Adaptive Roles of SSY1 and SIR3 During Cycles of Growth and Starvation in Saccharomyces cerevisiae Populations Enriched for Quiescent or Nonquiescent Cells

Abstract

Over its evolutionary history, Saccharomyces cerevisiae has evolved to be well-adapted to fluctuating nutrient availability. In the presence of sufficient nutrients, yeast cells continue to proliferate, but upon starvation haploid yeast cells enter stationary phase and differentiate into nonquiescent (NQ) and quiescent (Q) cells. Q cells survive stress better than NQ cells and show greater viability when nutrient-rich conditions are restored. To investigate the genes that may be involved in the differentiation of Q and NQ cells, we serially propagated yeast populations that were enriched for either only Q or only NQ cell types over many repeated growth-starvation cycles. After 30 cycles (equivalent to 300 generations), each enriched population produced a higher proportion of the enriched cell type compared to the starting population, suggestive of adaptive change. We also observed differences in each population's fitness suggesting possible tradeoffs: clones from NQ lines were better adapted to logarithmic growth, while clones from Q lines were better adapted to starvation. Whole-genome sequencing of clones from Q- and NQ-enriched lines revealed mutations in genes involved in the stress response and survival in limiting nutrients (ECM21, RSP5, MSN1, SIR4, and IRA2) in both Q and NQ lines, but also differences between the two lines: NQ line clones had recurrent independent mutations affecting the Ssy1p-Ptr3p-Ssy5p (SPS) amino acid sensing pathway, while Q line clones had recurrent, independent mutations in SIR3 and FAS1 Our results suggest that both sets of enriched-cell type lines responded to common, as well as distinct, selective pressures.

Keywords: SIR3; SPS pathway; SSY1; evolution; quiescence.

Figure 1

(A) Experimental design and (B) procedure shown for the separation of NQ and Q line populations. (A) We started the experiment with one clone, which was grown to stationary phase. This population was then diluted and plated onto three plates. The initial density of each population on the plate was ∼2 × 10⁵/ml. Plates were incubated for 3.5 d and all the cells were harvested. Each plate population was separately fractionated in Percoll solution. From each of the three plates we obtained two distinct fractions of cells that were founders of the six experimental lines. NQ, lines of NQ cell selection, Q, lines of Q cell selection. (B) Continuation of experiment shown via the example of one NQ and one Q line. We progressed the experiment by using only NQ or only Q cells from fractionated cultures, diluted them, and plated at an initial density of ∼2 × 10⁵/ml. Plates were incubated for 3.5 d, then cells were harvested, and fractionated. For the NQ lines we propagated only NQ cells, for Q lines only Q cells. We carried out the experiment for 30 cycles of growth and starvation. From the final fractionation, we isolated 15–16 clones from each experimental population for further analysis. NQ, nonquiescent; o/e, overnight; Q, quiescent.

Figure 2

Average proportions of the quiescent (Q) cells (red bars) and nonquiescent (NQ) cells (blue bars) in the populations at the selected experimental cycles of growth and starvation in: (A) NQ lines (average of I–III) where only NQ cells were selected for following cycles of growth and starvation, and (B) Q lines (average I–III) where only Q cells were selected for the following cycle. (C) Average proportions of the quiescent (Q) cells (red bars) and nonquiescent (NQ) cells (blue bars) after one cycle of growth and starvation in the populations derived from the clones isolated after 30 cycles, at the end of the experiment, and in the ancestral strain (Figure 1B).

Figure 3

Test of trade-offs in fitness of evolved clones in three different environmental conditions. Each clone was measured at least three times. Error bars represent 95% C.I. (A) average maximum growth rate (MGR) of the ancestral clone and all clones from the final populations of each experimental line; NQ I: 16 clones, NQ II: 15 clones; NQ III: 16 clones; Q I: 16 clones; Q II: 13 clones; and Q III: 15 clones. (B) Fitness of the 16 selected clones from ancestral and experimental lines NQ I–III (NQI 11 and 14; NQII 3 and 15; NQIII 6, 8, 11, and 12; QI 1 and 13; QII 5, 6, and 12; QIII 8, 9, and 11) relative to yellow fluorescent protein (YFP)-marked ancestral strain. The result of the competition of ancestral and YFP-marked ancestral clone is standardized to one. (C) Colony-forming capacity of starved cultures determined by plating assay. Values are expressed as the percentage of colony forming units (CFU) to the total number of cells in the population. Gray bar represents ancestral population, blue and red stand for NQ and Q, respectively. NQ, nonquiescent; Q, quiescent.

Figure 4

Presumably adaptive mutations identified in the whole-genome sequenced, nonmutator clones isolated from experimental Q and NQ lines on the 30th experimental cycle. A complete list of all identified mutations is in Table S1. NQ, nonquiescent; Q, quiescent.