The rate of compensatory mutation in the DNA bacteriophage phiX174

Abstract

A compensatory mutation occurs when the fitness loss caused by one mutation is remedied by its epistatic interaction with a second mutation at a different site in the genome. This poorly understood biological phenomenon has important implications, not only for the evolutionary consequences of mutation, but also for the genetic complexity of adaptation. We have carried out the first direct experimental measurement of the average rate of compensatory mutation. An arbitrary selection of 21 missense substitutions with deleterious effects on fitness was introduced by site-directed mutagenesis into the bacteriophage phiX174. For each deleterious mutation, we evolved 8-16 replicate populations to determine the frequency at which a compensatory mutation, instead of the back mutation, was acquired to recover fitness. The overall frequency of compensatory mutation was approximately 70%. Deleterious mutations that were more severe were significantly more likely to be compensated for. Furthermore, experimental reversion of deleterious mutations revealed that compensatory mutations have deleterious effects in a wild-type background. A large diversity of intragenic compensatory mutations was identified from sequencing fitness-recovering genotypes. Subsequent analyses of intragenic mutation diversity revealed a significant degree of clustering around the deleterious mutation in the linear sequence and also within folded protein structures. Moreover, a likelihood analysis of mutation diversity predicts that, on average, a deleterious mutation can be compensated by about nine different intragenic compensatory mutations. We estimate that about half of all compensatory mutations are located extragenically in this organism.

Figure 1.—

Log-log plot of average plaque size against relative fitness. Average plaque size is in square millimeters. Relative fitness is estimated by the average yield per plaque normalized by the ancestor 300aI. The top-rightmost data point represents the 300aI high-fitness ancestor. A solid line is drawn to illustrate a linear fit to the log-transformed data (R² = 0.85). The two fitness measures are strongly correlated (Pearson's r = 0.92; 95% C.I. = 0.83–0.96; P < 10⁻⁵), which thereby implies that plaque size is a justifiable proximate measure of fitness. In generating this plot, we have included fitness measures from two additional genotypes not used in this study.

Figure 2.—

Plot of the observed proportion of replicate populations that independently acquire a compensatory mutation, instead of the back mutation, as a function of the log-transformed relative fitness of the deleterious mutation. Log relative fitness is a significant factor (P < 10⁻⁴) in a binomial regression model, which predicts that the proportion of replicates with compensatory mutations increases as the deleterious mutation becomes more severe. The solid line indicates the expected proportion predicted by the binomial regression model as a function of log relative fitness. A regression analysis carried out for the estimated number of intragenic compensatory mutations, rather than the proportion of replicates that acquire any compensatory mutation, obtains a similar result (discussed in text).

Figure 3.—

Compensatory mutations of G1423C mapped to residues in the F protein structure. The residue affected by the deleterious mutation G1423C (i.e., F140) is highlighted in yellow, and compensatory residues are highlighted in blue. Each pair of residues is no more than 15 Å apart. These residues form a group on the external face of the F protein, located above the β-barrel indicated by the purple ribbon. This image was generated in UCSF Chimera v. 1.2 (Computer Graphics Laboratory, UCSF) using the published structure PDB ID 1AL0 (see materials and methods).

Figure 4.—

Fitness estimates for sets of deleterious and compensatory mutant genotypes, with respect to average yield per plaque. Each set of genotypes for a pair of deleterious and compensatory mutations is denoted by the location of the deleterious mutation and replicate population designation letter. Specifically, 3524a refers to the deleterious mutation G3524C and the intragenic compensatory mutation G3562A, which appeared in replicate population a. The other compensatory mutations are as follows: 3524h, T3696C; 2746a, A2652T; 1757b, A2404G (an extragenic mutation in gene G isolated in two independent replicates); and 5337b, A121G. Fitness estimates are averaged over six or more replicate assays for each genotype. The symbol “+” denotes the wild-type allele that occupies the site of either the deleterious (d) or the compensatory (c) mutation. Fitness of the ancestor 300aI (++) is represented by a solid bar. Open bars represent deleterious mutations (d+), thinly hatched columns are compensated deleterious genotypes (dc), and thickly hatched columns are isolated compensatory mutations (+c).