Cyc8p and Tup1p transcription regulators antagonistically regulate Flo11p expression and complexity of yeast colony biofilms

<div><p>Yeast biofilms are complex multicellular structures, in which the cells are well protected against drugs and other treatments and thus highly resistant to antifungal therapies. Colony biofilms represent an ideal system for studying molecular mechanisms and regulations involved in development and internal organization of biofilm structure as well as those that are involved in fungal domestication. We have identified here antagonistic functional interactions between transcriptional regulators Cyc8p and Tup1p that modulate the life-style of natural <i>S</i>. <i>cerevisiae</i> strains between biofilm and domesticated mode. Herein, strains with different levels of Cyc8p and Tup1p regulators were constructed, analyzed for processes involved in colony biofilm development and used in the identification of modes of regulation of Flo11p, a key adhesin in biofilm formation. Our data show that Tup1p and Cyc8p regulate biofilm formation in the opposite manner, being positive and negative regulators of colony complexity, cell-cell interaction and adhesion to surfaces. Notably, in-depth analysis of regulation of expression of Flo11p adhesin revealed that Cyc8p itself is the key repressor of <i>FLO11</i> expression, whereas Tup1p counteracts Cyc8p’s repressive function and, in addition, counters Flo11p degradation by an extracellular protease. Interestingly, the opposing actions of Tup1p and Cyc8p concern processes crucial to the biofilm mode of yeast multicellularity, whereas other multicellular processes such as cell flocculation are co-repressed by both regulators. This study provides insight into the mechanisms regulating complexity of the biofilm lifestyle of yeast grown on semisolid surfaces.</p></div

Yeast strains.

<p>Yeast strains.</p

Development and architecture of colonies with altered levels of Cyc8p and Tup1p.

<p>A, Development of colonies grown on GMA at a density of ~10³ colonies per plate (left panel). Two photon excitation confocal microscopy (2PE-CM) of colony cross-sections stained with Calcofluor white (false green color); colonies were grown for 5 days on GMA at a density of ~3 x 10³ colonies per plate (right panel). B, 2PE-CM of colony cross-sections stained with Calcofluor white (false green color). Colonies were grown for 3 days on GMA at a density of ~3 x 10³ colonies per plate. Upper part, whole colonies (20x objective); lower part, central colony parts shown at a higher magnification (63x objective), insets: details of aerial and subsurface cells (strains BR-F and p_GAL-<i>CYC8</i>); detail of central part (p_TEF-<i>CYC8</i>); detail of the colony bottom (<i>tup1</i>). White bar, 100 μm; yellow bar, 20 μm. Arrow indicates chains of rounded cells invading the agar.</p

Model schematic of the regulatory functions of Cyc8p and Tup1p.

<p>In colony biofilms, levels of Cyc8p and Tup1p are balanced in such a way that Tup1p inhibits Cyc8p repressor (by forming Cyc8p-Tup1p complex), thus preventing <i>FLO11</i> repression. In addition, free Tup1p contributes to repression of putative extracellular protease that degrades Flo11p. The Cyc8p-Tup1p complex represses other cellular functions, such as cell flocculation.</p

Levels of <i>CYC8</i>, <i>TUP1</i> and <i>FLO11</i> mRNAs and respective proteins in strains with differently altered levels of Cyc8p and Tup1p regulators.

<p>A. Northern blot of <i>CYC8</i>, <i>TUP1</i> and <i>FLO11</i> mRNAs from 3-day-old colonies grown on GMA and then induced/uninduced 4 h with galactose and/or copper. Representative result of the 4 experiments is shown. Northern blot loading controls are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007495#pgen.1007495.s004" target="_blank">S4 Fig</a>. Asterisks indicate reduction of transcription from p_GAL promoter in the presence of copper. B. Levels of Tup1p and Cyc8p quantified by LC-MS/MS. Colonies were grown 3 days on GMA and then were induced/uninduced by galactose for 4 h. Protein amounts were related to the amount of Tup1p in BR-F colonies. C. Levels of Flo11p-GFP and free GFP in lysates from 3-day-old colonies (prepared from whole colony biomass including extracellular material) of respective strains grown on GMA, induced/uninduced for 18 h by galactose and/or copper. Arrows points to total degradation of Flo11p-GFP to GFP in particular samples. D. Levels of Flo11p-GFP and free GFP in the extracellular fluid extracted from 50 mg of wet weight biomass of 3-day-old colonies grown on GMA without galactose or copper induction.</p

Cell morphology and adhesion characteristics within colonies of strains with altered levels of Cyc8p and Tup1p.

<p>A, Cell invasion (right picture) shown after washing the cells grown on GMA plates for 3 days (left picture). B, Morphology of cells from the aerial parts of 3-day-old colonies grown on GMA. C, Cell flocculation in liquid cultures grown for 2 days in GM (graph); examples of flocculation in tubes (right) and D, microscopic pictures of free cells and flocs.</p

Extracellular fibrillar material between cells within colonies of strains with altered levels of Cyc8p and Tup1p.

<p>A, Electron microscopy of Velcro connections between cells from 3-day-old (a, b, e) and 5-day-old (c, d) colonies grown on GMA. B, The average length of the fibers is shown in the graph. Black columns, structured colony biofilms; white columns, smooth colonies; bars, SD; ***, P<0.001. C, Examples of extracellular material in higher magnification from colony biofilms (BR-F, inset a1 from Aa) and from smooth colonies (<i>tup1</i>, inset d1 from Ad). Velcro-like connections in BR-F are schematically indicated in yellow color, the red color indicates N-terminal Flo11A domains [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007495#pgen.1007495.ref048" target="_blank">48</a>], potentially involved in the interaction.</p

Additional file 2: of Metabolic differentiation of surface and invasive cells of yeast colony biofilms revealed by gene expression profiling

Table S1. Log ratios and p-values from DESeq analysis of read counts. Table S2. Genes and lncRNAs expressed at a higher level in aerial cells. Table S3. Genes and lncRNAs expressed at a higher level in roots. Table S4. S. cerevisiae orthologs of C. albicans secreted proteins. Table S5. Mapping and relative expression of lncRNA to lncRNA loci in GTF file. Table S6. All lncRNAs upregulated 2-fold in aerial or root and lying within 1.5 kB of a coding gene. Table S7. Yeast strains used in this study. Table S8. Primers and plasmids used in this study. (XLSX 2047 kb