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

Abstract

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.

The distribution of fitness effects among synonymous mutations in a gene under directional selection

“…It is notable that promoter-associated effects on transcription have been shown to underlie the fitness effects of synonymous mutations in two other microbial systems (Ando et al, 2014; Kershner et al, 2016), suggesting this mechanism may be quite general, at least for organisms with operon-like genetic architectures. Nevertheless, others have pointed to changes in translational efficiency associated with the accessibility of mRNA near a start codon as the primary mediator of fitness in Salmonella enterica (Kristofich et al, 2018) and synonymous mutations are known to impact fitness in a wide range of organisms beyond prokaryotes (Lawrie et al, 2013; Cuevas et al, 2012; Kashiwagi et al, 2014). Uncovering the full spectrum of mechanisms by which synonymous mutations impact fitness, and how often they contribute to adaptation, remains a major task for the future.…”

“…Our ability to use DNA sequence data to make inferences about the evolutionary process from genes or genomes often relies on the assumption that synonymous mutations, those that do not result in an amino acid change, are neutral with respect to fitness. Yet there is compelling evidence that this assumption is sometimes wrong: comparative (Lawrie et al, 2013) and experimental (Lind et al, 2010) data show that synonymous mutations can have a range of fitness effects from negative to positive, and can even contribute to adaptation (Bailey et al, 2014; Agashe et al, 2016; Kristofich et al, 2018; She and Jarosz, 2018). A range of mechanisms including codon usage bias, altered mRNA structure, and the creation of promoter sequences could lead to changes in the rate or efficiency of transcription, translation, and/or protein folding and/or expression that, in turn, impact fitness (Plotkin and Kudla, 2011).…”

Synonymous genome recoding: a tool to explore microbial biology and new therapeutic strategies

“…These studies illustrate the possible fitness costs of synonymous genome recoding, which can be under strong selection. As suggested previously, their importance in evolution may be under-appreciated (57). Synonymous mutations influence fitness in bacteria (8,11,18,57–67) and viruses (36,68–89).…”

“…As suggested previously, their importance in evolution may be under-appreciated (57). Synonymous mutations influence fitness in bacteria (8,11,18,57–67) and viruses (36,68–89). In addition, synonymous rewriting of protein-coding regions allows the encoded amino acid sequences to be retained, but it may remove other genetic information, including hidden control elements embedded within amino acid coding regions (56).…”

ΦX174 Attenuation by Whole Genome Codon Deoptimization

“…At any given site, natural selection may act on one or more of these features to favor the use of certain codons over other synonymous ones (34) . While the strength of selection on synonymous mutations is generally considered weak to neutral and genetic drift should therefore also exert a strong influence, the overall signature of selection in the form of non-random codon usage is broadly observed across life (34)(35)(36) where selection has been shown to strongly favor one synonymous mutation over others (37)(38)(39)(40) .…”