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.

When microbes are faced with an environmental challenge or opportunity, preexisting enzymes with promiscuous secondary activities can be recruited to provide newly important functions. Mutations that increase the efficiency of a new activity often compromise the original activity, resulting in an inefficient bifunctional enzyme. We have investigated the mechanisms by which growth of Escherichia coli can be improved when fitness is limited by such an enzyme, E383A ProA (ProA*). ProA* can serve the functions of both ProA (required for synthesis of proline) and ArgC (required for synthesis of arginine), albeit poorly. We identified four genetic changes that improve the growth rate by up to 6.2-fold. Two point mutations in the promoter of the proBA* operon increase expression of the entire operon. Massive amplification of a genomic segment around the proBA* operon also increases expression of the entire operon. Finally, a synonymous point mutation in the coding region of proB creates a new promoter for proA*. This synonymous mutation increases the level of ProA* by 2-fold but increases the growth rate by 5-fold, an ultrasensitive response likely arising from competition between two substrates for the active site of the inefficient bifunctional ProA*. IMPORTANCEThe high-impact synonymous mutation we discovered in proB is remarkable for two reasons. First, most polar effects documented in the literature are detrimental. This finding demonstrates that polar effect mutations can have strongly beneficial effects, especially when an organism is facing a difficult environmental challenge for which it is poorly adapted. Furthermore, the consequence of the synonymous mutation in proB is a 2-fold increase in the level of ProA* but a disproportionately large 5.1-fold increase in growth rate. While ultrasensitive responses are often found in signaling networks and genetic circuits, an ultrasensitive response to an adaptive mutation has not been previously reported. Metabolic enzymes, although prodigious catalysts, are not perfectly specific for their physiological substrates. They typically possess secondary activities as a consequence of the assemblage of highly reactive functional groups, metal ions, and cofactors in their active sites. Secondary activities that are physiologically irrelevant, either because they are too inefficient to contribute to fitness or because the enzyme never encounters the substrate, are termed promiscuous activities (1).Promiscuous activities are important from an evolutionary standpoint because they provide a reservoir of catalytic potential within a proteome that can be drawn upon when the environment changes. A promiscuous activity may become important for fitness when a new source of carbon, nitrogen, or phosphorous appears in the environment or when a previously available compound, such as an amino acid or cofactor, becomes unavailable. A promiscuous activity may also become critical when the organism is exposed to a novel toxin, such as an antibiotic or pesticide.A newly recruited pro...

SignificanceThe evolution of new metabolic pathways has been a driver of diversification from the last universal common ancestor 3.8 billion y ago to the present. Bioinformatic evidence suggests that many pathways were assembled by recruiting promiscuous enzymes to serve new functions. However, the processes by which new pathways have emerged are lost in time. We have little information about the environmental conditions that fostered emergence of new pathways, the genome context in which new pathways emerged, and the types of mutations that elevated flux through inefficient new pathways. Experimental laboratory evolution has allowed us to evolve a new pathway and identify mechanisms by which mutations increase fitness when an inefficient new pathway becomes important for survival.