M. R. Badgett

Experimental molecular evolution of bacteriophage T7

We present an analysis of molecular evolution in a laboratory‐generated phylogeny of the bacteriophage T7, a virus of 40 kilo‐base pairs of double‐stranded DNA. The known biology of T7 is used in concert with observed changes in restriction sites and in DNA sequences to produce a model of restriction‐site convergence and divergence in the experimental lineages. During laboratory propagation in the presence of a mutagen, the phage lineages changed an estimated 0.5%‐1.5% in base pairs; most change appears to have been G → A or C → T, presumably because of the mutagen employed. Some classes of restriction‐site losses can be explained adequately as simple outcomes of random processes, given the mutation rate and the bias in mutation spectrum. However, some other classes of sites appear to have undergone accelerated rates of loss, as though the losses were selectively favored. Overall, the wealth of knowledge available for T7 biology contributes only modestly to these explanations of restriction‐site evolution, but rates of restriction‐site gains remain poorly explained, perhaps requiring an even deeper understanding of T7 genetics than was employed here. Having measured these properties of molecular evolution, we programmed computer simulations with the parameter estimates and pseudo‐replicated the empirical study, thereby providing a data base for statistical evaluation of phylogeny reconstruction methods. By these criteria, replicates of the experimental phylogeny would be correctly reconstructed over 97% of the time for the three methods tested, but the methods differed significantly both in their ability to recover the correct topology and in their ability to predict branch lengths. More generally, the study illustrates how analyses of experimental evolution in bacteriophage can be exploited to reveal relationships between the basics of molecular evolution and abstract models of evolutionary processes.

Genome properties and the limits of adaptation in bacteriophages.

Abstract Eight bacteriophages were adapted for rapid growth under similar conditions to compare their evolved, endpoint fitnesses. Four pairs of related phages were used, including two RNA phages with small genomes (MS2 and Q(3), two single‐stranded DNA phages with small genomes (174 and G4), two T‐odd phages with medium‐sized, double‐stranded DNA genomes (T7 and T3), and two T‐even phages with large, double‐stranded DNA genomes (T6 and RB69). Fitness was measured as absolute growth rate per hour under the same conditions used for adaptation. T7 and T3 achieved the highest fitnesses, able to increase by 13 billionfold and three‐quarters billionfold per hour, respectively. In contrast, the RNA phages achieved low fitness maxima, with growth rates approximately 400‐fold and 4000‐fold per hour. The highest fitness limits were not attributable to high mutation rates or small genome size, even though both traits are expected to enhance adaptation for fast growth. We suggest that major differences in fitness limits stem from different‘global” constraints, determined by the organization and composition of the phage genome affecting whether and how it overcomes potentially rate‐limiting host processes, such as transcription, translation, and replication. Adsorption rates were also measured on the evolved phages. No consistent pattern of adsorption rate and fitness was observed across the four different types of phages, but within each pair of related phages, higher adsorption was associated with higher fitness. Different adsorption rate limits within pairs may stem from “local” constraints–sequence differences leading to different local optima in the sequence space.

Experimental Evolution Yields Hundreds of Mutations in a Functional Viral Genome

Two lines of the bacteriophage T7 were grown to fix mutations indiscriminately, using a combination of population bottlenecks and mutagenesis. Complete genome sequences revealed 404 and 299 base substitutions in the two lines, the largest number characterized in functional microbial genomes so far. Missense substitutions outnumbered silent substitutions. Silent substitutions occurred at similar rates between essential and nonessential genes, but missense substitutions occurred at a higher rate in nonessential genes than in essential genes, as expected if they were less deleterious in the nonessential genes. Viral fitness declined during this protocol, and subsequent passaging of each mutated line in large population sizes restored some of the lost fitness. Substitution levels during these recoveries were less than 6% of those during the bottleneck phase, and only two changes during recovery were reversions of the original mutations. Exchanges of genomic fragments between the two recovered lines revealed that fitness effects of some substitutions were not additive—that interactions were accumulating which could lead to incompatibility between the diverged genomes. Based on these results, unprecedented high rates of nucleotide and functional divergence in viral genomes should be attainable experimentally by using repeated population bottlenecks at a high mutation rate interspersed with recovery.

Viral escape from antisense RNA

RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31–270 of the positive‐stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3–4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5′ region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson–Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition.