447229.
1 M. Schepper, H. F. M. Boelens, and M. J. Teixeira de Mattos, manuscript in preparation.
2 R. van Rooijen and P. Klaassen, unpublished results.
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