Genotypic and Phenotypic Changes in Yeast Related to Selective Growth Pressures Unique to Microgravity

NASA Award # NAG9-1559

The major goal of this project is to investigate how cells adapt to the unique aspects of the space environment (i.e.: microgravity), using the model eukaryotic organism, Saccharomyces cerevisiae.

To mimic some of the elements of microgravity on the ground we utilize a rotating wall culture vessel, a High Aspect Rotational Vessel (HARV). Cells (in media suspension) are inoculated into the HARV in a manner that reduces air bubbles inside the vessel, which can disturb the convection currents. The rotation of the vessel ensures the media suspended cells do not settle over time, but rather are in a continuous state of free-fall. The configuration of the Synthecon Cell Culture Rotary System allows the HARVs to be placed vertically so that the cells rotate around a vertical axis (perpendicular to the ground), thus placing the contents parallel to the gravitational force vector, resulting in a randomized gravitational effect. This randomized effect in combination with the minimized turbulence over the cells (due to the constant state of free-fall) creates the net effect of “functional weightlessness” or “low shear modeled microgravity”. (Klaus et al. 2001)

We have examined the behavior of S. cerevisiae as grown in mimicked microgravity, and have found that the cells themselves do not differ from controls in regards to growth rate (with the exception of a shortened lag phase by 90 minutes), size, shape, or viability. However, yeast cells grown under the influence of microgravity exhibit aberrant budding and cell clumping in contrast to typical bipolar budding found in controls. Significant changes (= 2x) in expression of genes responsible for polarity establishment ( BUD 5), bipolar budding (RAX1, RAX2, and BUD 25), as well as cell separation (DSE1, DSE2, and EGT2) were found. (Purevdorj-Gage, B. 2006 Effects of Low-Shear Modeled Microgravity on Cell Function, Gene Expression, and Phenotype in Saccharomyces cerevisiae . Appl Environ Microbiol 72:4569-4575)

In addition, we have identified, by microarray analysis, a number of genes that either are up or down regulated by at least two fold over the course of time when exposed to microgravity conditions. Cultures were grown and examined at 5 and 25 generations in an effort to determine the ability of the cells to adapt over time to their environment. Of the genes we identified, the top five categories in numerical order were metabolism, unclassified, transcription, protein with binding function, and protein fate. These genes were compared to previously identified sets of genes associated with other Environmental Stress Responses (ESR) by Gash et al.;2000. The comparison of gene sets showed a 26% correlation of LSMMG affected genes to ESR genes. The remaining 74% of the up/down regulated genes under LSMMG may represent a unique response to microgravity. (Manuscript In Press – Sheehan, K. 2006 Yeast Genomic Expression Patterns in Response to Low-Shear Modeled Microgravity)

Over the course of the previous work, we obtained preliminary data that suggested an increase in biofilm formation during growth in the HARVs by S. cerevisiae . This was an exciting finding because S. cerevisiae is not commonly associated with biofilm studies, but would be an excellent eukaryotic model organism due to it's ease of use (non-pathogenic, easy to handle, well characterized genome, and ease of manipulation) in contrast to other commonly used organisms such as Candida albicans . We investigated the role of FLO 11 (MUC1) in S. cerevisiae biofilm development in a flow-cell system based on the findings of Reynolds and Fink in 2001. They reported that FLO 11p was required for the attachment of the cells to liquid-hydrophobic-solid interfaces. We found that cells with increased expression of FLO 11 formed larger cellular aggregates and were able to adhere to not only liquid-hydrophobic, but also liquid-hydrophilic surfaces. (Manuscript in Press – Purevdorj-Gage, B. 2006 The Role of FLO 11 in Saccharomyces cerevisiae Biofilm Development in Laboratory Flow-Cell System)

Future goals of the project include:

A. Examine the localization of BUD 5, RAX1, RAX2, and DSE1 in yeast cells grown under LSMMG conditions vs. control growth by means of GFP tagging.

B. Examine the fitness profile of strains containing deletions in the genes discussed above.

C. Examine the behavior of the genes identified by microarray analysis that are involved in the cell wall integrity sensing pathways.

D. Investigate the growth of C. albicans in HARVs under LSMMG conditions. Determine if biofilm formation and/or pathogenic morphological forms are increased under these altered growth conditions.