Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Abstract

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.

Figure 1.—

Colony morphology and fluorescence level. (A) Deletion of FLO11F in 133d generates smooth colonies. (B) Deletion of MSS11 in 133d pFLT_133dGFP generates smooth nonfluorescent colonies in contrast to the fluffy fluorescent colonies for 133d pFLT_133dGFP. The smooth colonies are semitransparent in contrast to the opaque, dark, fluffy colonies (133d).

Figure 2.—

FLO11F expression levels in the deleted mutants. Northern blot analysis was performed to determine FLO11F expression level. ACT1 was used as probe for loading control.

Figure 3.—

FLO11F-related phenotypes in the deleted mutants. (A) Hydrophobicity. Cells cultures were overlaid with octane and mixed. The OD₆₀₀ of the aqueous layer was taken and the relative difference with the initial OD₆₀₀ was used to determine the percentage of hydrophobicity. (B) Biofilm on solid surface. Exponentially growing cells were placed in microtiter plate wells and incubated for 1 hr at 28°. The cells were then stained with 1% crystal violet, and the wells were washed repeatedly with water and photographed. For biofilm quantification, the crystal violet was solubilized using SDS (10%), and the absorbance at 530 nm (A530) was measured. Data presented represent averages of three independent assays. Error bars correspond to standard deviation. (C) Invasive growth. Dots of exponentially growing cells were spotted on YPED solid medium and photographed before (unwashed) and after (washed) washing under a stream of water.

Figure 4.—

FLO11F expression and related phenotypes in mutants for SWI/SNF complex members. (A) FLO11F expression. Northern blot analysis using FLO11F as a probe. ACT1 was used as a loading control. B–D represent hydrophobicity, biofilm on solid surface, and invasive growth, respectively, performed as mentioned in Figure 3. For hydrophobicity and biofilm on solid surface, data presented represent averages of three independent assays. Error bars correspond to standard deviation. swi1(T) and arp7(T) are the original transposon insertion mutants isolated during the screen, because full deletion of these genes caused lethality.

Figure 5.—

FLO11F expression in rim20Δ, rim101Δ, and rim20Δrim101Δ mutants. FLO11F expression was measured by Northern blot using ACT1 as probe for loading control.

Figure 6.—

Expression of FLO11 in L5684 (Σ1278b background) and 133d derivative strains. A gene representing any of the main group of genes identified in the screening was analyzed. Cells were grown on YEPD with a low amount of glucose to mimic the natural derepressed state of the FLO11 promoter in the 133d strain. Expression was measured by Northern blot using ACT1 as a probe for loading control.