Adaptive Prediction Emerges Over Short Evolutionary Time Scales

Abstract

Adaptive prediction is a capability of diverse organisms, including microbes, to sense a cue and prepare in advance to deal with a future environmental challenge. Here, we investigated the timeframe over which adaptive prediction emerges when an organism encounters an environment with novel structure. We subjected yeast to laboratory evolution in a novel environment with repetitive, coupled exposures to a neutral chemical cue (caffeine), followed by a sublethal dose of a toxin (5-FOA), with an interspersed requirement for uracil prototrophy to counter-select mutants that gained constitutive 5-FOA resistance. We demonstrate the remarkable ability of yeast to internalize a novel environmental pattern within 50-150 generations by adaptively predicting 5-FOA stress upon sensing caffeine. We also demonstrate how novel environmental structure can be internalized by coupling two unrelated response networks, such as the response to caffeine and signaling-mediated conditional peroxisomal localization of proteins.

Keywords: adaptive prediction; conditioned fitness; peroxisomal translocation; structured environments; variant analysis.

Fig. 1.

—Experimental design for investigating laboratory evolution of AP. (a) Yeast cultures were subjected to 30 cycles of laboratory evolution in a novel structured environment, with 10 generations between cycles. Each cycle had three phases: in phase A, cultures were exposed to 3 mM caffeine for 30 min; followed by 3-h exposure to 3 mg/ml 5-FOA in phase B; thereafter, an aliquot of the culture was transferred to fresh medium for overnight growth without uracil (phase C). To prevent temporal conditioning, short random intervals (< 2 h) were introduced between cycles. (b) Conditioned fitness (CF), defined as relative change in survival to 5-FOA given caffeine as a cue, was calculated using a culture aliquot that was exposed to caffeine or just growth medium (control), prior to treatment with 5-FOA. Survival was assessed by sampling immediately before or after adding 5-FOA, by counting colony formation units (CFUs) on five replicate plates (see Materials and Methods).

Fig. 2.

—Rapid emergence of AP in an engineered yeast strain. (a) Schematic diagram for mechanistic model: caffeine induces a global pleiotropic response in yeast bringing cells into a different state. A mutation(s) causing a network rewiring linking the caffeine-induced response and the signaling network for peroxisomal localization of the engineered Gpd1-EGFP-Ura3 construct could potentially generate AP. (b) Laboratory evolution of AP in six mutagenized, engineered yeast lines. Significant CF (Mann–Whitney U test, P value < 0.05, indicated by asterisks) was observed in at least one time point in each evolution cell line, reaching a maximum of CF = 0.390 (cell line E2, n = 190), which indicates that caffeine pre-treatment resulted in 39% increase in 5-FOA survival. Error bars indicate standard deviation. Symbols mark when transcriptomics, whole genome re-sequencing and image analysis were performed (see key at the top for details). (c) Fluorescence in cytoplasm and peroxisomes was quantified from at least 50 cells in each condition. Examples of representative segmented cells are shown (left) and each microscopy image was segmented and quantified (right, see supplementary methods, Supplementary Material online, for details). (d) Bars represent changes in relative peroxisomal fluorescence signal before and after caffeine exposure, asterisks indicate significance. Specifically to cell line E4, exposure to caffeine shifted Gpd1-EGFP-Ura3 to the peroxisome at generations n = 150 and n = 200. Significant CF was observed also at n = 200. Both caffeine-conditioned phenotypes, increase in 5-FOA survival and peroxisomal translocation of Gpd1-EGFP-Ura3, disappeared at n = 250.

Fig. 3.

—Rapid emergence of AP in mutagenized and clonal lines of yeast. (a) Laboratory evolution of AP in M cell lines. AP emerged within 100–150 generations, reaching a maximum CF of 0.213 (M1 at n = 200). AP was sustained over at least 50–100 generations and it consistently emerged in other cell lines. (b) Sustained AP emerged only in C1 cell line and appeared in at least one time point in all cell lines. Asterisks indicate significant CF (Mann–Whitney U test, P value < 0.05), error bars indicate standard deviation. (c) Unique and shared caffeine-induced transcript changes before and after AP emerged in three evolution lines. Novel caffeine-responsive transcript changes included downregulation of URA3 in line E2, and also downregulation of several transcription factors in lines E1 and E3 (indicated in red font). (d) Distribution of CF values for clonal isolates before and after emergence of AP; lines C1–3 are isogenic cultures derived from single colonies of BY4741 URA3; blue lines represent CF values from BY4741 GPD1-EGFP-URA3 mixed populations (as in fig. 2b).