title: Turbidostat culture of Saccharomyces cerevisiae W303-1A under selective pressure elicited by ethanol selects for mutations in SSD1 and UTH1.
abstract: We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in a medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 to 0.32 h(-1) . Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within 6 days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within 2 days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of six clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 of 19 tolerant clones, the stop codon in ssd1-d was replaced with an amino acid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol.
authors.affiliation: Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
authors.forename: Liat
authors.initials: L
authors.lastname: Avrahami-Moyal
authors.forename: David
authors.initials: D
authors.lastname: Engelberg
authors.forename: Jared W
authors.initials: JW
authors.lastname: Wenger
authors.forename: Gavin
authors.initials: G
authors.lastname: Sherlock
authors.forename: Sergei
authors.initials: S
authors.lastname: Braun
mesh_terms: D002473:Cell Wall / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004252:DNA Mutational Analysis; D000431:Ethanol / Q000633:toxicity*; D016681:Genome, Fungal; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; C485219:Ssd1 protein, S cerevisiae; C116477:UTH1 protein, S cerevisiae; D000431:Ethanol; D004693:Glucan Endo-1,3-beta-D-Glucosidase