Physiological and transcriptional responses of...
Transcript of Physiological and transcriptional responses of...
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Physiological and transcriptional responses of Saccharomyces cerevisiae to zinc limitation in 1
chemostat cultures 2
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Raffaele De Nicola1§**
, Lucie A. Hazelwood2,5,§
, Erik A. F. De Hulster2,5
, Michael C. Walsh3, 4
Theo A. Knijnenburg4,5
, Marcel J.T. Reinders4,5
, Graeme M. Walker1, Jack T. Pronk
2,5, Jean-5
Marc Daran2,5
, Pascale Daran-Lapujade2,5*
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§Both authors contributed equally to this work 7
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Running title: Yeast transcriptional response to Zn limitation 9
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1Yeast Research Group, Division of Molecular and Life Sciences, University of Abertay, 11
Dundee, DD1 1HG, Scotland 12
2Department of Biotechnology, Delft University of Technology, Julianalaan 67, 26 28 BC Delft, 13
The Netherlands 14
3Heineken Supply Chain, Research and Innovation, Burgemeester Smeetsweg 1, 23 80 BB 15
Zoeterwoude, The Netherlands 16
4Information and Communication Theory Group, Delft University of Technology, Mekelweg 4, 17
26 28 CD Delft, The Netherlands 18
5Kluyver Centre for Genomics of Industrial Fermentations 19
*Corresponding author: Pascale Daran-Lapujade, Department of Biotechnology, Delft University 20
of Technology, Julianalaan 67, 26 28 BC Delft, The Netherlands. Telephone: +31 15 278 24 10; 21
Fax: +31 15 278 23 55 22
**Current address: DSM Nutritional Products AG, Center of Biotechnology, Basel CH-4002, 23
Switzerland 24
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Manuscript for submission in Applied and Environmental Microbiology 26
Section: Physiology and Biotechnology 27
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Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01445-07 AEM Accepts, published online ahead of print on 12 October 2007
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Abstract 1
Transcriptional responses of Saccharomyces cerevisiae to Zn availability were investigated at a 2
fixed specific growth rate under limiting and abundant Zn concentrations in chemostat culture. 3
To investigate the context-dependency of this transcriptional response and eliminate growth rate-4
dependent variations in transcription, yeast was grown under several chemostat regimes resulting 5
in various carbon (glucose), nitrogen (ammonium), zinc and oxygen supplies. A robust set of 6
genes that responded consistently to Zn limitation was identified and enabled the definition of the 7
Zn-specific Zap1 regulon comprising of 26 genes and characterized by a broader ZRE consensus 8
(MHHAACCBYNMRGGT) than so far described. Most surprising was the Zn-dependent 9
regulation of genes involved in storage carbohydrate metabolism. Their concerted down-10
regulation was physiologically relevant as revealed by a substantial decrease in glycogen and 11
trehalose cellular content under Zn limitation. An unexpectedly large amount of genes were 12
synergistically or antagonistically regulated by oxygen and Zn availability. This combinatorial 13
regulation suggested a more prominent involvement of Zn in mitochondrial biogenesis and 14
function than hitherto identified. 15
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Introduction 1
Zinc is a cofactor of many proteins and is indispensable for their catalytic activity and/or 2
structural stability. Zn is also a ubiquitous component of enzymes involved in transcription and 3
of the Zn finger proteins that regulate gene expression (11). In the yeast Saccharomyces 4
cerevisiae, zinc is estimated to be required for the function of nearly 3% of the proteome (11). 5
Besides its involvement in protein structure and function (73, 53), interaction of zinc with lipids 6
contributes to regulation of membrane fluidity (7) and its interaction with nucleic acids helps to 7
prevent deleterious radical reactions (5). Deficiency of this essential trace element can have 8
severe consequences. For example, in beer fermentation, zinc depletion in wort leads to 9
‘sluggish’ fermentation and thus to deterioration of beer quality (34). While accurate monitoring 10
of the zinc concentration in such industrial fermentations is important, formation of complexes 11
with polyphenols, proteins and other compounds (41) implies that the concentration of zinc per 12
se does not always accurately predict its bioavailability to yeast. 13
Excess zinc is toxic. It can compete with other metal ions for the active sites of enzymes 14
or intracellular transport proteins (26, 37, 57, 54, 65). For this reason, organisms have evolved 15
mechanisms that tightly control intracellular zinc levels. Zinc homeostasis in yeast can be 16
mediated via i) control of zinc uptake, ii) storage of zinc in vacuoles, iii) intracellular binding of 17
zinc by metallothioneins and iv) efflux of zinc from the cells. In S. cerevisiae, various proteins 18
involved in zinc uptake and storage have been identified in the last decade. Zinc uptake across the 19
plasma membrane mainly occurs via the transporters Zrt1p and Zrt2p (83, 84). Fet4p and 20
Pho84p, low-affinity and broad substrate range transporters of heavy metals, can also transport 21
zinc (79). Zinc storage occurs in the vacuole and transport of zinc into this compartment is 22
mediated by Cot1p and Zrc1p (46, 58) while release of zinc from vacuolar storages is mediated 23
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by Zrt3p (51, 52). Msc2p (46) and Yke4p (43) are implicated in transport of Zn into the lumen of 1
the endoplasmic reticulum and perhaps an additional organelle involved in the secretory pathway. 2
The genes encoding these transporters are transcriptionally induced by Zap1p (Zinc Activated 3
Protein) under conditions of zinc limitation or deficiency (85). Contrary to the situation in 4
mammalian cells, no plasma membrane transporter dedicated to zinc export from yeast cells has 5
been identified so far (63). Two cytosolic metallothioneins (Cup1-1p and Cup1-2p) involved in 6
copper chelation can also bind zinc (80). However, the expression of these proteins is not zinc-7
dependent, and involvement in zinc detoxification has not yet been demonstrated (80). 8
In order to better define the Zap1p regulon, Lyons and co-workers analysed the genome-9
wide transcriptional response of a S. cerevisiae Zap1 mutant strain and a control strain to zinc 10
abundance or depletion (50). A combinatorial analysis identified a subset of 46 zinc-responsive 11
genes whose expression was reduced in the Zap1p mutant and that possessed a Zinc-Responsive 12
Element (ZRE, 5’-ACCYYNAAGGT-3’). Among the members of this updated defined Zap1p 13
regulon were the well-characterised plasma membrane, vacuolar and endoplasmic reticulum zinc 14
transporters. However, involvement of many of the proposed Zap1p targets in zinc homeostasis 15
was difficult to interprete and, as suggested by the authors, may be due to contribution of factors 16
other than zinc depletion. Indeed, these experiments were performed in shake flask in which the 17
growth conditions cannot be strictly monitored and maintained at constant level as the pH, the 18
dissolved oxygen and nutrient concentrations change during growth. Furthermore, zinc depletion 19
and ZAP1 deletions are bound to reduce the specific growth rate as compared to zinc sufficient 20
cultures of a wild-type strain. The regulation of gene expression is therefore affected not only by 21
the difference in growth conditions but also by the specific growth rate (66). This variation in 22
gene regulation can obscure the interpretation of the results. 23
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The goal of the present study was to investigate physiological and transcriptional 1
responses of S. cerevisiae to zinc limitation, while minimizing the impact of secondary effects of 2
zinc limitation. To this end, S. cerevisiae was grown at a fixed specific growth rate, oxygen 3
availability, temperature and pH under zinc limitation in chemostat cultures. Comparing the 4
transcriptome of zinc-limited cultures to those of carbon and nitrogen limited cultures identified 5
sets of genes that responded uniquely to zinc limitation. Furthermore, these cultures were grown 6
both in the presence and the complete absence of oxygen, in order to identify genes that are 7
subjected to combinatorial control by oxygen and zinc availability. 8
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Material and methods 10
Yeast strain and maintenance. The haploid prototrophic S. cerevisiae strain CEN.PK 113-7D 11
(MATa) was obtained from Dr. P. Kötter, Frankfurt, Germany. Zinc-depleted cells were obtained 12
by four serial transfers of yeast cells in shake flasks containing synthetic medium (77) from 13
which zinc was omitted, and subsequently mixed with glycerol (final concentration 20%), 14
aliquoted and stored at -80 °C. 15
Minimizing Zn contamination of culture vessels. To minimize zinc contamination, all glassware 16
(including shake flasks for pre-cultivation), tubing and fermenters were subjected to an overnight 17
soak in 2 % nitric acid, followed by two washes with deionised water, one wash with 0.1 M 18
EDTA and four further washes with deionised water. 19
Media for chemostat cultivation. The synthetic medium composition was based on that 20
described by Verduyn (77). The modifications introduced for carbon, nitrogen and zinc limited 21
growth are listed in Table 1. In all chemostats except for those limited by carbon, the residual 22
glucose concentration was targeted to 17 g/l (95 mM) in order to have the same degree of glucose 23
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repression (Table 2). Under anaerobic glucose-limited conditions, the glucose concentration was 1
increased to compensate for a low biomass yield. The decreased sulfate concentration (resulting 2
from the reduced (NH4)2SO4 concentration under nitrogen limitation) was compensated by 3
K2SO4 addition. The zinc replete cultures (carbon and nitrogen-limited) contained excess zinc 4
concentration, but at sub-toxic levels (36). In anaerobic zinc-limited cultures, a minute zinc 5
contamination (probably leaking from the metal fermenter parts) was enough to sustain growth. 6
Conversely aerobic zinc-limited cultures could not grow at a dilution rate of 0.10 h-1
without the 7
addition of zinc as 0.05 µM zinc sulfate. For anaerobic cultivations, the reservoir medium was 8
supplemented with the anaerobic growth factors Tween-80 and ergosterol (76). 9
Chemostat cultivation. Zinc-depleted pre-cultures were obtained by inoculating shake flasks that 10
contained 100 ml zinc-free synthetic medium with zinc-depleted cells (obtained as described 11
above). After overnight cultivation, these zinc depleted precultures were inoculated in 2-liter 12
fermenters (Applikon) with a working volume of 1 l (74). Chemostat cultures were fed with 13
synthetic medium (as described in the previous section) that limited growth by carbon, nitrogen 14
or zinc with all other growth requirements in excess and at constant residual concentration (9). 15
The dilution rate was set at 0.10 h-1
. Cultures were assumed to be in steady-state when, after at 16
least five volume changes, culture dry-weight, glucose concentration, carbon-dioxide production 17
rate and oxygen consumption rate varied by less than 2 % during one additional volume change 18
(21). Steady-state samples were taken after 10 generations at the latest to avoid strain adaptation 19
due to long-term cultivation (35). Each cultivation condition was performed in triplicate. 20
The pH was measured on-line and kept constant at 5.0 by the automatic addition of 2 M 21
KOH using an Applikon ADI 1030 Biocontroller. The stirrer speed was set at 800 rpm. 22
Anaerobic conditions were maintained by sparging the medium reservoir (0.05 liter.min-1
) and 23
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the fermenter (0.5 liter.min-1
) with pure nitrogen gas. Norprene tubing and butyl rubber septa 1
were used to minimize oxygen diffusion into the anaerobic cultures (78). The off-gas was cooled 2
by a condenser connected to a cryostat set at 2 °C. Oxygen and carbon dioxide were measured 3
off-line with an NGA 2000 Rosemont gas analyzer. 4
Analytical methods. Culture supernatants were obtained after centrifugation of samples from the 5
chemostats. For the purpose of glucose determination and carbon recovery, culture supernatant 6
and media were analyzed by high performance liquid chromatography (HPLC) on an AMINEX 7
HPX-87H ion exchange column using 5 mM H2SO4 as the mobile phase. Culture dry weights 8
were determined via filtration as described by Postma et al. (64). Trehalose and glycogen 9
measurements were adapted according to François et al .(22). Trehalose was determined in 10
triplicate measurements for each chemostat. Glycogen was determined in duplicate for each 11
chemostat. Glucose was determined using the UV-method based on Roche kit no. 0716251. 12
Microarray analysis. Sampling of cells from chemostats and total RNA extraction was 13
performed as previously described (1). Probe preparation and hybridization to Affymetrix 14
Genechip® microarrays were performed following Affymetrix instructions. The one-cycle 15
eukaryotic target labelling assay was used, starting with 15 µg of total RNA. The quality of total 16
RNA, cDNA, cRNA and fragmented cRNA were checked using the Agilent Bioanalyzer 2100 17
(Agilent Technologies). Results for each growth condition were derived from three independent 18
culture replicates. 19
Transcriptomics data acquisition and statistical analysis. Acquisition and quantification of array 20
images and data filtering were performed using Affymetrix GeneChip® Operating Software 21
version 1.2. Before comparison, all arrays were globally scaled to a target value of 150 using the 22
average signal from all gene features using GeneChip® Operating Software (GCOS), version 1.2. 23
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To eliminate insignificant variations, genes with expression values below 12 were set to 12 as 1
previously described (9). 2
To detect genes that exhibited differential expression in at least one of the experimental 3
conditions, an in-house version of SAM (Significance Analysis of Microarrays) (72) was 4
employed using the multiclass setting. Genes with a Q-value below the median FDR (false 5
discovery rate) of 1.5·10-4
were considered differentially expressed. 6
Transcript data can be downloaded from GEO under the following series accession 7
numbers: zinc-limited chemostats GSE8035; carbon-limited chemostats GSE8088 and GSE5326; 8
nitrogen-limited chemostats GSE8089. 9
Grouping of genes into modules. The continuous expression levels of all (1500) differentially 10
expressed genes were discretized, as described in Knijnenburg et al. (39). Resultantly, each gene 11
is represented by a discretized expression pattern of length six, indicating whether the gene is not 12
differentially expressed (0), up-regulated (1) or down-regulated (-1) under each of the six 13
cultivation conditions. For example, a gene that has the following discretized expression pattern: 14
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C-Ana N-Ana Zn-Ana C-Aer N-Aer Zn-Aer
0 1 0 0 0 -1
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is up-regulated when grown anaerobically under a nitrogen limitation (N-Ana) and down-17
regulated when grown aerobically under a zinc limitation (Zn-Aer), while the four other 18
conditions do not exhibit differential expression. Genes are grouped into modules based on this 19
discretized representation by imposing certain constraints on the discretized expression pattern of 20
a gene in order for it to be part of a particular module. For example, a module could be formed by 21
grouping all genes that have a higher discretized expression level under the zinc limitation, when 22
compared to the other two limitations, both for aerobic and anaerobic growth. This approach 23
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provides a coherent and meaningful way to create modules of genes, since the expression 1
behaviour of the genes in a module is directly related to the cultivation conditions, allowing for a 2
straightforward interpretation. In our study, six modules were created. The exact constraints on 3
the discretized expression pattern of a gene to be included in one of the six modules are found in 4
the Appendix. Table 3 gives a short verbal description for each of the modules. 5
Hypergeometric tests. The six modules were consulted for enrichment in functional annotation 6
and significant transcription factor (TF) binding. To test for significant relations the 7
hypergeometric test was employed. In the case of the TF binding data, the largest available TF 8
binding dataset for yeast in its most conservative setting (highest binding confidence) was used 9
(29). This dataset, which originally indicates the number of binding sites for each of 102 TFs in 10
the promoter region of each gene, was binarized, such that the data indicates whether a TF can 11
bind a gene (upstream) or not. Then, the hypergeometric test assesses if a TF (or a TF pair) can 12
bind the promoter region of the genes in a module much more frequently than in a randomly 13
selected set of genes. In case of the employed gene annotation information (MIPS (56) and 14
KEGG (38)) it assesses if the number of genes in a module that belongs to a particular functional 15
category is much larger than would be expected by chance. The P-value cut-off to decide whether 16
a relation is significant is P ≤ 1/(ncnx), where nc is the number of modules and nx is the number of 17
TFs (or TF pairs) or the number of MIPS or KEGG annotation categories. This adjustment for 18
multiple testing, corresponds with a per comparison error rate (PCER) of one (24). 19
Motif discovery. The promoters (from -800 to -1) of the genes in each module were analyzed for 20
over-represented regulatory motifs using the web-based software MEME (2). The p-value cut-off 21
to consider a motif significant was 10-4
. Other parameter settings included a motif width from 6 22
to 15 nucleotides, that could be repeated any number of times. 23
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Comparison with the transcriptome study from Lyons and co-workers. The data from the Lyons 1
and co-workers (50) were downloaded from http://genome-www.stanford.edu/zinc/rawdata.html. 2
As this website only provides raw data, the array data were processed following the instructions 3
described in their publication and 496 genes that were up-regulated in response to zinc depletion 4
were thus isolated. The slightly larger size of this gene set compared to the one isolated by Lyons 5
et al. (458 genes) probably results from a few differences in data handling. 6
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Results 8
Establishing Zn-limited chemostat cultures of S. cerevisiae 9
While macronutrient limitation in chemostats can be achieved in a straightforward 10
manner, establishing micronutrient limitation still presents an experimental challenge. This holds 11
especially for metals (Zn, Fe, Cu) that are present in laboratory equipment and that can sustain 12
growth at extremely low concentrations (typically in the µM range). Despite thorough and 13
repeated washing steps and use of high-grade medium components, we did not achieve 14
completely Zn-free cultivation conditions, presumably due to Zn leakage from the metal parts 15
(fermenter lid, pipes and connections). This contamination was sufficient to allow for anaerobic 16
Zn-limited growth at a steady-state biomass concentration of 2.5 g.L-1
. However, 0.05 µM ZnSO4 17
had to be added to the Zn-deficient medium to enable aerobic Zn-limited growth (steady-state 18
biomass concentration 4.2 g.L-1
). Addition of 15 µM Zn to anaerobic and aerobic Zn-limited 19
cultures resulted in a large increase of the biomass concentration, thus confirming that growth 20
was solely limited by Zn availability (data not shown). The Zn content of biomass from Zn-21
limited cultures was up to five-fold lower than that of carbon- and nitrogen-limited cultures 22
(Table 2). Consistent with a higher Zn requirement for aerobic cultivation, the Zn content of 23
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biomass from aerobic Zn-limited cultures was two-fold higher than that of anaerobic Zn-limited 1
cultures (Table 2). Since genes encoding Zn transporters were not differentially transcribed in the 2
presence and absence of oxygen, this difference is unlikely to be due to a different affinity for Zn 3
uptake. 4
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Physiology of Zn, glucose- and ammonia-limited chemostat cultures 6
Zn-limited cultures were grown at a high residual glucose concentration. Comparison of 7
their physiology and transcriptome with those of glucose-limited cultures will therefore also 8
identify changes caused by the different glucose concentrations in the cultures. Therefore, 9
nitrogen-limited cultures, grown at the same residual glucose concentration as the Zn-limited 10
cultures, were included as an additional reference situation. The combination of three nutrient 11
limitations under aerobic and anaerobic conditions resulted in six unique physiological situations 12
(Table 2). 13
Only in the carbon-limited aerobic cultures, a completely respiratory sugar metabolism 14
was observed, resulting in a high biomass yield on glucose (Table 2). In the anaerobic cultures, 15
glucose metabolism was fully fermentative, the main products of glucose dissimilation being 16
ethanol and carbon dioxide. Finally, in glucose-sufficient (i.e. N- or Zn-limited) aerobic cultures, 17
a mixed respiro-fermentative metabolism was observed. The Zn-limited cultures strongly 18
resembled the nitrogen-limited cultures with respect to biomass yields and rates of product 19
formation. Even under anaerobic conditions, the biomass yield on glucose of these glucose 20
sufficient cultures was lower than that of glucose-limited cultures, indicating a partial uncoupling 21
of dissimilation and biomass formation under these ‘energy excess’ conditions. The only notable 22
difference was a slightly higher specific rate of acetate and glycerol production in the Zn-limited 23
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cultures, which may be related to a reduced in vivo activity of Zn-dependent alcohol 1
dehydrogenases. 2
3
Overall transcriptional responses to Zn limitation 4
For all six culture conditions described above, microarray analysis was performed on 5
three independent replicate cultures. Statistical analysis (see Material and Methods section) 6
identified 1500 genes that were differentially transcribed in at least one cultivation condition. 381 7
of these genes responded specifically to Zn-limited growth. Of these Zn-responsive genes, 81 8
proteins do not yet have an assigned cellular function. The 381 Zn-responsive genes were 9
subjected to a further analysis to identify combinatorial effects of Zn and oxygen availability 10
(Table 3). A majority of the genes that showed a transcriptional response to Zn-limitation (248 11
genes, Modules 3-6 in Table 3) did so in an oxygen-dependent manner. The remainder (133 12
genes, Modules 1-2 in Table 3) of the Zn-responsive genes showed a consistent response to Zn 13
limitation that was independent of oxygen availability. The identity and transcript levels of the 14
genes contained in the six modules are available in Supplementary Material 1. Below, we will 15
analyse these sets of Zn-responsive genes for over-representation of genes involved in specific 16
functional categories and/or controlled by specific transcription factors (see Material and 17
Methods section). 18
19
Zinc homeostasis and the Zap1p regulon 20
The MIPS functional category ‘heavy metal transport’ was over-represented among the 93 21
genes that were transcriptionally up-regulated in response to Zn limitation irrespective of oxygen 22
availability (Table 3, Module 1). Of the seven genes belonging to this category found in Module 23
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1, six are directly involved in Zn homeostasis. ZRT1, encoding the plasma-membrane high-1
affinity Zn transporter, was strongly induced (average fold-change of 13, Table 4). Transcript 2
levels of ZRT2, ZRT3, ZRC1 and ZRG17, involved in Zn transport and homeostasis, were also 3
increased but to a lesser extent than those of ZRT1 (fold-changes ranging from two to seven). 4
FET4 (up-regulated 3 to 43 fold under Zn limitation) encodes a protein involved in iron transport 5
that has been demonstrated to also be a physiologically relevant Zn carrier (79). The comparison 6
of aerobic and anaerobic cultures confirmed the previously described combinatorial regulation of 7
FET4 by Zn and oxygen availability (79). In addition, a clear hierarchy was observed: while 8
FET4 was strongly regulated by oxygen availability under Zn sufficient conditions (79), its 9
transcript level in Zn-limited cultures was consistently high regardless of oxygen supply (Fig. 2). 10
The transcriptional regulation of these six genes was in agreement with previous studies (30, 50), 11
and so was the up-regulation of ZAP1, the transcriptional activator of these six transporters (8 to 12
20 fold increase relative to Zn-sufficient cultures). FRE1, which also belongs to the ‘heavy metal 13
transport’ category, encodes a protein specifically involved in ferric iron transport (25, 82). FRE1 14
does not contain a ZRE and its increased transcript levels under Zn-limited conditions suggest an 15
indirect effect. 16
Previous reports have investigated the role of MSC2 in Zn transport into the endoplasmic 17
reticulum (19, 46) and have found that mutations in the latter affect the cellular distribution of 18
zinc (46). In our study, MSC2 was not found among the genes that were transcriptionally induced 19
under Zn limitation. Instead, its transcript levels remained low under the conditions tested. 20
Consistent with this observation, transcription of MSC2 was not affected in a zap1 mutant (50). 21
ZRG17 encodes a protein that has been proposed to act as a complex with Msc2p (18, 46). The 22
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promoter of ZRG17 does contain a ZRE and its transcript levels were increased in Zn-limited 1
cultures, suggesting that this protein could be the regulatory sub-unit of the complex. 2
In an attempt to further define the Zap1p regulon, the promoter regions of the 93 genes 3
that showed a robust, oxygen-independent response to Zn limitation (Module 1, Table 3) were 4
searched for over-represented motifs. The web-based software MEME (2), which enables 5
unbiased probability-based motif discovery, identified 26 genes with a 15-nucleotide motif that 6
strongly resembled the previously published ZRE Zap1p-binding consensus sequence (Figure 1, 7
Table 4). In agreement with previous reports on Zap1p regulation, all six Zn transporters in 8
Module 1, as well as ZAP1 itself, harboured this element. Twelve additional genes (Table 4) have 9
been previously proven or proposed to be Zap1p targets. An additional 7 genes that harboured the 10
15-nucleotide motif had not previously been implicated as Zap1p targets (50) (Table 4). The 11
detailed ZRE sequences and positions are listed in Supplementary Material 2. 12
13
Comparison with previous Zn-related transcriptome studies 14
Two previous transcriptome studies investigated yeast adaptation to Zn depletion in batch 15
cultures of an industrial (30) and a laboratory strain of S. cerevisiae (50). Using maltose-grown 16
cultures, Higgins et al. observed a down-regulation of maltose-permease and maltase genes 17
(MAL12, MAL32 and MAL31) in Zn-depleted cultures. In the present study, growth on glucose 18
resulted in the absence of MAL gene transcripts, thus masking transcriptional responses of these 19
genes to Zn availability. Lyons et al. identified a Zap1p regulon consisting of 46 genes by 20
comparing the transcriptional responses to Zn depletion of a zap1∆ mutant and its parental strain. 21
Three of these 46 genes (COS2, COS4 and COS6) were not represented on the microarrays used 22
in our study. Of the remaining 43 genes, 25 showed increased transcript levels in Zn-limited 23
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chemostat cultures (Fig. 2A). The large majority of these (21 genes) were consistently induced in 1
response to Zn limitation irrespective of oxygen availability (Module 1, Table 3; Fig. 2A). 2
MEME failed to identify a ZRE sequence in 3 of these 21 genes (RAD27, YJL132W and 3
YOL131W), which are therefore absent from Table 4. Four genes from the Zap1p regulon 4
defined by Lyons and co-workers (IZH1, IZH2, NRG2 and PST1) were found in Module 3 (Table 5
3), indicating that their transcription was induced under Zn limitation but only when oxygen was 6
absent. Their identification by Lyons et al. may have been caused by the poor oxygen transfer 7
characteristics of shake flask cultures (68, 28, 55). Two additional genes (ADE17 and GPG1) 8
identified as Zap1p-targets by Lyons et al. were up-regulated in Zn-limited chemostat cultures, 9
however their expression resulted from an intricate regulation by Zn, glucose and oxygen 10
availability. Both genes responded to zinc-limitation under aerobic and anaerobic conditions. 11
However, they also responded to limiting glucose supply but this response was oxygen specific; 12
while ADE17 was up-regulated under glucose-limitation in the presence of oxygen, GPG1 13
expression increased under glucose limited anaerobic growth. The remaining 16 of the 43 genes 14
identified as Zap1p targets by Lyons et al. and included on our microarrays did not respond to Zn 15
availability in our chemostat study. 16
Eight potential Zap1p-targets identified in the present study (Table 4) were not found in 17
the study of Lyons et al.. However, of these 8 genes, HOR2 and TEX1 were found to be 18
transcriptionally induced by Zn depletion in their study. Furthermore, Zap1p was shown to bind 19
TEX1 on ChIP on chip experiments (29). Seven genes (HOR2, FLO11, KTR6, KTI12, VTC3, 20
MUP1 and YER130C; Table 4) are here for the first time proposed to be Zap1p targets. HOR2 21
encodes a glycerol-3-phosphate phosphatase involved in glycerol biosynthesis (62), which may 22
account for the slightly, but significantly (Student’s t-Test, p-value < 0.05) elevated glycerol 23
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production observed under zinc-limited growth. VTC3 encodes a vacuolar transport chaperone 1
involved in inorganic ion transport (13). Although it has been shown to be involved in 2
polyphosphate transport, it may also participate in vacuolar Zn transport (61). Alternatively, Zn 3
may be involved in polyphosphate accumulation or react with polyphosphates. Like the 4
previously identified Zap1p target MNT2 (50) (Table 4), KTR6 encodes a mannosyl transferase 5
involved in glycosylation of cell wall proteins (49). It can be speculated that they play a role in 6
mannosylation of Zn-scavenging cell wall proteins. For instance ZPS1, a Zap1p target also up-7
regulated under Zn limitation (Table 4), encodes a cell wall mannoprotein with high similarity to 8
Zn metalloproteinases from filamentous fungi (44, 50). The yeast cell wall, and more specifically 9
mannoproteins, has been shown to fix a substantial fraction of the cellular zinc (15). Zinc fixation 10
by mannoproteins may represent an efficient mechanism to scavenge low zinc concentrations 11
(59). The up-regulation of mannoproteins such as ZPS1 under zinc limitation would support this 12
zinc scavenging function of the cell wall. The consistent up-regulation of FLO11, KTI12, MUP1 13
and YER130C in Zn-limited cultures and the presence of a ZRE-like motif in their promoters 14
suggest that the encoded proteins have some as yet unknown role under Zn-limited conditions, 15
too. For example, Flo11p is known to play an essential role in biofilm formation, filamentation 16
and invasive growth (47). In addition, studies on Candida albicans have demonstrated that 17
dimorphic switching from budding growth to mycelium formation is regulated by zinc (69, 4). 18
However, in the present study, we did not observe any difference in morphology between the 19
different culture conditions. 20
When the 289 genes in Modules 1, 3 and 5 that were induced under Zn limitation in 21
chemostat cultures, either in an oxygen-dependent or in an oxygen-independent manner, were 22
compared to the 493 genes that were induced upon Zn depletion in shake flasks (50), 73 genes 23
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overlapped between the two studies. These were for the most part clustered in Modules 1 and 3 1
(Fig. 2B, Supplementary Material 3). Only a small overlap was observed with Module 5 2
(representing only 8% of the genes in this module), which includes genes that are only induced 3
by Zn limitation under aerobic conditions. As mentioned above, this small overlap may reflect a 4
limiting oxygen supply in the shake flask studies. 5
6
Transcriptional regulation of structural genes for zinc-dependent proteins 7
S. cerevisiae contains multiple alcohol dehydrogenases. While the enzymes encoded by 8
ADH1, 2, 3 and 5 all require Zn as a cofactor, Adh4p uses Mg. ADH4 has been shown to be 9
regulated by Zap1p, while expression of the Zn-requiring isoenzymes has been reported to be 10
decreased upon Zn depletion (presumably via Rap1p) (8). In agreement with earlier findings, 11
ADH4 was strongly up-regulated in response to Zn limitation irrespective of the aeration 12
conditions. Transcript levels of other, Zn-dependent alcohol dehydrogenase genes were either 13
unchanged or reduced. In addition to alcohol dehydrogenases, many other yeast proteins use Zn 14
as structural component or cofactor. Regalla and Lyons (54, 65) separated the Zn dependent 15
protein in two distinct classes, i) the proteins that use zinc in a catalytic capacity (105 genes) and 16
ii) the proteins with a structural Zn binding domain (360 genes). Of 105 S. cerevisiae proteins 17
that use Zn as a cofactor (54, 65), none of the structural genes were found to be transcriptionally 18
regulated in response to Zn availability in chemostat cultures (with the clear exception of alcohol 19
dehydrogenases). On the other hand, out of the 360 S. cerevisiae proteins that contain a structural 20
Zn binding domain, 16 genes were up-regulated in response to Zn limitation (Modules 1, 2 and 5) 21
while 7 were down-regulated (Modules 2, 4 and 6). Most of these Zn-responsive genes encoded 22
proteins that have a function in nucleic acid binding (transcription factors, chromatin 23
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reorganizing activity, mRNA binding). The two homologous transcription factors Met31p and 1
Met32p that induce the expression of genes involved in methionine biosynthesis were only 2
affected by Zn availability in the presence of oxygen. While MET32 expression increased two-3
fold, MET31 expression decreased two-fold. These changes in gene expression probably resulted 4
in modifications of the transcriptional regulation of these transcriptional activators as their target 5
genes displayed a slightly higher expression under conditions of aerobic Zn limitation. This 6
antagonistic regulation of MET31 and MET32 remains difficult to relate to Zn supply as both 7
proteins contain two Zn finger domains and do not have a different Zn content. 8
In agreement with previous reports (81), SOD1, which encodes the cytosolic Zn-Cu 9
superoxide dismutase, showed a two fold reduction of its transcript level under conditions of low 10
Zn supply. However, SOD2, which encodes mitochondrial manganese-containing superoxide 11
dismutase, did not show an increased transcript level in Zn-limited cultures. In fact, SOD2, which 12
was only transcribed in aerobic cultures, was also down-regulated by ca. two-fold under Zn 13
limitation. As proposed previously (81), reduced expression of superoxide dismutase may affect 14
resistance to oxidative stress. A more direct involvement of zinc in oxidative stress resistance was 15
previously suggested via the transcriptional regulation of TSA1, encoding a Zn-dependent 16
peroxiredoxin, by Zap1p (81). Unfortunately, in our experiments TSA1 expression was 17
independent of zinc and oxygen availability. This difference with earlier work may be attributed 18
either to the difference between complete Zn depletion (81) and Zn-limited growth (this study) or 19
to a different strain background. However, close scrutiny of the transcript levels revealed no 20
oxidative stress response (AAD3, AAD6, AAD10, AAD14, AAD15, ATR1, CCP1, GTT2, GRE2, 21
LYS20, OYE2, OYE3, TRR1, TRX2, YDR453C, YLR460C, YNL134C, YMR318C and 22
YML131W) (40). Although the transcript levels of both SOD1 and SOD2 were reduced, their 23
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levels (748 and 295 respectively under aerobic zinc-limitation) may still be high enough to enable 1
efficient processing of ROS and thereby to prevent oxidative stress. 2
Finally, while we cannot exclude the possibility that Zn sparing and/or mobilisation mechanisms 3
occurs at (a) post-transcriptional level(s), these results indicate that a general ‘Zn sparing’ 4
regulation at the transcriptional level is most probably absent in S. cerevisiae. The exceptions of 5
alcohol dehydrogenase and superoxide dismutase may be related to the relative abundance of 6
these proteins and their pivotal role in fermentative and respiratory metabolism, respectively. 7
8
Combinatorial response of mitochondrial function to oxygen and zinc availability. 9
Aerobic Zn-limitation of S. cerevisiae resulted in the up-regulation of 119 genes and the 10
down-regulation of 16 genes (Table 3, Modules 5 and 6). However, hypergeometric distribution 11
analysis did not reveal clear trends in the identity and function of these oxygen-responsive 12
proteins. In order to better investigate the potential synergetic effects between oxygen and zinc 13
availability, different discretized patterns were considered. As described in Fig. 4 for the 14
aerobically up-regulated genes, the applied constraints selected genes for which the expression 15
under carbon limitation was unaffected by oxygen, the expression under nitrogen limitation was 16
also oxygen-insensitive, but for which the response to zinc limitation was oxygen-dependent. 196 17
genes respecting these constraints were identified, 130 being up-regulated in the presence of 18
oxygen in a Zn-dependent manner and 66 down-regulated (given in Supplementary Material 4). 19
Fisher’s exact statistics was then applied to search for over-representation of genes involved in 20
specific functional categories and/or controlled by specific transcription factors. While no 21
enrichment was found within the genes that were down-regulated, the module containing the up-22
regulated showed interesting trends. This module was characterized by enrichment for two 23
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functional categories: ‘respiration’ (10 genes) and ‘mitochondrial biogenesis’ (14 genes). The 1
category of ‘respiration’ comprised genes encoding various subunits of the Fo (ATP4, ATP14, 2
ATP18 and ATP20) and F1 (ATP3 and ATP15) domains of mitochondrial ATP synthase (16) but 3
also COX23, COX14, MAM33 and MBA1 involved in the assembly of respiratory complexes in 4
mitochondria (3, 27, 60, 67). The relation between Zn availability and these proteins remains 5
unclear, although cytochrome c oxydase activity has been shown to be inhibited by Zn. Most of 6
the genes in the ‘mitochondrial biogenesis’ category encoded mitochondrial ribosomal proteins 7
(MRPL10, MRPL11, MRPL37, MNP1, RSM19 and MRPS16), but also MSS116, a gene involved 8
in the splicing of mitochondrial group I and II introns (33). Finally, also TIM10/MRS11 9
responded synergistically to Zn and oxygen availability. TIM10 encodes a protein involved in the 10
translocation of mitochondrial proteins from the cytoplasm to the mitochondria. For instance 11
Aac1p and Aac2p, encoding ADP/ATP mitochondrial carrier cannot be translocated in a tim10 12
mutant (75). This translocation process, also identified in plant (6), requires Zn (48). The present 13
study reveals a more important role for Zn in mitochondrial function and biogenesis than so far 14
assumed. Although still not clearly understood this role could, at least in part, explain the higher 15
Zn requirement for cells grown in the presence of oxygen, condition where mitochondria are 16
essential for respiration. 17
18
Zn limitation and storage carbohydrate metabolism 19
Genes from both glycogen biosynthesis (GSY2, GAC1, GLC3) and degradation pathways 20
(GDB1, GPH1) were down-regulated by up to 22-fold in Zn-limited chemostat cultures, 21
regardless of oxygen availability (Fig. 3A). Several additional genes involved in glycogen 22
metabolism that did not pass the very stringent statistical test used in genome-wide analysis, 23
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displayed a decreased expression upon closer inspection (Fig. 3A). To investigate whether these 1
transcriptional modifications resulted in phenotypic differences, glycogen contents were analysed 2
in the chemostat cultures on which the transcriptome analyses had been performed (Fig. 3B). 3
Indeed, glycogen accumulation was strongly (10 to 20-fold) reduced in Zn-limited cultures. 4
Genes involved in glycogen metabolism are known to be transcriptionally regulated in response 5
to a wide variety of environmental conditions and signalling pathways (20) (temperature, nutrient 6
supply, oxidative stress). This regulation is mediated by the general environmental stress 7
response (ESR) and HOG pathways (22, 23). However, no other target genes of these signalling 8
pathways were found to be differentially transcribed in response to Zn limitation. This suggests 9
that the regulation of glycogen metabolism by Zn occurs via another, hitherto unknown signal 10
transduction mechanism. 11
Several genes involved in trehalose metabolism were also significantly down-regulated in 12
Zn limited cultures (PGM1, PGM2, TPS1, TPS2 and TPS3, see Fig. 3A). These down-regulations 13
coincided with substantially lower trehalose biomass contents, an effect that was most 14
pronounced in aerobic cultures (Fig. 3B). These results clearly demonstrated, for the first time the 15
impact of Zn availability on reserve carbohydrate accumulation. As the genes involved in 16
glycogen and trehalose metabolism do not contain ZREs, their transcriptional regulation is 17
unlikely to be directly mediated by Zap1p. In addition, their down-regulation probably occurs via 18
a STRE-independent mechanism (we did not find over-represented STRE in the promoters of 19
down-regulated genes). Among the above-mentioned Zap1p regulon, YER130C, encoding a 20
protein of unknown function containing two tandem Zn-finger domains, was up-regulated under 21
zinc limitation. This putative transcription factor may be involved in a Zap1p-dependent 22
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regulation of genes involved in trehalose and glycogen and is an interesting candidate for further 1
functional analysis. 2
3
Discussion 4
Analysis of Zn limitation in chemostat cultures 5
The unique option of chemostat cultures to control specific growth rate prevented 6
occurrence of specific-growth-rate-related responses. For example, in a previous study in batch 7
cultures of S. cerevisiae (30), the observed down-regulation of ribosomal proteins in low-Zn 8
cultures is likely to have been caused by a decrease in specific growth rate rather than directly by 9
Zn depletion. 10
The use of different aeration regimes showed that yeast responses to Zn limitation are 11
strongly context dependent. This notwithstanding, a set of genes was identified whose specific 12
transcriptional regulation by Zn availability was independent of the oxygen supply. This enabled 13
us to propose a more precise definition of the Zap1p regulon. Most of these 26 potential Zap1p 14
targets overlapped with those proposed in a previous batch-cultivation study (50). The present 15
study demonstrated that responses of several of the previously identified putative Zap1p targets 16
were not Zn-specific. Instead, they were synergistically or antagonistically regulated by carbon, 17
nitrogen and/or oxygen supply. 18
As compared to the transcriptional responses observed in chemostat cultures under other 19
nutrient limitations (9, 10, 14, 70), transcriptional responses to Zn limitation were strikingly 20
pleiotropic. Genes involved in a large variety of cellular functions, apparently unrelated to Zn 21
availability, showed marked differences to Zn limitation. Statistical analysis of co-regulated 22
genes identified only a very limited number of over-represented functional categories or DNA 23
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binding proteins, with the clear exception of the Zap1p regulon. These observations suggest that 1
the only direct effect of Zn limitation on transcriptional regulation is mediated by Zap1p. 2
Although no concerted transcriptional regulation was observed for genes encoding proteins that 3
contain Zn as a catalytic or structural component, Zn availability is likely to influence the in vivo 4
activity of such proteins, many of which are transcription factors. The apparently ‘scattered’ 5
transcriptional responses to Zn limitation may further be due to the fact that new roles of Zn in 6
yeast physiology continue to be discovered. For instance, the involvement of Zn in protein 7
translocation by the Tim10p/Tim9p complex has only been recently revealed (48). 8
9
Effects of Zn limitation on storage carbohydrate accumulation: a possible cause for stuck 10
fermentations in beer fermentation? 11
Zn used by yeast during the beer fermentation process comes from barley malt and is 12
extracted during the mashing procedure (starch conversion and extraction). However, Zn content 13
varies largely between fermentations as its concentration is dependent on the crop quality (32) 14
and is partly removed from wort during lautering (41) or wort separation. Insufficient Zn supply 15
during brewing results in ‘sluggish’ fermentations characterized by a slow fermentation rate (12). 16
The metabolic and/or regulatory processes in yeast cells that underlie such retarded fermentations 17
are incompletely understood. Yeast crops are commonly re-used four to ten times for inoculating 18
succeeding brews and are generally stored around 2°C under starvation (54). Under such 19
conditions, high reserve carbohydrates contents have been shown to be critical for the survival 20
and recovery of metabolic activity of yeast (54). To our knowledge, no published study has 21
investigated how storage carbohydrate metabolism might be affected by Zn deficiency. This 22
present study demonstrates for the first time that Zn limitation causes a strong transcriptional 23
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down-regulation of genes involved in reserve carbohydrate accumulation. The physiological 1
relevance of this response was verified by analysis of intracellular glycogen and trehalose 2
contents, which were strongly reduced in Zn limited cultures. Comparative studies with nitrogen-3
limited cultures showed that the decreased accumulation of storage carbohydrates was specific 4
for Zn limitation and not merely a consequence of glucose-excess conditions. Furthermore, this 5
effect was independent of the aeration of the cultures and the expression profiles of several genes 6
involved in reserve carbohydrate metabolism perfectly matched the profile of trehalose and 7
glycogen accumulation (Figure 3B). 8
While our hypothesis remains to be tested under brewing conditions and with brewing 9
strains of S. cerevisiae, it seems highly probable that the fermentation performance of Zn-limited 10
brewers’ yeast will be strongly compromised. Additionally, follow-up research should focus on 11
the molecular mechanisms that link reserve carbohydrate metabolism and Zn availability. 12
13
Potential implication of Zn-limitation for flavour formation 14
Another consequence of limiting Zn supply during the course of beer fermentation might 15
be related to flavour formation. Indeed, three genes involved in the biosynthesis of the branched-16
chain amino acids leucine, valine and isoleucine (ILV2, ILV3 and BAT2) were consistently down-17
regulated under Zn-limited growth, both in the presence and in the absence of oxygen (Table 3). 18
The flux through the branched chain amino acids synthetic pathways has been shown to have a 19
positive impact on desirable flavour compound production, such as isoamyl acetate and isobutyl 20
acetate (45) and Zn supplementation to wort results in increased production of the acetate esters 21
of higher alcohols (31). The present data suggests that this effect of zinc availability on flavour 22
formation may be mediated by the transcriptional regulation of ILV2, ILV3 and BAT2. 23
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Maintaining a sufficiently high zinc level during beer fermentation is clearly critical to maintain 1
the desired balance between several flavour compounds. 2
3
Signature transcripts for diagnosing Zn bio-availability in industrial media 4
In complex industrial fermentation media such as wort or other plant biomass 5
hydrolysates, Zn can form complexes with several medium components, thereby reducing its 6
bioavailability for yeast (41, 34, 42). This limits the relevance of chemical analyses of the Zn 7
content to test the bioavailability of zinc in wort and other industrial media. Addition of Zn in the 8
form of salt or trub is a common practice to prevent Zn depletion during the brewing process 9
(71). Especially in beer brewing, this is not risk-free as excess Zn leads to the modification of 10
flavour compound formation (17). Molecular markers can be used to monitor fermentation 11
processes through transcript profiling (30). For such diagnostic purposes, it would be preferable 12
to construct small, cost-effective microarrays that contain a limited number of ‘signature 13
transcripts’. A prerequisite of these signature transcripts is that they are specific to one 14
environmental parameter and show a robust response in various environmental (process) 15
contexts. Comparison of multiple chemostat regimes enabled the identification such Zn-specific 16
signature transcripts. For instance, ZAP1 and ZRT1 would be very good signature transcripts. 17
Also YOR387C and YGL258W, encoding proteins that have not been characterized yet and that 18
have been previously proposed as potential signature transcripts for Zn depletion (50, 30), were 19
specifically and consistently induced under Zn limitation in chemostat cultures. Conversely 20
NRG2 and PST1, potential Zap1p-targets (50) were here shown to be also regulated by oxygen 21
availability and are therefore not recommended for diagnostic purposes. 22
23
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Acknowledgements 1
The research group of J. T. Pronk is part of the Kluyver Centre for Genomics of Industrial 2
Fermentation, which is supported by The Netherlands Genomics Initiative. Raffaele De Nicola 3
would like to thank FEMS for the scholarship that allowed him to support his stay in Delft during 4
the execution of this work. 5
6
Appendix 7
Constraints imposed to group zinc responsive genes into modules. 8
Let the discretized expression pattern of a gene be denoted by vector x of length six. The values 9
of the elements of x can either be 0 (no differential expression), 1 (up-regulated) or -1 (down-10
regulated). The elements of x correspond to the cultivation conditions as follows: 11
12
x(1) x(2) x(3) x(4) x(5) x(6)
C-Ana N-Ana Zn-Ana C-Aer N-Aer Zn-Aer
13
Below, we state the constraints on x that must be satisfied in order for a gene to be part of a 14
particular module. Note that all constraints must be met to suffice. 15
Module 1 Upregulated regardless of aeration
constraints:
x(3)>x(1)
x(3)>x(2)
x(3)>x(4)
x(3)>x(5)
x(6)>x(1)
x(6)>x(2)
x(6)>x(4)
x(6)>x(5)
16
Module 2 Downregulated regardless of aeration
constraints:
x(3)<x(1)
x(3)<x(2)
x(3)<x(4)
x(3)<x(5)
x(6)<x(1)
x(6)<x(2)
x(6)<x(4)
x(6)<x(5)
17
Module 3 Anaerobically upregulated
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constraints:
x(3)>x(1)
x(3)>x(2)
x(3)>x(4)
x(3)>x(5)
x(3)>x(6)
1
Module 4 Anaerobically downregulated
constraints:
x(3)<x(1)
x(3)<x(2)
x(3)<x(4)
x(3)<x(5)
x(3)<x(6)
2
Module 5 Aerobically upregulated
constraints:
x(6)>x(1)
x(6)>x(2)
x(6)>x(3)
x(6)>x(4)
x(6)>x(5)
3
Module 6 Aerobically downregulated
constraints:
x(6)<x(1)
x(6)<x(2)
x(6)<x(3)
x(6)<x(4)
x(6)<x(5)
4
5
6
7
8
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List of Tables: 1
2
Table 1: Composition of the media used to perform carbon, nitrogen and zinc limitation under 3
aerobic and anaerobic environment. Numbers in bold indicate the modifications introduced to the 4
synthetic media described by Verduyn (77) in order to obtain the relevant nutrient limitations. 5
Table 2: Physiological characteristics of CEN.PK113-7D grown in aerobic and anaerobic 6
carbon-, nitrogen-, or zinc-limited chemostat cultures (dilution rate of 0.1 h-1
). 7
DW: biomass dry weight; NA: not applicable; ND: not determined; BD: below detection 8
aBiomass yield on glucose 9
bSpecific consumption rates of glucose and oxygen and specific production rates of ethanol, 10
glycerol, acetate and carbon dioxide 11
cRespiratory quotient: qCO2/qO2 12
Table 3: Clustering of the zinc-responsive genes 13
aExpression pattern of genes of each module along with their averaged expression and standard 14
deviation. Here, the expression levels of each gene are normalized to have zero mean and unit 15
variance. (Y axis: normalized expression, X axis: culture condition, from left to right: anaerobic 16
carbon, nitrogen, zinc limitation , and aerobic counterparts). 17
bEach data-set was analysed individually for enrichment of transcription factor binding and 18
functional categories as described in the Material and Methods section. 19
cTF: Transcription Factor 20
dMIPS: Munich Information Center for Protein Sequences 21
eKEGG: Kyoto Encyclopedia of Genes and Genomes 22
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Table 4: Identity and expression levels of the genes from Module 1 consistently up-regulated in 1
response to zinc limitation and containing ZRE sequences (Zinc Responsive Element). Genes 2
indicated in bold were also part of the regulon defined by Lyons and co-workers. 3
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List of Figures: 1
2
Figure 1: Consensus ZRE sequence identified by MEME using Module 1 as input. 3
Figure 2: Venn diagram of chemostat based transcriptome data in comparison with data obtained 4
by Lyons and co-workers. A: Zap1p-regulon (Modules 1 and 3 in comparison with 46 genes from 5
Lyons and co-workers). B: genome-wide comparison (Modules 1, 3 and 5 in comparison with all 6
up-regulated genes from Lyons and co-workers). 7
Figure 3: A: Glycogen and trehalose metabolism in S. cerevisiae. Genes indicated in green are 8
clustered in Module 2. The four boxes indicate the following fold-changes from left to right: zinc 9
vs carbon anaerobic, zinc vs nitrogen anaerobic, zinc vs carbon aerobic, zinc vs nitrogen aerobic. 10
Intensities of fold changes are indicated by the colour map in the legend. B: normalized 11
expression profile of genes involved in glycogen and trehalose metabolism and intracellular 12
glycogen and trehalose concentrations. 13
Figure 4: Combinatorial regulation of gene expression by Zn and oxygen availability: constraints 14
for the selection of the discretized patterns and average expression profile of these selected 15
patterns. Only the oxygen-induced genes are represented. 16 ACCEPTED
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Table 1: 1
Limiting
nutrient
Glucose
g/l
(NH4)2SO4
g/l
K2SO4
g/l
ZnSO4·7H2O
mg/l
Carbon 7.5 5 - 4.5
Nitrogen 59 1 5.3 4.5 Aerobic
Zinc 66 5 - 0.014
Carbon 25 5 - 4.5
Nitrogen 46 1 5.3 4.5 Anaerobic
Zinc 58 5 - 0
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Table 2:
Ratesb, mmol.gDW
-1.h
-1 Growth
condition
Residual
glucose
mM
Zn in
biomass
µmol.gDW-1
YSXa
gDW.gglucose-1
qglucose qEthanol qGlycerol qAcetate qO2 qCO2 RQ
c
Carbon
recovery
%
C BD 2.36 ± 0.4 0.49 ± 0.00 - 1.1 ± 0.0 0.0 ± 0.0 BD BD - 2.8 ± 0.3 2.8 ± 0.3 1.0± 0.0 98 ± 3
N 92.7 ± 5.5 2.74 ± 1.3 0.09 ± 0.00 - 5.8 ± 0.1 8.0 ± 0.1 0.08 ± 0.01 0.03 ± 0.01 - 2.7 ± 0.1 12.1 ± 0.2 4.5 ±0.2 96 ± 1
AE
RO
BIC
Zn 102.4 ± 6.4 0.9 ± 0.2 0.10 ± 0.00 - 5.3 ± 0.0 8.1 ± 0.2 0.08 ± 0.01 0.07 ± 0.01 - 2.8 ± 0.0 12.3 ± 0.2 4.5 ±0.1 105 ± 2
C BD 2.1 ± 0.4 0.09 ± 0.00 - 6.0 ± 0.0 9.6 ± 0.1 0.81 ± 0.06 0.01 ± 0.00 NA 10.3 ± 0.4 NA 101 ± 2
N 100.8 ± 8.6 2.74 ± 1.3 0.07 ± 0.00 - 8.4 ± 0.0 13.5 ± 0.6 0.76 ± 0.04 0.06 ± 0.05 NA 14.8 ± 0.3 NA 101 ± 2
AN
AE
RO
BIC
Zn 110.4 ± 3.9 0.52 ± 0.03 0.07 ± 0.00 - 8.4 ± 0.0 13.7 ± 0.2 1.09 ± 0.01 0.16 ± 0.02 NA 15.5 ± 0.5 NA 100 ± 1
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Table 3:
Number in
Module Description Expression patterna
Over-represented categoriesb genom
e
module
p-value
1
(93 genes)
Genes up-
regulated under
the zinc limitation
regardless of
aeration. C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
TFc: Zap1p
MIPSd: heavy metal ion transport (Cu,
Fe, etc.)
8
52
6
7
2.2.10-10
8.7.10-6
2
(40 genes)
Genes down-
regulated under
the zinc limitation
regardless of
aeration. C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
TFc: Msn2
MIPSd: C-compound and carbohydrate
metabolism
Metabolism of energy reserves
(e.g. glycogen, trehalose)
Metabolism of Leu and Val
KEGGe: Val, Leu and Ile biosynthesis
Panthotenate and CoA
biosynthesis
65
508
53
7
16
10
11
3
5
3
3
3
6.8.10-4
2.0.10-4
1.7.10-5
7.8.10-6
1.2.10-4
2.7.10-5
3
(77 genes)
Genes up-
regulated only
under anaerobic
zinc limitation
C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
TFc: Hsf1
Skn7
MIPSd: Homeostasis of cations
133
156
162
7
8
9
1.0.10-3
5.4.10-4
1.3.10-4
4
(36 genes)
Genes down-
regulated only
under anaerobic
zinc limitation
C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
MIPSd: proteasomal degradation
(ubiquitin/proteasamol pathway)
KEGGe: TCA cycle
Proteasome
191
30
34
7
3
4
7.7.10-5
6.0.10-4
3.5.10-5
5
(119
genes)
Genes up-
regulated only
under aerobic zinc
limitation
C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
TFc: Yap7 158 12 3.4.10
-5
6
(16 genes)
Genes down-
regulated only
under aerobic zinc
limitation
C-anae N-anae Zn-anae C-ae N-ae Zn-ae
-3
-2
-1
0
1
2
MIPSd: METABOLISM 1526 11 1.8.10
-4
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Table 4
Transcript level
Anaerobic Aerobic Gene Description
Number
of ZREs C N Zn C N Zn
ZAP1 Zn-responsive TF 1 23.2 41.5 479.9 33.8 43.5 356.6
ZRT1 High affinity Zn transporter 3 175.6 286.0 2004.1 68.3 240.6 1867.5
ZRT2 Low affinity Zn transporter 2 108.6 124.0 739.4 102.8 162.8 551.5
ZRT3 Vacuolar Zn efflux 1 236.9 257.2 1665.5 313.4 282.9 1708.5
ZRC1 Vacuolar Zn influx 1 237.8 367.3 712.2 261.3 337.8 811.6
ZRG17 Putative Zn transporter 1 84.7 91.9 446.3 142.6 97.9 527.4
FET4 Low affinity Fe transporter 1 235.0 223.8 743.3 12 89.9 443.5
ADH4 Alcohol dehydrogenase 3 153.3 206.3 2889.8 76.6 118.1 2872.1
HOR2 Glycerol-P phosphatase 1 45.1 63.3 141.3 97.3 84.9 198.5
DPP1 DAGPP phosphatase 1 307.3 517.8 1019.4 294.4 636.8 1233.3
URA10 Pyrimidine biosynthesis 1 18.7 25.1 81.3 30.4 16.7 50.9
FLO11 Cell surface flocculin 1 1150.8 1293.3 1935.4 42 60.2 2105.0
ZPS1 Cell surface mannoprotein 2 74.7 225.8 3385.4 139.5 119 3349.2
MNT2 Mannosyl transferase 3 18.1 12 51.6 12 12 40.9
KTR6 Mannosyl transferase 1 449.7 388.4 537.8 314.0 253.5 620.4
MCD4 Transferase required for GPI
anchor synthesis 1 337.0 323.3 996.5 232.2 279.2 1190.3
ZIP1 Synaptonemal complex 1 12 12 46.2 12.5 12 24.6
KTI12 tRNA modification 1 157.9 134.1 218.4 120.3 121.5 278.3
VTC3 Vacuolar transporter chaperone 1 66.4 95.7 175.0 51.1 85.8 272.8
TEX1 TREX complex 1 29.4 29.9 91.5 25.1 24.4 177.9
MUP1 Methionine transporter 1 69.6 38.7 636.5 128.8 211.9 912.5
YNL254C Unknown 1 22.2 27.5 342.0 17.4 32.3 354.6
YER130C Unknown 1 32.2 34.5 179.1 30.7 28.1 66.8
ICY2 Unknown 2 290.5 204.4 1939.2 568.3 143.8 1436.1
VEL1 Unknown – similar to YOR387C 3 12 12 1047.3 12 12 858.6
YOR387C Unknown – similar to VEL1 3 12 12 2803.0 12 12 2612.0 ACCEPTED
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Figure 1
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Lyons et al.
21
214
7273
0
0
ADH4, DPP1, FET4, ICY2, MCD4, MNT2, RAD27, URA10, YGL258W, YJL132W,
YNL254C, YOL131W, YOR387C, ZAP1,
ZIP1, ZPS1, ZRC1, ZRG17, ZRT1, ZRT2, ZRT3
IZH1, IZH2, PST1, NRG2
Module 1Module 3
A (Zap1 regulon) B (genome-wide comparison)
Lyons et al.
420 73 214
Modules 1, 3 & 5
Module 1: 36 genes
Module 3: 26 genes
Module 5: 11 genes
C-A
nae
N-A
nae
Zn-A
nae
C-a
eN
-ae
Zn-ae
-2.5
-1.5
-0.5
0.5
1.5
2.5
No
rmal
ized
ex
pre
ssio
n
C-A
nae
N-A
nae
Zn-A
nae
C-a
eN
-ae
Zn-ae
-2.5
-1.5
-0.5
0.5
1.5
2.5
No
rmal
ized
ex
pre
ssio
n
Figure 2
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Glucose
Glucose-6-P
ATP
ADP
UDP-Glucose
Trehalose
TPS2
NTH1
NTH2
ATH1
(Glycogen)n
UDP
Glucose-1-P Glucose
GPH1 GDB1
-6-P
Glucose-1-P-1- -GlucoseUGP1
PGlucose-6-- -
Trehalose-6-P- -
PGM1
PGM2
(Glycogen)n-1
(
PPi
Glucose-1-P Glucose
UTP
HXK1
HXK2
GLK1
TSL1
TPS1
TPS3
GLG1
GLG2
GLC3
GSY1
GSY2
GLG2
GAC1
Legend: fold changes
-1.5 to +1.5
+1.5 to 2.0
-1.5 to -3
-3 to -6
< -6
Expression profile of genes involved inglycogen metabolism
C-a
nae
N-a
nae
Zn-an
aeC-a
eN
-ae
Zn-ae
-2
-1
0
1
2PGM1
PGM2
UGP1
GLG1
GLG2
GLC3
GSY1
GSY2
GPH1
GDB1
Glycogen0
10
20
30GAC1
Norm
aliz
ed e
xpre
ssio
n
Gly
cogen
(mg
equiv
glu
cose
. gD
W-1
Expression profile of genes involved intrehalose metabolism
C-a
nae
N-a
nae
Zn-an
aeC-a
eN
-ae
Zn-ae
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
PGM1
PGM2
UGP1
TPS1
TPS3
TSL1
TPS2
NTH1
NTH2
ATH1
Trehalose0
5
10
15
20
25
30
35
40
45N
orm
aliz
ed e
xpre
ssio
n
Treh
alose (m
gequiv
glu
cose .g
DW
-1)
A.
B.
Figure 3
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Zn-Ae > Zn-Ana
C-Ae =C-Ana
N-Ae = N-Ana
Zinc-dependent oxygen
response (130 genes)
Zn-Ae > C-Ana
Zn-Ae > N-Ana
Zn-Ae > Zn-Ana
Zn-Ae > C-Ae
Zn-Ae > N-Ae
Module 5 (119 genes)
Aerobically up-regulated
Constraints
C-li
m
N-li
m
Zn-lim
-1.5
-0.5
0.5
1.5
2.5
Nutrient limitation
Dif
fere
nce
in n
orm
alis
ed e
xpre
ssio
n
bet
wee
n a
erobic
and a
nae
robic
cult
ure
s
Figure 4
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ay 16, 2018 by guesthttp://aem
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Dow
nloaded from