Identification of a complex genetic network underlying Saccharomyces cerevisiae colony morphology.

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.

Variable tandem repeats accelerate evolution of coding and regulatory sequences.

Genotype-to-phenotype mapping commonly focuses on two major classes of mutations: single nucleotide polymorphisms (SNPs) and copy number variation (CNV). Here, we discuss an underestimated third class of genotypic variation: changes in microsatellite and minisatellite repeats. Such tandem repeats (TRs) are ubiquitous, unstable genomic elements that have historically been designated as nonfunctional "junk DNA" and are therefore mostly ignored in comparative genomics. However, as many as 10% to 20% of eukaryotic genes and promoters contain an unstable repeat tract. Mutations in these repeats often have fascinating phenotypic consequences. For example, changes in unstable repeats located in or near human genes can lead to neurodegenerative diseases such as Huntington disease. Apart from their role in disease, variable repeats also confer useful phenotypic variability, including cell surface variability, plasticity in skeletal morphology, and tuning of the circadian rhythm. As such, TRs combine characteristics of genetic and epigenetic changes that may facilitate organismal evolvability.

Unstable tandem repeats in promoters confer transcriptional evolvability.

Relative to most regions of the genome, tandemly repeated DNA sequences display a greater propensity to mutate. A search for tandem repeats in the Saccharomyces cerevisiae genome revealed that the nucleosome-free region directly upstream of genes (the promoter region) is enriched in repeats. As many as 25% of all gene promoters contain tandem repeat sequences. Genes driven by these repeat-containing promoters show significantly higher rates of transcriptional divergence. Variations in repeat length result in changes in expression and local nucleosome positioning. Tandem repeats are variable elements in promoters that may facilitate evolutionary tuning of gene expression by affecting local chromatin structure.

FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast.

The budding yeast, Saccharomyces cerevisiae, has emerged as an archetype of eukaryotic cell biology. Here we show that S. cerevisiae is also a model for the evolution of cooperative behavior by revisiting flocculation, a self-adherence phenotype lacking in most laboratory strains. Expression of the gene FLO1 in the laboratory strain S288C restores flocculation, an altered physiological state, reminiscent of bacterial biofilms. Flocculation protects the FLO1 expressing cells from multiple stresses, including antimicrobials and ethanol. Furthermore, FLO1(+) cells avoid exploitation by nonexpressing flo1 cells by self/non-self recognition: FLO1(+) cells preferentially stick to one another, regardless of genetic relatedness across the rest of the genome. Flocculation, therefore, is driven by one of a few known "green beard genes," which direct cooperation toward other carriers of the same gene. Moreover, FLO1 is highly variable among strains both in expression and in sequence, suggesting that flocculation in S. cerevisiae is a dynamic, rapidly evolving social trait.

The morphogenetic regulator Czf1p is a DNA-binding protein that regulates white opaque switching in Candida albicans.

Czf1p has been demonstrated to regulate the switch between the yeast-cell morphology and filamentous morphologies of the human fungal pathogen Candida albicans. The predicted amino acid sequence of Czf1p contains a zinc-cluster motif similar to the DNA-binding domains of proteins such as Saccharomyces cerevisiae Gal4p, suggesting that Czf1p is a DNA-binding protein. Czf1p also demonstrates genetic interaction and a two-hybrid interaction with a second regulator of C. albicans cellular morphology, Efg1p. During growth in contact with an agar matrix, Efg1p has a negative effect on filamentation and Czf1p antagonizes this effect. In addition to regulating cellular morphology, Efg1p plays a role in regulating the cell-type switch between the commonly observed white phase of C. albicans and the opaque, mating-competent phase. While overexpression of EFG1 stimulates the switch from opaque to white, the results reported here demonstrate that overexpression of CZF1 promotes the reverse switch, from white to opaque. We also demonstrate that Czf1p binds CZF1 promoter DNA in vitro. Therefore, for the regulation of both contact-dependent filamentation and white-opaque switching, Czf1p and Efg1p have opposing functions.

Expression of the Candida albicans morphogenesis regulator gene CZF1 and its regulation by Efg1p and Czf1p.

The ability of Candida albicans to transit between different cellular morphologies is believed to be important for virulence. Morphological transitions occur in response to a variety of environmental signals. One such signal is encountered when cells are grown in a semisolid matrix. An important regulator of cellular morphology is the putative transcription factor CZF1. Here we demonstrate that transcription of CZF1 is responsive to growth parameters such as the temperature, carbon source, growth phase of cells, and the physical environment. In wild-type cells, a CZF1 transcript of about 4 kb was expressed when cells were grown embedded in semisolid agar medium, as well as in late exponential phase when cells were grown in liquid medium. Deletion of EFG1, a key regulator of morphogenesis, abolished CZF1 expression. Overexpression of CZF1 revealed that this gene also autoregulates its expression. Efg1p and Czf1p were shown by chromatin immunoprecipitation to act by binding to the promoter of CZF1. The coupling of environmental cues to the expression of a morphogenetic transcription factor may allow C. albicans to coordinate morphogenesis in response to specific conditions encountered in the human host.

Contributions of hyphae and hypha-co-regulated genes to Candida albicans virulence.

The fascinating ability of Candida albicans to undergo dramatic changes in cellular morphology has invited speculation that this plasticity in form contributes to the virulence of the organism. Molecular genetic analyses have confirmed this hypothesis and further demonstrated that genes that govern cellular morphology are co-regulated with genes encoding conventional virulence factors such as proteases and adhesins. The transcriptional regulatory networks of C. albicans thus ensure that hyphae are produced concomitantly with virulence factors, resulting in cells that are adapted for invading the tissues of an immunocompromised host. Hyphae are able to exert mechanical force, aiding penetration of epithelial surfaces, and hyphae damage endothelial cells, aiding escape of C. albicans from the host bloodstream into deeper tissue. Hyphal morphogenesis is thus an integral part of the overall virulence strategy of C. albicans.

Alternative Candida albicans lifestyles: growth on surfaces.

Candida albicans, an opportunistic fungal pathogen, causes a wide variety of human diseases such as oral thrush and disseminated candidiasis. Many aspects of C. albicans physiology have been studied during liquid growth, but in its natural environment, the gastrointestinal tract of a mammalian host, the organism associates with surfaces. Growth on a surface triggers several behaviors, such as biofilm formation, invasion, and thigmotropism, that are important for infection. Recent discoveries have identified factors that regulate these behaviors and revealed the importance of these behaviors for pathogenesis.