Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds
through specific adaptive cellular programmes, in particular by modifying gene expression.
Many adaptive changes affect the physico-chemical properties of the cell wall, and several
mechanisms that specifically affect the expression levels of genes that encode for cell wall
components have been described previously. Cell wall modification directly impacts on general
cell wall properties and cell-cell and cell-surface interactions. Many of these properties have
been directly linked to families of cell wall proteins referred to as adhesins. In particular
members of the Flocculation (FLO) gene family have been shown to play a crucial role in
adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion,
plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears
to control flocculation. The regulation of FLO11 expression is well documented and is mainly
controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A
(cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is
central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to
activate transcription. In this study we further explore additional gene targets of Flo8p and
Mss11p, as well as their regulation and their impact on cell wall characteristics and associated
adhesion phenotypes.
Our analysis shows that Mss11p is also required for FLO1 expression, and functions
together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene
expression analysis further reveals that altered Mss11p levels leads to the change in the
expression of various cell membrane and cell wall genes, notably AQY2 and members of the
DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes
display an almost exclusive dependence on FLO gene expression. We also demonstrate that
these phenotypes require Flo10p and are thus dependent on the specific balance of Flo
proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of
FLO10 shares signalling components with FLO11, but that the two genes are differentially
regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for
Mss11p but rather for the MAPK component Ste12p.
Whole genome expression analysis were also performed on strains with altered levels of
Flo8p which were compared with the above mentioned transcriptome data set. This analysis
shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also
have significant unique gene targets. The combination of transcriptome data with current
information concerning transcription factor (TF) interaction networks reveals the importance of
network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we
constructed a TF interaction model in which Flo8p acts as the predominantly activating TF
component, whereas Mss11p function as a target hub TF, possibly as a mediator- or
polymerase II holo-enzyme component.
Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and
show that by adjusting FLO11 expression in this strain we are able to significantly change this
phenotypic behaviour. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae neem veranderinge in sy omgewing waar en reageer daarop
deur middel van spesifieke sellulêre programme, in die besonder deur geenuitdrukking aan te
pas. Verskeie aanpasbare veranderinge beïnvloed die fisieke, asook chemiese eienskappe van
die selwand, en talle meganismes is al beskryf wat die uitdrukkingsvlakke beïnvloed van gene
wat vir selwandkomponente kodeer. Die modifikasie van die selwand het ’n direkte impak op
selwand-eienskappe, asook die sel-sel- en sel-oppervlak-interaksies. Verskeie van hierdie
eienskappe word direk gekoppel aan die selwandproteïenfamilies, wat ook as adhesie-faktore
bekend staan. Veral lede van die Flokkulasie (FLO) -geenfamilie het ’n noodsaaklike funksie in
adhesie-fenotipes. Flo11p speel ’n rol in verskeie fenotipes, wat insluit die indringende groei van
agar, plastiekaanhegting en die vorming van pseudohifes, “flor“ en “matte“, terwyl Flo1p
flokkulasie beheer. Die regulering van FLO11-uitdrukking is deeglik gedokumenteerd en dit
word hoofsaaklik gereguleer deur die mitogeen-geaktiveerde proteïenkinase (MAPK) en sikliese
AMP-proteïenkinase A (cAMP-PKA) seintransduksiekaskades. Genetiese analises toon dat
Mss11p stroom-af en sentraal tot hierdie kaskades funksioneer, en dit aktiveer transkripsie deur
interaksie met die cAMP-PKA-komponent, Flop8. In hierdie studie word ’n ondersoek gedoen
na addisionele teikengene van Flo8p en Mss11p, en hoe hierdie gene gereguleer word, asook
hul impak op selwandeienskappe en geassosieerde adhesie-fenotipes.
Ons analises toon dat Mss11p ook benodig word vir die ekspressie van FLO1 en dat dit,
tesame met Flo8p, beheer uit oefen oor verskeie Flo-afhanklike fenotipes. Genoomwye
geenekspressie-analises wys verder daarop dat veranderde Mss11p-vlakke lei tot die
aanpassing van die ekspressie van verskeie selmembraan- en selwandgene, naamlik AQY2
asook lede van die DAN- en TIR-geenfamilies. Verdere genetiese analise dui daarop dat
adhesie-fenotipes byna eksklusief afhanklik is van FLO-geenekspressie. Daar is verder getoon
dat hierdie fenotipes ook Flo10p benodig en dus afhanklik is van die spesifieke balans van Floproteïene
in die selwand. Die analise van seintransduksiemutante demonstreer dat FLO10 en
FLO11 seintransduksie-komponente deel, maar dat hierdie gene verskillend gereguleer word.
Anders as FLO11, toon FLO10 nie ’n absolute noodsaaklikheid vir Mss11p nie, maar eerder vir
die MAPK-komponent, Ste12p.
Totale genoomekspressie-analises is ook gedoen op gisrasse met aangepaste vlakke van
Flo8p en dis vergelyk met bogenoemde transkripsiedatastel. Hierdie analise wys dat Flo8p and
Mss11p die FLO-gene, asook AQY2 en TIR3, koreguleer, maar ook beduidende unieke
teikengene het. Die kombinasie van transkripsiedata met huidig beskikbare informasie
betreffende transkripsiefaktor (TF) -interaksienetwerke dui op die relevansie van
netwerkinteraksie tussen Cin5p, Flo8p, Mga1p en Mss11p. Hiervan is daar ’n model opgestel
waarin Flo8p in die meeste gevalle as die aktiverende TF-komponent optree, terwyl Mss11p as
TF-teiken dien, moontlik as ’n mediator- of polimerase II holoënsiemkomponent.
Laatens word daar vir die eerste keer verslag gedoen van ”mat”-vorming deur ’n industriële
wyngisras en toon ons verder dat hierdie fenotipe beduidend verander word deur middel van die
aanpassing van FLO11-uitdrukking.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/4026 |
| Date | 03 1900 |
| Creators | Bester, Michael Christiaan |
| Contributors | Bauer, Florian F., University of Stellenbosch. Faculty of Agrisciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 197 p. : ill. |
| Rights | University of Stellenbosch |
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