Evolution of phage with chemically ambiguous proteomes

Abstract

Background: The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.

Results: The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.

Conclusions: A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.

Figure 1

Fitness of ancestral and selected phage on various tryptophan analogues. Ancestral and round 25 selected phage were tested for fitness on eight additional tryptophan analogues (95% analogue, 5% W) for Line 1 (left) and Line 2 (right). Analogues used were 4-fluorotryptophan (4fW), 5-fluorotryptophan (5fW), 4-methyltryptophan (4MeW), 5-methyltryptophan (5MeW), 6-methyltryptophan (6MeW), 7-methyltryptophan (7MeW), 5-hydroxytryptophan (5OHW) and 5-methoxytryptophan (5MeOW). Data for fitness on 95% 6fW and W are taken from Figures 3 and 5 respectively. Error bars represent standard deviations of at least three replicates.

Figure 2

Genotype changes over the course of the selection. A phylogeny of the two lines, along with associated mutations, is shown. Evolution on W is in black, while selection carried out on 6fW is indicated in red; the number of cycles of selection are indicated beside the lines. Mutations are color coordinated as indicated at the bottom of the figure; mutations in green are in A2, black is the coat protein, orange indicates a mutation in A1, and green in the replicase; genotypes with no mutations are indicated by 'wt.' Lower case mutations correspond to base changes; upper case indicates protein substitutions.

Figure 3

Fitness of selected populations on 95%6fW. Populations were tested for fitness on 95%6fW at various points over the course of the selection. Error bars represent standard deviations of at least three replicates.

Figure 4

Specific mutations found in Qβ phage selected on W and 6fW. Of the upper portion of the figure, the first column indicates the protein affected by mutations, the second column indicates the specific genetic mutation and the third column indicates the expected protein mutation. Later columns represent specific populations or clones as indicated. Mutations indicated in the second and third columns are present in the phage in question if a particular cell is filled with the color corresponding to the specific protein in question, as indicated in the first column. The lower portion of the figure presents a summary of the mutations found.

Figure 5

Fitness of selected populations on W. Populations were tested for fitness on W at various points over the course of the selection. Error bars represent standard deviations of at least three replicates.

Figure 6

Distribution of mutations in the Qβ genome. (A) Missense and (B) Silent mutations are mapped onto the genome of Qβ. Short bars represent locations of codons for tryptophan, mid-sized bars represent mutations found only in clones and full-height bars represent mutations found in populations. Mutations extending upwards show mutations found in Line 1, while mutations extending downwards from the genome represent mutations found in Line 2.