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1. Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae

   https://doi.org/10.1093/molbev/msac202  

   Krause, David J ; Hittinger, Chris Todd ( October 2022 , Molecular Biology and Evolution)

   Zhang, Jianzhi (Ed.)

   Abstract The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts. 

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2. Pyruvate decarboxylase and thiamine biosynthetic genes are regulated differently by Pdc2 in S. cerevisiae and C. glabrata

   https://doi.org/10.1371/journal.pone.0286744  

   Iosue, Christine L. ; Ugras, Julia M. ; Bajgain, Yakendra ; Dottor, Cory A. ; Stauffer, Peyton L. ; Hopkins, Rachael A. ; Lang, Emma C. ; Wykoff, Dennis D. ( June 2023 , PLOS ONE)

   Misra, Hari S. (Ed.)

   Understanding metabolism in the pathogen Candida glabrata is key to identifying new targets for antifungals. The thiamine biosynthetic (THI) pathway is partially defective in C . glabrata , but the transcription factor Cg Pdc2 upregulates some thiamine biosynthetic and transport genes. One of these genes encodes a recently evolved thiamine pyrophosphatase ( CgPMU3 ) that is critical for accessing external thiamine. Here, we demonstrate that Cg Pdc2 primarily regulates THI genes. In Saccharomyces cerevisiae , Pdc2 regulates both THI and pyruvate decarboxylase (PDC) genes, with PDC proteins being a major thiamine sink. Deletion of PDC2 is lethal in S . cerevisiae in standard growth conditions, but not in C . glabrata . We uncover cryptic cis elements in C . glabrata PDC promoters that still allow for regulation by Sc Pdc2, even when that regulation is not apparent in C . glabrata . C . glabrata lacks Thi2, and it is likely that inclusion of Thi2 into transcriptional regulation in S . cerevisiae allows for a more complex regulation pattern and regulation of THI and PDC genes. We present evidence that Pdc2 functions independent of Thi2 and Thi3 in both species. The C-terminal activation domain of Pdc2 is intrinsically disordered and critical for species differences. Truncation of the disordered domains leads to a gradual loss of activity. Through a series of cross species complementation assays of transcription, we suggest that there are multiple Pdc2-containing complexes, and C . glabrata appears to have the simplest requirement set for THI genes, except for CgPMU3 . CgPMU3 has different cis requirements, but still requires Pdc2 and Thi3 to be upregulated by thiamine starvation. We identify the minimal region sufficient for thiamine regulation in CgTHI20 , CgPMU3 , and ScPDC5 promoters. Defining the cis and trans requirements for THI promoters should lead to an understanding of how to interrupt their upregulation and provide targets in metabolism for antifungals. 

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3. Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function

   https://doi.org/10.1371/journal.pgen.1011082  

   Fletcher, Jessica ; O’Connor-Moneley, James ; Frawley, Dean ; Flanagan, Peter R. ; Alaalm, Leenah ; Menendez-Manjon, Pilar ; Estevez, Samuel Vega ; Hendricks, Shane ; Woodruff, Andrew L. ; Buscaino, Alessia ; et al ( December 2023 , PLOS Genetics)

   Forche, Anja (Ed.)

   TheCandida albicansgenome contains between ten and fifteen distinctTLOgenes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicanswe deleted all fourteenTLOgenes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in thetloΔ mutant strain compared to the parent, while RNA-seq analysis showed that thetloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, thetloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLOclades resulted in the complementation of the mutant phenotypes, but to different degrees.TLOα1could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLOgenes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression ofTLOβ2in thetloΔmutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegratedTLOgenes in atloΔ/med3Δdouble mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range ofC.albicanscellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.

    

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4. A Novel cis Element Achieves the Same Solution as an Ancestral cis Element During Thiamine Starvation in Candida glabrata

   https://doi.org/10.1534/g3.119.400897  

   Iosue, Christine L. ; Gulotta, Anthony P. ; Selhorst, Kathleen B. ; Mody, Alison C. ; Barbour, Kristin M. ; Marcotte, Meredith J. ; Bui, Lilian N. ; Leone, Sarah G. ; Lang, Emma C. ; Hughes, Genevieve H. ; et al ( January 2020 , G3: Genes|Genomes|Genetics)

   Regulatory networks often converge on very similar cis sequences to drive transcriptional programs due to constraints on what transcription factors are present. To determine the role of constraint loss on cis element evolution, we examined the recent appearance of a thiamine starvation regulated promoter in Candida glabrata . This species lacks the ancestral transcription factor Thi2, but still has the transcription factor Pdc2, which regulates thiamine starvation genes, allowing us to determine the effect of constraint change on a new promoter. We identified two different cis elements in C. glabrata - one present in the evolutionarily recent gene called CgPMU3 , and the other element present in the other thiamine (THI) regulated genes. Reciprocal swaps of the cis elements and incorporation of the S. cerevisiae Thi2 transcription factor-binding site into these promoters demonstrate that the two elements are functionally different from one another. Thus, this loss of an imposed constraint on promoter function has generated a novel cis sequence, suggesting that loss of trans constraints can generate a non-convergent pathway with the same output. 

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5. The Ndr/LATS Kinase Cbk1 Regulates a Specific Subset of Ace2 Functions and Suppresses the Hypha-to-Yeast Transition in Candida albicans

   https://doi.org/10.1128/mBio.01900-20  

   Wakade, Rohan S. ; Ristow, Laura C. ; Stamnes, Mark A. ; Kumar, Anuj ; Krysan, Damian J. ( August 2020 , mBio)

   Kronstad, James W. (Ed.)

   ABSTRACT The r egulation of A ce2 and m orphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans . The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2 , only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae , Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast-phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hypha transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hypha-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hypha-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans . IMPORTANCE The r egulation of A ce2 and m orphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis: suppression of the hypha-to-yeast transition. Very few regulators of this transition have been described, and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2. 

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