Mutational robustness is defined as the constancy of a phenotype in the face of deleterious mutations. Whether robustness can be directly favored by natural selection remains controversial. Theory and in silico experiments predict that, at high mutation rates, slow-replicating genotypes can potentially outcompete faster counterparts if they benefit from a higher robustness. Here, we experimentally validate this hypothesis, dubbed the “survival of the flattest,” using two populations of the vesicular stomatitis RNA virus. Characterization of fitness distributions and genetic variability indicated that one population showed a higher replication rate, whereas the other was more robust to mutation. The faster replicator outgrew its robust counterpart in standard competition assays, but the outcome was reversed in the presence of chemical mutagens. These results show that selection can directly favor mutational robustness and reveal a novel viral resistance mechanism against treatment by lethal mutagenesis.
It is often argued that high mutation rates are advantageous for RNA viruses, because they confer elevated rates of adaptation. However, there is no direct evidence showing a positive correlation between mutation and adaptation rates among RNA viruses. Moreover, theoretical work does not argue in favor of this prediction. We used a series of vesicular stomatitis virus clones harboring single amino acid substitutions in the RNA polymerase to demonstrate that changes inducing enhanced fidelity paid a fitness cost, but that there was no positive correlation between mutation an adaptation rates. We demonstrate that the observed mutation rate in vesicular stomatitis virus can be explained by a trade-off between replication rate and replication fidelity.fitness trade-off ͉ mutation rate ͉ adaptation ͉ experimental evolution M utation provides the allelic variation that natural selection can act on. Conversely, mutation rates show phenotypic variability, which turns out to be a target for selection. RNA viruses are characterized by high mutation rates compared with most DNA systems (1), due mainly to the lack of exonuclease proofreading activity displayed by their RNA polymerases (2). Current evidence suggests that this high mutation rate cannot simply be attributed to biochemical restrictions: in HIV-1, several antimutator retrotranscriptases have been described (3-6); Pfeiffer and Kierkegaard (7) showed that a single-residue mutation in the RNA polymerase gene of type 1 poliovirus can confer resistance to ribavirine through a 3-fold increase in fidelity; Pugachev et al. (8) suggested that mutation rate in the yellow fever virus could be as low as 2 ϫ 10 Ϫ7 mutations per nucleotidic site per replication round. Such evidence demonstrates the need for an evolutionary model that accounts for high mutation rates in RNA viruses beyond a purely mechanistic level.It is often argued that high mutation rates in RNA viruses are favored, because they confer a greater adaptive capacity (9, 10). However, there is still no experimental proof for this selective advantage: no evidence has been found to support a positive correlation between mutation and adaptation rates, as one would expect according to this hypothesis. A substantial part of our knowledge on the nexus between mutation and adaptation comes from experiments exploring the dynamics of mutator and antimutator genotypes in Escherichia coli (e.g., ref. 11), where high mutation rates have been associated with increased population fitness, because of the genetic hitchhiking of the mutator allele with beneficial changes produced at other loci. The hitchhiking hypothesis (12) might explain why high error rates could have risen and could have been maintained in RNA viruses, especially in the absence of recombination. On the other hand, the vast majority of mutations having a phenotypic effect are deleterious. Then, short-term selection should favor lower mutation rates, to minimize the genetic load (Fig. 1). Moreover, under high mutation rates, favorable alleles will frequently...
Parallel evolution is the independent evolution of the same phenotype or genotype in response to the same selection pressure. There are examples of parallel molecular evolution across divergent genetic backgrounds, suggesting that genetic background may not play an important role in determining the outcome of adaptation. Here, we measure the influence of genetic background on phenotypic and molecular adaptation by combining experimental evolution with comparative analysis. We selected for resistance to the antibiotic rifampicin in eight strains of bacteria from the genus Pseudomonas using a short term selection experiment. Adaptation occurred by 47 mutations at conserved sites in rpoB, the target of rifampicin, and due to the high diversity of possible mutations the probability of within-strain parallel evolution was low. The probability of between-strain parallel evolution was only marginally lower, because different strains substituted similar rpoB mutations. In contrast, we found that more than 30% of the phenotypic variation in the growth rate of evolved clones was attributable to among-strain differences. Parallel molecular evolution across strains resulted in divergent phenotypic evolution because rpoB mutations had different effects on growth rate in different strains. This study shows that genetic divergence between strains constrains parallel phenotypic evolution, but had little detectable impact on the molecular basis of adaptation in this system.
Introductory paragraphThere is an urgent need to develop novel approaches for predicting and preventing the evolution of antibiotic resistance. Here we show that the ability to evolve de novo resistance to a clinically important β-lactam antibiotic, ceftazidime, varies drastically across the genus Pseudomonas. This variation arises because strains possessing the ampR global transcriptional regulator evolve resistance at a high rate. This does not arise because of mutations in ampR. Instead, this regulator potentiates evolution by allowing mutations in conserved peptidoglycan biosynthesis genes to induce high levels of β-lactamase expression. Crucially, blocking this evolutionary pathway by co-administering ceftazidime with the β-lactamase inhibitor avibactam can be used to eliminate pathogenic P. aeruginosa populations before they can evolve resistance. In summary, our study shows that identifying potentiator genes that act as evolutionary catalysts can be used to both predict and prevent the evolution of antibiotic resistance.
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