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.

New enzymes often evolve by gene amplification and divergence. Previous experimental studies have followed the evolutionary trajectory of an amplified gene, but have not considered mutations elsewhere in the genome when fitness is limited by an evolving gene. We have evolved a strain of Escherichia coli in which a secondary promiscuous activity has been recruited to serve an essential function. The gene encoding the ‘weak-link’ enzyme amplified in all eight populations, but mutations improving the newly needed activity occurred in only one. Most adaptive mutations occurred elsewhere in the genome. Some mutations increase expression of the enzyme upstream of the weak-link enzyme, pushing material through the dysfunctional metabolic pathway. Others enhance production of a co-substrate for a downstream enzyme, thereby pulling material through the pathway. Most of these latter mutations are detrimental in wild-type E. coli, and thus would require reversion or compensation once a sufficient new activity has evolved.

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16New enzymes often evolve by amplification and divergence of genes encoding enzymes 17 with a weak ability to provide a new function. Experimental studies to date have followed the 18 evolutionary trajectory of an amplified gene, but have not addressed other mutations in the 19 genome when fitness is limited by an evolving gene. We have adapted Escherichia coli in which 20 an enzyme's weak secondary activity has been recruited to serve an essential function. While the 21 gene encoding the "weak-link" enzyme amplified in all eight populations, mutations improving 22 the new activity occurred in only one. This beneficial allele quickly swept the amplified array, 23 Results 93Growth rate of ∆argC proA* E. coli increased 3-fold within a few hundred generations of 94 adaptation in M9/glucose/proline 95We generated a progenitor strain for laboratory evolution by replacing argC with the kan r 96 antibiotic resistance gene, modifying proA to encode ProA*, and introducing a mutation in the -97 10 region of the promoter of the proBA operon that was previously shown to occur frequently 98 during adaptation of the ∆argC strain (Kershner et al., 2016). We also introduced yfp 99 downstream of proA* and deleted several genes (fimAICDFGH and csgBAC, which are required 100 for the formation of fimbriae and curli, respectively (Barnhart & Chapman, 2006; Proft & Baker, 101 2009)) to minimize the occurrence of biofilms. We evolved eight parallel lineages of this strain 102 (AM187, Table 1) in M9 minimal medium supplemented with 0.2% glucose, 0.4 mM proline, 103 and 20 µg/mL kanamycin in a turbidostat to identify mutations that improve synthesis of 104 arginine. We used a turbidostat rather than a serial transfer protocol because turbidostats can 105 maintain cultures in exponential phase and thereby avoid selection for mutations that simply 106 decrease lag phase or improve survival in stationary phase. Turbidostats also avoid population 107 bottlenecks during serial passaging that can result in loss of genetic diversity. 108Growth rate in each culture tube was averaged over each 24-hour period and was used to 109 calculate the number of generations each day. Each culture was maintained until a biofilm 110 formed (33-57 days, corresponding to 470-1000 generations). While it is possible to restart 111 cultures from individual clones after biofilm formation, this practice introduces a severe 112 population bottleneck. Thus, we decided to stop the adaptation for each population when a 113 biofilm formed. For this reason, every population was adapted for a different number of 114 generations. 115

New enzymes often evolve by duplication and divergence of genes encoding enzymes with promiscuous activities that have become important in the face of environmental opportunities or challenges. Amplifications that increase the copy number of the gene under selection commonly amplify many surrounding genes. Extra copies of these co-amplified genes must be removed, either during or after evolution of a new enzyme. Here we report that amplicon remodeling can begin even before mutations occur in the gene under selection. Amplicon remodeling and mutations elsewhere in the genome that indirectly increase fitness result in complex population dynamics, leading to emergence of clones that have improved fitness by different mechanisms. In this work, one of the two most successful clones had undergone two episodes of amplicon remodeling, leaving only four co-amplified genes surrounding the gene under selection. Amplicon remodeling in the other clone resulted in removal of 111 genes from the genome, an acceptable solution under these selection conditions, but one that would certainly impair fitness under other environmental conditions.

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