Table 1.
Yeast strains.
Fig 1.
Development and architecture of colonies with altered levels of Cyc8p and Tup1p.
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 pGAL-CYC8); detail of central part (pTEF-CYC8); detail of the colony bottom (tup1). White bar, 100 μm; yellow bar, 20 μm. Arrow indicates chains of rounded cells invading the agar.
Fig 2.
Expression of Flo11p adhesin in colonies with altered levels of Cyc8p and Tup1p.
A, Effects of TUP1 (left) and CYC8 (right) expression induced by galactose diffusing from the wells in the agar (marked in red) on colony morphology. 120 μl of 10% galactose was applied to each well. Morphologies of wt colonies treated in the same way are shown in S5 Fig. B, Flo11p-GFP in 3-day-old colonies of Flo11p-GFP strains grown on GMA. Representative result of the 4 experiments is shown. Western blot loading controls are in S4 Fig. C, 2PE-CM of cross-sections of 3-day-old colonies of BR-F-Flo11p-GFP strain grown on GMA. Green, Flo11p-GFP; red, cell autofluorescence (in upper picture). Plating density ~4–6 x 103 colonies per plate. D, 2PE-CM of cross-sections of colonies from the plates with galactose induction (panel A). Induced colonies localized close to the galactose source and uninduced colonies localized to the plate margin are shown. Green, Flo11p-GFP; red, cell autofluorescence. Plating density ~4–6 x 103 colonies per plate.
Fig 3.
Levels of CYC8, TUP1 and FLO11 mRNAs and respective proteins in strains with differently altered levels of Cyc8p and Tup1p regulators.
A. Northern blot of CYC8, TUP1 and FLO11 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 S4 Fig. Asterisks indicate reduction of transcription from pGAL 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.
Fig 4.
Model schematic of the regulatory functions of Cyc8p and Tup1p.
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 FLO11 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.
Fig 5.
Extracellular fibrillar material between cells within colonies of strains with altered levels of Cyc8p and Tup1p.
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 (tup1, inset d1 from Ad). Velcro-like connections in BR-F are schematically indicated in yellow color, the red color indicates N-terminal Flo11A domains [48], potentially involved in the interaction.
Fig 6.
Cell morphology and adhesion characteristics within colonies of strains with altered levels of Cyc8p and Tup1p.
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.