Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae
to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity
pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast
cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.
Yeast cells display various differentiation modes according to nutrient availability and environmental conditions, including spore formation under starvation and stress conditions, filamentation under various nutrient stresses, and flocculation. Various yeasts, including S. cerevisiae and C. albicans, can also be found in biofilms formed by a diverse set of microorganisms. Though there is some correlation with filamentation and invasive behavior, it is not clear exactly how filamentation might cause invasion and colonization of surfaces and tissues. Yeast can certainly be found in both vegetative and filamentous forms in biofilms in nature as well as places where they threaten human health, such as catheters and infected human organs. 10-13 In order to understand the signaling pathways utilized by yeasts to infect animals and to participate in harmful and beneficial biofilms, we must develop accessible and reliable assays. Here we have developed an assay, adopted from already existing adhesion and invasion assays available for yeast, which allow us to qualitatively determine the adhesive and invasive phenotypes of yeast strains and mutants in various conditions. The assay presented here eliminates the requirement for streaking yeast cells onto the agar, where the mere action of streaking the agar surface changes the invasive and adhesive qualities of yeast. Digital imaging of especially the invading cells by a microscope allows for semi-quantitative assessment of the degree of invasion and adhesion. Such detection of invasive and adhesive cells is complimentary the single cell agar invasion assay developed by the Sprague lab 9 and can be adapted to do time course experiments.
1. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R. & Lappin-Scott, H. M. Microbial biofilms. Annu Rev Microbiol 49, 711-45 (1995).
2. Elortondo, F. J. P., Salmeron, J., Albisu, M. & Casas, C. Biofilms in the food industry. Food Science and Technology International 5, 25-30 (1999).
3. Keinanen, M. M., Martikainen, P. J. & Kontro, M. H. Microbial community structure and biomass in developing drinking water biofilms. Can J Microbiol 50, 183-91 (2004).
4. Biffinger, J. C., Pietron, J., Ray, R., Little, B. & Ringeisen, B. R. A biofilm enhanced miniature microbial fuel cell using Shewanella oneidensis DSP10 and oxygen reduction cathodes. Biosens Bioelectron 22, 1672-9 (2007).
5. Kim, G. T. et al. Bacterial community structure, compartmentalization and activity in a microbial fuel cell. J Appl Microbiol 101, 698-710 (2006).
6. Kim, J. R., Jung, S. H., Regan, J. M. & Logan, B. E. Electricity generation and microbial community analysis of alcohol powered microbial fuel cells. Bioresour Technol 98, 2568-77 (2007).
7. Picioreanu, C., Head, I. M., Katuri, K. P., van Loosdrecht, M. C. & Scott, K. A computational model for biofilm-based microbial fuel cells. Water Res 41, 2921-40 (2007).
8. Singh, R., Paul, D. & Jain, R. K. Biofilms: implications in bioremediation. Trends in Microbiology 14, 389-397 (2006).
9. Cullen, P. J. & Sprague, G. F., Jr. Glucose depletion causes haploid invasive growth in yeast. Proc Natl Acad Sci U S A 97, 13619-24 (2000).
10. Gimeno, C. J., Ljungdahl, P. O., Styles, C. A. & Fink, G. R. Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, 1077-90 (1992).
11. Blankenship, J. R. & Mitchell, A. P. How to build a biofilm: a fungal perspective. Curr Opin Microbiol 9, 588-94 (2006).
12. Reynolds, T. B. & Fink, G. R. Bakers' yeast, a model for fungal biofilm formation. Science 291, 878-81 (2001).
13. Verstrepen, K. J. & Klis, F. M. Flocculation, adhesion and biofilm formation in yeasts. Mol Microbiol 60, 5-15 (2006).
14. Liu, H., Styles, C. A. & Fink, G. R. Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262, 1741-4 (1993).
15. Madhani, H. D. & Fink, G. R. The control of filamentous differentiation and virulence in fungi. Trends Cell Biol 8, 348-53 (1998).
16. Mosch, H. U., Kubler, E., Krappmann, S., Fink, G. R. & Braus, G. H. Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae. Mol Biol Cell 10, 1325-35 (1999).
17. Mosch, H. U., Roberts, R. L. & Fink, G. R. Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 93, 5352-6 (1996).
18. Pan, X. & Heitman, J. Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19, 4874-87 (1999).
19. Roberts, R. L. & Fink, G. R. Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev 8, 2974-85 (1994).
20. Robertson, L. S. & Fink, G. R. The three yeast A kinases have specific signaling functions in pseudohyphal growth. Proc Natl Acad Sci U S A 95, 13783-7 (1998).
21. Reynolds, T. B., Jansen, A., Peng, X. & Fink, G. R. Mat formation in Saccharomyces cerevisiae requires nutrient and pH gradients. Eukaryot Cell (2007).