From the Kluyver Laboratory of Biotechnology,
Technical University of Delft, Julianalaan 67, 2628BC Delft, The
Netherlands and § DSM Life Sciences, Division of Bakery
Ingredients, Technology Cluster, 2600MA Delft, The Netherlands
Received for publication, September 23, 2002
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ABSTRACT |
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Profiles of genome-wide transcriptional events
for a given environmental condition can be of importance in the
diagnosis of poorly defined environments. To identify clusters of genes
constituting such diagnostic profiles, we characterized the specific
transcriptional responses of Saccharomyces cerevisiae
to growth limitation by carbon, nitrogen, phosphorus, or sulfur.
Microarray experiments were performed using cells growing in
steady-state conditions in chemostat cultures at the same dilution
rate. This enabled us to study the effects of one particular limitation
while other growth parameters (pH, temperature, dissolved oxygen
tension) remained constant. Furthermore, the composition of the media
fed to the cultures was altered so that the concentrations of excess nutrients were comparable between experimental conditions. In total,
1881 transcripts (31% of the annotated genome) were significantly changed between at least two growth conditions. Of those, 484 were
significantly higher or lower in one limitation only. The functional
annotations of these genes indicated cellular metabolism was altered to
meet the growth requirements for nutrient-limited growth. Furthermore,
we identified responses for several active transcription factors with a
role in nutrient assimilation. Finally, 51 genes were identified that
showed 10-fold higher or lower expression in a single condition only.
The transcription of these genes can be used as indicators for the
characterization of nutrient-limited growth conditions and provide
information for metabolic engineering strategies.
Growth of microorganisms in their natural environment and in many
industrial applications is often limited by nutrient availability (1,
2). In these situations the specific growth rate of the organism is
determined by the low (non-saturating) concentration of a single
nutrient. For example, in the industrial production of bakers' yeast
sugar-limited, aerobic cultivation at relatively low specific growth
rates is essential to achieve high biomass yields. On the other hand
processes such as beer fermentation occur at high concentrations of
fermentable sugars and are limited by other nutrients (e.g.
oxygen, nitrogen). As a result the yeast's metabolic activities are
altered. This situation is different from nutrient starvation in which
the absence of a nutritional component is often the cause of stress
responses that result in growth arrest or cell death (3, 4).
In the laboratory, cultivation of microorganisms is predominantly
performed in shake-flasks, in which all relevant nutrients are at least
initially present in excess. During the course of batch cultivation the
physical and chemical environment constantly changes, which directly
affects the specific growth rate and the regulation of many metabolic
processes (5). With the use of chemostat cultures, it is possible to
study steady-state physiological adaptations to nutrient-limited
growth. The medium that is continuously fed into the culture can be
designed such that growth is limited by a single, defined nutrient,
whereas all other nutrients remain present in excess. In conjunction
with this continuous feed of fresh media into the vessel, waste media
and cells are removed at the same rate. This results in a constant
dilution rate (h Microorganisms have evolved a multitude of strategies to cope with
nutrient limitations. Low, growth-limiting amounts of important nutrients often lead to the induction of high affinity transport systems and/or metabolic systems that allow more efficient
incorporation of the nutrients into biomass constituents. A classical
example is the high affinity glutamine synthetase/glutamine
oxoglutatrate aminotransferase system from Klebsiella
pneumoniae (formerly Aerobacter aerogenes) that can
replace glutamate dehydrogenase as the primary ammonia-assimilating
enzyme system during ammonia-limited growth (7). In other cases, the
final biomass composition itself has a reduced content of the
growth-limiting nutrient. For example, in Saccharomyces
cerevisiae and Escherichia coli, it has been observed
that the amino acid composition of the subset of structural enzymes
used in the assimilation of sulfur, carbon, or nitrogen have a reduced
content of the respective element compared with their average content
in the predicted proteome (8).
The physiological responses of microorganisms to different nutrient
limitation regimes can be regulated at various levels. At the level of
transcription, DNA microarrays allow the accurate, genome-wide mapping
of regulatory responses. With few exceptions (9-12) DNA microarray
analyses have been performed using cells grown in shake flasks. With
the use of chemostats, detailed analyses of the transcriptional
responses of S. cerevisiae to nutrient limitations may aid
in the development of new, DNA array-based approaches for diagnosis of
industrial fermentation processes. Furthermore, such studies provide
valuable information for the functional analysis of genes whose encoded
protein has no known or only poorly defined function. However, as yet
there have been no systematic investigations into the impact on
genome-wide transcriptional regulation of different nutrient limitation
regimes in chemostat cultures.
The main chemical elements that are assimilated into yeast biomass are
carbon, hydrogen, oxygen, nitrogen, sulfur, and phosphorus (13).
Previous work from our laboratory already addressed the impact of
oxygen supply on the transcriptome of S. cerevisiae (9, 12).
The aim of the present study was to determine which of the currently
recognized genes of S. cerevisiae have uniquely higher or
lower expression when growth is limited for each of the macro
nutrients, carbon, nitrogen, sulfur, or phosphorus. To this end, a wild
type strain of S. cerevisiae was grown on glucose under
strictly defined conditions in aerobic, nutrient-limited chemostat
cultures at a constant growth rate. These experiments revealed the
transcriptional differences that contribute to altered yeast metabolism
and so can serve as molecular identifiers to diagnose the status of
nutrient-limited fermentations or refine metabolic engineering
strategies. The complete data set is available for download at
www.nutrient-limited.bt.tudelft.nl.
Strain and Growth Conditions--
Wild type S. cerevisiae strain CEN.PK113-7D (MATa) (14)
was grown at 30 °C in 2-liter chemostats (Applikon) with a working
volume of 1.0 liter as described in van den Berg et al.
(15). Cultures were fed with a defined mineral medium that limited
growth by either carbon, nitrogen, phosphorus, or sulfur with all other
growth requirements in excess and at a constant residual concentration.
The dilution rate was set at 0.10 h Media--
The defined mineral medium composition was based on
that described by Verduyn et al. (17). In all limitations
except for carbon, the residual glucose concentration was targeted to
17 g·liter Analytical Methods--
Culture supernatants were obtained after
centrifugation of samples from the chemostats or by a rapid sampling
technique using steel balls precooled to
Microarray Analysis--
Sampling of cells from chemostats,
probe preparation, and hybridization to Affymetrix
GeneChip® microarrays was performed as described
previously (12). The results for each growth condition were derived
from three independently cultured replicates.
Data Acquisition and Analysis--
Acquisition and
quantification of array images and data filtering were performed using
the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0,
and Data Mining Tool v3.0. For further statistical analyses, Microsoft
Excel Significance Analysis of Microarrays (SAM; v1.12) add-in was used
(20). The data representation used in Figs. 2 and 3 were generated
using the mean and variance normalize function of the software
J-Express v2.1.
Before comparison, all arrays were globally scaled to a target value of
150 using the average signal from all gene features using Microarray
Suite v5.0. From the 9335 transcript features on the YG-S98 arrays a
filter was applied to extract 6383 yeast open reading frames of which
there were 6084 different genes. This discrepancy was due to several
genes being represented more than once when suboptimal probe sets were
used in the array design.
To represent the variation in triplicate measurements, the coefficient
of variation (standard deviation divided by the mean) was first
calculated for each transcript. When the genes were ordered by
increasing average signal, the average coefficient of variation
displayed a sharp increase for the 900 genes with the lowest abundance.
The average coefficient of variation for the remaining 5483 signals was
used to represent the average error for each condition (for further
explanation and use of these values, see Piper et al. 12).
Because the lowest 900 transcripts were unable to be reliably measured,
their level was set to a value of 12 (see "Lowest measurable level"
in Table II) for the comparison analyses.
Clusters of expression profiles were identified from all possible
pairwise comparisons of the four data sets. A transcript fell into one
of the eight expression clusters (significantly higher or lower in only
one condition) if it was called significantly changed using
Significance Analysis of Microarrays (expected median false positive
rate of 1%) by at least 2-fold from each other condition. In our
experience, these criteria establish a data set able to be reproduced
by an independent laboratory (12).
Promoter analyses were performed using the web-based software
Regulatory Sequence Analysis Tools
(bioinformatics.bmc.uu.se/~jvanheld/rsa-tools) (21). The promoters
(from Physiology and Global Transcriptional Responses of S. cerevisiae
during Nutrient-limited Growth--
Metabolic changes, mediated partly
by transcriptional regulation, are required for successful adaptation
to environmental changes. Here we have measured the genome-wide
transcriptional responses of S. cerevisiae to four different
macronutrient limitations during steady-state growth in chemostats. To
verify that each of the nutrient limitations was appropriately
achieved, we measured the concentrations of nutrients in the culture
supernatants (Table I). This showed that
when a nutrient was growth-limiting, its residual concentration was
below the detection limit, whereas each other nutrient was in excess.
Furthermore, the concentrations of each excess nutrient were comparable
between cultures. This ability to control the concentrations of
excess nutrients is a unique feature of chemostat cultivation and
one that is especially important for ammonia and glucose in light of
their impact on transcriptional regulation via sensors of extracellular
nutrients (22). Indeed, from work in our laboratory beyond the scope of this paper, we have noted systematic alterations in global
transcription as a result of differences in excess extracellular
glucose concentrations (data not shown).
For each culture, the rates of glucose and oxygen consumption as
well as carbon dioxide and ethanol evolution were determined (Table I).
In the glucose-limited culture, no ethanol was produced, and cells grew
with a biomass yield on glucose of 0.5 g·g
For the transcriptome analyses, the variation for each condition was
measured from the three independent array replicates performed (Table
II). The average coefficient of variation
was no more than 0.21 and for sulfur limitation was as low as 0.13, reflecting the high reproducibility between replicate arrays. Furthermore, the level of the ACT1 transcript and the signal
from the gene with lowest measurable expression were both unchanged between culture conditions. This indicated that the transcriptomes from
each condition were similar in their overall magnitude of expression,
thus supporting the use of global scaling for these comparisons.
In total 1881 genes (31% of the genome) had altered expression levels
in at least one condition, whereas 3558 (58%) were unchanged, and 645 (11%) remained below reliable detection in all four conditions (Fig.
1). It is not surprising that such a
large proportion of the genome was altered across our experiments since
the four limiting nutrients are major constituents of biomass. For each
nutrient it is believed that metabolic changes occur in the cell that
result in both sparing of accessible nutrients and initiation of
methods to make alternative forms of that nutrient available. In both cases, this requires the differential regulation of many genes involved
in transport and metabolism of the required compounds.
Among the changes observed, a division was made to separate the genes
that had a significantly higher or lower abundance in only one
condition when compared with each other limitations and those with more
complex regulatory patterns (Fig. 1). Within the former class, there
were four regulatory patterns that showed higher expression under only
one limitation and four patterns that showed lower expression in only
one limitation (Fig. 2). We rationalize
that these eight patterns of expression should be the most informative
for promoter and functional analysis studies as well as forming the
basis for the list of transcripts that could be used as specific
molecular identifiers to characterize specific growth limitations.
Because our aim was to define transcripts that could be used for this
purpose, we chose to only concentrate on the 484 open reading frames
that fell into these eight classes.
Identification of Putative Regulatory Elements Responsible for
Transcriptional Regulation during Nutrient-limited
Growth--
Coordinated regulation of global transcription is
driven by the action of transcription factors that generally act
once bound to short elements in gene promoters. Searching the promoters
of co-regulated genes for short sequences that are over-represented can
identify these elements. We analyzed the genes from the eight regulatory classes defined above using the web-based tool RSAT (21).
Because our classifications selected for genes that were differentially
regulated under only one of the nutrient limitations tested, each group
should have been enriched for the regulatory elements that are
specifically required for an appropriate response.
Several significantly over-represented elements were recovered
from each group of genes except for those that had specifically lower
expression under nitrogen or phosphorus limitation (Table III). Because it is known that many
transcriptionally active elements have an enhanced effect when
present in more than one copy (for example, see Ref. 25), the
proportion of gene promoters in each subgroup with at least two
elements was compared with the proportion found in all promoters of the
genome (Table III). For each regulatory profile in which a known
transcriptional regulator is thought to act, its corresponding binding
sequence was found among the multiple elements recovered. In addition,
several unknown elements were found that could not be associated with
the binding of known transcription factors. To define unique
transcriptional responses to distinct nutrient limitations, only the
known elements are discussed further below.
The Regulatory Response of S. cerevisiae Specific to
Carbon-limited Growth--
S. cerevisiae has the
ability to use several different carbohydrate molecules as its sole
source of carbon and energy. The carbon source that is most directly
incorporated into central metabolism is glucose, which is often
referred to as a preferred carbon source. At the molecular level,
the availability of glucose to cells signals repression of many genes
involved in the utilization of alternative carbon sources via a complex
of signals collectively known as carbon catabolite repression (for
reviews, see Refs. 26 and 27). We found 163 transcripts with
significantly higher levels and 62 transcripts with significantly
lower levels in a manner specific to glucose-limited growth. This set
of genes is the largest of each of the four groups represented here
(Fig. 1), reflecting the central importance of adaptation to changes in
carbon source availability.
Among the genes that had higher transcript levels, there were 47 known
to be involved in uptake and phosphorylation of glucose (4 genes),
uptake and metabolism of fatty acids and storage carbohydrates (17 genes), glyoxylate cycle and gluconeogenesis (5 genes), uptake and
utilization of alternative carbon sources (7 genes), energy generation
(6 genes), and other areas of carbohydrate metabolism (8 genes). When
also including the associated regulators ADR1, CAT8, SIP2, SIP4, and GAL4,
the alterations to cellular metabolism that allow carbon and energy
scavenging become apparent. Interestingly, there are also 4 genes
(CTA1, GPX1, GTT1, and
TSA2) that were specifically higher with a role in
protecting cells against oxidative stress that is known to arise at
least partially from respiratory metabolism and the oxidation of fatty
acids. For the remaining 104 transcripts, there were 25 of known
function that could not be directly related to carbohydrate metabolism
and 82 of only poorly defined or no known function (Fig.
3).
Using the promoters of these co-regulated genes to look for
over-represented sequences, several potential regulatory elements were
found (Table III). Because this comparison identified genes that were
specifically higher when glucose was limiting when compared with
conditions in which glucose was in excess, it was no surprise to find
the sequences for the glucose-sensitive repressor Mig1p (28) and the
glucose-repressible activator Adr1p (29). Although the element for the
meiosis-specific transcriptional repressor/activator Ume6p was also
found (30), its relatively low coverage of the regulated genes
indicates it is not critical for their coordinated regulation. A
notable omission from this list is the Cat8p/Sip4p binding site or
carbon source-responsive element (5'-CCrTyCrTCCG-3', r is A or
G, and y is C or T (31)). Because only a relatively small number of
genes have been shown to be regulated through this element (26), its
retrieval from among these 162 gene promoters is unlikely. There is a
weak possibility that the element 5'-drCGGCT-3' (d is A, G, or T, and y
is C or T), which was retrieved represents this site.
The largest functional category of the 62 genes that were specifically
lower during glucose-limited growth was those concerned with transport
(10 genes). This included three hexose transporters of low or moderate
affinity (HXT1, HXT3, and HXT4) that
would not be required when external glucose concentrations are low. There were also three genes required for the regulation of glucose repression (MIG2, STD1, and TUP1) as
well as two isogenes of components of central carbon metabolism
(PFK27 and TDH3). Finally, there were 18 transcripts of various (known) functions that were specifically down-regulated during glucose-limited growth. It is of note that this
group contained several genes involved in cell proliferation and
differentiation (TPO1, TPO2, TPO3,
PHD1, BUD9, GIC2, SST2, STE2, STE6, and SUN4). The remaining
31 transcripts had no known or only poorly defined function.
The promoter analysis of these genes revealed the binding site for the
regulatory proteins Med8p and Hxk2p (Table III) that play an important
role in the repression and activation of genes during glucose
(de)repressing conditions (32). Although an abundant sequence in the
genome, its presence is consistent with the observation that the
glucose-limited chemostats were fully respiratory and that Hxk2p plays
a critical role in the control of respiration (23).
The Regulatory Response of S. cerevisiae Specific to
Nitrogen-limited Growth--
From our experiments, there were 51 transcripts whose level was significantly higher in a manner that was
specific for growth limitation by the nitrogen source. Of these, 21 had
no known functions or only poorly defined functions, whereas the
remainder were principally involved in the metabolism of
nitrogen-containing compounds (Fig. 3). Not surprisingly, the gene
encoding the high affinity ammonia permease (and signal transducer),
MEP2, showed a strong increase in expression. It was also
notable that the multicopy suppressor of the differentiation defect in
a mep2 null strain, HMS1, was increased as well
as four other genes involved in cellular differentiation. Of the
structural genes for catabolism of nitrogenous compounds, the complete
set of genes encoding the uptake and utilization of allantoin
(DAL genes), proline (PUT genes), and urea
(DUR genes) were represented as well as two genes encoding
peptidases that are involved in recycling proteins targeted to the
vacuole. Other genes whose enzyme activities yield ammonia (from
serine, cystathionine, or amide bonds) or glutamate (from glutathione)
were also induced as well as GAP1 for the uptake of all
amino acids. The functional relevance of the regulation of the
remaining three genes (FET4, PDC5, and
ALD2) that were specifically higher is somewhat more difficult to explain. For PDC5 and ALD2 it is
possible they have a role in catabolism of the branched chain and
aromatic amino acids via the Ehrlich pathway. Evidence for this role of
PDC5 has been found in our laboratory when cells were grown
with phenylalanine as the sole nitrogen
source.2
In the first 800 nucleotides of the promoters of these co-regulated
genes, we found the core sequence for binding the transcriptional activators Gln3p and Gat1p and repressors Dal80p and Gzf3p (Table III
(33)). These regulators are central to the control of genes under
nitrogen catabolite repression (34, 35), and therefore, it is
understandable that this regulatory system has an important role when
cellular growth is limited by the nitrogen source. Furthermore, it is
interesting to note that the six known genes (MLS1,
PDC5, MF
During nitrogen-limited growth there were also 15 genes whose
expression was significantly lower compared with the other three conditions. On close inspection of expression of these genes across conditions, it was apparent that as well as being significantly lower
during nitrogen-limited growth, all but three (NSR1,
FET3, and HIP1) varied between two other
conditions as well (see Fig. 3, top right panel). This
together with the fact that the group was small and that the promoter
analysis failed to detect an over-represented sequence indicated that
they were subject to more complex regulation than simply repression
under nitrogen limitation. If this were true, their down-regulation was
probably not unique to nitrogen-limited growth but rather the
downstream consequences of the physiological changes that occur when
nitrogen was limiting.
The Regulatory Response of S. cerevisiae Specific to
Phosphorus-limited Growth--
The range of substrates S. cerevisiae can use as a source of phosphate is relatively limited
when compared with carbon or nitrogen. The two principle sources are
inorganic phosphate that can be scavenged and taken up from the growth
medium and glycerophosphoinositol, which is imported then degraded
(36). In support of this, it has been shown previously that when cells
were grown in low phosphate-containing media, many genes required for
the uptake and assimilation of these molecules changed (37).
When we grew cells under phosphate-limiting conditions, we found there
were 62 genes significantly higher and 14 genes significantly lower
than the other three limitations (Fig. 3). Of the genes that were
increased, there were 35 that had no known or only poorly defined
functions, including several genes implicated in phosphate metabolism
by sequence similarity (SPL2 and YPL110c) or
expression pattern (PHM genes). Of the remaining 27, there
were 6 PHO genes that function in the regulation and
utilization of inorganic phosphate from the medium as well as 3 genes
(GIT1, KCS1, and INM1) involved in the
uptake and metabolism of inositol phosphates. Three additional open
reading frames known to be involved in the accumulation of polyphosphates in the vacuole (VTC genes) were present as
well as PLB3, HOR2, PYK2,
PRS4, and DDP1, which are directly involved in
the metabolism of phosphorylated metabolites. Finally there were 10 genes for which there is no simple explanation for their specifically
higher level under hosphate-limiting conditions.
From the promoter analyses, there was a clear over-representation of a
redundant sequence that is inclusive of the binding site for the
transcriptional activator Pho4p (38), which is nuclear-localized in
response to phosphate starvation (Table III; for review, see Ref. 39).
This sequence was also reported in the study of Ogawa et al.
(37), who reported a good correlation between the presence of the
element and induction during growth in low phosphate-containing media.
It is also apparent here that the Pho4p regulator plays an important
role for optimal growth under phosphate limitation.
Phosphate-limited growth also resulted in significantly lower levels in
the expression of 14 genes. Like those that were low under nitrogen
limitation, the small size of this group and the failure of the
promoter analysis to detect over-represented sequences argues for more
complex mechanisms of control than a single regulator. However, unlike
those that were lower during nitrogen-limited growth, the nature of the
repression here was more specific since there were six transcripts
(RPS22B, YBR099c, YBL049w,
YIP3, YLR264c, and IPT1) whose levels
were not significantly changed between any other two conditions.
However, a promoter analysis of the genes in this sub-group revealed no
significant elements, consistent with the earlier conclusion for
nitrogen limitation that the group is not under the specific control of
a single transcriptional regulator.
The Regulatory Response of S. cerevisiae Specific to Sulfur-limited
Growth--
Sulfur is principally used by S. cerevisiae for
the synthesis of the amino acids cysteine and methionine and the
closely related metabolite S-adenosylmethionine. From these
molecules, the important redox properties of sulfur are exploited by
the cell in diverse roles such as cellular defense and detoxification
as well as being a structural component of prosthetic groups,
cofactors, and proteins. Several of these roles are represented among
the 68 yeast genes that were specifically higher during sulfate limitation.
Transcripts encoding proteins for the uptake of sulfate
(SUL2) and sulfur-containing molecules (MMP1,
MUP1, MUP3, SAM3, GNP1, and
HGT1) were specifically higher. Furthermore, genes whose
products are involved in acquiring and generating intermediates and
cofactors for sulfate assimilation (serine: AGP3,
SER33; siroheme: MET1, MET8) were
found along with structural components of the assimilation pathway
(MET3, MET16, MET22, MET10,
MET2, STR3, CYS3, and MHT1) and their transcriptional regulators (MET28 and
MET32). In terms of the wider role of sulfur in the cell,
transcripts related to glutathione metabolism (GTT2) and its
redox cycling (YHR176w and OYE3) were found to be
higher in this condition as well as genes required for iron-sulfur
cluster generation (ATM1 and ARN1). We also found
the pyruvate decarboxylase isozyme PDC6 dramatically induced
in agreement with a recent hypothesis that yeast can remodel its
biomass composition to spare sulfur (40). Finally there were seven
known genes whose increase could not be easily rationalized during
sulfur-limited growth as well as 30 genes of unknown or only poorly
defined function.
Two promoter sequences have been identified as important in the
regulation of genes for sulfate assimilation in S. cerevisiae. These are the binding sites for the Cbf1p-Met4p-Met28p
transcriptional activation complex (41) and that for the Met31p and
Met32p transcription factors (42). Together, these drive the
coordinated expression of the genes encoding components of the sulfur
amino acid biosynthetic pathway. Redundant forms of both of these
elements were found specifically over-represented in the promoters of
the genes expressed at a higher level during sulfur limitation (Table
III).
In contrast with the lower abundance transcripts for nitrogen and
phosphate limitations, the genes specifically lower under sulfur
limitation formed a relatively large group. Of 49 genes, 19 were
unknown whereas the next largest group (4 genes) was that involved in
glycogen metabolism. One open reading frame (SSU1) is
required for the export of (and resistance to) sulfite, an intermediate
in the reduction of sulfate to sulfide for homocysteine synthesis. This
may result in conservation of intracellular sulfite that results in
sulfur sparing. Two other interesting genes (CTR1 and
CTR3) encode copper transporters whose down-regulation could be important to reduce the import of metal ions that are potentially harmful to thiol-compromised cells. Although the remainder of the genes
cover a broad range of functional categories, they appear to be
transcriptionally unified through the presence of stress-responsive elements in their promoters for the binding of transcription factors Msn2p and Msn4p (43).
These two transcription factors are responsible for transcriptional
induction of many genes when cells are exposed to a variety of
stresses, including nutritional (glucose starvation) stress (reviewed
by Estruch (44)). The specific reduction in expression of
stress-responsive element-driven transcripts seen here is difficult to
rationalize since none of the cultures in our comparisons was challenged with stresses that are known to modulate the activity of
these factors. One explanation is that the promoter context is
especially important here. This is tentatively supported by the
presence of unknown elements also found over-represented in the
promoters (Table III).
During macronutrient-limited growth of steady-state cultures of
S. cerevisiae we measured the differential expression of
1881 different genes (31% of the predicted genome). From this group of
transcripts, we studied 484 that were found to have significantly higher or significantly lower abundance in one condition when compared
with the other three conditions tested. The information distilled in
the functions and promoters of these open reading frames offers us
insights into the metabolic responses of the cell that result in more
efficient utilization of the growth limiting nutrient. Furthermore,
because these regulatory events were coupled to highly specific
environmental changes using the controlled environment of the
chemostat, these genes were used to generate a set of molecular
"indicators." It is proposed that this list can be used for the
diagnosis of media deficiencies from fermentations using undefined
media or regulatory anomalies arising during rounds of strain
improvement by metabolic engineering.
One consequence of our specific goal to study these limited regulatory
events is that many interesting phenomena were excluded from the
present work (such as the previously described connection between
phosphorus and sulfur metabolism (45) or carbon and nitrogen metabolism
(46)). These analyses are, however, beyond the scope of this paper and
will, therefore, be investigated in future studies.
Global Transcriptional Regulation to Optimize Assimilation of
Growth-limiting Nutrients--
Almost all nutrients enter the cell via
transport proteins whose capacity and affinity properties vary. When
the growth of a microorganism is limited by the low abundance of a
nutrient in the medium, an increase in high affinity uptake systems is essential to cell survival. This situation is exemplified in S. cerevisiae by the hexose transport family of proteins (encoded by
the HXT genes) that contains as many as 20 members (47). In
our study, when growth was limited by the carbon source (glucose) we
found a gene for high affinity glucose uptake (HXT2) and a reserve transporter (HXT5) elevated, whereas genes encoding
low affinity uptake (HXT1, HXT3, and
HXT4) were reduced relative to conditions of glucose excess.
This was in agreement with previous chemostat studies by Diderich
et al. (48, 49). Furthermore, the genes encoding high
affinity transport proteins of the other nutrients (MEP2,
PHO84/PHO89, and SUL2 for ammonium,
phosphorus, and sulfate, respectively) were all relatively increased in
a manner specific to the appropriate nutrient-limited culture. In the
case of phosphorus limitation, the genes for vacuolar polyphosphate synthesis were also transcribed at a higher level. This agreed with the
hypothesis that polyphosphate synthesis is a mechanism to maintain low
intracellular free phosphate levels to enhance high affinity uptake
(37).
The second strategy that was identified to enhance survival during
nutrient-limited growth is to exploit other possible sources of that
nutrient. Amino acids can serve to supply the cell with carbon,
nitrogen, and sulfur, which was reflected by elevated levels of several
amino acid transporter genes in these three limitations. Furthermore,
strategies to scavenge carbon (in the form of maltose, sucrose, fatty
acids, and carboxylic acids), energy (from lactate and formate),
nitrogen (from allantoin, proline, and urea), phosphorus (from
glycerophosphoinositol), and sulfur (from
S-adenosylmethionine, isethionate, taurine, and glutathione) were reflected in the specific transcript changes for each limitation.
The third regulatory outcome was the elevation of genes whose products
are involved in the mobilization and utilization of storage compounds.
This was principally seen during nitrogen-limited and sulfate-limited
cultivation. Both protein degradation (51) and allantoin utilization
(52) are recognized routes for the mobilization of stored nitrogen,
whereas glutathione catabolism can be used as a source of sulfur for
S. cerevisiae (53). Several genes involved in these pathways
were elevated in a manner specific to their respective limitations.
Finally, biomass remodeling for nutrient sparing has also been proposed
as a mechanism for successful adaptation to nutrient-limiting conditions. Previous reports of the preferential expression of low
sulfur-containing proteins during sulfur-limited growth in other
microbes (54, 55) have recently been complemented by a yeast study that
analyzed the proteome (8). This work proposed a model for the
evolutionary adaptation of proteins to low nutrient levels through
their reduced carbon, nitrogen, or sulfur content following a model
recognized by Pardee (50) for a sulfur-binding protein from
Salmonella typhimurium. More recently still, sulfur conservation in yeast by differential gene expression has been observed
in response to sulfur demand induced by cadmium stress (40). Our
results corroborate these reports because we found a (2-fold)
overrepresentation of genes that code for proteins with low methionine
content (below 1%) among those elevated specifically during sulfur
limitation. This differential regulation was most dramatically
illustrated by the PDC6 transcript that increased some
10-50-fold when compared with the other three conditions. The protein
encoded by this gene has 6 sulfur-containing amino acids, in contrast
to its isozymes Pdc1p and Pdc5p, which contain 17 and 18 sulfur-containing amino acids, respectively. However, in contrast to
the report of Fauchon et al. (40), we only found the
relationship to be true for the methionine content in proteins and not
for cysteine. It is possible that this is a function of the importance
of cysteine to protein stability. This important distinction may only
be detectable in our experiments since we used steady-state
sulfur-limited chemostat cultures and not stress-induced sulfur depletion.
In addition to these regulatory events that reflect adaptation to
nutrient limitations, we observed disproportionately low biomass yields
on glucose of the nitrogen-, phosphorus-, and sulfur-limited cultures
for the rates of respiration. The significantly lower yield per ATP has
previously been explained for nitrogen-limited chemostat cultures by
increased uncoupling between ATP synthesis and biomass production (24).
This interesting observation is under further investigation in our laboratory.
The Activities of Specific Transcriptional Regulons during
Nutrient-limited Growth--
Genome-wide regulation of transcription
in response to environmental change is an essential component of the
rearrangement of metabolic fluxes. However, because of the introduction
of microarray technologies, it is apparent that the functional
relationships of many changed transcripts are difficult if not
impossible to explain. One factor that often contributes to this
ambiguity is experimental design since the changes observed for a given
experimental condition are defined relative to an unavoidably imperfect
reference. Our experiments illustrate this point clearly since for each
experimental culture (carbon-, nitrogen-, phosphorus-, or
sulfur-limited) each of the other three cultures could serve as a
reference condition because they contained each others' nutrients in
excess at a constant residual concentration. The most informative group
of genes for specific regulatory information, therefore, are the 484 genes identified here that were specifically up- or down-regulated in response to change of only a single nutrient.
We found several over-represented sequences in the promoters of the
co-regulated genes; however, the relationship between these putative
transcription factor binding sites and gene regulation was imperfect.
The explanation for this is 2-fold; (i) changes in transcript abundance
can be achieved indirectly through the combined actions of other
regulatory events, and (ii) for most transcription factors, the
sequence(s) of their binding site is poorly described. We have tried to
minimize the occurrence of the former possibility by using multiple
comparisons as described above; however, this can be further improved
by the addition of more reference situations (e.g. trace
element or vitamin growth limitations) and selecting the specifically
regulated gene sets. Improvement on the issue raised by the second
possibility is, however, more complicated, as shown by a study of the
DAL1/DAL4 promoter that showed active promoter
elements were extremely difficult to predictably identify (18). The
promoter analysis data presented here therefore holds its greatest
value in simply identifying over-represented sequences to indicate
which transcription factors are likely to be active. This can be used
to indicate what regulons underlie the observed metabolic adaptations
to nutrient-limited growth. This knowledge can be useful for the
metabolic engineering of global gene regulation.
Transcriptional Information to Identify Nutrient-limited
Growth--
Development of array-based diagnostic approaches requires
clear definitions of the nature of gene changes in response to
environmental conditions. Our experimental approach forms the basis for
this information by studying growth that was limited by each of four nutrients that are assimilated by the cell to form the principle components of biomass. Although each of the 484 genes identified above
is subject to regulation that is specific for one nutrient limitation,
further refinement of this list can provide identifier genes whose
changes are clearly recognizable even in the absence of reference
situations. We therefore searched these genes for specific increases or
decreases whose magnitude was at least 10-fold in comparisons with each
others' nutrient limitation. This resulted in 54 transcripts, of which
48 were specifically higher, and 6 were specifically lower in a single
limitation (Table IV). With the ease of
custom array design and array technologies that hugely reduce the time
from sample to signal, it is conceivable that transcript signals using
these open reading frames can be used in a control loop to ensure
successful fermentations when media composition is unknown.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1) which in steady-state cultures is
equal to the specific growth rate µ (6). This offers the unique
possibility to study metabolism and its regulation at a fixed and
constant specific growth rate under tightly defined nutritional conditions.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. The pH was measured
online and kept constant at 5.0 by the automatic addition of 2 M KOH with the use of an Applikon ADI 1030 bio
controller. Stirrer speed was 800 rpm, and the airflow was 0.5 liters·min
1. Dissolved oxygen tension was measured on
line with an Ingold model 34-100-3002 probe and was above 50%
of air saturation. The off-gas was cooled by a condenser connected to a
cryostat set at 2 °C, and oxygen and carbon dioxide were measured
off line with an ADC 7000 gas analyzer. Steady-state samples were taken after ~10-14 volume changes to avoid strain adaptation due to long
term cultivation (16). Dry weight, metabolite, dissolved oxygen and gas
profiles had to be constant over at least 3 volume changes before
sampling for RNA extraction.
1 to sustain glucose repression at the same
level. For each limitation, the medium contained the following
components (per liter). For carbon-limited, the composition was
5.0 g of (NH4)2SO4, 3.0 g of KH2PO4, 0.5 g of
MgSO4·7H2O, and 7.5 g of glucose. For
nitrogen-limited, the composition was 1.0 g of
(NH4)2SO4, 5.3 g of
K2SO4, 3.0 g of
KH2PO4, 0.5 g of
MgSO4·7H2O, and 59 g of glucose. For
phosphorus-limited, the composition was 5.0 g of
(NH4)2SO4, 1.9 g of
K2SO4, 0.12 g of
KH2PO4, 0.5 g of
MgSO4·7H2O, and 59 g of glucose . For
sulfur-limited, the composition was 4.0 g of NH4Cl,
0.05 g of MgSO4·7H2O, 3.0 g of
KH2PO4, 0.4g of MgCl2, and 42 g of glucose.
20 °C.1 For the
purpose of glucose determination and carbon recovery, culture
supernatants and media were analyzed by high performance liquid
chromatography fitted with an AMINEX HPX-87H ion exchange column using
5 mM H2SO4 as the mobile phase.
Residual glucose in the glucose-limited chemostats was determined
enzymatically using a commercial glucose determination kit from Roche
Molecular Biochemicals. Ammonium concentrations were determined by a
modified method of the Boehringer ureum test. Phosphate and
sulfate were determined with the use of cuvette tests from DRLANGE
(Düsseldorf, Germany). Culture dry weights were determined via
filtration as described by Postma et al. (19).
800 to
50) of each set of co-regulated genes were analyzed
for over-represented hexanucleotides. When hexanucleotide sequences
shared largely common sequences, they were aligned to form longer
conserved elements. All the individual promoter sequences contributing
to these elements were then aligned, and redundant elements were
determined by counting the base representation at each position. The
relative abundance of these redundant elements was then determined from
a new enquiry of the co-regulated gene promoters and the entire set of
yeast promoters in the genome.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Nutrient concentrations and physiological parameters of cultures used
in this study
1, reflecting complete respiratory
catabolism. This is typical of steady-state growth of S. cerevisiae strain CEN.PK113-7D under glucose limitation at
dilution rates below 0.3 h
1 (23). The three
nutrient-limited cultures containing residual glucose exhibited mixed
respiratory (indicated by high qO2) and fermentative glucose catabolism, indicating that excess glucose in the
medium does not fully repress respiration. Similarly high rates of
oxygen consumption in the presence of excess glucose have been noted
previously (24).
Summary of microarray experiment quality parameters for each growth
limitation
View larger version (36K):
[in a new window]
Fig. 1.
Summary of the global transcriptional
responses to growth in nutrient-limited chemostats. The global
transcript profiles from yeast grown under limitation for either
carbon, nitrogen, phosphorus, or sulfur were compared, and the classes
of expression profiles were scored. More than two-thirds of the
predicted genome (69%) were either not measurable or unchanged across
all four conditions. The remaining (significantly changed) genes were
either specifically higher or lower in a single condition (484 transcripts) or subject to more complex regulation (1397 transcripts).
Those genes subject to unique regulation are further subdivided into
the eight possible expression profiles.
View larger version (36K):
[in a new window]
Fig. 2.
Representative regulatory profiles of genes
with specifically higher (black squares) or lower
(gray circles) transcript abundance in a single
growth limitation. The transcript level across the four conditions
for each gene was mean and variance normalized to give an
average of 0 and variance of 1. The data plotted here represent
the average abundance of all transcripts in a class for each growth
limitation. Error bars represent the S.D.
Gene coverage of over-represented sequences retrieved from the
promoters of co-regulated genes
View larger version (83K):
[in a new window]
Fig. 3.
The transcript profiles and identities of the
genes that were specifically up- or down-regulated in each of the four
limitations. The average of three replicate genome-wide transcript
profiles were averaged for each condition and then compared.
Green (relatively low expression) and red
(relatively high expression) squares are used to represent
the transcription profiles of genes deemed specifically changed. The
full data set containing all transcript abundance measurements as well
as those for the eight categories of changes can be found at
www.nutrient-limited.bt.tudelft.nl.
, FUS1, HMS1,
and FET4) whose promoters do not contain this element are
less directly related by function to metabolism involved in scavenging
nitrogen. These data indicate that GATA-mediated regulation is the
principle requirement for the observed regulatory profile.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
List of diagnostic genes that are indicative of growth limited by
either carbon, nitrogen, phosphorus, or sulfur
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ACKNOWLEDGEMENTS |
---|
We thank Arjen van Tuijl for technical assistance, Pascale Daran-Lapujade for the use of data from chemostat cultures and microarrays, and Hans van Dijken for helpful comments on the manuscript.
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FOOTNOTES |
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* This work was financially supported by the Board of the Delft University of Technology (Beloning Excellent Onderzoek program) and the Dutch Ministry of Economic Affairs.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.
¶ To whom correspondence should be addressed. Tel.: 31-15-2782410; Fax: 31-15-2782355; E-mail: m.piper@tnw.tudelft.nl.
Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M209759200
1 M. R. Mashego, W. M. van Gulik, J. L. Vinke, and J. J. Heijnen (2002) Biotechnol. Bioeng., in press.
2 Z. Vuralhan, M. A. Morais, S. L. Tai, M. D. W. Piper, and J. T. Pronk, unpublished data.
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