The goal of this project is to understand how closely related fungal species have changes in their genome specific to nutrient starvation, and how those subtle genomic differences allow for changes in each species' behavior. This work studies the thiamine signal transduction (THI) pathway in the yeast species Candida glabrata, Saccharomyces cerevisiae, and other related species, to determine if pathway function is predictable from genomic sequence. Thiamine (vitamin B1) is required by all living organisms to metabolize sugars, is synthesized de novo in most microorganisms, and it is a dietary requirement in all animals. In this project, researchers at Villanova University, including undergraduate and Master's students, will characterize in detail how vitamin B1 is synthesized in various yeast species, with a focus on two species - Saccharomyces cerevisiae (or budding yeast) and Candida glabrata. Using this detailed characterization, the investigators will then use this information to develop predictions as to how uncharacterized yeast species will behave in response to thiamine starvation. This project will educate and motivate the next generation of scientists by training them in modern molecular biological techniques. Undergraduates working on this project will, on average, spend over two and a half years working on their research, will take responsibility for their projects and present their data at professional meetings and in scientific journals, giving them a rigorous foundation to continue STEM studies after graduation. In the long-term, because thiamine is essential for growth, gaining a deep understanding of how thiamine is made and acquired will be critical for the development of interventions to prevent fungal growth.
C. glabrata requires thiamine to be supplied for growth, unlike many yeast species that can synthesize it de novo. During thiamine starvation, S. cerevisiae upregulates ~10 genes (called THI genes) using the transcriptional regulators Thi2, Thi3, and Pdc2. C. glabrata lacks 1) one half of the thiamine biosynthetic pathway, 2) the ancestral phosphatase required for thiamine recycling, and 3) the conserved transcription factor Thi2. Because these species have differences in how they acquire and synthesize thiamine, a detailed understanding of the THI pathway in both species is required to use genomic sequence to predict behaviors of uncharacterized, sequenced yeast species, and to understand the evolution of the THI pathway more generally. This project will identify DNA elements essential for the thiamine starvation response using promoter truncation and promoter fusion experiments. After identification of TREs (or thiamine responsive elements), the researchers will determine whether these TREs are sufficient for thiamine starvation regulation; will use a SEL-seq approach to identify the sequence determinants of the TREs; use ChIP-seq to confirm Pdc2 binding to the TREs; and determine the commonalities of regulation with the two species S. cerevisiae and C. glabrata. After a detailed characterization of these two species, the researchers will reconstitute the C. glabrata pathway into S. cerevisiae (and the reverse - the S. cerevisiae THI pathway into C. glabrata). This reconstitution will allow investigators to validate that all components necessary for regulation of THI genes have been identified, and to test the predictive power of the results in other evolutionarily related species (focusing on the Nakaseomyces clade) where only DNA sequence is known. When this work is completed, researchers should be able to predict which yeast species 1) can synthesize thiamine, 2) are altered in their ability to recycle thiamine, and 3) may have come to other evolutionary solutions to surviving when thiamine is not present.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
