Data from: Too much of a good thing: The unique and repeated paths toward copper adaptation

Copper is a micronutrient essential for growth due to its role as a co-factor in enzymes involved in respiration, defense against oxidative damage, and iron uptake. Yet too much of a good thing can be lethal, and yeast cells typically do not have tolerance to copper levels much beyond the concentration in their ancestral environment. Here, we report a short-term evolutionary study of Saccharomyces cerevisiae exposed to levels of copper sulfate that are inhibitory to the initial strain. We isolated and identified adaptive mutations soon after they arose, reducing the number of neutral mutations, to determine the first genetic steps that yeast take when adapting to copper. We analyzed 34 such strains through whole-genome sequencing and by assaying fitness within different environments; we also isolated a subset of mutations through tetrad analysis of four lines. We identified a multi-layered evolutionary response. In total, 57 single base-pair mutations were identified across the 34 lines. In addition, gene amplification of the copper metallothionein protein, CUP1, was rampant, as was chromosomal aneuploidy. Four other genes received multiple, independent mutations in different lines (the vacuolar transporter genes VTC1 and VTC4; the plasma membrane H+-ATPase PMA1; and MAM3, a protein required for normal mitochondrial morphology). Analyses indicated that mutations in all four genes, as well as CUP1 copy number, contributed significantly to explaining variation in copper tolerance. Our study thus finds that evolution takes both common and less trodden pathways toward evolving tolerance to an essential, but highly toxic, micronutrient.

铜作为参与呼吸作用、抗氧化损伤防御以及铁摄取过程的酶类辅因子，是生物体生长所必需的微量营养素。然而凡事过犹不及，过量铜会产生致命毒性，而酿酒酵母（Saccharomyces cerevisiae）通常无法耐受远超其祖先环境浓度的铜离子水平。本研究针对暴露于会抑制初始菌株生长的硫酸铜浓度环境中的酿酒酵母，开展了一项短期进化研究。我们在适应性突变出现后第一时间对其进行分离与鉴定，以减少中性突变的干扰，进而明确酿酒酵母适应铜胁迫时的首批遗传步骤。我们通过全基因组测序（whole-genome sequencing）与不同环境下的适合度测定，分析了34株此类进化菌株；同时通过4个谱系的四分体分析（tetrad analysis），分离得到了部分突变位点。研究揭示了多层级的进化应答机制。在34个菌株谱系中共鉴定出57个单碱基突变。此外，铜金属硫蛋白基因（CUP1）的扩增与染色体非整倍体现象均广泛存在。另有4个基因在不同谱系中发生了多起独立突变：液泡转运蛋白基因VTC1与VTC4、质膜H+-ATP酶基因PMA1，以及维持线粒体正常形态所需的MAM3基因。分析结果显示，上述4个基因的突变以及CUP1的拷贝数变异，均对铜耐受性的个体差异具有显著的解释力。综上，本研究发现，在应对这种既是必需又兼具高毒性的微量营养素时，进化既会采用常见的通路，也会开辟较少被探索的路径。