Large-Effect Beneficial Synonymous Mutations Mediate Rapid and Parallel Adaptation in a Bacterium

Abstract

Contrary to previous understanding, recent evidence indicates that synonymous codon changes may sometimes face strong selection. However, it remains difficult to generalize the nature, strength, and mechanism(s) of such selection. Previously, we showed that synonymous variants of a key enzyme-coding gene (fae) of Methylobacterium extorquens AM1 decreased enzyme production and reduced fitness dramatically. We now show that during laboratory evolution, these variants rapidly regained fitness via parallel yet variant-specific, highly beneficial point mutations in the N-terminal region of fae These mutations (including four synonymous mutations) had weak but consistently positive impacts on transcript levels, enzyme production, or enzyme activity. However, none of the proposed mechanisms (including internal ribosome pause sites or mRNA structure) predicted the fitness impact of evolved or additional, engineered point mutations. This study shows that synonymous mutations can be fixed through strong positive selection, but the mechanism for their benefit varies depending on the local sequence context.

Keywords: Shine–Dalgarno sequence; codon use; epistasis; fitness; mRNA folding..

Fig. 1.

Populations rapidly evolve increased fitness on methylamine. Summary of population level initial and final growth rate of fae synonymous variants in methylamine (±SD). Dashed lines = large population size (n = 3–4 replicate populations per strain, with three technical replicates each); solid lines = small population size (n = 5–6 populations per strain, with three technical replicates each).

Fig 2.

Evolved coding and noncoding mutations associated with fae. (A) The first 14 amino acid residues of the fae coding and relevant upstream sequence are shown, indicating nucleotide positions that acquired mutations during experimental evolution. Coding mutations are shown above the gene sequence and noncoding mutations are shown below. No mutations were observed in the rest of the fae gene, and each tested clone had a maximum of one mutation. Alleles are named as follows: “e” indicates evolved allele, letters identify the ancestral fae strain, and the number differentiates multiple evolved alleles for each ancestor. (B) Each evolved mutation is described, along with its final frequency in replicate evolved populations (n = 5 isolated clones per large population; n = 10 clones for small populations). The frequency of clones without a fae-associated mutation is also indicated for each ancestor. Each column represents an independent population, with the cell value and color intensity indicating the final frequency of the corresponding allele in that population (maximum color intensity indicates a fixed mutation with frequency 1). In some cases, there were fewer replicate populations, and these “missing” populations are marked with an X.

Fig 3.

Impact of evolved mutations on fitness, gene expression, protein production and enzyme activity. Average (±SEM) values of (A) growth rate, (B) mRNA, (C) protein, and (D) enzyme activity per milligram total cell protein are shown for the ancestor (“anc”) and an ancestor carrying only the evolved SNP (“snp”). For panels A and B, n = 3–10 biological replicates per strain; for panel C, n = 3–6 biological replicates per strain; for panel D, n = 3 biological replicates with three technical replicates each. In panel B, the split y-axis allows visualization of the high impact of SNPs in strain VA as well as the lower impact of SNPs in other strains. In panels B and C, error bars on only one side are shown for clarity. Asterisks mark significant differences between “anc” – “snp” pairs after correcting for multiple comparisons using the Benjamini–Hochberg method that controls for false discovery rate (*P = 0.05; **P < 0.05). (E and F) Growth rate as a function of (E) FAE protein production and (F) enzyme activity. (G) Impact of evolved SNPs on mean enzyme levels versus mean mRNA levels. The dashed line indicates y = x. (H) Amount of FAE protein produced during growth on succinate versus methylamine. The dashed line indicates y = x, and the solid line indicates the best-fit linear regression with associated correlation strength and P value. In panels E–H, circles = “anc” and triangles = “snp” strains; open triangles = synonymous SNPs. mRNA and protein values were calculated relative to WT (e.g., mRNA_anc/mRNA_WT). In all panels, strains are colored as in figure 1 and dashed lines and open triangles indicate synonymous mutations.

Fig. 4.

Effect of N-terminal insert on fitness. Mean Growth rate (±SEM; n = 3 biological replicates) of strains carrying plasmid-borne fae alleles induced with 1 μM cumate is shown as a function of (A) 5′ MFE and (B) anti-SD affinity. Filled circles indicate ancestral fae alleles, and open inverted triangles indicate alleles carrying the N-terminal insert rich in frequent codons. Points are colored as in figure 1. For MFE calculations we used 100 bp long sequences (−50 to +50 bp relative to the fae start site). For anti-SD affinity calculations, we used 55 bp long sequences (−5 to +50 bp relative to the fae start site).

Fig. 5.

(A–D) Predicted effect of fae coding SNPs on MFE and anti-SD binding affinity of the ancestral alleles (mutant – ancestor; kcal/mol in both cases). Dashed lines indicate no effect on anti-SD affinity and MFE. In each panel, coordinates (0,0) refer to the respective ancestral strain (indicated with a filled circle colored according to the strain). Hence, the actual MFE and affinity values at this point differ across panels. In panel A, the difference in MFE and anti-SD affinity is also shown for ancestral AF, VA, and AC with respect to the WT, leading to the predicted fitness effects of point mutants in these strains. Open circles = all possible mutations in the first 50 bases of the coding sequence; red triangles = evolved mutations in each strain; colored circles = evolved mutations from other strains that could have (but did not) occur in the focal strain; grey points = engineered mutations (highlighted in subsequent panels). (E–H) Fitness impact of a subset of mutations from panels A–D. Growth rate (±SEM) conferred by plasmid-borne alleles induced with 100 ng/ml anhydrous tetracycline. Points are shaded according to growth rate, with darker color indicating faster growth as indicated by the bar in each panel. Triangles = evolved mutations; filled circles = tested mutations; open circles = untested possible mutations. For MFE calculations we used 100 bp long sequences (−50 to +50 bp relative to the fae start site). For anti-SD affinity calculations, we used 55 bp long sequences (−5 to +50 bp relative to the fae start site). (I and J) Growth rate (±SEM) of strains carrying point mutations, as a function of predicted (I) anti-SD affinity and (J) MFE. Triangles = evolved fae alleles; filled circles = engineered mutations (from panels D–F). Points are colored according to each ancestral allele as indicated in panels A–D.

Fig. 6.

Fitness effect of evolved mutations in different allelic backgrounds. Open circles = ancestral fae alleles; triangles = evolved fae alleles; filled circles = ancestral fae alleles with evolved mutations from a different strain (labeled). Cross-swaps conferring significantly higher fitness than actually evolved alleles are marked with an asterisk. Overlapping points are displaced slightly along the x-axis for visualization. Points are colored according to the ancestral fae allele as indicated in figure 1.