Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme

Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme’s ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.同义突变不会改变所编码的氨基酸，但可能通过多种途径改变mRNA的结构与功能，进而影响进化适合度（fitness）。然而，现有文献中鲜有报道定量测定同义突变对微生物生长速率的影响，且阐明其内在分子机制的案例更是寥寥无几。 我们以一株肠炎沙门氏菌（Salmonella enterica）为研究对象，对其四组种群开展实验室进化实验：该菌株通过招募一种杂泛酶（promiscuous enzyme）替代了自身的必需酶（essential enzyme）。此前已鉴定出一处点突变，该突变可增强该酶催化精氨酸生物合成所需反应的能力，但同时削弱其催化天然反应（脯氨酸生物合成所需）的活性。该酶的催化低效性限制了菌株在葡萄糖培养基上的生长速率。 经过260代传代进化后，我们在编码该弱效酶（weak-link enzyme）的基因的前六个密码子中，鉴定出两处同义突变，可分别使菌株生长速率提升41%与67%。随后我们在该基因前六个密码子中引入所有可能的同义突变，发现其对生长速率存在显著调控效应：其中一处突变可使生长速率翻倍，而另一处则完全抑制了生长。 计算机模拟分析显示，这些同义突变要么影响了隔离起始密码子的茎环结构（stem-loop structure）的稳定性，要么改变了Shine-Dalgarno序列（Shine-Dalgarno sequence，简称SD序列）与起始密码子之间区域的可及性。据此可推测，此类突变会影响翻译效率，并间接调控mRNA稳定性——因为正在进行翻译的核糖体（ribosome）可保护mRNA免受降解。后续实验数据验证了上述假说。 综上，我们认为此类同义突变的效应源于对mRNA稳定性与翻译效率的联合调控，这一过程会改变该弱效酶的表达水平。本研究结果表明，在强选择压力下，同义突变可对进化适合度产生深远影响，其在生物进化过程中的重要性或许被严重低估了。