Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology

Abstract

When grown on solid substrates, different microorganisms often form colonies with very specific morphologies. Whereas the pioneers of microbiology often used colony morphology to discriminate between species and strains, the phenomenon has not received much attention recently. In this study, we use a genome-wide assay in the model yeast Saccharomyces cerevisiae to identify all genes that affect colony morphology. We show that several major signalling cascades, including the MAPK, TORC, SNF1 and RIM101 pathways play a role, indicating that morphological changes are a reaction to changing environments. Other genes that affect colony morphology are involved in protein sorting and epigenetic regulation. Interestingly, the screen reveals only few genes that are likely to play a direct role in establishing colony morphology, with one notable example being FLO11, a gene encoding a cell-surface adhesin that has already been implicated in colony morphology, biofilm formation, and invasive and pseudohyphal growth. Using a series of modified promoters for fine-tuning FLO11 expression, we confirm the central role of Flo11 and show that differences in FLO11 expression result in distinct colony morphologies. Together, our results provide a first comprehensive look at the complex genetic network that underlies the diversity in the morphologies of yeast colonies.

Fig. 1

Yeast colony morphology depends on strain background and environment. Different media confer different morphologies in the same background and different strains yield different morphologies in the same media. Strains were grown in media with different carbon sources as described in Experimental procedures. Glu, glucose; Mal, maltose; Gal, galactose; EtOH, ethanol; Gly, glycerol; Suc, sucrose.

Fig. 2

Physical interaction network visualization of genes involved in colony morphology. Genes with mutations resulting in altered colony morphology are mapped onto a physical interaction network as round nodes. To improve clarity, protein complexes containing more than 2 genes are visualized as single coloured octagonal nodes. Small protein complexes containing genes associated with colony morphology are complemented with their corresponding gene members which were not directly associated with altered colony morphology, these are visualized as a parallelogram if the mutation of this gene is lethal, or as a triangle if the mutation resulted in decreased fitness. FLO11 is indicated as a large round yellow node. The edges between the nodes indicate physical interactions and specifically green edges indicate protein–protein interactions, blue phosphorylation interactions, orange de-phosphorylation interactions and red protein–DNA interactions. The direction if applicable for an interaction is indicated with an arrow. Genes with mutations resulting in altered colony morphology which are not connected to other smooth/semi-smooth genes or associated complexes are omitted from this figure.

Fig. 3

Variation of FLO11 levels and colony morphology. A. Strains from single tetrads can also exhibit great variety in colony morphology and gene expression. Top, FLO11 gene expression of KV34, KV35, KV36 and KV37, haploids derived from a single tetrad of EM93 diploid strain. Bottom, corresponding photos of the same strains. Both photos and gene expression levels are of colonies grown on YPS agar medium. Scale bar represents 5 mm. B. De-repressing FLO11 expression increases wrinkliness of a smooth strain. KV34 and KV35 are sister haploid strains derived from the same tetrad of natural isolate strain EM93. KV34 is wrinkly and KV35 is smooth, and this is reflected in the levels of FLO11 expression, with KV34 having higher levels of FLO11. Deletion of SFL1, a repressor of FLO11 expression, raises levels of FLO11 and makes KV35 as wrinkly as KV34. Scale bar represents 5 mm. C. Increasing FLO11 expression correlates with increasing colony wrinkliness. Replacement of the native FLO11 promoter by a series of constitutive promoters of increasing strength results in a series of strains with increasing wrinkliness. The TEF1prm::FLO11 series was made in the EM93 haploid background. Scale bar represents 5 mm. D. Flo11 expression correlates with wrinkliness, but is uniform within wrinkly areas of colony. A FLO11–YFP construct was made that incorporated a self-cleaving viral sequence, such that simultaneous expression of Flo11 and YFP was assured without causing interference of Flo11 function. Wrinkly colonies often spawn variants or mutants with smooth morphologies, which results in smooth sectors in growing colonies (arrow in panel D). These sectors are associated with low Flo11 (low YFP) levels. However, closer inspection of wrinkly parts of colonies shows rather homogenous expression of YFP, suggesting that differential expression of FLO11 does not account for patterned growth within a colony (bottom panel).

Fig. 4

Overview of genes differentially expressed between a flo11 deletion mutant and a wild-type strain grown on liquid and solid medium. The colour of the core of the genes indicates the differential expression of the genes in liquid, while the colour of the border indicates the differential expression on solid medium. Green indicates under-expression and red overexpression of the flo11 mutant compared with the wild type. Overrepresented GO biological process terms were categorized and overlain onto the network as grey shaded areas. Red edges indicate protein–DNA interactions while green edges indicate protein–protein interactions.