Genomewide patterns of substitution in adaptively evolving populations of the RNA bacteriophage MS2

Abstract

Experimental evolution of bacteriophage provides a powerful means of studying the genetics of adaptation, as every substitution contributing to adaptation can be identified and characterized. Here, I use experimental evolution of MS2, an RNA bacteriophage, to study its adaptive response to a novel environment. To this end, three lines of MS2 were adapted to rapid growth and lysis at cold temperature for a minimum of 50 phage generations and subjected to whole-genome sequencing. Using this system, I identified adaptive substitutions, monitored changes in frequency of adaptive mutations through the course of the experiment, and measured the effect on phage growth rate of each substitution. All three lines showed a substantial increase in fitness (a two- to threefold increase in growth rate) due to a modest number of substitutions (three to four). The data show some evidence that the substitutions occurring early in the experiment have larger beneficial effects than later ones, in accordance with the expected diminishing returns relationship between the fitness effects of a mutation and its order of substitution. Patterns of molecular evolution seen here--primarily a paucity of hitchhiking mutations--suggest an abundant supply of beneficial mutations in this system. Nevertheless, some beneficial mutations appear to have been lost, possibly due to accumulation of beneficial mutations on other genetic backgrounds, clonal interference, and negatively epistatic interactions with other beneficial mutations.

Figure 1.—

Locations of substitutions and high-frequency mutations in the MS2 genome. All four genes are shown, with the direction of translation indicated by the shaded arrows. Substitutions are indicated with black arrows and high-frequency mutations with gray arrows.

Figure 2.—

Frequencies of mutations vs. passage number for (A) line 1, (B) line 2, and (C) line 3. Only mutations counted as substitutions and those that reached high frequencies are shown. Frequencies were determined by sequencing ∼10 single plaques, with each plaque representing an individual phage from the evolving populations. The identity of the mutation represented is indicated by the site number; the full sequenced region and exact sample sizes are shown in the supplemental tables.

Figure 3.—

(A) Fitness plotted against substitution number. Each point represents the mean of ≥10 replicate growth assays performed on a single genotype. “Substitution number” is the order in which a mutation that is ultimately fixed appears. The straight line shows the best fit of a linear regression model; the curve is the best fit of a hypergeometric model [y = x₀ + (a × x)/(x + b), where x₀ = 1 and a and b are estimated]. (B) The magnitude of fitness “jumps” plotted against the order in which they occur. The categories on the x-axis indicate which fitness jump is measured, e.g, the first data point is the difference in fitness between the first substitution and the ancestor.

Figure 4.—

Mutations in regulatory regions of MS2. (A) Three mutations in the region containing the RNA secondary structure regulating lysis expression. This structure overlaps coding regions of both the coat and the lysis genes and in its folded state prevents ribosomes from binding to the lysis start. The structure can be disrupted, however, allowing lysis translation, by ribosomes translating the coat gene. The three adaptive mutations in this region are indicated by arrows pointing from the ancestral nucleotide to the derived state. (B) Two nucleotide mutations and one amino acid mutation in regions involved in regulation of the replicase gene. Two local RNA secondary structures, the coat protein (which translationally represses replicase) and a long-range RNA interaction (the Min-Jou LRI) are shown. The RNA structures initially prevent ribosomes from binding to the replicase start, but ribosomes translating the coat gene disrupt the structure shown, causing the region to reform into a different configuration that allows translation of the replicase gene. As coat protein concentration increases, it binds to the rightmost secondary structure shown here, stabilizing the inhibitory conformation of the region and preventing further replicase translation. (See text for citations.)