Transcript Diapositiva 1

Study of the adaptation of
S. cerevisiae strains to
winemaking conditions by means
of directed evolution and
competition experiments with
bar-coded YKO strains
Long-term objective
Comprehensive identification of genes involved in
the adaptation of Saccharomyces cerevisiae to
the winemaking environment.
Some limitations of transcriptomic approaches
•Genes relevant for many biological processes are not
subject to transcriptional regulation in response to
environmental conditions that influence these processes
(Birrell et al. 2002; PNAS).
•Not all genes showing a transcriptional change in
response to a given culture condition are required for
fitness under these conditions (Tai et al., 2007;
Microbiology SGM).
Some alternative genome-wide approaches (wine)
Proteomics
•Complementary information
•Usually no direct correlation with transcription data
Comparative genomics by hybridization
(aCGH or low coverage sequencing)
•Strains showing different fermentation phenotypes
•Wine vs. non-wine strains
Whole genome sequencing
•Horizontal transfer
•¿New mobile elements?
HaploInsuficiency Profiling/HOmozygous Profiling
HIP/HOP
Construction of YKO S. cerevisiae collections
X
Heterozygous strains
Homozygous strains
x6000
x4500
HaploInsuficiency Profiling/HOmozygous Profiling
HIP/HOP
Some considerations about HIP/HOP analysis of
wine fermentation
•Environmental conditions experiment dramatic changes
•Low number of generations in similar conditions
Alternative approach
Continuous culture
Simulation of wine fermentation in continuous culture
Continuo EC1118 CENIT 24%
25,00
600,00
CTR3
VCT3
500,00
20,00
300,00
10,00
200,00
5,00
100,00
0,00
0:00:00
0,00
4:48:00
9:36:00
14:24:00
19:12:00
24:00:00
Inoculation Time
28:48:00
33:36:00
38:24:00
43:12:00
VCT [mM]
CTR [mM/h]
400,00
15,00
Simulation of first step of wine fermentation in
continuous culture
•10 generation times for homozygous competition (SM)
•20 generation times for heterozygous competition (SM)
•Controls for 10 and 20 generation times in YPD
3 biological replicates for each of the above
HIP-HOP results for the first step of alcoholic
fermentation
At least 150 heterozygous deleted strains showed
deficient growth in synthetic must after 20 generations
(>2-fold reduced fitness as compared to fitness in YPD)
At least 126 homozygous deleted strains showed deficient
growth in synthetic must after 10 generations
(>2-fold reduced fitness as compared to fitness in YPD)
Individual phenotypic characterization of selected
strains.
Relevant functions from HIP analysis
•Vacuolar functions, including autophagy
•Different functions in the “DNA-to-protein” pathway
omRNA processing and stability
oProtein synthesis
oSecretion (ER functions)
Relevant functions from HOP analysis
•Adenine and lysine biosynthesis
•Inositol biosynthesis
•Biosynthesis of phospholipids
Apparent limitations of the HIP/HOP approach
•Limited to loss-of-function phenotypes
•Difficulty to estimate wine-related phenotypes in a BY4743
background
Complementary approaches
•Directed evolution of laboratory strains
•QTL mapping by high throughput methods
Directed evolution of laboratory strains
•Haploid laboratory strain (BY4741)
•Continuous culture in conditions emulating the first step of
alcoholic fermentation
•Working volume 40-50 ml
•150-250 generations (three biological replicates)
•Verification of the “evolved” phenotype
•Whole genome sequence analysis of the evolved strains
Phenotype of evolved strains. Batch culture
Strain
Growth rate
BY 4741
0,1683
AV 8
0,2254
BV 19
0,2569
E18
0,2760
AV 16
0,2779
1,4
1,2
w t-1
w t-2
w t-3
1
AV16.1
AV 16.2
AV 16.3
0,8
AV 18.1
AV 18.2
AV 18.3
0,6
AV 21.1
AV 21.2
AV 21.3
0,4
AV 22.1
AV 22.2
AV 23.3
0,2
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
36
38
Adaptation to first steps does not involve improved overall
fermentation performance, rather the opposite
Phenotype of evolved strains. Continuous culture
OD 600
D=0,20 h-1
D=0,25 h-1
BY4741
0.69
Av16
1.28
BY4741
0.28
Av16
0.74
Summary of mutations already identified
•SNPs in non-coding regions
Strain
SNPs
50%
•Nonsense mutations
E18
4
4
•Missense mutations
BV19
2
1
•50% mutations
AV8
6
2
AV16*
1
0
Mutations requiring further confirmation
•Deletions/insertions
•Changes in copy-number
•Chromosomal rearrangements
RSP5: E3 Ubiquitin ligase
Ubiquitin-proteasome pathway mutants
Gene
Strain
Mutation
RSP5
E18
Asn>Lys
E3 Ubiquitin ligase
BV19
Glu>Asp
E3 Ubiquitin ligase
AV16
Asn>Thr
E3 Ubiquitin ligase
CDC4
E18
Ser>Leu
(50%)
Part of a complex with ubiquitin ligase activity on a CDK
inhibitor
BRE5
E18
Glu>STOP
Ubiquitin protease cofactor
UBC6
BV19
Small
deletion*
Ubiquitin-conjugating enzyme
BUL1
AV8
Asp>His
Ubiquitin-binding component of the Rsp5p E3-ubiquitin ligase
complex
Pilar Morales
Maite Novo
Manuel Quirós
Zoel Salvadó
Martijn Wapenaar
Ana Mangado
www.icvv.es/winehiphop
Coincidences with previous studies
28 overlapping HOP genes
19 overlapping HIP genes
3 overlapping HIP genes
Previous reports of HIP/HOP analysis of wine
fermentation
Delneri et al. 2008
•Commercial grape must (100 g/L sugar) (among several other media)
•Chemostat
•Not supplemented with uridine. Aerobic.
•Only HIP analysis
•No unstressed contrast
•Concluded all nutritional requirements were provided by must
Previous reports of HIP/HOP analysis of wine
fermentation
Piggott et al. 2011
•Synthetic must (200 g/L sugar)
•Single biological replicate each (HIP and HOP analyses)
•Time-course
•YPD amplification of samples
•No unstressed contrast
•Autophagy and ubiquitin-proteasome functions required
•Proficient deleted strains also identified (ribosomal and peroxisomal
functions)
•FUR4
Some genes to watch
From the HIP analysis
SAM1 and SAM2; URE2; DUR1,2; MAL12, OCA6; CDC19; genes
involved in Gap1p sorting; genes involved in chromatin remodeling
and histone modification
From the HOP analysis
NPR2, NPR3 and RTC1; CAR1 and CAN1; GPD1 y GPD2; UBR1;
STB5; BCK1; BUL2; ADH3; AQR1; genes coding for ribosomal
proteins; genes involved in protein folding in the ER
HIP
SAM1
SAM2
URE2
DUR1,2
MAL12
OCA6
GTR1
GTR2
SWD3
SGF29
RPH1
NHP6B
ITC1
GTR2
CAC2
RLF2
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two
differentially regulated isozymes (Sam1p and Sam2p)
S-adenosylmethionine synthetase, catalyzes transfer of the adenosyl group of ATP to the sulfur atom of methionine; one of two
differentially regulated isozymes (Sam1p and Sam2p)
Nitrogen catabolite repression transcriptional regulator that acts by inhibition of GLN3 transcription in good nitrogen source; has
glutathione peroxidase activity and can mutate to acquire GST activity; altered form creates [URE3] prion
Urea amidolyase, contains both urea carboxylase and allophanate hydrolase activities, degrades urea to CO2 and NH3;
expression sensitive to nitrogen catabolite repression and induced by allophanate, an intermediate in allantoin degradation
Maltase (alpha-D-glucosidase), inducible protein involved in maltose catabolism; encoded in the MAL1 complex locus; hydrolyzes
the disaccharides maltose, turanose, maltotriose, and sucrose
Cytoplasmic protein required for replication of Brome mosaic virus in S. cerevisiae, which is a model system for studying positivestrand RNA virus replication; null mutation confers sensitivity to tunicamycin and DTT
Cytoplasmic GTP binding protein and negative regulator of the Ran/Tc4 GTPase cycle; component of GSE complex, which is
required for sorting of Gap1p; involved in phosphate transport and telomeric silencing; similar to human RagA and RagB
Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE
complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagD
Essential subunit of the COMPASS (Set1C) complex, which methylates histone H3 on lysine 4 and is required in transcriptional
silencing near telomeres; WD40 beta propeller superfamily member and ortholog of mammalian WDR5
Component of the HAT/Core module of the SAGA, SLIK, and ADA complexes; HAT/Core module also contains Gcn5p, Ngg1p,
and Ada2p; binds methylated histone H3K4; involved in transcriptional regulation through SAGA recruitment to target promoters
and H3 acetylation
JmjC domain-containing histone demethylase; specifically demethylates H3K36 tri- and dimethyl modification states; associates
with actively transcribed (RNA polymerase II) regions in vivo and specifically targets H3K36 in its trimethylation state as its
substrate; transcriptional repressor of PHR1; Rph1p phosphorylation during DNA damage is under control of the MEC1-RAD53
pathway
High-mobility group (HMG) protein that binds to and remodels nucleosomes; involved in recruiting FACT and other chromatin
remodelling complexes to the chromosomes; functionally redundant with Nhp6Ap; homologous to mammalian HMGB1 and
HMGB2
Subunit of the ATP-dependent Isw2p-Itc1p chromatin remodeling complex, required for repression of a-specific genes, repression
of early meiotic genes during mitotic growth, and repression of INO1; similar to mammalian Acf1p, the regulatory subunit of the
mammalian ATP-utilizing chromatin assembly and modifying factor (ACF) complex
Putative GTP binding protein that negatively regulates Ran/Tc4 GTPase cycle; activates transcription; subunit of EGO and GSE
complexes; required for sorting of Gap1p; localizes to cytoplasm and to chromatin; homolog of human RagC and RagD
Subunit of chromatin assembly factor I (CAF-1), with Rlf2p and Msi1p; chromatin assembly by CAF-1 is important for multiple
processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the
DNA damage checkpoint after DNA repair; and chromatin dynamics during transcription
Largest subunit (p90) of the Chromatin Assembly Complex (CAF-1); chromatin assembly by CAF-1 is important for multiple
processes including silencing at telomeres, mating type loci, and rDNA; maintenance of kinetochore structure; deactivation of the
DNA damage checkpoint after DNA repair; and chromatin dynamics during transcription
HOP
GPD1
GPD2
NPR2
NPR3
RTC1
CAR1
CAN1
UBR1
STB5
STB5
BUL2
ADH3
AQR1
NAD-dependent glycerol-3-phosphate dehydrogenase, key enzyme of glycerol synthesis, essential for growth under osmotic
stress; expression regulated by high-osmolarity glycerol response pathway; homolog of Gpd2p
NAD-dependent glycerol 3-phosphate dehydrogenase, homolog of Gpd1p, expression is controlled by an oxygen-independent
signaling pathway required to regulate metabolism under anoxic conditions; located in cytosol and mitochondria
Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity
upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p
complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; Iml1p interacts primarily with
phosphorylated Npr2p; homolog of human NPRL2; target of Grr1p; required for growth on urea and proline
Subunit of SEA (Seh1-associated), Npr2/3, and Iml1p complexes; Npr2/3 complex mediates downregulation of TORC1 activity
upon amino acid limitation; SEA complex is a coatomer-related complex that associates dynamically with the vacuole; Iml1p
complex (Iml1p-Npr2p-Npr3p) is required for non-nitrogen-starvation (NNS)-induced autophagy; required for Npr2p
phosphorylation and Iml1p-Npr2p interaction; null mutant shows delayed meiotic DNA replication and double-strand break repair
Subunit of the SEA (Seh1-associated) complex, a coatomer-related complex that associates dynamically with the vacuole; null
mutation suppresses cdc13-1 temperature sensitivity; has N-terminal WD-40 repeats and a C-terminal RING motif
Arginase, responsible for arginine degradation, expression responds to both induction by arginine and nitrogen catabolite
repression; disruption enhances freeze tolerance
Plasma membrane arginine permease, requires phosphatidyl ethanolamine (PE) for localization, exclusively associated with lipid
rafts; mutation confers canavanine resistance
E3 ubiquitin ligase (N-recognin), forms heterodimer with Rad6p to ubiquitinate substrates in the N-end rule pathway; regulates
peptide transport via Cup9p ubiquitination; mutation in human UBR1 causes Johansson-Blizzard Syndrome (JBS)
Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p;
contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitro
Transcription factor, involved in regulating multidrug resistance and oxidative stress response; forms a heterodimer with Pdr1p;
contains a Zn(II)2Cys6 zinc finger domain that interacts with a pleiotropic drug resistance element in vitro
Component of the Rsp5p E3-ubiquitin ligase complex, involved in intracellular amino acid permease sorting, functions in heat
shock element mediated gene expression, essential for growth in stress conditions, functional homolog of BUL1
Mitochondrial alcohol dehydrogenase isozyme III; involved in the shuttling of mitochondrial NADH to the cytosol under anaerobic
conditions and ethanol production
Plasma membrane multidrug transporter of the major facilitator superfamily, confers resistance to short-chain monocarboxylic
acids and quinidine; involved in the excretion of excess amino acids