Laboratory evolutions lead to reproducible mutations in PDR3 conferring resistance to MCHM

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Ayers, Taizina Momtareen, Dionysios Patriarcheas, Liam McCarthy, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4548300/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Discover Genetics and Evolution → Version 1 posted 4 You are reading this latest preprint version Abstract The solubility of protein complexes and membraneless compartments is maintained by liquid-liquid phase separation (LLPS). Phase transition is induced or dissolved by biological hydrotropes such as ATP and RNA. 4-methylcyclohexane methanol (MCHM), an alicyclic alcohol, is a synthetic hydrotrope that induces a starvation response by upregulation of biosynthetic pathways despite the availability of nutrients. To investigate how cellular metabolism can tolerate changes in LLPS, we evolved eight MHCM-resistant strains of S. cerevisiae . We identified thousands of SNPs and indel variants per strain, which was a consistent number between strains that evolved resistance and control strains that remained sensitive. These variants did not show a pattern that would cluster resistant strains together. The many background mutations likely masked any pattern from few large-effect loci or implicated an epistatic effect of many small mutations spread throughout the genome that was undetectable. Among coding variants in the strains that change protein sequence and thereby may alter function, only one gene showed a protein-coding mutation in every resistant strain while showing no variants at all in the control strains. This gene, PDR3 , controls transcription for the pleiotropic drug response and is the most significant driver of adaptive MCHM resistance in yeast. While many of the evolved alleles of PDR3 would likely produce functional proteins, a knockout in the parent YJM789 strain was sufficient to produce resistance to MCHM. Normal catabolism of amino acids uses the Pleiotropic Drug Response (PDR) pathway to export breakdown products. The pdr3 resistance is mediated through Med15, a component of the Mediator complex which regulates activation by transcription factors of RNA pol II. Pdr3 can homodimerize or dimerize with Pdr1, another transcription factor and loss of Pdr1 also confers MCHM resistance. Knockouts of other mutated genes in flocculation, glutathione, SAM, and sugar transport mildly affected growth in the ancestral strain. Mutations in PDR3 are first known to increase resistance to this novel hydrotropic chemical. In-Lab Evolution evolution MCHM Saccharomyces cerevisiae PDR3 MED15 pleiotropic drug response hydrotrope cell wall transporters Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Hydrotropes, such as ATP and RNA, increase the solubility of organic compounds by inducing liquid-liquid phase condensates. Hydrotropes are not classified as detergents because detergents are effective at lower concentrations for solubilizing compounds. MCHM acts as a hydrotrope in vitro and prevents protein aggregation (Pupo et al. 2019a ). In contrast to ATP (Patel et al. 2017 ; Hayes et al. 2018 ; Kang et al. 2018 ) and RNA (Lin et al. 2015 ), MCHM is not readily metabolized in the cell and can serve as a model to study the effect of hydrotropes on biological systems. MCHM is a cyclic hydrocarbon with saturated bonds that are difficult to break. MCHM is an exotic hydrotrope, to which yeast are not ordinarily exposed in nature. MCHM induces G1 arrest (Ayers et al. 2020 ) and amino acid biosynthesis (Pupo et al. 2019b ). Intrinsically disordered regions (IDR) of proteins frequently drive liquid-liquid phase separation (LLPS). These IDRs may represent a more general mechanism to increase the local concentration of proteins within condensates, adding complexity to regulating cellular metabolism and environmental responses. By their nature, structures of intrinsically disordered regions are difficult to determine but are important for changes in interacting with other proteins within protein complex conformations (Patel et al. 2017 ; Cho et al. 2018 ; Sabari et al. 2018 ; Boehning et al. 2018 ; Boija et al. 2018 ). Notably, proteins involved in transcription also have IDRs. Med15 contains three IDRs of variable length and differences in IDR length contribute to sensitivity to MCHM (Gallagher et al. 2020 ). Med15, a component of the tail subcomplex of the Mediator directly interacts with numerous transcription factors and forms protein condensates with Gcn4 (Boija et al. 2018 ). Changes in the conformation of these proteins can profoundly increase phenotypic diversity by their effect on the protein composition of translation regulation machinery. Recent work has produced genomic datasets to determine the cellular pathways involved in MCHM response. MCHM, as a hydrotrope that affects protein folding, has been implicated to have a role in zinc ion concentration changes in the cell to alleviate this stress (Pupo et al. 2019a ). Lipid biosynthetic changes induced by MCHM may also be a source of stress on the plasma membrane (Pupo et al. 2019b ). MCHM also causes amino acid accumulation according to metabolomic data, while exhibiting a nutrient starvation signal activating the environmental stress response (Pupo et al. 2019b , a ; Ayers et al. 2020 ). One early study using stress gene reporters showed oxidative stress and DNA damage response activation in response to MCHM and its potential metabolites (Lan et al. 2015 ). The more recent work using yeast genomic approaches confirmed that MCHM causes the production of reactive oxygen species (ROS) with a small percentage of yeast cells in a population, as well as DNA damage, making yeast strains sensitive to ROS, such as petite yeast, unable to grow (Lan et al. 2015 ; Pupo et al. 2019b ; Ayers et al. 2020 ). These findings highlight the diverse cellular pathways important for MCHM tolerance. In-Lab evolution (ILE) of yeast is a tool that allows for the genetic analysis of many phenotypes and processes of evolution. The short generation time of yeast allows for the quick production of large populations that accumulate mutations that may respond to selection or drift in the lab. This tool has been used to experimentally observe evolution in industrial environments with yeast hybrid strains, determining whether they evolve similarly in terms of aneuploidy and copy number to historical lager brewing yeast hybrids (Gorter De Vries et al. 2019). ILEs have also been utilized with wine strains, where ploidy events control significant changes in fermentation efficiency (Mangado et al. 2018 ). Experiments also explore processes such as yeast and bacterial populations in competition and yeast populations that converge towards phenotypes with similar fitness due to epistatic interactions of beneficial mutations (Kryazhimskiy et al. 2014 ; Zhou et al. 2018 ). To understand stress resistance, ILE has been used to produce yeast adapted to a multitude of stressors. Adaptation to copper has been produced in Saccharomyces cerevisiae and Candida humilis , revealing mechanisms such as overexpression of superoxide dismutases and catalases to detoxify ROS and overexpression of proteins that bind excess copper (Adamo et al. 2012 ). Thermotolerance was also evolved in yeast stains to explore mechanisms that could improve industrial biomass to ethanol conversion processes. Cellular changes involving sterol components of the membrane and concentrations of glycerol also provided increased tolerance to stresses from osmolarity and excess glucose and ethanol (Caspeta and Nielsen 2015 ). The evolution of resistance to a glyphosate-based herbicide in the lab involves copy number changes in proteins affecting cell wall stability in response to the presumably inert non-glyphosate additives in the herbicide (Ravishankar et al. 2020b ). ILE experiments are an unbiased and insightful method for understanding specific mechanisms and processes of adaptation to environments. Yeast cells use the pleiotropic drug response to detoxify and remove a multitude of general xenobiotics and chemical stressors. Much of the work of this response is performed by a highly conserved class of proteins called ATP-binding cassette (ABC) transporters that translocate chemicals out of the cell (Jungwirth and Kuchler 2006 ). Some important stress-responsive yeast ABC transporters include Pdr5, Snq2, Pdr15, and Yor1, all of which have roles in exporting drugs or various chemically unrelated toxic chemicals out of the cell in response to stress (Servos et al. 1993 ; Balzi et al. 1994 ; Wolfger et al. 1997 ; Rogers et al. 2001 ; Jungwirth and Kuchler 2006 ; Tsujimoto et al. 2015 ). The transcriptional control of the genes encoding these proteins involves the interplay of the transcription factors Pdr1 and Pdr3, two paralogs sharing 36% amino acid sequence similarity along the entire length of the proteins (Delaveau et al. 1994 ). These proteins form heterodimers and homodimers that compete to occupy cis- regulatory elements termed PDREs upstream of pleiotropic drug response genes (Katzmann et al., 1996 ; Mamnun et al., 2002 ). Their interaction in different combinations and on PDREs of different genes likely plays a role in the relative inhibition and activation of these same genes (Mamnun et al., 2002 ). The role of the ABC transporters in a cell’s response to chemical stressors is important for evolved stress adaptation. In particular, induced mutagenesis experiments on PDR3 have produced activated gain-of-function alleles where Pdr3 increases the expression of PDR5 and SNQ2 and increases resistance to a multitude of chemicals (Nourani et al., 1997 ; Simonics et al., 2000 ). The role of the ABC transporters in the response to chemical stress makes them and the proteins that control the expression drug responsive genes targets for adaptation to chemicals such as MCHM. In this study, we utilized In-Lab evolutions to produce strains adapted to the hydrotrope MCHM. The goals of the study were to identify mechanisms as yet uncovered in the cellular response to MCHM as well as explore the evolutionary process under novel stress that yeast were unlikely to have encountered in their environmental niche. Eight strains evolved in MCHM became resistant to high dosages of the chemical, while eight control strains evolved under similar conditions without MCHM remained sensitive. All sixteen strains accumulated similar amounts of mutations overall. There were no clear patterns in mutations that led to resistance, except for variants appearing in the gene PDR3 . The mutations in PDR3 were the major reproducible drivers of resistance to MCHM mediated by interactions with Med15. Other mutations spread across the genome and pathways partly contributed to the resistance but could also contribute to convergent phenotype through epistatic interactions of small effects. Materials and Methods In-lab evolutions The YJM789 strain (MATalpha, lys2 ∆) (Tawfik et al. 1989 ; McCusker et al. 1994 ; Wei et al. 2007 ) was grown in biological quadruplicate in 2mL liquid cultures of YPD (1% yeast extract, 2% peptone, 2% dextrose) media with and without 700ppm crude MCHM at 30C. Cultures were grown for two days before 1% of the culture was used to inoculate fresh tubes containing 2mL of YPD or YPD + 700ppm MCHM media. This passaging process was performed for six times before plating serial dilutions onto YPD plates containing ranges of 0-1000ppm MCHM. Two colonies were isolated from each final evolved population. The isolates were grown for 2–3 passages in liquid YPD without MCHM to allow for removal of epigenetic memory of resistance that may impact growth on MCHM media. The resistance of isolated strains were retested on 1000ppm MCHM solid. Following the epigenetic check, genomic DNA was then isolated from the strains and submitted for sequencing at the WVU Genomics Core (Ravishankar et al. 2020b ). Sequencing and analysis Strains were sequenced on an Illumina MiSeq producing 151bp paired-end reads of between 1,638,426 and 3,445,498 reads per strain. Sequences are available at Genbank (BioProject ID PRJNA1111078) and are available at http://www.ncbi.nlm.nih.gov/bioproject/1111078 . Reads were aligned to the S288c reference genome R64.2.1 release obtained from yeastgenome.org (Engel and Cherry 2013 ) using BWA version 0.7.17-r1188 (Li and Durbin 2009 ), giving coverage of approximately 20-30x across the genome for each strain. SAM and BAM files were created using samtools version 1.7 (Li and Durbin 2009 ; Li 2011 ). Variants were called using the HaplotypeCaller function of GATK 4.1.6.0 (Van der Auwera et al. 2013 ) on Java Runtime Environment 11.0.6. Variants were filtered by removing all variants that did not pass GATK quality measures. As YJM789 has approximately 60,000 SNPs (Wei et al. 2007 ) when compared with the S288c reference, these variants were removed by filtering all variants that were shared amongst all 16 evolved strains (control and MCHM) and using an existing SNP list for the strains. Any variants shared by all 16 strains were assumed to be existing variation between S288c and YJM789, as opposed to random mutations that happened in every sample. The 6874 remaining variants in the 16 strains were analyzed for presence in coding regions and the intersection between strains as below. The numbers of variants per strain in the 8 control strains and 8 MCHM strains were compared with a two-tailed Student’s T-test to determine if there was any difference in total number of variants between resistant phenotypes. Variants were analyzed further using R version 3.6.3 and the following packages: VariantAnnotation version 1.32.0(Obenchain et al. 2014 ), pcadapt 4.3.1 (Luu et al. 2017 ), BSgenome.Scerevesiae.UCSC.sacCer3_1.4.0 (Huang et al. 2009a , b ; Team 2014 ; Pagès 2019 ), TxDb.Scerevisiae.UCSC.sacCer3.sgdGene_3.2.2 (Carlson and Maintainer 2015 ), and all dependencies are available in Supplemental Table 1. In short, the 6800 variants that remained following filtering were analyzed for PCA clustering analysis using the pcadapt package with a k = 3 based on screenplot. Variants then were filtered to remove all those found in any of the control strains, regardless of presence in the MCHM strains (Supplemental Table 2). This left 1171 variants existing only in at least one of the 8 MCHM evolved strains. These variants were analyzed for their effect on coding sequences using the predictCoding command of the VariantAnnotation package and the UCSC sacCer3 genome and transcript packages mentioned above. There are three protein/ORF annotation differences between the sacCer3 version of the genome and the S288c R64.2.1 version of the genome used to map the original sequences and call variants that did not affect the coding predictions of this dataset. The SIFT 4G variant annotator software was also used to determine the predicted tolerance score for each variant from the original ILE vcf files (Vaser et al. 2016 ). The Saccharomyces cerevisiae R64-1-1.23 database included with the SIFT4G jar was used to annotate the variants. GO terms and intersection of strain variants: GO terms for the 107 genes that contained nonsynonymous and synonymous variants in the MCHM evolved strains were determined using the DAVID bioinformatic database at https://david.ncifcrf.gov (Huang et al. 2009a , b ). Default options were used for the analysis. The intersection analysis of strains containing the same genes was performed using the shiny app tool version of UpSetR at https://gehlenborglab.shinyapps.io/upsetr/ (Lex et al. 2014 ). Strain construction The YJM789 PDR3 :: NAT knockout was produced by transforming PCR construct containing the NAT R gene amplified from the pAG25 plasmid (Goldstein and McCusker 1999 ). Other knockout cassettes of genes were amplified from the BY4742 knockout collection using the G418 R or amplified from pAG32 plasmid (HYG R ). Transformations were performed using lithium acetate, as previously described (Gietz and Schiestl 2007 ). Colony PCR confirmation of the knockout cassette integration was performed. Serial dilution growth assays for resistance phenotyping of the knockout were performed on solid MCHM YPD media as above and compared to growth of the BY4742 knockout collection knockout of PDR3 as well (Brachmann et al. 1998 ; Giaever et al. 2002 ). Reciprocal hemizygosity assay used YJM789 pdr3::Nat R and BY4741 pdr3::Kan R and their respective parents to test the impact of the PDR3 allele on growth on MCHM (Rong-Mullins et al. 2017 ). The YJM789 pdr3::Nat R was crossed with YJM789K5a med15::Hyg R and the diploid was sporulated (Winans et al. 2020 ). Tetrads were dissected and haploid double mutants selected based on markers. ROS assay Overnight cultures of the strains YJM789 WT, YJM789 pdr3 , S12, and S12 gex2 were grown in YPD medium, then diluted and grown to log phase (OD 600 0.4). The log phase cultures were further diluted to OD 600 0.2 and 100ul of cells were added into each well of a flat transparent 96-well plate. This was followed by adding 100ul YPD for the negative control group, and 100ul of MCHM (800ppm, 1000ppm, and 1200ppm) for the experimental groups. For the positive control, 100ul of the log phase cultures were treated with 4mM H 2 O 2 for 30 min and then added to a well containing 100ul YPD. Each strain under each condition was performed in triplicate. The plate was then incubated at 30°C for 22 hours, with absorbance measurements taken every hour using the TECAN Infinite M Nano plate reader. ROS levels were quantified using the H2DCF-DA assay as previously described (James et al. 2015 ). The plate was centrifuged to remove the media, followed by two washes with 100 µL mixture of sorbitol (1.2 M), EDTA (50 mM), and mercaptoethanol (2%). 100ul of lyticase (25 U/ml) was added to each well and incubated at 37°C for 30min. This was followed by another round of centrifugation to remove the lyticase solution. Next, 100 µL of the H2DCF-DA solution was added, gently pipetted up and down and then placed in the incubator, covered with foil, for 30 minutes. After this incubation, the plate was centrifuged, the liquid was discarded, and the resulting pellets were resuspended in 100 µL of 1x PBS by pipetting. ROS levels were measured at OD 504nm using the TECAN plate reader. Invasion Assay Yeast were serially diluted and then grown for seven days. Cells were then washed off under running water with a gentle circular pressure from a gloved finger. Structural Modeling Prediction of the impact of SNPs and variants on Pdr3 structure was performed by generating de novo structure predictions using the RoseTTAFold method in the Robetta server (Kim et al. 2004 ). Viability Assay The viability of YJM789 WT, pdr3 , and pdr1 strains was determined using the Nexcelom Cellometer X2. Cells were cultured in YPD flasks on a shaker for 12 days, with viability readings recorded every third day using the Nexcelcom ViaStain ™ viability kit. Elemental Analysis Four biological replicates were treated with 650 ppm of MCHM for 30 minutes and then cells were digested and elements analyzed as previous carried out (Ravishankar et al. 2020a ; Winans and Gallagher 2020 ). Results and Discussion In-lab evolution of YJM789 in MCHM Previous studies on the effects of MCHM on yeast used genetic knockouts, transcriptomics, metabolomics, and biochemical assays to determine that MCHM resistance is dependent on a complex interaction of many cellular and genetic networks (Pupo et al. 2019b ; Gallagher et al. 2020 ; Ayers et al. 2020 ). To expand upon this knowledge, resistant strains were developed through In-Lab Evolution (ILE) experiments to help elucidate genetic changes that might produce a new resistant genotype. There is considerable genetic variation across different yeast strains to MCHM (Pupo et al. 2019b ). Previously a mapping population between S96 (an S288c laboratory strain) and YJM789 (a clinical isolate) uncovered standing genetic variation that is linked to MCHM response (Pupo et al. 2019b ). S96 is more MCHM resistant than YJM789 and we used these strains to select for increased MCHM resistance. Four replicates of each strain were evolved in rich liquid YPD media with and without 700ppm MCHM (Fig. 1 A). The lineages of the original eight samples (four control, four treated) were passaged every two days for a total of six passages. Control and treated lineages were plated after the sixth passage, and two single colonies were isolated from each lineage to produce eight total control strains and eight total treated strains. After six passages, the MCHM treated strains grew faster than the earlier passaged. However, this was only seen in the YJM789 cultures, whereas MCHM resistance in S96 strains did not develop after multiple attempts (not shown). S96 was already more MCHM resistant than YJM789 and further resistance could not be achieved in the ILEs. We therefore continued with YJM789 strains. These YJM789 strains were named S1-16, with S1-8 being the control strains (and S1 and S2 being from the same evolution experiment), and S9-16 being the treated strains (and S9 and S10 being from the same original lineage). The sixteen strains were tested for their MCHM resistance at 1000ppm on YPD media. The eight control strains showed wildtype levels of resistance, while all eight treated strains showed increased resistance as compared to wildtype (Fig. 1 B). There was plate to plate variation in MCHM at 1000ppm, likely due to nearing the solubility limit of the chemical (Sain et al. 2015 ; Phetxumphou et al. 2016 ). The wildtype strain was plated on each plate to control for plate to plate variation (Fig. 1 B). Principal component analysis of mutations After successfully evolving resistant strains from YJM789 yeast, the sixteen samples were sequenced at approximately 25x coverage using Illumina paired-end reads. The reads of all 16 strains were mapped to the S288c reference genome and variants were called using GATK HaplotypeCaller. After removing the variants shared between all 16 strains (Supplemental Table 2), considered to be the existing variants between the reference genome and the YJM789 genetic background, variants were clustered on three principal components (Fig. 2 A-C). To analyze whether there may be a general pattern of evolved resistance in the remaining variants, a PCA analysis was performed. However, the strains did not cluster according to resistance on any of these components. As an aside, they also did not cluster by original lineage, so none of the pairs (individual isolates from the same population), such as S1 and S2 or S9 and S10, clustered. This indicates the colonies isolated from the same cultures were genetically distinct. A large number of remaining background variants, approximately 6900 (Table 2 ), likely prevented clustering by resistance phenotype. A few high effect loci would be masked by large numbers of non-causative variants. Similarly, resistance spread among many lower effect loci spread throughout the genome would also be masked if resistance in each strain was due to different combinations of these small effects. In total, looking at the sum of variants produced by the in-lab evolution conditions did not produce clear patterns of resistance. The mutation patterns of the MCHM and control strains showed that there was no significant difference in the number of variants in control vs. MCHM ILEs (p = 0.15) and the evolved strains had between 2326 and 2681 variants per strain (Table 1 ). Although MCHM is known to induce DNA damage (Ayers et al. 2020 ), which could possibly be a mechanism for mutation, the control strains accumulated similar numbers of mutations. Therefore, it likely played a minor role other than a selective pressure on mutations occurring normally in the growth conditions. The total number of mutations does not seem to be from any effect of the chemical on the number of mutations. There was no copy number variation noted in resistant strains. Table 1 Summary of mutated genes in ILE strains. This table contains the number of overall mutations and mutated genes in each ILE strain. Ty retrotransposon genes such as Gag/Pol domains were excluded from this analysis. Strain Total mutations (6874 total) MCHM specific mutations only (1171 total) Mutations in coding regions – Excluding Ty Genes with mutations (107) Non-synonymous mutations only Genes with Non-synonymous mutations (75) S9 2579 212 54 25 33 16 S10 2496 187 63 28 31 18 S11 2493 180 43 24 20 16 S12 2415 187 53 25 28 16 S13 2512 189 59 33 35 23 S14 2475 170 64 33 42 20 S15 2572 180 53 24 34 18 S16 2326 170 45 25 25 15 Filtering MCHM evolved variants to those involved in coding sequence changes To find patterns of mutations that contributed to MCHM resistance, mutants in both the control ILE and the MCHM resistant strains were assumed to be passenger mutations and not causative for the MCHM resistance phenotype. These mutations in the control ILE strains were filtered from the variants in the eight resistant ILEs. This left 1172 variants between the eight treated ILE strains (Table 1 ). Many of these variants were in non-coding regions of the genome and while they could contribute to resistance to MCHM through changes in expression levels of genes, mutations in intergenic regions were filtered out, leaving 485 variants in genes between the eight strains (Table 1 ). The final filter used to narrow the focus to those that may be causative of resistance was to remove variants in Ty retrotransposon Gag/Pol proteins. The YJM789 strain does not contain the same Ty complement as the S288c reference strain (Wei et al. 2007 ). Therefore, the detection of these variants is likely due to the existing variation between the parent strain of the ILEs and the reference. The persistence of these variants, despite the prior utilization of filters to remove all variants shared between all 16 ILEs, as well as a filter for known SNPs in YJM789, can be attributed to the limitations of 25x coverage. Specifically, short-read sequencing is often unable to detect repetitive regions and large deletions. After removing Gag/Pol Ty retrotransposon detected variants, there were between 45 and 64 variants per strain in the eight strains, contained in 107 total genes (Table 1 ). Finally, limiting our analysis to nonsynonymous coding changes left between 20 and 42 variants per strain, contained in only 75 genes (Table 1 ). SNPs were concentrated in the subtelomeric regions (Supplemental Fig. 1). In order to analyze the intersection of these 75 genes with nonsynonymous changes between strains, UpSetR software was utilized (Lex et al. 2014 ). Each strain contained nonsynonymous variants in three to seven genes that were exclusive to that individual strain (Fig. 3 A, Supplemental Table 3). This accounted for 42 of the genes containing variants in the MCHM evolved strains, so more than half overlap of genes with variants were in multiple strains. An additional 18 genes contained variants in two strains. The remaining 15 genes contained variants within at least three strains, but only one gene was found to have a nonsynonymous variant in all eight strains (Fig. 3 A). This gene was PDR3 (Fig. 3 A), a transcription factor that primarily controls the expression of ABC transporters involved in the pleiotropic drug response which exports chemicals from the cell that are involved in general stress (Delaveau et al. 1994 ). When the intersection of all 107 genes containing nonsynonymous or synonymous variants was analyzed, PDR3 remained the only gene with a variant in all eight MCHM ILE strains (Fig. 3 , Table 2 ). MCHM is known to act as a hydrotrope (Pupo et al. 2019b ), so variants that may stabilize the plasma membrane or cell wall could be likely resistance mechanisms. Despite consistency with previous work, these patterns also have issues that reduced confidence that they were causative variants. In particular, the variants included synonymous mutations and genes that were accumulating mutations in control strains as well. Synonymous mutations do not change protein sequence and are unlikely to significantly affect the function of that protein unless altering expression levels. The plentiful mutations in the same genes in control strains may indicate that the genes are hotspots for mutation accumulation, reducing the likelihood any particular mutation was found due to selection for a competitive advantage in MCHM. However, based on the QTL study previously done in MCHM, associative loci that failed to reach LOD score significance were spread relatively evenly throughout the genome (Pupo et al. 2019b ). This indicates that the presence of small effect loci contributing to a combined pleiotropic effect on resistance phenotype is likely outside mutations in PDR3 which will be discussed in more detail below. These variants may be contributing small phenotypic effects even if they are not sufficient to produce resistance on their own. Table 2 Summary of genes containing unique variants in at least four MCHM ILE strains . Uncharacterized ORFs have no annotated function in yeast but may produce functional proteins. Dubious ORFs are unlikely to produce functional proteins. The last two columns show how many MCHM, and control ILE strains contain any variant at all, including variants that appear in both MCHM and control strains. These columns are included to show to what extent this gene seems to accumulate variants overall. Total number of variants is in parentheses in the last two columns, as some strains contain multiple variants in that gene. Gene Name MCHM ILEs Containing Unique Variant(s) in this Gene ORF Character-ization Chr Start End MCHM ILEs - any Variant [genes (variants)] Control ILEs - any Variant [genes (variants)] AGP3 4 Verified chrVI 17004 18680 4 (13) 3 (10) COS4 5 Verified chrVI 6426 7565 7 (36) 6 (22) FLO1 4 Verified chrI 203403 208016 8 (187) 8 (280) HPF1 4 Verified chrXV 28703 31606 8 (96) 8 (111) HXT13 4 Verified chrV 21537 23231 8 (61) 8 (60) HXT17 5 Verified chrXIV 772657 774351 6 (16) 4 (5) HXT9 5 Verified chrX 19497 21200 7 (13) 5 (19) PAU24 4 Verified chrII 809057 809419 7 (24) 7 (10) PDR3 8 Verified chrII 217470 220400 8 (8) 0 (0) SAM3 4 Verified chrXVI 22938 24701 7 (20) 8 (29) TPO2 5 Verified chrVII 763762 765606 5 (6) 0 (0) YAL069W 7 Dubious chrI 335 649 8 (91) 8 (97) YCR108C 6 Uncharacterized chrIII 315997 316188 8 (36) 8 (26) YEL077C 4 Uncharacterized chrV 264 4097 8 (44) 8 (63) YEL077W-A 4 Dubious chrV 630 1112 5 (16) 3 (7) YER188C-A 4 Uncharacterized chrV 569608 569907 8 (68) 8 (56) YNL018C 4 Uncharacterized chrXIV 599936 601774 8 (67) 8 (64) YNL034W/ PAR1 Uncharacterized chrXIV YNL337W 4 Dubious chrXIV 7165 7419 7 (18) 6 (12) To determine if mutations in any cellular process might be important for evolved MCHM resistance, the 107 genes containing at least one variant in at least one MCHM ILE strain, including synonymous variants, were analyzed via GO Term analysis (Fig. 3 B). Most GO terms pointed to mutations in plasma membrane (FDR = 0.0096%) or cell wall proteins (FDR = 5.85 x 10 − 5 %), including those important for sugar transport or flocculation (Fig. 3 B). In agreement with previous genomic work with MCHM, the vacuole (FDR = 0.21%) and response to stress (FDR = 0.19%) also contained mutations that may impact resistance (Ayers et al. 2020 ). To identify mutations that directly contributed to MCHM resistance, we focused on genes that contained mutations in multiple isolates. There were 18 genes with at least one synonymous or nonsynonymous variant not found in control strains within their coding regions in four or more MCHM ILEs (Table 2 ). However, many of these genes also seem to accumulate a high number of mutations in the rich liquid media culture environment. This is exemplified by FLO1 , where all sixteen strains contained variants, 467 variants total. Some genes in the list accumulate more variants, possibly through their position near the ends of chromosomes where mutation rates are known to be higher (Lang and Murray 2011 ; Agier and Fischer 2012 ; Ivanova et al. 2020 ) and/or reduced selective pressure on their function in laboratory growth media. Only two genes showed no mutations in any of the eight control ILE strains while also showing mutations in at least half of the MCHM ILEs, PDR3 , and TPO2 . The list included seven uncharacterized or dubious ORFs and were mostly encoded at the ends of their respective chromosomes (Table 2 ). The unknown or nonexistent function of these genes made them unlikely candidates for adaptive resistance mechanisms. The remaining genes’ functions varied, but most were membrane or cell wall proteins ( FLO1, HPF1, PAU24 ), often involved in sugar or nutrient transport ( AGP3, HXT9, HXT13, HXT17, TPO2 ). These cell wall genes, as well as stress response genes such as PDR3 , may be situated as targets of adaptive changes for MCHM resistance due to their changes that may decrease the internal cellular concentration of the chemical. Prediction analysis for the tolerance of protein-coding changes from variants in MCHM ILEs Because there were thousands of variants per strain, including dozens of variants in coding regions of each MCHM ILE, all mutants unique to the MCHM resistant strains were scored either as deleterious or well tolerated for protein function. SIFT analysis predicts whether amino acid substitutions from single nucleotide polymorphisms will be tolerable for protein function using multiple sequence alignment of homologous protein sequences (Vaser et al. 2016 ). The variants in our dataset included both SNPs and indels, but indel variants could not be scored, because SIFT exclusively analyzes SNPs. CNVs were analyzed but not detected in the evolved strains. Most of the variants were predicted to be tolerated (Table 3 and Supplemental File 2). There are two reasons this may be the case. First, many of these variants are synonymous with coding changes, therefore SIFT analysis aligns proteins and predict no deleterious effect based on conservation. That does not eliminate the possibility that the proteins’ expression levels change due to codon bias changes (Letzring et al. 2010 ; Presnyak et al. 2015 ), but these synonymous changes are less likely to be the major drivers of resistance change than protein sequence changes. The second reason most of these variants would be tolerated is selective pressures to keep conserved functions of proteins with nonsynonymous changes. The nonsynonymous changes would be more likely to replace similar amino acids that do not disrupt protein function, or in regions of the protein less likely to disrupt function. Variants in this dataset that were predicted to be tolerated were deemed less likely to be causative of the evolved resistance to MCHM. The deleterious and NA scored variants all include mutations that change the protein sequences. A variant given a score of NA (not applicable) was usually an indel variant that could not be scored, but occasionally was a SNP variant that did not appear in the SIFT database. Many of the deleterious and NA scored variants appear in proteins that are uncharacterized or dubious ORFs (Supplemental File 1). Notably, the variants in PDR3 in all eight MCHM ILEs received a score of NA. The only pattern of genes with these scores amongst the ILEs was the PDR3 mutations. Other deleterious and NA scored mutations may provide unique effects on MCHM resistance in individual strains but did not represent a consistent pathway to resistance in different strains. Table 3 Summary of predicted tolerance for coding variants found only in MCHM ILE strains. The eight MCHM ILE strains (S9-S16) are represented from left to right in columns. Each strain is summarized for whether its coding variants are predicted to be tolerated, deleterious, or NA based on SIFT4G analysis. NA scores mean a classification as either tolerated or deleterious was not possible to determine, or not applicable (NA). SIFT4G scores approaching 0 are expected to alter protein function to be deleterious based on amino acid substitutions. The NA results are usually due to the inability of SIFT to predict the effects of indels or occasionally SNPs that do not appear in the SIFT database. Coding variants in this table exclude those in Ty regions. MCHM ILE Strain S9 S10 S11 S12 S13 S14 S15 S16 Total Variants 54 63 43 53 59 64 53 45 Tolerated 38 41 30 35 42 30 31 23 Deleterious 7 2 5 2 8 12 6 4 NA 9 20 8 16 9 22 16 18 Comparison of MCHM evolved genes with genes appearing in other genomic datasets from MCHM studies Previous work has been done to produce genomic datasets that implicate genes involved in MCHM resistance mechanisms, so it hypothesized that some variants may target these same genes to adapt to MCHM treatment. The list of 107 genes containing at least one variant in an MCHM ILE strain was compared to a genetic screen of the BY4742 knockout collection for MHCM sensitivity and transcriptomic dataset (Fig. 4 ) (Ayers et al. 2020 ). There was very little overlap with the genetic screen (Fig. 4 A). This is expected, as the knockout screen selected for mutants with reduced MCHM resistance, revealing genes that are required for resistance. Therefore, in the 329 screen hits required for MCHM resistance, any variants would necessarily have to not reduce, perhaps even improve, the function of any proteins produced by those ORFs in order to create adaptive resistance. Yeast were treated with MCHM and the changes in gene expression were quantified. The transcriptomic dataset included a list of 592 upregulated genes and a list of 576 downregulated genes. There was more overlap with these lists and the 107 ILE genes, including 14 upregulated (Fig. 4 B) and eight downregulated genes (Fig. 4 C). The overlapping genes in these cases include several of the genes with variants in many ILE strains. DOG1 , which encodes a deoxyglucose phosphatase and is involved in resistance to 2-deoxyglucose (Sanz et al. 1994 ; Randez-Gil et al. 1995 ; Soncini et al. 2020 ), had mutations in the ILE and was upregulated in MCHM exposure. Several genes encoding cell wall proteins appear as well, including PIR3, TIR1, SCW10 , and FLO1 , each with varying functions from cell wall stability ( PIR3 ) to flocculation ( FLO1 ) (Hodgson et al. 1985 ; Kitagaki et al. 1997 ; Cappellaro et al. 1998 ; Doolin et al. 2001 ; Rossouw et al. 2015 ). We tested the requirement of several of these genes for resistance to MCHM. First, we tested the growth of the knockouts in the S288c background on MCHM. The ynr065c , ynl034w ( par1) , and hxt17 mutants in S288c background did not show appreciable change in growth on MHCM (Fig. 5 A). YNR065c, YNL034w (PAR1) , and COS4 were selected for further study by knocking them out in the YJM789 strain. The ynl034c and cos4 mutants grew slightly slower than YJM789 (Fig. 5 B). COS4 is required for the multivesicular vesicle body sorting pathway and provides ubiquitin in trans for non-ubiquitinated cargo proteins (MacDonald et al. 2015). There was not much difference visually in the growth of the YJM789 wild type and that of the COS4 knockout. It is possible that the knockout had no increased sensitivity compared to the wild type, so the mutation was not activating and cos4 knockout growth was not affected by MCHM exposure. We uncovered nonsynomous SNPs in an unknown ORF YNL034c that has no predicted transmembrane domains or GPI anchor site but is associated with the cell membrane (Weill et al. 2018 ). Ynl034c has a paralog, Ynl018c, that is also localized on the cell membrane (Weill et al. 2018 ). YNL034c has 24 nonsynonymous SNPs between YJM789 and S288c and 15 synonymous SNPs suggesting it is under positive selective pressure. Two different SNPs were detected in different ILE strains, K554R and H541N. We have named it PAR1 ( P atches A round R adius). Examining the genomes of the ILEs uncovered other ILE induced SNPs in genes encoding membrane associated proteins. We tested knockouts of flo1 and flo10 and loss of these genes conferred sensitivity to MCHM in S288c (Fig. 5 C), we knocked out these genes in YJM789 and the ILE strain (S14, Fig. 5 D). In the ancestral strain, loss of either flocculin did not change growth but in S14, the flo1 mutant was less sensitive and flo10 was slightly more resistant. Flo1 expressing cells excrete glucose and mannose polysaccharides that limit the size of chemicals that interact with cell wall and membrane. The ILE SNP in Flo1 was S1092A and in Flo10 was T402T, both outside the flocculin repeat. Flo5 and Flo9 also contained nonsynonymous SNPs int the ILE strains. In S288c, the pir3 knockout was also MCHM sensitive (Supplemental Fig. 2B) which supports that cell wall structure perturbations alter cellular tolerance to MCHM. Flocculins are cell wall proteins that bind mannose chains on other cells causing the cells to stick together (Rossouw et al. 2015 ) and flocculate. Flocculation enhances survival during starvation and is regulated by the cell wall integrity pathway (Sariki et al. 2019 ). Flo1 regulates cell surface hydrophobicity (Sariki et al. 2019 ). Flocculation also aids in directed cell growth into the agar as cells consume the available nutrients. This invasion growth is assessed by determining if cells can be washed off the surface of the plates. MCHM inhibited invasion (Fig. 5 E). YJM789 liquid cultures flocculate, rapid sediment when not shaking and saturated cultures but flo mutant cultures remain suspended as well as ILE S14 strain (Fig. 5 F). Expression of some FLO genes is dependent on amino acid transporters (Torbensen et al. 2012 ). These datasets continue to point to the cell wall and sugar metabolism as functions that are important ways to adapt resistance to MCHM. Numerous mutations in transporters were identified in the genomic analysis of the ILE strains. TPO2 belongs to a family of transporters that are part of the multidrug resistance pathway. The protein localizes to the plasma membrane and has been characterized as an exporter of polyamines (Tomitori et al. 2001 ), which could be related to the amino acid biosynthesis pathways implicated in MCHM resistance in previous work (Ayers et al. 2020 ). However, the mutations in TPO2 are all synonymous (Supplemental File 1). The codon changes could potentially affect protein levels if they affect translation rates. The S288c knockout showed no change in resistance but the tpo3 knockout was more sensitive (Supplemental Fig. 2A). Tpo3 is a homolog of Tpo2 and both are polyamine transporters localized to the vacuole (Tomitori et al. 2001 ). Mal31 is a maltose permease regulated by Mal13, a transcription factor (Orikasa et al. 2018 ). The mal31 mutant in BY4742 was sensitive to MCHM (Supplemental Fig. 2A). We then knocked out MAL31 in YJM789 and ILE strains with a nonsynonymous SNP, I415V in Mal31. I415V did not alter the transmembrane domain prediction. Unlike the BY4742 mutant there was no change in growth in response to MCHM in any of the mutants (Supplemental Fig. 2B). We also grew these strains on maltose, disaccharide of dextrose instead of dextrose and while cells grew slower there was no impact of genotype on growth (Supplemental Fig. 2B). In ILE strains with the MAL31 mutation there was also a mutation in MAL13 , which encodes a transcription factor that regulates MAL genes (Supplemental File 1). The mutation was outside the DNA binding domain. Yeast resistant to 2-deoxyglucose increase expression of MAL31 (Orikasa et al. 2018 ). Slowing yeast growth by changing the carbon source from dextrose to maltose reduces the ability of MCHM to slow growth. Flux balance analysis on MCHM which integrates transcriptomic and metabolomic data identified CYT1 as a key step in metabolic regulation during MCHM (Pupo et al. 2019b ). Sugars such as maltose can inhibit flocculation (Stratford 1989 ). There were mutations in other types of transporters. GEX2 encodes a glutathione transporter important for oxidative stress resistance, a known source of stress from MCHM treatment (Dhaoui et al. 2011 ; Ayers et al. 2020 ). At early growth stages, glutathione is brought into the cell's vacuole, while in later stages it is exported to the cytosol surrounding the cell (Outten et al. 2018 ). Glutathione has been known to reduce reactive oxygen species (ROS) by acting as an antioxidant, however it is unsure whether in these cases it is acting more so as a nitrogen source or an antioxidant when exposed to MCHM (Ayers et al. 2020 ). Gex2 transports glutathione while Sam3 transports S-adenosylmethionine (SAM) and polyamines such as spermidine and putrescence. Gex2 YJM789 has five naturally occurring nonsynonymous SNPs and the ILE induced SNP, D570G, is within the last transmembrane domain (aa 578 to 615) but does not alter the transmembrane domain prediction. The transmembrane domain predictions for Sam3 were also not altered with ILE SNPs K566R and V339I. The Agp3 ILE SNP was V212I, which was also in a transmembrane domain, but it did not change its prediction. While there are no SNPs between YJM789 and S288c, the APG3 deletion mutant in BY4742 was sensitive to MCHM (Fig. 6 A) but the apg3 knockout was likely lethal in YJM789 because it could not be generated. There are examples of other genes that become essential in different strain backgrounds; the aro1 knockout in RM11 was lethal while it was not essential in other strains (Rong-Mullins et al. 2017 ). In YJM789, the gex2 mutant was slightly more sensitive to MCHM while the sam3 mutant growth was not different from the parent (Fig. 6 B). However, knocking out these genes in the ILE strain that they were identified from increased the MCHM sensitivity which suggests that the ILE induced SNPs were activating mutations. Due to the involvement of Gex2 in ROS response, we further measured the ROS production in YJM789 gex2 after MCHM exposure (Fig. 6 C). Because MCHM slowed growth, we measured correlated growth rate with ROS production (Fig. 6 D). Production of ROS was proportional to growth so that the small population that had increased ROS when previously measured at the single cell level (Ayers et al. 2020 ) and consistent with bulk measurements ROS levels decreased. Analysis of evolved alleles in PDR3 The PDR3 variants in the MCHM ILEs showed a pattern of evolution that pointed to a reproducible pathway to resistance. We decided to look more closely at the individual mutations. Previous research has been done with PDR3 mutagenesis to produce gain-of-function alleles that improve resistance to different chemicals (Nourani et al. 1997 ). For instance, amino acid mutations in the region from approximately residues 220–280 created alleles that increased the expression of multiple ABC transporters that pump chemicals out of cells, specifically SNQ2 and PDR5 (Nourani et al. 1997 ). The mutations in PDR3 in the S12, S13, and S15 ILEs mutated single amino acids at residues 288, 209, and 229 respectively (Fig. 7 A). It is possible that these mutations are creating gain-of-function alleles that increase the expression of ABC transporters, thereby conveying resistance to MCHM. The gene SNQ2 is required for resistance to MCHM (Ayers et al. 2020 ), but it has yet to be shown if overexpression would be sufficient to produce resistance. The remaining five mutations resulted in changes to the C-terminal portion of the protein, where an activation domain homologous to Gal4-like transcriptional activators is located (Delaveau et al. 1994 ). The S9 mutation in Pdr3 was a M842L single amino acid change (Fig. 7 A and Supplemental Fig. 3A). One study produced six different gain-of-function mutations in this region with single nucleotide changes that increased expression of PDR3 , PDR5 , and SNQ2 and conferred resistance to several chemicals (Simonics et al. 2000 ). None of the mutations were the same as found in the ILE strains, but they implicate this region of the protein for possible gain-of-function resistance mutations. The S16 mutation was an insertion and frameshift occurring at the amino acid 972 that altered and extended the remaining four amino acids into an extra 9 amino acids before the new stop codon (Fig. 7 A). Figure 7 cont . shown by a slash and dashed line where the missing portion of the protein coding sequence will not be translated. The final strain S16 contains an insertion that alters the last five amino acids of the protein to a new 13 amino acid sequence. The asterisks at the top represent the interface residues found on the Pdr3 homodimer structure prediction. B. Serial dilution growth assays of one of the evolved resistant strains (S11), the YJM789 parent strain, the YJM789 PDR3 knockout strain, the BY4742 wildtype strain, and BY4742 PDR3 knockout were carried out. The growth assays are on increasing concentrations of MCHM from 0ppm (YPD) to 1000ppm. C. Serial dilution reciprocal hemizygosity growth assays of S288c (BY) and YJM789 hybrid mutants of PDR3. The growth of the wildtype hybrid was compared to hybrids lacking PDR3 BY , PDR3 YJM , or both (homozygous diploid) on MCHM. D. Serial dilution single pdr3, med15 , the double pdr3, med15 mutant and yke4 in YJM789 grown on MCHM or rapamycin. E. Percent change of elements potassium (K), magnesium (Mg), phosphorus (P), sulfur (S), and zinc (Zn) when yeast from part D were exposed to MCHM. The S10 and S14 mutations truncated the protein by 415 and 406 amino acids respectively (Fig. 7 A). The S11 mutation also truncated the protein, but by 82 amino acids (Fig. 7 A). One hypothesis is that deletions of such a large part of the protein sequence would produce nonfunctional products, effectively acting as a knockout. Another possibility is that these truncations could still produce proteins with some function, considering the DNA binding domain is at the N-terminus like in other Gal4-like transcription factors (Mamnun et al. 2002 ). Furthermore, the domains implicated in the homo- and heterodimer interactions of Pdr3 and Pdr1 are also in the N-terminal 400 amino acids. With dimerization and DNA interacting regions of the protein products intact, the truncated alleles of S10, S11, and S14 could be functional proteins. A side-by-side comparison of the predicted structure of Pdr3 from YJM789, S10, S11, and S14 shows considerable differences in structure upon truncations in the C-terminus (Supplemental Fig. 3B). Interface residues in the predicted Pdr3 homodimer structure revealed contact points in the N- and C- terminals (Fig. 7 A and Supplemental Fig. 3B) but none of these residues had ILE induced mutations. To test the more fundamental hypothesis that a knockout-like truncation may confer resistance to MCHM, we knocked the PDR3 gene out of the wildtype parent of the ILEs, YJM789. The pdr3∆ strain showed similar resistance as the S11 resistant MCHM strain (Fig. 7 B). S11 corresponds to the strain containing the shortest truncation of the Pdr3 protein, only 82 amino acids. If the mutations to PDR3 in the evolved strains are mimicking a knockout by making Pdr3 nonfunctional, that would be sufficient to produce the resistant phenotype. During the ILE experiment both YJM789 and S288c were passaged in MCHM but no resistance could be detected in the S288c strain. A Quantitative Trait Loci (QTL) analysis of S288c and YJM789 failed to identify PDR3 and there are four nonsynonymous SNPs Q56R, T102I, A885T, and N916S (Pupo et al. 2019a ). The first two SNPs are in the Zn finger domain and the last two are in the regulatory domain. To assess if these SNPs affected growth on MCHM, PDR3 knockout in YJM789 and S288c (BY) were mated together to assess the individual contribution in the hybrid with only the allele of PDR3 being different between strains (Fig. 7 C). Hybrid yeast expressing only PDR3 YJM789 were very sensitive to MCHM. Yeast expressing both PDR3 alleles were slightly less sensitive, while the double mutant or yeast expressing only PDR3 BY were more tolerant. To assess which genes are differentially regulated in pdr1 and pdr3 deletion strains, we determined the promoter binding signal of Pdr3 and Pdr1 targets using a published dataset in the S288C background (Supplemental Table 4, (Gera et al. 2022 )). Notably, Pdr3 appears to exhibit a weaker binding signal overall, which strengthens considerably in absence of PDR1 (Supplemental Fig. 4). Upon closer examination, deletion of PDR3 primarily results in stronger Pdr1 binding upstream of GAC1 , a regulatory subunit of a phosphatase involved in glycogen accumulation (Supplemental Fig. 5A) (Wu et al. 2001 ). Additionally, instances of Pdr1 binding strength increasing upon PDR3 deletion include TPO1, SPO24 and LDB7 (Supplemental Fig. 5B). To test the hypothesis that loss of Pdr3 function changed Pdr1 function we tested if the YJM789 pdr1 mutant was also MCHM sensitive. However, the YJM789 pdr1 was just as MCHM resistant as the pdr3 knockout (Supplemental Fig. 6A). Interestingly, mutations in PDR1 were not detected in the ILE and one possibility is that pdr1 mutant has a defect in long term survival during starvation. We measured viability of YJM789 pdr3 and pdr1 mutants during long term starvation in YPD, but no significant differences were found (p value 0.79) in 12 days of starvation. Both Pdr1 and Pdr3 strongly bind upstream of PDR5. While binding of Pdr1 was not affected in the pdr3 knockout, binding of Pdr3 was decreased in the pdr1 knockout (Supplemental Fig. 5). Pdr5 is an ABC transporter facilitating the export of diverse chemicals out of the cells. Consequently, we tested the YJM789 pdr5 growth on MCHM (Supplemental Fig. 5). The pdr5 mutant was not more sensitive to MCHM but the yrr1 mutant which is sensitive to 4NQO (Gallagher et al. 2014 ; Rong-Mullins et al. 2018 ) was also MCHM sensitive and resistant to rapamycin (Supplemental Fig. 6B). Yrr1 is also involved in response to vanillin (Cao et al., 2021; Zhao et al., 2023) and the knockout is vanillin sensitive (Supplemental Fig. 6B). Interestingly, Pdr3, Pdr1 and Yrr1 have all been described as transcriptional activators of SNQ2 , an ABC transporter (Cui et al. 1998 ). The BY4742 SNQ2 knockout was previously determined to be MCHM sensitive (Fig. 6 C) (Ayers et al. 2020 ). Yrr1 also has a paralog, Pdr8 and both do not appear to strongly bind PDR5 (Supplemental Fig. 4). The yrr1 mutant was also MCHM resistant but does not bind AGP3 promoter. The AGP3 knockout was MCHM sensitive in the S288c background (Fig. 6 A) but we could not generate the YJM789 knockout. Four ILE mutations in AGP3 were the most activating mutations and increased amino acid import. PDR3 is the first gene to be identified in yeast as a target to induce sensitivity to MCHM. Interestingly, four different wine strains contained genomic deletions of the PDR3 (Dunn et al. 2005 ). Previous work, such as the genetic screen, focused only on finding genes required for tolerance (Ayers et al. 2020 ). The screen was not designed to detect an increased resistance phenotype in any of the mutants tested. The model for this resistance involves the importance of Pdr3 in controlling the activation of multiple ABC transporters that pump stress-inducing chemicals out of cells. Pdr3 and its paralog Pdr1 form homo- and heterodimers and then bind to transcriptional response regions termed PDREs where they can inhibit or activate transcription of genes such as PDR5 , SNQ2 , YOR1 , PDR15 , PDR10 , and other transporters (Supplement Fig. 4 ). We tested via knockout in the YJM789 parent strain if inactivating mutations could be sufficient to produce MCHM resistance (Pupo et al. 2019b ). The mutations in PDR3 could also be activating mutations that make Pdr3 increase transcription of all or some transporters, such as SNQ2 or PDR5 . Having identified mutations in PDR3 that directly affect response to MCHM across all ILE strains, we aimed to delve deeper into its regulatory mechanism. Previous work further uncovered genetic variation in Med15, a component of the Mediator complex, that contributed to variation in yeast MCHM response between YJM789 and S288c (Gallagher et al. 2020 ). As part of the tail in the Mediator complex, Med15 directly interacts with Pdr3 (Shahi et al. 2010 ). Therefore, in order to test if loss of Med15 could suppress the pdr3 MCHM resistance we generated double mutants (Fig. 7 D). The med15 knockouts are slow growing and while not specifically sensitive to MCHM their slow growth is not altered in the presence of MCHM. However, the med15, pdr3 double mutant appeared just as sensitive to MCHM as the parental strain (Fig. 7 D). Given the impact of MCHM on protein solubility, a property regulated by Intrinsically Disordered Regions (IDRs), and the known regulation of MCHM response by Med15 IDRs, we determined the IDR prediction of Pdr3 alleles (Erdős and Dosztányi 2020 ) and found no difference between strains (Supplemental Fig. 3C). Med15 interacts with multiple transcription factors and to test whether Med15 mediates the MCHM resistance due to loss of Pdr3 function we generated double mutants in the YJM789 background. Loss of med15 represses the MCHM resistance of the pdr3 knockout (Fig. 7 D). Due to the effect of MCHM on growth through induced amino acid starvation response, we tested whether rapamycin inhibits TORC1 directly. The pdr3 and pdr3, med15 double mutant showed no change in growth on rapamycin (Fig. 7 D). The med15 mutant is slow growing on YPD but its growth rate is not affected on rapamycin. It is possible that in the absence of pdr3 , Med15 is free to recruit other transcription factors or Pdr1, would only be present as a homodimer since it dimerizes with Pdr3. Previous work has shown that zinc levels are increased in MCHM exposure and excess zinc suppresses S288c MCHM sensitivity (Pupo et al. 2019a ). We found that zinc levels were not increased in the pdr3 mutant upon MCHM exposure, and neither were the levels of other metals tested (Fig. 7 E). Dysregulation of the metallome correlated with yeast sensitivity to MCHM. In a QTL analysis, YKE4 , a zinc transporter was linked to MCHM resistance. We tested the ability of zinc to suppress MCHM response, but we only noted mild exacerbation of growth inhibition of all YJM789 (Fig. 7 E). The SNQ2 gene is required for MCHM resistance, so it is a likely candidate for this increased expression (Ayers et al. 2020 ). SNQ2 does not have SNPs between YJM789 and S288c. The YJM789 allele of PDR5 is divergent from the reference and other yeast strains, with a 5.3% amino acid difference compared to the reference strain (Wei et al. 2007 ; Guan et al. 2010 ). The YJM789 allele has been shown to alter the strain’s resistance to antifungals (Guan et al. 2010 , p. 201), increasing or decreasing resistance dependent on the chemical. PDR5 is not required for resistance to MCHM like SNQ2 according to previous work, but this work was done in the BY4742 strain, which has the same allele as the reference strain (Supplemental Fig. 6C) (Ayers et al. 2020 ). It is possible that the YJM789 PDR5 allele can provide resistance to MCHM if the expression is increased. PDR5 is significantly upregulated by gain-of-function mutations in PDR3 in the same regions as the MCHM ILE variants (Nourani et al. 1997 ; Simonics et al. 2000 ), but the pdr5 mutant did not change MCHM resistance. From previous analysis, Pdr1 binding at GAC1 decreases in the pdr3 mutant and Pdr3 binding also decreases in the pdr1 mutant at GAC1 (Gera et al. 2022 ). Gac1 binds Hsf1, a stress induced transcription factor, and it is induced as dextrose is consumed and glycogen accumulates (Lin and Lis 1999 ). However, GAC1 expression did not change in S288c (Pupo et al. 2019a ; Gallagher et al. 2020 ). Conclusion The In-Lab evolution of the YJM789 strain of S. cerevisiae produced thousands of mutations in each strain. The mutations were similar in number from strain to strain, including both control media and MCHM treated conditions. There was no clear pattern of mutations resulting in MCHM resistance across strains, though the sheer number of mutations could mask any pattern with background variants. When filtered for mutations unique to MCHM ILEs that were only found in coding sequences, patterns such as cell wall and responses to stress did begin to emerge. These patterns are consistent with previous knowledge of MCHM effects on cells, including oxidative stress activation (Ayers et al. 2020 ). Transcriptional analysis, genetic screens, and metabolomic data showed that diverse pathways are affected by crude MCHM (Pupo et al. 2019b , a ; Gallagher et al. 2020 ; Ayers et al. 2020 ; Perfetto et al. 2021 ). The pleiotropic effects of MCHM likely stem from its ability to act as a hydrotrope, altering protein structures and solubility (Pupo et al. 2019b ). Hydrotropes in cells prevent protein aggregation, but unlike surfactants, work at millimolar concentrations and display low cooperativity. These contribute to liquid-liquid phase separation (LLPS) that explains how membraneless organelles form in the cell. MCHM is a hydrophobic chemical and likely alters membrane dynamics. Multiple amino acid biosynthetic pathways are upregulated signaling inhibition of TORC1, but levels of several amino acid are leveled after only 30 minutes of exposure. Excess amino acids are stored in the vacuole and reduction in vacuolar acidification increases MCHM sensitivity. We propose a model in which Pdr3 is the major driver of resistance to MCHM through regulating transcription of ABC transporters, such as Pdr5, Pdr10, Pdr15, Snq2 and Yor1. Loss of mutations are far more common than gain of mutations and through the ILE loss of other transcription factors such as Pdr1 or Yrr1 were not selected for because those mutants would have a decrease in fitness during the competition during nutrient starvation. Yrr1 mutants have decreased growth during respiration (Rong-Mullins et al. 2018 ). Loss of Pdr3, Pdr1, or Yrr1 enables the mediator complex component Med15 to function elsewhere through an unknown mechanism and contribute to MCHM resistance (Fig. 8 ). The variable length of Med15’s IDRs are likely affected by MCHM and its ability to form condensates would be altered. Med15 induces protein condensates with Gcn4, and it has yet to be seen if other transcription factors also this characteristic liquid-liquid phase separation have to regulate transcription. Declarations Acknowledgments: The MCHM sample used was obtained as a gift from Eastman Chemical Company. The yeast knockout collection was a gift from Angela Lee. Mohammad Rahman assisted in Pdr3 interface interaction prediction. We would like to acknowledge the WVU Genomics Core Facility, Morgantown WV for the support provided to help make this publication possible and CTSI Grant #U54 GM104942 which in turn provides financial support to the Core Facility. Amaury Pupo provided invaluable help in setting up many of the bioinformatic analyses running GATK. This work was supported by NIH NIEHS R15ES026811-01A1. MCA was supported by a WVU STEM Mountains of Excellence Fellowship. DJ was supported by the SyTox training grant NIH NIEHS 1T32ES032920-01A1. Author Contributions: MCA designed and carried out yeast experiments and wrote the paper. JEGG designed the study and supervised the project. DP analyzed transcription factor binding while LM tested effects of zinc on mutants. TM modeled transcription factor structures, measured element levels, and worked with SP and GL on ROS assay. TM and DJ supervised the experiments knocking out genes in YJM789. MQ and NW analyzed the FLO genes, ND characterized PAR1 and SM assessed PRM9. FJ aided in Chec-seq analysis. Competing Financial Interests The authors declare no competing interests. References Adamo GM, Lotti M, Tamas MJ, Brocca S (2012) Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae. Microbiology 158:2325–2335 Agier N, Fischer G (2012) The mutational profile of the yeast genome is shaped by replication. 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Supplementary Files SupplementalFigureandtablelegends.docx AyersMCHMILEssupfigures.pdf AyersSupplementalTable1.xlsx AyersSupplementalTable2.csv AyersSupplementalTable3.xlsx AyersSupplementalTable4ChIPSec.csv Cite Share Download PDF Status: Published Journal Publication published 19 Nov, 2025 Read the published version in Discover Genetics and Evolution → Version 1 posted Editorial decision: Revision requested 17 Jun, 2024 Editor assigned by journal 10 Jun, 2024 Submission checks completed at journal 10 Jun, 2024 First submitted to journal 07 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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G.","lastName":"Gallagher","suffix":""}],"badges":[],"createdAt":"2024-06-07 23:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4548300/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4548300/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00294-025-01333-w","type":"published","date":"2025-11-19T15:57:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59003797,"identity":"8cd5d5c1-719d-409e-8110-5c92b3eb074b","added_by":"auto","created_at":"2024-06-25 07:38:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn Lab Evolutions produce MCHM resistant yeast strains.\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003eFour biological replicates of YJM789 yeast were grown in liquid rich media (YPD) with and without 700ppm MCHM. Each replicate was grown for two days, then passaged by inoculating 1% of the culture into fresh media. Six total passages were done, testing growth by serial diluting the cultures onto solid media containing 1000ppm MCHM to determine that resistance was achieved. Resistant isolates were passaged on solid rich media plates without MCHM to check for loss of epigenetically inherited resistance mechanism. Two single colony isolates from each of the four controls and four MCHM replicates were selected for analysis. Genomic DNA was extracted and sequenced with Illumina. \u003cstrong\u003eB.\u003c/strong\u003eResistance of isolates from the In-Lab evolutions were examined by serial dilution assay on solid YPD with and without 1000ppm MCHM. The eight control strains were labeled S1-S8. Pairs of strains (S1 and S2, S3 and S4, etc.) were isolated from the same original biological replicate. Pairs were plated on the same plates with a control YJM789 parent strain for growth assays. The YJM789 parent appears on each plate to control for plate to plate variation in MCHM dosage due to volatility and the high concentration of MCHM approaching its limits. The eight MCHM evolved strains are labeled S9-S16, and pairs such as S9 and S10 were isolated from the same biological replicate.\u003c/p\u003e","description":"","filename":"Figure1Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/237f458712d44603f84cfe07.png"},{"id":59003049,"identity":"b844d203-18aa-4a77-94d9-da766f71e2a4","added_by":"auto","created_at":"2024-06-25 07:30:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePCA analysis of evolved variants in in-lab evolution strains according to resistance phenotype.\u003c/strong\u003e The 6874 variants found in the control and MCHM strains after sequencing, aligning to the reference yeast genome, and filtering of existing variants in the YJM789 parent genome were considered to constitute the entirety of variants produced through the in-lab evolution passaging experiment. There were approximately 2500 variants per strain, many variants appeared in more than one strain. The sixteen strains were clustered by PCA analysis according to their variant content. Strains are colored by their resistance phenotype, with the eight control strains in red and the eight MCHM resistant strains in blue. Samples are also labeled S1-S16 corresponding to strain names from Figure 1. The first three principal components are plotted against each other as follows: \u003cstrong\u003eA. \u003c/strong\u003eprincipal component 1 vs principal component 2, \u003cstrong\u003eB.\u003c/strong\u003e principal component 1 vs. principal component 3, and\u003cstrong\u003e C.\u003c/strong\u003e principal component 2 vs. principal component 3.\u003c/p\u003e","description":"","filename":"Figure2Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/a7f419c31069772027c2ee49.png"},{"id":59003039,"identity":"183ff262-8e09-4d41-8f35-0d7dbe42b4f8","added_by":"auto","created_at":"2024-06-25 07:30:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":114375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntersection and functional analysis of genes containing variants in the MCHM evolved strains. A.\u003c/strong\u003e Variants were filtered for their presence in coding sequences and for their presence exclusively in MCHM evolved strains. The variants from this list that resulted in nonsynonymous changes were found in 75 genes. Each of the eight MCHM strains contained nonsynonymous variants in between 15 and 23 genes, so there was some overlap in genes between strains. The intersection of the genes between the strains was analyzed by UpSetR. The bar graph vertical axis shows the intersection size, or the number of genes present in each intersection. The definition of each intersection appears in the filled and connected ovals below each bar. The eight strains are organized from smallest to largest (top to bottom) number of genes (set size) on the left. \u003cstrong\u003eB.\u003c/strong\u003e A similar set of genes as in part A, but containing both synonymous and nonsynonymous variants, consisted of 107 genes containing coding variants for the MCHM strains. These genes were analyzed for function, process, and cellular component terms. The GO Term and the False Discovery Rate (FDR) for each term is shown on the yeast cell diagram. FDR is represented as percentages, not ratios.\u003c/p\u003e","description":"","filename":"Figure3Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/487ebb55d1083eff2a3fd673.png"},{"id":59003799,"identity":"1010f74f-7ed9-4c34-82a7-0b1b1603eb3e","added_by":"auto","created_at":"2024-06-25 07:38:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVenn diagrams show overlap between the genes with variants in MCHM evolved strains with genes in other MCHM genomic datasets. A. \u003c/strong\u003eThis Venn Diagram shows the overlap of the gene lists from the MCHM evolution strains (ILE Variant Genes) and the genetic screen of the BY4742 strain knockout collection showing genes required for MCHM resistance (knockout screen genes). The number of genes found only in each list and in the overlap are shown in the diagram. The one gene in the overlap of the lists is written below the diagram. \u003cstrong\u003eB.\u003c/strong\u003e This Venn Diagram shows the overlap of the MCHM evolution strains genes and the upregulated genes from a transcriptome of MCHM treated BY4741 strain. The 14 genes in the overlap of the datasets are listed below the diagram. \u003cstrong\u003eC. \u003c/strong\u003eThis Venn Diagram shows the overlap of the MCHM evolution strains genes and the downregulated genes from the same transcriptomic analysis as B. The eight genes in the overlap of the datasets are listed below the diagram\u003c/p\u003e","description":"","filename":"Figure4Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/f23fb2f3158c164ba55dc0af.png"},{"id":59003796,"identity":"e1e01f1f-845f-48d2-8148-d1dd41236cbc","added_by":"auto","created_at":"2024-06-25 07:38:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":170489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative growth assays of yeast strains with various genes knocked out on different concentrations of MCHM A. \u003c/strong\u003eBY4742 knockouts of \u003cem\u003eynl065w, par1 (ynl034c), \u003c/em\u003eand \u003cem\u003ehxt17 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eB. \u003c/strong\u003eYJM789 knockouts of \u003cem\u003eynl065w, par1 (ynl034c), cos4, \u003c/em\u003eand \u003cem\u003epdr3 hxt17 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eC. \u003c/strong\u003eBY4742 knockouts of \u003cem\u003eflo1 \u003c/em\u003eand \u003cem\u003eflo10 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eD. \u003c/strong\u003eYJM789 and S14 knockouts of \u003cem\u003eflo1 \u003c/em\u003eand \u003cem\u003eflo10 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eE. \u003c/strong\u003eInvasion assay of \u003cem\u003eflo \u003c/em\u003emutants grown with and without MCHM for seven days and then photographed. \u003cstrong\u003eF. \u003c/strong\u003eSaturated cultures sediment at different rates after being removed from the shaker for 5 minutes.\u003c/p\u003e","description":"","filename":"Figure5Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/723fd0ad077da04ec098b6b3.png"},{"id":59003055,"identity":"f0002508-e5c0-4c83-a22b-7b897c25abb3","added_by":"auto","created_at":"2024-06-25 07:30:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":128358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantitative growth assays of yeast strains with transporter genes knocked out on different concentrations of MCHM A. \u003c/strong\u003eBY4742 knockouts of \u003cem\u003esam3, gex2, \u003c/em\u003eand \u003cem\u003eagp3 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eB. \u003c/strong\u003eYJM789 knockouts of \u003cem\u003eynl065w, par1 (ynl034c), cos4, \u003c/em\u003eand \u003cem\u003epdr3 hxt17 \u003c/em\u003ewere serial diluted and plated on YPD plates with and without MCHM. \u003cstrong\u003eC. \u003c/strong\u003eROS levels were measured in YJM789 WT, YJM789 \u003cem\u003epdr3\u003c/em\u003e, S12 WT, and S12 \u003cem\u003egex2\u003c/em\u003e treated with 400ppm, 500ppm, 600ppm MCHM, and 4mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as positive control. \u003cstrong\u003eD. \u003c/strong\u003eGraphing cell count to ROS levels of strains.\u003c/p\u003e","description":"","filename":"Figure6Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/562a0fe26e45d456cff27626.png"},{"id":59003052,"identity":"d4a50112-1953-492c-bbe1-e498e0643fa0","added_by":"auto","created_at":"2024-06-25 07:30:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":291193,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePdr3 alleles in the MCHM evolved strains are genetically controlled by Med15. A.\u003c/strong\u003e The eight MCHM evolved strains each contained exactly one mutation in the \u003cem\u003ePDR3\u003c/em\u003e gene, all of which created protein sequence changes. The alleles are diagrammed alongside the wildtype YJM789 allele. The names of each strain are the left of each allele. The Zn Finger DNA-binding domain and Gal4-like Activation Domains are highlighted with green boxes in the N-terminal and C-terminal regions respectively. The residue number for the start of each of the eight mutations is at the bottom of the diagram. Single letter amino acid abbreviations are used to indicate the identity of the residues in the parental allele. Each of the evolved strains (S9-S16) have exactly one mutation, so S9, S12, S13, and S15 show their single residue changes from parent. The S10, S11, and S14 strains have early stop codons shown by a slash and dashed line where the missing portion of the protein coding sequence will not be translated. The final strain S16 contains an insertion that alters the last five amino acids of the protein to a new 13 amino acid sequence. The asterisks at the top represent the interface residues found on the Pdr3 homodimer structure prediction. \u003cstrong\u003eB. \u003c/strong\u003eSerial dilution growth assays of one of the evolved resistant strains (S11), the YJM789 parent strain, the YJM789 \u003cem\u003ePDR3 \u003c/em\u003eknockout strain, the BY4742 wildtype strain, and BY4742 \u003cem\u003ePDR3 \u003c/em\u003eknockout were carried out. The growth assays are on increasing concentrations of MCHM from 0ppm (YPD) to 1000ppm. \u003cstrong\u003eC. \u003c/strong\u003eSerial dilution reciprocal hemizygosity growth assays of S288c (BY) and YJM789 hybrid mutants of \u003cem\u003ePDR3. \u003c/em\u003eThe growth of the wildtype hybrid was compared to hybrids lacking \u003cem\u003ePDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBY\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, PDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eYJM\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e, \u003c/em\u003eor both (homozygous diploid) on MCHM. \u003cstrong\u003eD. \u003c/strong\u003eSerial dilution single \u003cem\u003epdr3, med15, \u003c/em\u003ethe double \u003cem\u003epdr3, med15 \u003c/em\u003emutant and \u003cem\u003eyke4 \u003c/em\u003ein YJM789 grown on MCHM or rapamycin. \u003cstrong\u003eE.\u003c/strong\u003e Percent change of elements potassium (K), magnesium (Mg), phosphorus (P), sulfur (S), and zinc (Zn) when yeast from part D were exposed to MCHM.\u003c/p\u003e","description":"","filename":"Figure7Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/9f84caff38b16731cf0385b3.png"},{"id":59003053,"identity":"5d107427-fdae-4712-b2c7-55caab0626a0","added_by":"auto","created_at":"2024-06-25 07:30:15","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":122598,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of Pdr1, Pdr3, and Yrr1 transcription factors controlling the expression of ABC transporters involved in the export of chemicals in response to stress.\u003c/strong\u003e As cells are exposed to drug the transcription factor dimerizes which to bind \u003cem\u003ecis-\u003c/em\u003eregulatory elements called PDREs upstream of genes encoding various ABC transporters. The major ABC and hexose transporters (Pdr5, Snq2, etc.) contain PDREs bound by Pdr1 and Pdr3 are shown in the plasma membrane and labeled. In absence of Pdr1, Pdr3 or Yrr1, the mediator complex component Med15 is released and interacts with other elements orchestrating resistance to MCHM through unknown targets.\u003c/p\u003e","description":"","filename":"Figure8Ayers.png","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/52637f3f54082e5546d8d4be.png"},{"id":96650227,"identity":"e8ae3bd4-67f6-42ee-b74d-cdc9465b0b8f","added_by":"auto","created_at":"2025-11-24 16:10:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3057549,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/a1a0da95-1bb4-45be-8866-3a3c3f726b0c.pdf"},{"id":59003048,"identity":"0d2c6a86-42ac-4211-af5f-783560a15606","added_by":"auto","created_at":"2024-06-25 07:30:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":508060,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigureandtablelegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/d3784879fd2dab4972be25b2.docx"},{"id":59003043,"identity":"fade6c60-d213-41fa-a229-321fe2de8a4a","added_by":"auto","created_at":"2024-06-25 07:30:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1232555,"visible":true,"origin":"","legend":"","description":"","filename":"AyersMCHMILEssupfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/f1590fe304753d3003ee3539.pdf"},{"id":59003045,"identity":"e218a461-75ac-4c56-8728-0fc2adf9d060","added_by":"auto","created_at":"2024-06-25 07:30:14","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14715,"visible":true,"origin":"","legend":"","description":"","filename":"AyersSupplementalTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/ba39d9e5719f7eb6effc8013.xlsx"},{"id":59003056,"identity":"bbb77ab2-7dfa-4175-8923-55ae3c469753","added_by":"auto","created_at":"2024-06-25 07:30:16","extension":"csv","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":560231,"visible":true,"origin":"","legend":"","description":"","filename":"AyersSupplementalTable2.csv","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/dc8a8cd401d6c758b514047f.csv"},{"id":59003057,"identity":"44864c27-b6ab-46ae-831b-d1314045e67d","added_by":"auto","created_at":"2024-06-25 07:30:16","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":77866,"visible":true,"origin":"","legend":"","description":"","filename":"AyersSupplementalTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/b08292747b1a05bdf6b1443d.xlsx"},{"id":59003798,"identity":"8dc4970d-edd7-4d4c-9da9-b70e53c490c5","added_by":"auto","created_at":"2024-06-25 07:38:15","extension":"csv","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":494257,"visible":true,"origin":"","legend":"","description":"","filename":"AyersSupplementalTable4ChIPSec.csv","url":"https://assets-eu.researchsquare.com/files/rs-4548300/v1/7685edfc949379f067d939b5.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Laboratory evolutions lead to reproducible mutations in PDR3 conferring resistance to MCHM","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrotropes, such as ATP and RNA, increase the solubility of organic compounds by inducing liquid-liquid phase condensates. Hydrotropes are not classified as detergents because detergents are effective at lower concentrations for solubilizing compounds. MCHM acts as a hydrotrope \u003cem\u003ein vitro\u003c/em\u003e and prevents protein aggregation (Pupo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). In contrast to ATP (Patel et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hayes et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and RNA (Lin et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), MCHM is not readily metabolized in the cell and can serve as a model to study the effect of hydrotropes on biological systems. MCHM is a cyclic hydrocarbon with saturated bonds that are difficult to break. MCHM is an exotic hydrotrope, to which yeast are not ordinarily exposed in nature. MCHM induces G1 arrest (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and amino acid biosynthesis (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIntrinsically disordered regions (IDR) of proteins frequently drive liquid-liquid phase separation (LLPS). These IDRs may represent a more general mechanism to increase the local concentration of proteins within condensates, adding complexity to regulating cellular metabolism and environmental responses. By their nature, structures of intrinsically disordered regions are difficult to determine but are important for changes in interacting with other proteins within protein complex conformations (Patel et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Cho et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sabari et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Boehning et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Boija et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Notably, proteins involved in transcription also have IDRs. Med15 contains three IDRs of variable length and differences in IDR length contribute to sensitivity to MCHM (Gallagher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Med15, a component of the tail subcomplex of the Mediator directly interacts with numerous transcription factors and forms protein condensates with Gcn4 (Boija et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Changes in the conformation of these proteins can profoundly increase phenotypic diversity by their effect on the protein composition of translation regulation machinery.\u003c/p\u003e \u003cp\u003eRecent work has produced genomic datasets to determine the cellular pathways involved in MCHM response. MCHM, as a hydrotrope that affects protein folding, has been implicated to have a role in zinc ion concentration changes in the cell to alleviate this stress (Pupo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). Lipid biosynthetic changes induced by MCHM may also be a source of stress on the plasma membrane (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). MCHM also causes amino acid accumulation according to metabolomic data, while exhibiting a nutrient starvation signal activating the environmental stress response (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003ea\u003c/span\u003e; Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). One early study using stress gene reporters showed oxidative stress and DNA damage response activation in response to MCHM and its potential metabolites (Lan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The more recent work using yeast genomic approaches confirmed that MCHM causes the production of reactive oxygen species (ROS) with a small percentage of yeast cells in a population, as well as DNA damage, making yeast strains sensitive to ROS, such as petite yeast, unable to grow (Lan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These findings highlight the diverse cellular pathways important for MCHM tolerance.\u003c/p\u003e \u003cp\u003eIn-Lab evolution (ILE) of yeast is a tool that allows for the genetic analysis of many phenotypes and processes of evolution. The short generation time of yeast allows for the quick production of large populations that accumulate mutations that may respond to selection or drift in the lab. This tool has been used to experimentally observe evolution in industrial environments with yeast hybrid strains, determining whether they evolve similarly in terms of aneuploidy and copy number to historical lager brewing yeast hybrids (Gorter De Vries et al. 2019). ILEs have also been utilized with wine strains, where ploidy events control significant changes in fermentation efficiency (Mangado et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Experiments also explore processes such as yeast and bacterial populations in competition and yeast populations that converge towards phenotypes with similar fitness due to epistatic interactions of beneficial mutations (Kryazhimskiy et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). To understand stress resistance, ILE has been used to produce yeast adapted to a multitude of stressors. Adaptation to copper has been produced in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and \u003cem\u003eCandida humilis\u003c/em\u003e, revealing mechanisms such as overexpression of superoxide dismutases and catalases to detoxify ROS and overexpression of proteins that bind excess copper (Adamo et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Thermotolerance was also evolved in yeast stains to explore mechanisms that could improve industrial biomass to ethanol conversion processes. Cellular changes involving sterol components of the membrane and concentrations of glycerol also provided increased tolerance to stresses from osmolarity and excess glucose and ethanol (Caspeta and Nielsen \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The evolution of resistance to a glyphosate-based herbicide in the lab involves copy number changes in proteins affecting cell wall stability in response to the presumably inert non-glyphosate additives in the herbicide (Ravishankar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). ILE experiments are an unbiased and insightful method for understanding specific mechanisms and processes of adaptation to environments.\u003c/p\u003e \u003cp\u003eYeast cells use the pleiotropic drug response to detoxify and remove a multitude of general xenobiotics and chemical stressors. Much of the work of this response is performed by a highly conserved class of proteins called ATP-binding cassette (ABC) transporters that translocate chemicals out of the cell (Jungwirth and Kuchler \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Some important stress-responsive yeast ABC transporters include Pdr5, Snq2, Pdr15, and Yor1, all of which have roles in exporting drugs or various chemically unrelated toxic chemicals out of the cell in response to stress (Servos et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Balzi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wolfger et al. \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Rogers et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Jungwirth and Kuchler \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Tsujimoto et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The transcriptional control of the genes encoding these proteins involves the interplay of the transcription factors Pdr1 and Pdr3, two paralogs sharing 36% amino acid sequence similarity along the entire length of the proteins (Delaveau et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). These proteins form heterodimers and homodimers that compete to occupy \u003cem\u003ecis-\u003c/em\u003eregulatory elements termed PDREs upstream of pleiotropic drug response genes (Katzmann et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Mamnun et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Their interaction in different combinations and on PDREs of different genes likely plays a role in the relative inhibition and activation of these same genes (Mamnun et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The role of the ABC transporters in a cell\u0026rsquo;s response to chemical stressors is important for evolved stress adaptation. In particular, induced mutagenesis experiments on \u003cem\u003ePDR3\u003c/em\u003e have produced activated gain-of-function alleles where Pdr3 increases the expression of \u003cem\u003ePDR5\u003c/em\u003e and \u003cem\u003eSNQ2\u003c/em\u003e and increases resistance to a multitude of chemicals (Nourani et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Simonics et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The role of the ABC transporters in the response to chemical stress makes them and the proteins that control the expression drug responsive genes targets for adaptation to chemicals such as MCHM.\u003c/p\u003e \u003cp\u003eIn this study, we utilized In-Lab evolutions to produce strains adapted to the hydrotrope MCHM. The goals of the study were to identify mechanisms as yet uncovered in the cellular response to MCHM as well as explore the evolutionary process under novel stress that yeast were unlikely to have encountered in their environmental niche. Eight strains evolved in MCHM became resistant to high dosages of the chemical, while eight control strains evolved under similar conditions without MCHM remained sensitive. All sixteen strains accumulated similar amounts of mutations overall. There were no clear patterns in mutations that led to resistance, except for variants appearing in the gene \u003cem\u003ePDR3\u003c/em\u003e. The mutations in \u003cem\u003ePDR3\u003c/em\u003e were the major reproducible drivers of resistance to MCHM mediated by interactions with Med15. Other mutations spread across the genome and pathways partly contributed to the resistance but could also contribute to convergent phenotype through epistatic interactions of small effects.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn-lab evolutions\u003c/h2\u003e \u003cp\u003eThe YJM789 strain (MATalpha, \u003cem\u003elys2\u003c/em\u003e∆) (Tawfik et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; McCusker et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) was grown in biological quadruplicate in 2mL liquid cultures of YPD (1% yeast extract, 2% peptone, 2% dextrose) media with and without 700ppm crude MCHM at 30C. Cultures were grown for two days before 1% of the culture was used to inoculate fresh tubes containing 2mL of YPD or YPD\u0026thinsp;+\u0026thinsp;700ppm MCHM media. This passaging process was performed for six times before plating serial dilutions onto YPD plates containing ranges of 0-1000ppm MCHM. Two colonies were isolated from each final evolved population. The isolates were grown for 2\u0026ndash;3 passages in liquid YPD without MCHM to allow for removal of epigenetic memory of resistance that may impact growth on MCHM media. The resistance of isolated strains were retested on 1000ppm MCHM solid. Following the epigenetic check, genomic DNA was then isolated from the strains and submitted for sequencing at the WVU Genomics Core (Ravishankar et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSequencing and analysis\u003c/h2\u003e \u003cp\u003eStrains were sequenced on an Illumina MiSeq producing 151bp paired-end reads of between 1,638,426 and 3,445,498 reads per strain. Sequences are available at Genbank (BioProject ID PRJNA1111078) and are available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/bioproject/1111078\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/bioproject/1111078\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Reads were aligned to the S288c reference genome R64.2.1 release obtained from yeastgenome.org (Engel and Cherry \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) using BWA version 0.7.17-r1188 (Li and Durbin \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), giving coverage of approximately 20-30x across the genome for each strain. SAM and BAM files were created using samtools version 1.7 (Li and Durbin \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Li \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Variants were called using the HaplotypeCaller function of GATK 4.1.6.0 (Van der Auwera et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) on Java Runtime Environment 11.0.6. Variants were filtered by removing all variants that did not pass GATK quality measures. As YJM789 has approximately 60,000 SNPs (Wei et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) when compared with the S288c reference, these variants were removed by filtering all variants that were shared amongst all 16 evolved strains (control and MCHM) and using an existing SNP list for the strains. Any variants shared by all 16 strains were assumed to be existing variation between S288c and YJM789, as opposed to random mutations that happened in every sample. The 6874 remaining variants in the 16 strains were analyzed for presence in coding regions and the intersection between strains as below. The numbers of variants per strain in the 8 control strains and 8 MCHM strains were compared with a two-tailed Student\u0026rsquo;s T-test to determine if there was any difference in total number of variants between resistant phenotypes. Variants were analyzed further using R version 3.6.3 and the following packages:\u003c/p\u003e \u003cp\u003eVariantAnnotation version 1.32.0(Obenchain et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), pcadapt 4.3.1 (Luu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), BSgenome.Scerevesiae.UCSC.sacCer3_1.4.0 (Huang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Team \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Pag\u0026egrave;s \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), TxDb.Scerevisiae.UCSC.sacCer3.sgdGene_3.2.2 (Carlson and Maintainer \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and all dependencies are available in Supplemental Table\u0026nbsp;1. In short, the 6800 variants that remained following filtering were analyzed for PCA clustering analysis using the pcadapt package with a k\u0026thinsp;=\u0026thinsp;3 based on screenplot. Variants then were filtered to remove all those found in any of the control strains, regardless of presence in the MCHM strains (Supplemental Table\u0026nbsp;2). This left 1171 variants existing only in at least one of the 8 MCHM evolved strains. These variants were analyzed for their effect on coding sequences using the predictCoding command of the VariantAnnotation package and the UCSC sacCer3 genome and transcript packages mentioned above. There are three protein/ORF annotation differences between the sacCer3 version of the genome and the S288c R64.2.1 version of the genome used to map the original sequences and call variants that did not affect the coding predictions of this dataset. The SIFT 4G variant annotator software was also used to determine the predicted tolerance score for each variant from the original ILE vcf files (Vaser et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e R64-1-1.23 database included with the SIFT4G jar was used to annotate the variants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGO terms and intersection of strain variants:\u003c/h2\u003e \u003cp\u003eGO terms for the 107 genes that contained nonsynonymous and synonymous variants in the MCHM evolved strains were determined using the DAVID bioinformatic database at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Huang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009a\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003eb\u003c/span\u003e). Default options were used for the analysis. The intersection analysis of strains containing the same genes was performed using the shiny app tool version of UpSetR at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gehlenborglab.shinyapps.io/upsetr/\u003c/span\u003e\u003cspan address=\"https://gehlenborglab.shinyapps.io/upsetr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Lex et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStrain construction\u003c/h2\u003e \u003cp\u003eThe YJM789 \u003cem\u003ePDR3\u003c/em\u003e::\u003cem\u003eNAT\u003c/em\u003e knockout was produced by transforming PCR construct containing the NAT\u003csup\u003eR\u003c/sup\u003e gene amplified from the pAG25 plasmid (Goldstein and McCusker \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Other knockout cassettes of genes were amplified from the BY4742 knockout collection using the G418\u003csup\u003eR\u003c/sup\u003e or amplified from pAG32 plasmid (HYG\u003csup\u003eR\u003c/sup\u003e). Transformations were performed using lithium acetate, as previously described (Gietz and Schiestl \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Colony PCR confirmation of the knockout cassette integration was performed. Serial dilution growth assays for resistance phenotyping of the knockout were performed on solid MCHM YPD media as above and compared to growth of the BY4742 knockout collection knockout of \u003cem\u003ePDR3\u003c/em\u003e as well (Brachmann et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Giaever et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Reciprocal hemizygosity assay used YJM789 \u003cem\u003epdr3::Nat\u003c/em\u003e\u003csup\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sup\u003e and BY4741 \u003cem\u003epdr3::Kan\u003c/em\u003e\u003csup\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sup\u003e and their respective parents to test the impact of the \u003cem\u003ePDR3\u003c/em\u003e allele on growth on MCHM (Rong-Mullins et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The YJM789 \u003cem\u003epdr3::Nat\u003c/em\u003e\u003csup\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sup\u003e was crossed with YJM789K5a med15::Hyg\u003csup\u003eR\u003c/sup\u003e and the diploid was sporulated (Winans et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Tetrads were dissected and haploid double mutants selected based on markers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eROS assay\u003c/h2\u003e \u003cp\u003eOvernight cultures of the strains YJM789 WT, YJM789 \u003cem\u003epdr3\u003c/em\u003e, S12, and S12 \u003cem\u003egex2\u003c/em\u003e were grown in YPD medium, then diluted and grown to log phase (OD\u003csub\u003e600\u003c/sub\u003e 0.4). The log phase cultures were further diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.2 and 100ul of cells were added into each well of a flat transparent 96-well plate. This was followed by adding 100ul YPD for the negative control group, and 100ul of MCHM (800ppm, 1000ppm, and 1200ppm) for the experimental groups. For the positive control, 100ul of the log phase cultures were treated with 4mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 30 min and then added to a well containing 100ul YPD. Each strain under each condition was performed in triplicate. The plate was then incubated at 30\u0026deg;C for 22 hours, with absorbance measurements taken every hour using the TECAN Infinite M Nano plate reader. ROS levels were quantified using the H2DCF-DA assay as previously described (James et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The plate was centrifuged to remove the media, followed by two washes with 100 \u0026micro;L mixture of sorbitol (1.2 M), EDTA (50 mM), and mercaptoethanol (2%). 100ul of lyticase (25 U/ml) was added to each well and incubated at 37\u0026deg;C for 30min. This was followed by another round of centrifugation to remove the lyticase solution. Next, 100 \u0026micro;L of the H2DCF-DA solution was added, gently pipetted up and down and then placed in the incubator, covered with foil, for 30 minutes. After this incubation, the plate was centrifuged, the liquid was discarded, and the resulting pellets were resuspended in 100 \u0026micro;L of 1x PBS by pipetting. ROS levels were measured at OD 504nm using the TECAN plate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInvasion Assay\u003c/h2\u003e \u003cp\u003eYeast were serially diluted and then grown for seven days. Cells were then washed off under running water with a gentle circular pressure from a gloved finger.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStructural Modeling\u003c/h2\u003e \u003cp\u003ePrediction of the impact of SNPs and variants on Pdr3 structure was performed by generating de novo structure predictions using the RoseTTAFold method in the Robetta server (Kim et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eViability Assay\u003c/h2\u003e \u003cp\u003eThe viability of YJM789 WT, \u003cem\u003epdr3\u003c/em\u003e, and \u003cem\u003epdr1\u003c/em\u003e strains was determined using the Nexcelom Cellometer X2. Cells were cultured in YPD flasks on a shaker for 12 days, with viability readings recorded every third day using the Nexcelcom ViaStain\u003csup\u003e\u0026trade;\u003c/sup\u003e viability kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eElemental Analysis\u003c/h2\u003e \u003cp\u003eFour biological replicates were treated with 650 ppm of MCHM for 30 minutes and then cells were digested and elements analyzed as previous carried out (Ravishankar et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Winans and Gallagher \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn-lab evolution of YJM789 in MCHM\u003c/h2\u003e \u003cp\u003ePrevious studies on the effects of MCHM on yeast used genetic knockouts, transcriptomics, metabolomics, and biochemical assays to determine that MCHM resistance is dependent on a complex interaction of many cellular and genetic networks (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e; Gallagher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). To expand upon this knowledge, resistant strains were developed through In-Lab Evolution (ILE) experiments to help elucidate genetic changes that might produce a new resistant genotype. There is considerable genetic variation across different yeast strains to MCHM (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Previously a mapping population between S96 (an S288c laboratory strain) and YJM789 (a clinical isolate) uncovered standing genetic variation that is linked to MCHM response (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). S96 is more MCHM resistant than YJM789 and we used these strains to select for increased MCHM resistance. Four replicates of each strain were evolved in rich liquid YPD media with and without 700ppm MCHM (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The lineages of the original eight samples (four control, four treated) were passaged every two days for a total of six passages. Control and treated lineages were plated after the sixth passage, and two single colonies were isolated from each lineage to produce eight total control strains and eight total treated strains. After six passages, the MCHM treated strains grew faster than the earlier passaged. However, this was only seen in the YJM789 cultures, whereas MCHM resistance in S96 strains did not develop after multiple attempts (not shown). S96 was already more MCHM resistant than YJM789 and further resistance could not be achieved in the ILEs. We therefore continued with YJM789 strains. These YJM789 strains were named S1-16, with S1-8 being the control strains (and S1 and S2 being from the same evolution experiment), and S9-16 being the treated strains (and S9 and S10 being from the same original lineage). The sixteen strains were tested for their MCHM resistance at 1000ppm on YPD media. The eight control strains showed wildtype levels of resistance, while all eight treated strains showed increased resistance as compared to wildtype (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). There was plate to plate variation in MCHM at 1000ppm, likely due to nearing the solubility limit of the chemical (Sain et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Phetxumphou et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The wildtype strain was plated on each plate to control for plate to plate variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal component analysis of mutations\u003c/h2\u003e \u003cp\u003eAfter successfully evolving resistant strains from YJM789 yeast, the sixteen samples were sequenced at approximately 25x coverage using Illumina paired-end reads. The reads of all 16 strains were mapped to the S288c reference genome and variants were called using GATK HaplotypeCaller. After removing the variants shared between all 16 strains (Supplemental Table\u0026nbsp;2), considered to be the existing variants between the reference genome and the YJM789 genetic background, variants were clustered on three principal components (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). To analyze whether there may be a general pattern of evolved resistance in the remaining variants, a PCA analysis was performed. However, the strains did not cluster according to resistance on any of these components. As an aside, they also did not cluster by original lineage, so none of the pairs (individual isolates from the same population), such as S1 and S2 or S9 and S10, clustered. This indicates the colonies isolated from the same cultures were genetically distinct. A large number of remaining background variants, approximately 6900 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), likely prevented clustering by resistance phenotype. A few high effect loci would be masked by large numbers of non-causative variants. Similarly, resistance spread among many lower effect loci spread throughout the genome would also be masked if resistance in each strain was due to different combinations of these small effects. In total, looking at the sum of variants produced by the in-lab evolution conditions did not produce clear patterns of resistance. The mutation patterns of the MCHM and control strains showed that there was no significant difference in the number of variants in control vs. MCHM ILEs (p\u0026thinsp;=\u0026thinsp;0.15) and the evolved strains had between 2326 and 2681 variants per strain (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Although MCHM is known to induce DNA damage (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which could possibly be a mechanism for mutation, the control strains accumulated similar numbers of mutations. Therefore, it likely played a minor role other than a selective pressure on mutations occurring normally in the growth conditions. The total number of mutations does not seem to be from any effect of the chemical on the number of mutations. There was no copy number variation noted in resistant strains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of mutated genes in ILE strains.\u003c/b\u003e This table contains the number of overall mutations and mutated genes in each ILE strain. Ty retrotransposon genes such as Gag/Pol domains were excluded from this analysis.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal mutations (6874 total)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMCHM specific mutations only (1171 total)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMutations in coding regions \u0026ndash; Excluding Ty\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGenes with mutations (107)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNon-synonymous mutations only\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGenes with Non-synonymous mutations (75)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2579\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e212\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2496\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2493\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2415\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2326\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFiltering MCHM evolved variants to those involved in coding sequence changes\u003c/h2\u003e \u003cp\u003eTo find patterns of mutations that contributed to MCHM resistance, mutants in both the control ILE and the MCHM resistant strains were assumed to be passenger mutations and not causative for the MCHM resistance phenotype. These mutations in the control ILE strains were filtered from the variants in the eight resistant ILEs. This left 1172 variants between the eight treated ILE strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Many of these variants were in non-coding regions of the genome and while they could contribute to resistance to MCHM through changes in expression levels of genes, mutations in intergenic regions were filtered out, leaving 485 variants in genes between the eight strains (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The final filter used to narrow the focus to those that may be causative of resistance was to remove variants in Ty retrotransposon Gag/Pol proteins. The YJM789 strain does not contain the same Ty complement as the S288c reference strain (Wei et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Therefore, the detection of these variants is likely due to the existing variation between the parent strain of the ILEs and the reference. The persistence of these variants, despite the prior utilization of filters to remove all variants shared between all 16 ILEs, as well as a filter for known SNPs in YJM789, can be attributed to the limitations of 25x coverage. Specifically, short-read sequencing is often unable to detect repetitive regions and large deletions. After removing Gag/Pol Ty retrotransposon detected variants, there were between 45 and 64 variants per strain in the eight strains, contained in 107 total genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Finally, limiting our analysis to nonsynonymous coding changes left between 20 and 42 variants per strain, contained in only 75 genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). SNPs were concentrated in the subtelomeric regions (Supplemental Fig.\u0026nbsp;1).\u003c/p\u003e \u003cp\u003eIn order to analyze the intersection of these 75 genes with nonsynonymous changes between strains, UpSetR software was utilized (Lex et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Each strain contained nonsynonymous variants in three to seven genes that were exclusive to that individual strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Supplemental Table\u0026nbsp;3). This accounted for 42 of the genes containing variants in the MCHM evolved strains, so more than half overlap of genes with variants were in multiple strains. An additional 18 genes contained variants in two strains. The remaining 15 genes contained variants within at least three strains, but only one gene was found to have a nonsynonymous variant in all eight strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). This gene was \u003cem\u003ePDR3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), a transcription factor that primarily controls the expression of ABC transporters involved in the pleiotropic drug response which exports chemicals from the cell that are involved in general stress (Delaveau et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). When the intersection of all 107 genes containing nonsynonymous or synonymous variants was analyzed, \u003cem\u003ePDR3\u003c/em\u003e remained the only gene with a variant in all eight MCHM ILE strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMCHM is known to act as a hydrotrope (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e), so variants that may stabilize the plasma membrane or cell wall could be likely resistance mechanisms. Despite consistency with previous work, these patterns also have issues that reduced confidence that they were causative variants. In particular, the variants included synonymous mutations and genes that were accumulating mutations in control strains as well. Synonymous mutations do not change protein sequence and are unlikely to significantly affect the function of that protein unless altering expression levels. The plentiful mutations in the same genes in control strains may indicate that the genes are hotspots for mutation accumulation, reducing the likelihood any particular mutation was found due to selection for a competitive advantage in MCHM. However, based on the QTL study previously done in MCHM, associative loci that failed to reach LOD score significance were spread relatively evenly throughout the genome (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). This indicates that the presence of small effect loci contributing to a combined pleiotropic effect on resistance phenotype is likely outside mutations in \u003cem\u003ePDR3\u003c/em\u003e which will be discussed in more detail below. These variants may be contributing small phenotypic effects even if they are not sufficient to produce resistance on their own.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of genes containing unique variants in at least four MCHM ILE strains\u003c/b\u003e. Uncharacterized ORFs have no annotated function in yeast but may produce functional proteins. Dubious ORFs are unlikely to produce functional proteins. The last two columns show how many MCHM, and control ILE strains contain any variant at all, including variants that appear in both MCHM and control strains. These columns are included to show to what extent this gene seems to accumulate variants overall. Total number of variants is in parentheses in the last two columns, as some strains contain multiple variants in that gene.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMCHM ILEs Containing Unique Variant(s) in this Gene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eORF Character-ization\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChr\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStart\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEnd\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMCHM ILEs - any Variant\u003c/p\u003e \u003cp\u003e[genes (variants)]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eControl ILEs - any Variant\u003c/p\u003e \u003cp\u003e[genes (variants)]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAGP3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18680\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCOS4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6 (22)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eFLO1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e203403\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e208016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (187)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (280)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHPF1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e28703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (111)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHXT13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21537\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e23231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (60)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHXT17\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e772657\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e774351\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6 (16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHXT9\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19497\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e21200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (13)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (19)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePAU24\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e809057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e809419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7 (10)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePDR3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e217470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e220400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eSAM3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXVI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e24701\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (29)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTPO2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVerified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrVII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e763762\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e765606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYAL069W\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDubious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (97)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYCR108C\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUncharacterized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e315997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e316188\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (26)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYEL077C\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUncharacterized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (63)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYEL077W-A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDubious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e630\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 (16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYER188C-A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUncharacterized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e569608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e569907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (68)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (56)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYNL018C\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUncharacterized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e599936\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e601774\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (64)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYNL034W/ PAR1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUncharacterized\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eYNL337W\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDubious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003echrXIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7 (18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e6 (12)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo determine if mutations in any cellular process might be important for evolved MCHM resistance, the 107 genes containing at least one variant in at least one MCHM ILE strain, including synonymous variants, were analyzed via GO Term analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Most GO terms pointed to mutations in plasma membrane (FDR\u0026thinsp;=\u0026thinsp;0.0096%) or cell wall proteins (FDR\u0026thinsp;=\u0026thinsp;5.85 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e%), including those important for sugar transport or flocculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In agreement with previous genomic work with MCHM, the vacuole (FDR\u0026thinsp;=\u0026thinsp;0.21%) and response to stress (FDR\u0026thinsp;=\u0026thinsp;0.19%) also contained mutations that may impact resistance (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo identify mutations that directly contributed to MCHM resistance, we focused on genes that contained mutations in multiple isolates. There were 18 genes with at least one synonymous or nonsynonymous variant not found in control strains within their coding regions in four or more MCHM ILEs (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, many of these genes also seem to accumulate a high number of mutations in the rich liquid media culture environment. This is exemplified by \u003cem\u003eFLO1\u003c/em\u003e, where all sixteen strains contained variants, 467 variants total. Some genes in the list accumulate more variants, possibly through their position near the ends of chromosomes where mutation rates are known to be higher (Lang and Murray \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Agier and Fischer \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ivanova et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and/or reduced selective pressure on their function in laboratory growth media. Only two genes showed no mutations in any of the eight control ILE strains while also showing mutations in at least half of the MCHM ILEs, \u003cem\u003ePDR3\u003c/em\u003e, and \u003cem\u003eTPO2\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe list included seven uncharacterized or dubious ORFs and were mostly encoded at the ends of their respective chromosomes (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The unknown or nonexistent function of these genes made them unlikely candidates for adaptive resistance mechanisms. The remaining genes\u0026rsquo; functions varied, but most were membrane or cell wall proteins (\u003cem\u003eFLO1, HPF1, PAU24\u003c/em\u003e), often involved in sugar or nutrient transport (\u003cem\u003eAGP3, HXT9, HXT13, HXT17, TPO2\u003c/em\u003e). These cell wall genes, as well as stress response genes such as \u003cem\u003ePDR3\u003c/em\u003e, may be situated as targets of adaptive changes for MCHM resistance due to their changes that may decrease the internal cellular concentration of the chemical.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePrediction analysis for the tolerance of protein-coding changes from variants in MCHM ILEs\u003c/h2\u003e \u003cp\u003eBecause there were thousands of variants per strain, including dozens of variants in coding regions of each MCHM ILE, all mutants unique to the MCHM resistant strains were scored either as deleterious or well tolerated for protein function. SIFT analysis predicts whether amino acid substitutions from single nucleotide polymorphisms will be tolerable for protein function using multiple sequence alignment of homologous protein sequences (Vaser et al. \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The variants in our dataset included both SNPs and indels, but indel variants could not be scored, because SIFT exclusively analyzes SNPs. CNVs were analyzed but not detected in the evolved strains. Most of the variants were predicted to be tolerated (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplemental File 2). There are two reasons this may be the case. First, many of these variants are synonymous with coding changes, therefore SIFT analysis aligns proteins and predict no deleterious effect based on conservation. That does not eliminate the possibility that the proteins\u0026rsquo; expression levels change due to codon bias changes (Letzring et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Presnyak et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but these synonymous changes are less likely to be the major drivers of resistance change than protein sequence changes. The second reason most of these variants would be tolerated is selective pressures to keep conserved functions of proteins with nonsynonymous changes. The nonsynonymous changes would be more likely to replace similar amino acids that do not disrupt protein function, or in regions of the protein less likely to disrupt function. Variants in this dataset that were predicted to be tolerated were deemed less likely to be causative of the evolved resistance to MCHM.\u003c/p\u003e \u003cp\u003eThe deleterious and NA scored variants all include mutations that change the protein sequences. A variant given a score of NA (not applicable) was usually an indel variant that could not be scored, but occasionally was a SNP variant that did not appear in the SIFT database. Many of the deleterious and NA scored variants appear in proteins that are uncharacterized or dubious ORFs (Supplemental File 1). Notably, the variants in \u003cem\u003ePDR3\u003c/em\u003e in all eight MCHM ILEs received a score of NA. The only pattern of genes with these scores amongst the ILEs was the \u003cem\u003ePDR3\u003c/em\u003e mutations. Other deleterious and NA scored mutations may provide unique effects on MCHM resistance in individual strains but did not represent a consistent pathway to resistance in different strains.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eSummary of predicted tolerance for coding variants found only in MCHM ILE strains.\u003c/b\u003e The eight MCHM ILE strains (S9-S16) are represented from left to right in columns. Each strain is summarized for whether its coding variants are predicted to be tolerated, deleterious, or NA based on SIFT4G analysis. NA scores mean a classification as either tolerated or deleterious was not possible to determine, or not applicable (NA). SIFT4G scores approaching 0 are expected to alter protein function to be deleterious based on amino acid substitutions. The NA results are usually due to the inability of SIFT to predict the effects of indels or occasionally SNPs that do not appear in the SIFT database. Coding variants in this table exclude those in Ty regions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMCHM ILE Strain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS9\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS11\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS12\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS13\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS14\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS15\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS16\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Variants\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTolerated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDeleterious\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eComparison of MCHM evolved genes with genes appearing in other genomic datasets from MCHM studies\u003c/h2\u003e \u003cp\u003ePrevious work has been done to produce genomic datasets that implicate genes involved in MCHM resistance mechanisms, so it hypothesized that some variants may target these same genes to adapt to MCHM treatment. The list of 107 genes containing at least one variant in an MCHM ILE strain was compared to a genetic screen of the BY4742 knockout collection for MHCM sensitivity and transcriptomic dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There was very little overlap with the genetic screen (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This is expected, as the knockout screen selected for mutants with reduced MCHM resistance, revealing genes that are required for resistance. Therefore, in the 329 screen hits required for MCHM resistance, any variants would necessarily have to not reduce, perhaps even improve, the function of any proteins produced by those ORFs in order to create adaptive resistance.\u003c/p\u003e \u003cp\u003eYeast were treated with MCHM and the changes in gene expression were quantified. The transcriptomic dataset included a list of 592 upregulated genes and a list of 576 downregulated genes. There was more overlap with these lists and the 107 ILE genes, including 14 upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) and eight downregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The overlapping genes in these cases include several of the genes with variants in many ILE strains. \u003cem\u003eDOG1\u003c/em\u003e, which encodes a deoxyglucose phosphatase and is involved in resistance to 2-deoxyglucose (Sanz et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Randez-Gil et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Soncini et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), had mutations in the ILE and was upregulated in MCHM exposure.\u003c/p\u003e \u003cp\u003e Several genes encoding cell wall proteins appear as well, including \u003cem\u003ePIR3, TIR1, SCW10\u003c/em\u003e, and \u003cem\u003eFLO1\u003c/em\u003e, each with varying functions from cell wall stability (\u003cem\u003ePIR3\u003c/em\u003e) to flocculation (\u003cem\u003eFLO1\u003c/em\u003e) (Hodgson et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Kitagaki et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Cappellaro et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Doolin et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Rossouw et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). We tested the requirement of several of these genes for resistance to MCHM. First, we tested the growth of the knockouts in the S288c background on MCHM. The \u003cem\u003eynr065c\u003c/em\u003e, \u003cem\u003eynl034w\u003c/em\u003e (\u003cem\u003epar1)\u003c/em\u003e, and \u003cem\u003ehxt17\u003c/em\u003e mutants in S288c background did not show appreciable change in growth on MHCM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). \u003cem\u003eYNR065c, YNL034w (PAR1)\u003c/em\u003e, and \u003cem\u003eCOS4\u003c/em\u003e were selected for further study by knocking them out in the YJM789 strain. The \u003cem\u003eynl034c\u003c/em\u003e and \u003cem\u003ecos4\u003c/em\u003e mutants grew slightly slower than YJM789 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). \u003cem\u003eCOS4\u003c/em\u003e is required for the multivesicular vesicle body sorting pathway and provides ubiquitin \u003cem\u003ein trans\u003c/em\u003e for non-ubiquitinated cargo proteins (MacDonald et al. 2015). There was not much difference visually in the growth of the YJM789 wild type and that of the \u003cem\u003eCOS4\u003c/em\u003e knockout. It is possible that the knockout had no increased sensitivity compared to the wild type, so the mutation was not activating and \u003cem\u003ecos4\u003c/em\u003e knockout growth was not affected by MCHM exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe uncovered nonsynomous SNPs in an unknown ORF \u003cem\u003eYNL034c\u003c/em\u003e that has no predicted transmembrane domains or GPI anchor site but is associated with the cell membrane (Weill et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Ynl034c has a paralog, Ynl018c, that is also localized on the cell membrane (Weill et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003eYNL034c\u003c/em\u003e has 24 nonsynonymous SNPs between YJM789 and S288c and 15 synonymous SNPs suggesting it is under positive selective pressure. Two different SNPs were detected in different ILE strains, K554R and H541N. We have named it \u003cem\u003ePAR1\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003eatches \u003cem\u003eA\u003c/em\u003eround \u003cem\u003eR\u003c/em\u003eadius). Examining the genomes of the ILEs uncovered other ILE induced SNPs in genes encoding membrane associated proteins. We tested knockouts of \u003cem\u003eflo1\u003c/em\u003e and \u003cem\u003eflo10\u003c/em\u003e and loss of these genes conferred sensitivity to MCHM in S288c (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), we knocked out these genes in YJM789 and the ILE strain (S14, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In the ancestral strain, loss of either flocculin did not change growth but in S14, the \u003cem\u003eflo1\u003c/em\u003e mutant was less sensitive and \u003cem\u003eflo10\u003c/em\u003e was slightly more resistant. Flo1 expressing cells excrete glucose and mannose polysaccharides that limit the size of chemicals that interact with cell wall and membrane. The ILE SNP in Flo1 was S1092A and in Flo10 was T402T, both outside the flocculin repeat. Flo5 and Flo9 also contained nonsynonymous SNPs int the ILE strains. In S288c, the \u003cem\u003epir3\u003c/em\u003e knockout was also MCHM sensitive (Supplemental Fig.\u0026nbsp;2B) which supports that cell wall structure perturbations alter cellular tolerance to MCHM. Flocculins are cell wall proteins that bind mannose chains on other cells causing the cells to stick together (Rossouw et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and flocculate. Flocculation enhances survival during starvation and is regulated by the cell wall integrity pathway (Sariki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Flo1 regulates cell surface hydrophobicity (Sariki et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Flocculation also aids in directed cell growth into the agar as cells consume the available nutrients. This invasion growth is assessed by determining if cells can be washed off the surface of the plates. MCHM inhibited invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). YJM789 liquid cultures flocculate, rapid sediment when not shaking and saturated cultures but \u003cem\u003eflo\u003c/em\u003e mutant cultures remain suspended as well as ILE S14 strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Expression of some \u003cem\u003eFLO\u003c/em\u003e genes is dependent on amino acid transporters (Torbensen et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). These datasets continue to point to the cell wall and sugar metabolism as functions that are important ways to adapt resistance to MCHM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNumerous mutations in transporters were identified in the genomic analysis of the ILE strains. \u003cem\u003eTPO2\u003c/em\u003e belongs to a family of transporters that are part of the multidrug resistance pathway. The protein localizes to the plasma membrane and has been characterized as an exporter of polyamines (Tomitori et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), which could be related to the amino acid biosynthesis pathways implicated in MCHM resistance in previous work (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, the mutations in \u003cem\u003eTPO2\u003c/em\u003e are all synonymous (Supplemental File 1). The codon changes could potentially affect protein levels if they affect translation rates. The S288c knockout showed no change in resistance but the \u003cem\u003etpo3\u003c/em\u003e knockout was more sensitive (Supplemental Fig.\u0026nbsp;2A). Tpo3 is a homolog of Tpo2 and both are polyamine transporters localized to the vacuole (Tomitori et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Mal31 is a maltose permease regulated by Mal13, a transcription factor (Orikasa et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The \u003cem\u003emal31\u003c/em\u003e mutant in BY4742 was sensitive to MCHM (Supplemental Fig.\u0026nbsp;2A). We then knocked out \u003cem\u003eMAL31\u003c/em\u003e in YJM789 and ILE strains with a nonsynonymous SNP, I415V in Mal31. I415V did not alter the transmembrane domain prediction. Unlike the BY4742 mutant there was no change in growth in response to MCHM in any of the mutants (Supplemental Fig.\u0026nbsp;2B). We also grew these strains on maltose, disaccharide of dextrose instead of dextrose and while cells grew slower there was no impact of genotype on growth (Supplemental Fig.\u0026nbsp;2B). In ILE strains with the \u003cem\u003eMAL31\u003c/em\u003e mutation there was also a mutation in \u003cem\u003eMAL13\u003c/em\u003e, which encodes a transcription factor that regulates \u003cem\u003eMAL\u003c/em\u003e genes (Supplemental File 1). The mutation was outside the DNA binding domain. Yeast resistant to 2-deoxyglucose increase expression of \u003cem\u003eMAL31\u003c/em\u003e (Orikasa et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Slowing yeast growth by changing the carbon source from dextrose to maltose reduces the ability of MCHM to slow growth. Flux balance analysis on MCHM which integrates transcriptomic and metabolomic data identified \u003cem\u003eCYT1\u003c/em\u003e as a key step in metabolic regulation during MCHM (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Sugars such as maltose can inhibit flocculation (Stratford \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e1989\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere were mutations in other types of transporters. \u003cem\u003eGEX2\u003c/em\u003e encodes a glutathione transporter important for oxidative stress resistance, a known source of stress from MCHM treatment (Dhaoui et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At early growth stages, glutathione is brought into the cell's vacuole, while in later stages it is exported to the cytosol surrounding the cell (Outten et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Glutathione has been known to reduce reactive oxygen species (ROS) by acting as an antioxidant, however it is unsure whether in these cases it is acting more so as a nitrogen source or an antioxidant when exposed to MCHM (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e Gex2 transports glutathione while Sam3 transports S-adenosylmethionine (SAM) and polyamines such as spermidine and putrescence. Gex2\u003csup\u003eYJM789\u003c/sup\u003e has five naturally occurring nonsynonymous SNPs and the ILE induced SNP, D570G, is within the last transmembrane domain (aa 578 to 615) but does not alter the transmembrane domain prediction. The transmembrane domain predictions for Sam3 were also not altered with ILE SNPs K566R and V339I. The Agp3 ILE SNP was V212I, which was also in a transmembrane domain, but it did not change its prediction. While there are no SNPs between YJM789 and S288c, the \u003cem\u003eAPG3\u003c/em\u003e deletion mutant in BY4742 was sensitive to MCHM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) but the \u003cem\u003eapg3\u003c/em\u003e knockout was likely lethal in YJM789 because it could not be generated. There are examples of other genes that become essential in different strain backgrounds; the \u003cem\u003earo1\u003c/em\u003e knockout in RM11 was lethal while it was not essential in other strains (Rong-Mullins et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In YJM789, the \u003cem\u003egex2\u003c/em\u003e mutant was slightly more sensitive to MCHM while the \u003cem\u003esam3\u003c/em\u003e mutant growth was not different from the parent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, knocking out these genes in the ILE strain that they were identified from increased the MCHM sensitivity which suggests that the ILE induced SNPs were activating mutations. Due to the involvement of Gex2 in ROS response, we further measured the ROS production in YJM789 \u003cem\u003egex2\u003c/em\u003e after MCHM exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Because MCHM slowed growth, we measured correlated growth rate with ROS production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Production of ROS was proportional to growth so that the small population that had increased ROS when previously measured at the single cell level (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and consistent with bulk measurements ROS levels decreased.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of evolved alleles in\u003c/b\u003e \u003cb\u003ePDR3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePDR3\u003c/em\u003e variants in the MCHM ILEs showed a pattern of evolution that pointed to a reproducible pathway to resistance. We decided to look more closely at the individual mutations. Previous research has been done with \u003cem\u003ePDR3\u003c/em\u003e mutagenesis to produce gain-of-function alleles that improve resistance to different chemicals (Nourani et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). For instance, amino acid mutations in the region from approximately residues 220\u0026ndash;280 created alleles that increased the expression of multiple ABC transporters that pump chemicals out of cells, specifically \u003cem\u003eSNQ2\u003c/em\u003e and \u003cem\u003ePDR5\u003c/em\u003e (Nourani et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). The mutations in \u003cem\u003ePDR3\u003c/em\u003e in the S12, S13, and S15 ILEs mutated single amino acids at residues 288, 209, and 229 respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). It is possible that these mutations are creating gain-of-function alleles that increase the expression of ABC transporters, thereby conveying resistance to MCHM. The gene \u003cem\u003eSNQ2\u003c/em\u003e is required for resistance to MCHM (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but it has yet to be shown if overexpression would be sufficient to produce resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe remaining five mutations resulted in changes to the C-terminal portion of the protein, where an activation domain homologous to Gal4-like transcriptional activators is located (Delaveau et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The S9 mutation in Pdr3 was a M842L single amino acid change (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supplemental Fig.\u0026nbsp;3A). One study produced six different gain-of-function mutations in this region with single nucleotide changes that increased expression of \u003cem\u003ePDR3\u003c/em\u003e, \u003cem\u003ePDR5\u003c/em\u003e, and \u003cem\u003eSNQ2\u003c/em\u003e and conferred resistance to\u003c/p\u003e \u003cp\u003e several chemicals (Simonics et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). None of the mutations were the same as found in the ILE strains, but they implicate this region of the protein for possible gain-of-function resistance mutations. The S16 mutation was an insertion and frameshift occurring at the amino acid 972 that altered and extended the remaining four amino acids into an extra 9 amino acids before the new stop codon (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e \u003cb\u003econt\u003c/b\u003e. shown by a slash and dashed line where the missing portion of the protein coding sequence will not be translated. The final strain S16 contains an insertion that alters the last five amino acids of the protein to a new 13 amino acid sequence. The asterisks at the top represent the interface residues found on the Pdr3 homodimer structure prediction. \u003cb\u003eB.\u003c/b\u003e Serial dilution growth assays of one of the evolved resistant strains (S11), the YJM789 parent strain, the YJM789 \u003cem\u003ePDR3\u003c/em\u003e knockout strain, the BY4742 wildtype strain, and BY4742 \u003cem\u003ePDR3\u003c/em\u003e knockout were carried out. The growth assays are on increasing concentrations of MCHM from 0ppm (YPD) to 1000ppm. \u003cb\u003eC.\u003c/b\u003e Serial dilution reciprocal hemizygosity growth assays of S288c (BY) and YJM789 hybrid mutants of \u003cem\u003ePDR3.\u003c/em\u003e The growth of the wildtype hybrid was compared to hybrids lacking \u003cem\u003ePDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBY\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eYJM\u003c/em\u003e\u003c/sup\u003e, or both (homozygous diploid) on MCHM. \u003cb\u003eD.\u003c/b\u003e Serial dilution single \u003cem\u003epdr3, med15\u003c/em\u003e, the double \u003cem\u003epdr3, med15\u003c/em\u003e mutant and \u003cem\u003eyke4\u003c/em\u003e in YJM789 grown on MCHM or rapamycin. \u003cb\u003eE.\u003c/b\u003e Percent change of elements potassium (K), magnesium (Mg), phosphorus (P), sulfur (S), and zinc (Zn) when yeast from part D were exposed to MCHM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe S10 and S14 mutations truncated the protein by 415 and 406 amino acids respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The S11 mutation also truncated the protein, but by 82 amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). One hypothesis is that deletions of such a large part of the protein sequence would produce nonfunctional products, effectively acting as a knockout. Another possibility is that these truncations could still produce proteins with some function, considering the DNA binding domain is at the N-terminus like in other Gal4-like transcription factors (Mamnun et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Furthermore, the domains implicated in the homo- and heterodimer interactions of Pdr3 and Pdr1 are also in the N-terminal 400 amino acids. With dimerization and DNA interacting regions of the protein products intact, the truncated alleles of S10, S11, and S14 could be functional proteins. A side-by-side comparison of the predicted structure of Pdr3 from YJM789, S10, S11, and S14 shows considerable differences in structure upon truncations in the C-terminus (Supplemental Fig.\u0026nbsp;3B). Interface residues in the predicted Pdr3 homodimer structure revealed contact points in the N- and C- terminals (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supplemental Fig.\u0026nbsp;3B) but none of these residues had ILE induced mutations.\u003c/p\u003e \u003cp\u003eTo test the more fundamental hypothesis that a knockout-like truncation may confer resistance to MCHM, we knocked the \u003cem\u003ePDR3\u003c/em\u003e gene out of the wildtype parent of the ILEs, YJM789. The \u003cem\u003epdr3∆\u003c/em\u003e strain showed similar resistance as the S11 resistant MCHM strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). S11 corresponds to the strain containing the shortest truncation of the Pdr3 protein, only 82 amino acids. If the mutations to \u003cem\u003ePDR3\u003c/em\u003e in the evolved strains are mimicking a knockout by making Pdr3 nonfunctional, that would be sufficient to produce the resistant phenotype. During the ILE experiment both YJM789 and S288c were passaged in MCHM but no resistance could be detected in the S288c strain. A Quantitative Trait Loci (QTL) analysis of S288c and YJM789 failed to identify \u003cem\u003ePDR3\u003c/em\u003e and there are four nonsynonymous SNPs Q56R, T102I, A885T, and N916S (Pupo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). The first two SNPs are in the Zn finger domain and the last two are in the regulatory domain. To assess if these SNPs affected growth on MCHM, \u003cem\u003ePDR3\u003c/em\u003e knockout in YJM789 and S288c (BY) were mated together to assess the individual contribution in the hybrid with only the allele of \u003cem\u003ePDR3\u003c/em\u003e being different between strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Hybrid yeast expressing only \u003cem\u003ePDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eYJM789\u003c/em\u003e\u003c/sup\u003e were very sensitive to MCHM. Yeast expressing both \u003cem\u003ePDR3\u003c/em\u003e alleles were slightly less sensitive, while the double mutant or yeast expressing only \u003cem\u003ePDR3\u003c/em\u003e\u003csup\u003e\u003cem\u003eBY\u003c/em\u003e\u003c/sup\u003e were more tolerant.\u003c/p\u003e \u003cp\u003eTo assess which genes are differentially regulated in \u003cem\u003epdr1\u003c/em\u003e and \u003cem\u003epdr3\u003c/em\u003e deletion strains, we determined the promoter binding signal of Pdr3 and Pdr1 targets using a published dataset in the S288C background (Supplemental Table\u0026nbsp;4, (Gera et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)). Notably, Pdr3 appears to exhibit a weaker binding signal overall, which strengthens considerably in absence of \u003cem\u003ePDR1\u003c/em\u003e (Supplemental Fig.\u0026nbsp;4). Upon closer examination, deletion of \u003cem\u003ePDR3\u003c/em\u003e primarily results in stronger Pdr1 binding upstream of \u003cem\u003eGAC1\u003c/em\u003e, a regulatory subunit of a phosphatase involved in glycogen accumulation (Supplemental Fig.\u0026nbsp;5A) (Wu et al. \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Additionally, instances of Pdr1 binding strength increasing upon \u003cem\u003ePDR3\u003c/em\u003e deletion include \u003cem\u003eTPO1, SPO24\u003c/em\u003e and \u003cem\u003eLDB7\u003c/em\u003e (Supplemental Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cp\u003eTo test the hypothesis that loss of Pdr3 function changed Pdr1 function we tested if the YJM789 \u003cem\u003epdr1\u003c/em\u003e mutant was also MCHM sensitive. However, the YJM789 \u003cem\u003epdr1\u003c/em\u003e was just as MCHM resistant as the \u003cem\u003epdr3\u003c/em\u003e knockout (Supplemental Fig.\u0026nbsp;6A). Interestingly, mutations in \u003cem\u003ePDR1\u003c/em\u003e were not detected in the ILE and one possibility is that \u003cem\u003epdr1\u003c/em\u003e mutant has a defect in long term survival during starvation. We measured viability of YJM789 \u003cem\u003epdr3\u003c/em\u003e and \u003cem\u003epdr1\u003c/em\u003e mutants during long term starvation in YPD, but no significant differences were found (p value 0.79) in 12 days of starvation.\u003c/p\u003e \u003cp\u003eBoth Pdr1 and Pdr3 strongly bind upstream of \u003cem\u003ePDR5.\u003c/em\u003e While binding of Pdr1 was not affected in the \u003cem\u003epdr3\u003c/em\u003e knockout, binding of Pdr3 was decreased in the \u003cem\u003epdr1\u003c/em\u003e knockout (Supplemental Fig.\u0026nbsp;5). Pdr5 is an ABC transporter facilitating the export of diverse chemicals out of the cells. Consequently, we tested the YJM789 \u003cem\u003epdr5\u003c/em\u003e growth on MCHM (Supplemental Fig.\u0026nbsp;5). The \u003cem\u003epdr5\u003c/em\u003e mutant was not more sensitive to MCHM but the \u003cem\u003eyrr1\u003c/em\u003e mutant which is sensitive to 4NQO (Gallagher et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Rong-Mullins et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) was also MCHM sensitive and resistant to rapamycin (Supplemental Fig.\u0026nbsp;6B). Yrr1 is also involved in response to vanillin (Cao et al., 2021; Zhao et al., 2023) and the knockout is vanillin sensitive (Supplemental Fig.\u0026nbsp;6B).\u003c/p\u003e \u003cp\u003eInterestingly, Pdr3, Pdr1 and Yrr1 have all been described as transcriptional activators of \u003cem\u003eSNQ2\u003c/em\u003e, an ABC transporter (Cui et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The BY4742 \u003cem\u003eSNQ2\u003c/em\u003e knockout was previously determined to be MCHM sensitive (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Yrr1 also has a paralog, Pdr8 and both do not appear to strongly bind \u003cem\u003ePDR5\u003c/em\u003e (Supplemental Fig.\u0026nbsp;4). The \u003cem\u003eyrr1\u003c/em\u003e mutant was also MCHM resistant but does not bind \u003cem\u003eAGP3\u003c/em\u003e promoter. The \u003cem\u003eAGP3\u003c/em\u003e knockout was MCHM sensitive in the S288c background (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA) but we could not generate the YJM789 knockout. Four ILE mutations in \u003cem\u003eAGP3\u003c/em\u003e were the most activating mutations and increased amino acid import.\u003c/p\u003e \u003cp\u003e \u003cem\u003ePDR3\u003c/em\u003e is the first gene to be identified in yeast as a target to induce sensitivity to MCHM. Interestingly, four different wine strains contained genomic deletions of the \u003cem\u003ePDR3\u003c/em\u003e (Dunn et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Previous work, such as the genetic screen, focused only on finding genes required for tolerance (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The screen was not designed to detect an increased resistance phenotype in any of the mutants tested. The model for this resistance involves the importance of Pdr3 in controlling the activation of multiple ABC transporters that pump stress-inducing chemicals out of cells. Pdr3 and its paralog Pdr1 form homo- and heterodimers and then bind to transcriptional response regions termed PDREs where they can inhibit or activate transcription of genes such as \u003cem\u003ePDR5\u003c/em\u003e, \u003cem\u003eSNQ2\u003c/em\u003e, \u003cem\u003eYOR1\u003c/em\u003e, \u003cem\u003ePDR15\u003c/em\u003e, \u003cem\u003ePDR10\u003c/em\u003e, and other transporters (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We tested via knockout in the YJM789 parent strain if inactivating mutations could be sufficient to produce MCHM resistance (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). The mutations in \u003cem\u003ePDR3\u003c/em\u003e could also be activating mutations that make Pdr3 increase transcription of all or some transporters, such as \u003cem\u003eSNQ2\u003c/em\u003e or \u003cem\u003ePDR5\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHaving identified mutations in \u003cem\u003ePDR3\u003c/em\u003e that directly affect response to MCHM across all ILE strains, we aimed to delve deeper into its regulatory mechanism. Previous work further uncovered genetic variation in Med15, a component of the Mediator complex, that contributed to variation in yeast MCHM response between YJM789 and S288c (Gallagher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As part of the tail in the Mediator complex, Med15 directly interacts with Pdr3 (Shahi et al. \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Therefore, in order to test if loss of Med15 could suppress the \u003cem\u003epdr3\u003c/em\u003e MCHM resistance we generated double mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The \u003cem\u003emed15\u003c/em\u003e knockouts are slow growing and while not specifically sensitive to MCHM their slow growth is not altered in the presence of MCHM. However, the \u003cem\u003emed15, pdr3\u003c/em\u003e double mutant appeared just as sensitive to MCHM as the parental strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eGiven the impact of MCHM on protein solubility, a property regulated by Intrinsically Disordered Regions (IDRs), and the known regulation of MCHM response by Med15 IDRs, we determined the IDR prediction of Pdr3 alleles (Erdős and Doszt\u0026aacute;nyi \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) and found no difference between strains (Supplemental Fig.\u0026nbsp;3C). Med15 interacts with multiple transcription factors and to test whether Med15 mediates the MCHM resistance due to loss of Pdr3 function we generated double mutants in the YJM789 background. Loss of \u003cem\u003emed15\u003c/em\u003e represses the MCHM resistance of the \u003cem\u003epdr3\u003c/em\u003e knockout (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Due to the effect of MCHM on growth through induced amino acid starvation response, we tested whether rapamycin inhibits TORC1 directly. The \u003cem\u003epdr3\u003c/em\u003e and \u003cem\u003epdr3, med15\u003c/em\u003e double mutant showed no change in growth on rapamycin (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The \u003cem\u003emed15\u003c/em\u003e mutant is slow growing on YPD but its growth rate is not affected on rapamycin. It is possible that in the absence of \u003cem\u003epdr3\u003c/em\u003e, Med15 is free to recruit other transcription factors or Pdr1, would only be present as a homodimer since it dimerizes with Pdr3.\u003c/p\u003e \u003cp\u003ePrevious work has shown that zinc levels are increased in MCHM exposure and excess zinc suppresses S288c MCHM sensitivity (Pupo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). We found that zinc levels were not increased in the \u003cem\u003epdr3\u003c/em\u003e mutant upon MCHM exposure, and neither were the levels of other metals tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Dysregulation of the metallome correlated with yeast sensitivity to MCHM. In a QTL analysis, \u003cem\u003eYKE4\u003c/em\u003e, a zinc transporter was linked to MCHM resistance. We tested the ability of zinc to suppress MCHM response, but we only noted mild exacerbation of growth inhibition of all YJM789 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eSNQ2\u003c/em\u003e gene is required for MCHM resistance, so it is a likely candidate for this increased expression (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eSNQ2\u003c/em\u003e does not have SNPs between YJM789 and S288c. The YJM789 allele of \u003cem\u003ePDR5\u003c/em\u003e is divergent from the reference and other yeast strains, with a 5.3% amino acid difference compared to the reference strain (Wei et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The YJM789 allele has been shown to alter the strain\u0026rsquo;s resistance to antifungals (Guan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, p. 201), increasing or decreasing resistance dependent on the chemical. \u003cem\u003ePDR5\u003c/em\u003e is not required for resistance to MCHM like \u003cem\u003eSNQ2\u003c/em\u003e according to previous work, but this work was done in the BY4742 strain, which has the same allele as the reference strain (Supplemental Fig.\u0026nbsp;6C) (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It is possible that the YJM789 \u003cem\u003ePDR5\u003c/em\u003e allele can provide resistance to MCHM if the expression is increased. \u003cem\u003ePDR5\u003c/em\u003e is significantly upregulated by gain-of-function mutations in \u003cem\u003ePDR3\u003c/em\u003e in the same regions as the MCHM ILE variants (Nourani et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Simonics et al. \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), but the \u003cem\u003epdr5\u003c/em\u003e mutant did not change MCHM resistance. From previous analysis, Pdr1 binding at \u003cem\u003eGAC1\u003c/em\u003e decreases in the \u003cem\u003epdr3\u003c/em\u003e mutant and Pdr3 binding also decreases in the \u003cem\u003epdr1\u003c/em\u003e mutant at \u003cem\u003eGAC1\u003c/em\u003e (Gera et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Gac1 binds Hsf1, a stress induced transcription factor, and it is induced as dextrose is consumed and glycogen accumulates (Lin and Lis \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). However, \u003cem\u003eGAC1\u003c/em\u003e expression did not change in S288c (Pupo et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e; Gallagher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe In-Lab evolution of the YJM789 strain of \u003cem\u003eS. cerevisiae\u003c/em\u003e produced thousands of mutations in each strain. The mutations were similar in number from strain to strain, including both control media and MCHM treated conditions. There was no clear pattern of mutations resulting in MCHM resistance across strains, though the sheer number of mutations could mask any pattern with background variants. When filtered for mutations unique to MCHM ILEs that were only found in coding sequences, patterns such as cell wall and responses to stress did begin to emerge. These patterns are consistent with previous knowledge of MCHM effects on cells, including oxidative stress activation (Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Transcriptional analysis, genetic screens, and metabolomic data showed that diverse pathways are affected by crude MCHM (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003ea\u003c/span\u003e; Gallagher et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ayers et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Perfetto et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pleiotropic effects of MCHM likely stem from its ability to act as a hydrotrope, altering protein structures and solubility (Pupo et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e). Hydrotropes in cells prevent protein aggregation, but unlike surfactants, work at millimolar concentrations and display low cooperativity. These contribute to liquid-liquid phase separation (LLPS) that explains how membraneless organelles form in the cell. MCHM is a hydrophobic chemical and likely alters membrane dynamics. Multiple amino acid biosynthetic pathways are upregulated signaling inhibition of TORC1, but levels of several amino acid are leveled after only 30 minutes of exposure. Excess amino acids are stored in the vacuole and reduction in vacuolar acidification increases MCHM sensitivity.\u003c/p\u003e \u003cp\u003eWe propose a model in which Pdr3 is the major driver of resistance to MCHM through regulating transcription of ABC transporters, such as Pdr5, Pdr10, Pdr15, Snq2 and Yor1. Loss of mutations are far more common than gain of mutations and through the ILE loss of other transcription factors such as Pdr1 or Yrr1 were not selected for because those mutants would have a decrease in fitness during the competition during nutrient starvation. Yrr1 mutants have decreased growth during respiration (Rong-Mullins et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Loss of Pdr3, Pdr1, or Yrr1 enables the mediator complex component Med15 to function elsewhere through an unknown mechanism and contribute to MCHM resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The variable length of Med15\u0026rsquo;s IDRs are likely affected by MCHM and its ability to form condensates would be altered. Med15 induces protein condensates with Gcn4, and it has yet to be seen if other transcription factors also this characteristic liquid-liquid phase separation have to regulate transcription.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MCHM sample used was obtained as a gift from Eastman Chemical Company. The yeast knockout collection was a gift from Angela Lee. Mohammad Rahman assisted in Pdr3 interface interaction prediction. We would like to acknowledge the WVU Genomics Core Facility, Morgantown WV for the support provided to help make this publication possible and CTSI Grant #U54 GM104942 which in turn provides financial support to the Core Facility. Amaury Pupo provided invaluable help in setting up many of the bioinformatic analyses running GATK. This work was supported by NIH NIEHS R15ES026811-01A1. MCA was supported by a WVU STEM Mountains of Excellence Fellowship. DJ was supported by the SyTox training grant NIH NIEHS 1T32ES032920-01A1.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMCA designed and carried out yeast experiments and wrote the paper. JEGG designed the study and supervised the project. DP analyzed transcription factor binding while LM tested effects of zinc on mutants. TM modeled transcription factor structures, measured element levels, and worked with SP and GL on ROS assay. TM and DJ supervised the experiments knocking out genes in YJM789.\u0026nbsp;MQ and NW analyzed the \u003cem\u003eFLO\u0026nbsp;\u003c/em\u003egenes, ND characterized \u003cem\u003ePAR1\u0026nbsp;\u003c/em\u003eand SM assessed \u003cem\u003ePRM9.\u003c/em\u003e FJ aided in Chec-seq analysis.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting Financial Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdamo GM, Lotti M, Tamas MJ, Brocca S (2012) Amplification of the CUP1 gene is associated with evolution of copper tolerance in Saccharomyces cerevisiae. Microbiology 158:2325\u0026ndash;2335\u003c/li\u003e\n\u003cli\u003eAgier N, Fischer G (2012) The mutational profile of the yeast genome is shaped by replication. 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PloS One 13:. https://doi.org/10.1371/JOURNAL.PONE.0194911\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-genetics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cuge","sideBox":"Learn more about [Current Genetics](https://www.springer.com/journal/294)","snPcode":"294","submissionUrl":"https://submission.nature.com/new-submission/294/3","title":"Discover Genetics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"In-Lab Evolution, evolution, MCHM, Saccharomyces cerevisiae, PDR3, MED15, pleiotropic drug response, hydrotrope, cell wall, transporters","lastPublishedDoi":"10.21203/rs.3.rs-4548300/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4548300/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe solubility of protein complexes and membraneless compartments is maintained by liquid-liquid phase separation (LLPS). Phase transition is induced or dissolved by biological hydrotropes such as ATP and RNA. 4-methylcyclohexane methanol (MCHM), an alicyclic alcohol, is a synthetic hydrotrope that induces a starvation response by upregulation of biosynthetic pathways despite the availability of nutrients. To investigate how cellular metabolism can tolerate changes in LLPS, we evolved eight MHCM-resistant strains of \u003cem\u003eS. cerevisiae\u003c/em\u003e. We identified thousands of SNPs and indel variants per strain, which was a consistent number between strains that evolved resistance and control strains that remained sensitive. These variants did not show a pattern that would cluster resistant strains together. The many background mutations likely masked any pattern from few large-effect loci or implicated an epistatic effect of many small mutations spread throughout the genome that was undetectable. Among coding variants in the strains that change protein sequence and thereby may alter function, only one gene showed a protein-coding mutation in every resistant strain while showing no variants at all in the control strains. This gene, \u003cem\u003ePDR3\u003c/em\u003e, controls transcription for the pleiotropic drug response and is the most significant driver of adaptive MCHM resistance in yeast. While many of the evolved alleles of \u003cem\u003ePDR3\u003c/em\u003e would likely produce functional proteins, a knockout in the parent YJM789 strain was sufficient to produce resistance to MCHM. Normal catabolism of amino acids uses the Pleiotropic Drug Response (PDR) pathway to export breakdown products. The \u003cem\u003epdr3\u003c/em\u003e resistance is mediated through Med15, a component of the Mediator complex which regulates activation by transcription factors of RNA pol II. Pdr3 can homodimerize or dimerize with Pdr1, another transcription factor and loss of Pdr1 also confers MCHM resistance. Knockouts of other mutated genes in flocculation, glutathione, SAM, and sugar transport mildly affected growth in the ancestral strain. Mutations in \u003cem\u003ePDR3\u003c/em\u003e are first known to increase resistance to this novel hydrotropic chemical.\u003c/p\u003e","manuscriptTitle":"Laboratory evolutions lead to reproducible mutations in PDR3 conferring resistance to MCHM","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-25 07:30:06","doi":"10.21203/rs.3.rs-4548300/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-17T11:00:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-10T14:26:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T07:45:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Current Genetics","date":"2024-06-07T23:28:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-genetics-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cuge","sideBox":"Learn more about [Current Genetics](https://www.springer.com/journal/294)","snPcode":"294","submissionUrl":"https://submission.nature.com/new-submission/294/3","title":"Discover Genetics and Evolution","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"243514fb-8a77-49cb-b8a7-349df2f95681","owner":[],"postedDate":"June 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T16:04:22+00:00","versionOfRecord":{"articleIdentity":"rs-4548300","link":"https://doi.org/10.1007/s00294-025-01333-w","journal":{"identity":"discover-genetics-and-evolution","isVorOnly":false,"title":"Discover Genetics and Evolution"},"publishedOn":"2025-11-19 15:57:33","publishedOnDateReadable":"November 19th, 2025"},"versionCreatedAt":"2024-06-25 07:30:06","video":"","vorDoi":"10.1007/s00294-025-01333-w","vorDoiUrl":"https://doi.org/10.1007/s00294-025-01333-w","workflowStages":[]},"version":"v1","identity":"rs-4548300","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4548300","identity":"rs-4548300","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}