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.

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.

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

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