The control of cellular adhesion of Saccharomyces cerevisiae by the FLO gene regulator Mss11p

Bester, Michael Christiaan (2010-03)

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010.

Thesis

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.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/4026
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