1

Physiological and transcriptional responses of Saccharomyces cerevisiae to zinc limitation in 1

chemostat cultures 2

3

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*

6

§Both authors contributed equally to this work 7

8

Running title: Yeast transcriptional response to Zn limitation 9

10

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

25

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

9

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

15

C-Ana N-Ana Zn-Ana C-Aer N-Aer Zn-Aer

0 1 0 0 0 -1

16

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

7

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

5

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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Dow

nloaded from