Genome duplication and mutations in ACE2 cause multicellular, fast-sedimenting phenotypes in evolved Saccharomyces cerevisiae

Abstract

Laboratory evolution of the yeast Saccharomyces cerevisiae in bioreactor batch cultures yielded variants that grow as multicellular, fast-sedimenting clusters. Knowledge of the molecular basis of this phenomenon may contribute to the understanding of natural evolution of multicellularity and to manipulating cell sedimentation in laboratory and industrial applications of S. cerevisiae. Multicellular, fast-sedimenting lineages obtained from a haploid S. cerevisiae strain in two independent evolution experiments were analyzed by whole genome resequencing. The two evolved cell lines showed different frameshift mutations in a stretch of eight adenosines in ACE2, which encodes a transcriptional regulator involved in cell cycle control and mother-daughter cell separation. Introduction of the two ace2 mutant alleles into the haploid parental strain led to slow-sedimenting cell clusters that consisted of just a few cells, thus representing only a partial reconstruction of the evolved phenotype. In addition to single-nucleotide mutations, a whole-genome duplication event had occurred in both evolved multicellular strains. Construction of a diploid reference strain with two mutant ace2 alleles led to complete reconstruction of the multicellular-fast sedimenting phenotype. This study shows that whole-genome duplication and a frameshift mutation in ACE2 are sufficient to generate a fast-sedimenting, multicellular phenotype in S. cerevisiae. The nature of the ace2 mutations and their occurrence in two independent evolution experiments encompassing fewer than 500 generations of selective growth suggest that switching between unicellular and multicellular phenotypes may be relevant for competitiveness of S. cerevisiae in natural environments.

Keywords: reverse engineering; whole genome sequencing.

Fig. 1.

Sequential batch cultivation in bioreactors on glucose-galactose mixtures results in evolution of multicellular S. cerevisiae. (A) Maximum specific growth rate (μ_max) estimated from CO₂ production during glucose consumption in the glucose-galactose batch cultures (●); μ_max on galactose estimated from galactose batch cultures (○) in evolution experiment 1. Culture samples were taken at different stages of the evolution experiment, grown to stationary phase in shake flasks containing YP medium with 20 g⋅L⁻¹ glucose, and were left to settle for 30 min in a 1-mL cuvette. Sedimentation indices (■) were calculated as described in Materials and Methods. The data represent the average and the mean deviation of duplicate experiments. Microscopic pictures of evolution line 1 after (B) 0, (C) 1,196 (D) 2,105, (E) 3,209, and (F) 4,200 h of evolution. (G) Sedimentation of the reference strain CEN.PK113-7D and a culture sample of evolution lines 1 and 2 after 4,200 and 2,877 h of cultivation, respectively, photographed after 5 min of static incubation.

Fig. 2.

Ploidy of the evolved mutants IMS0267 and IMS0386. (A) Prediction of DNA content in the evolved strains S. cerevisiae IMS0267 (Upper) and IMS0386 (Lower), using the Magnolya algorithm (34). The numbers indicate chromosome position. + (red) indicates the ploidy of the ancestral genome (strain CEN.PK113-7D) and x (blue) indicates the ploidy of the evolved genome. (B) Determination of cell size (white bar) and DNA content measurements (black bar) of strains CEN.PK113-7D (MATa), CEN.PK122 (MATa/MATα), IMS0386, IMS0267, IMI220 (ACE2/ace2-1-HphNT1), and IMI221 (ACE2/ace2-2-HphNT1) by flow cytometry. Strains IMI220 and IMI221 are unicellular strains derived from IMS0267 and IMS0386 by reintroduction of a WT ACE2 allele. *For IMS0386 and IMS0267 the analysis was preceded by treatment with Trichoderma viride chitinase. Data are presented as average ± mean deviation of duplicate biological replicates.

Fig. 3.

Effect of mutations in ACE2 on gene expression and multicellularity. (A) Quantification of the expression of characterized Ace2 regulated genes (CTS1, SCW11, DSE1, and DSE2) in S. cerevisiae strains CEN.PK113-7D (black bar; ACE2), IMS0267 (white bar; ace2-1/ace2-1), and IMS0386 (gray bar; ace2-2/ace2-2). Samples were taken in midexponential phase from a shake flask culture grown on YPD medium. Relative gene expression data represent the expression of CTS1, SCW11, DSE1, and DSE2 normalized to ACT1. The expression ratios were further normalized relative to CEN.PK113-7D. The data represented are average ± mean deviation of duplicate biological replicates. (B) Calcofluor White staining of an IMS0267 multicellular cluster. This picture is representative for the entire culture as well as for the two other single-colony isolates obtained from evolved hyper-sedimenting cultures. Microscopic observations of a multicellular cluster of IMS0386 resuspended in 100 mM of potassium phosphate buffer (C) before and (D) after 7-h incubation with 60 units of chitinase at 25 °C.

Fig. 4.

Reverse engineering of the multicellular phenotype. Cellular morphology of different S. cerevisiae strains (A) loxP-HphNT1-loxP/ace2-2-loxP-KanMX CEN.PK113-7D (MATa ACE2), (B) IMK395 (MATa ace2Δ::loxP-HphNT1-loxP), (C) IMK245 (MATα ace2-1-loxP-HphNT1-loxP), (D) IMI197 (ace2-2), (E) CEN.PK122 (MATa/α ACE2/ACE2), (F) IMD014 (MATa/α ace2-2-loxP/ ace2-2-loxP), (G) IMS0267 (ace2-1/ace2-1), (H) IMI220* (ACE2/ace2-1-loxP-HphNT1-loxP), (I) IMS0386 (ace2-2/ace2-2), and (J) IMI221^# (ACE2/ace2-2-loxP-HphNT1-loxP). (K) Sedimentation indices (see Materials and Methods for definition) of the reference haploid strain CEN.PK113-7D, of the diploid reference CEN.PK122 (MATa/α), the evolved multicellular fast-sedimenting strains IMS0267 and IMS0386, and the reverse engineered mutants IMK395, IMK245, IMI197, IMD014, IMI220*, and IMI221^#. Data are represented as average ± mean deviation of duplicate biological replicates. *Strains constructed in the evolved IMS0267 background; ^#strains constructed in the evolved IMS0386 strain background.