Bacteriophages use an expanded genetic code on evolutionary paths to higher fitness

Abstract

Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a noncanonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquire neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability.

Figure 1. Genome evolution of a bacterial virus with a newly expanded genetic code

a, In the evolution experiment, six populations each of wild-type (WT) and hypermutator (Δ2) bacteriophage T7 were passaged on a Release Factor 1 knockout (RF0) E. coli host strain carrying an orthogonal tRNA and aminoacyl-tRNA synthetase system that recodes the amber stop codon to the non-natural amino acid 3-iodotyrosine. b, E. coli host strains used to test whether evolved phages required amber suppression by 3-iodotyrosine or tyrosine to replicate. c, Relative titer (number of plaques) formed by each evolved bacteriophage population on the host strains in b. Values are the average of three technical replicates with error bars that are 95% confidence limits. P-values for reduced phage number on the alternative hosts compared to RF0 IodoY are for Student's t-tests assuming equal variance and using the Bonferroni correction for multiple testing. d, Locations of point mutations with frequencies ≥5% observed in metagenomic DNA sequencing data for the four bacteriophage populations boxed in c. Red open reading frames encode T7 proteins with known functions. Mutations of interest in regions highlighted in yellow are discussed in the text and following figures. There were roughly 50 and 400 mutations present with frequencies ≥1% in the evolved WT and Δ2 T7 populations, respectively.

Figure 2. Beneficial amber mutation in the T7 holin II protein

a, The frequency of an amber mutation resulting in a tyrosine to 3-iodotyrosine substitution in the T7 type II holin (gene 17.5) can explain the reduced titer of population WT L6 on the standard genetic code host (Fig. 1c). Signatures of molecular evolution described in the text strongly suggest that this mutation increases phage fitness on the RF0 IodoY E. coli host. b, Rescue mutants of an evolved WT L6 phage isolate with this amber mutation were obtained by plating on E. coli BL21(DE3) hosts with the standard genetic code and isolating fast-growing plaques. Of the 8 possible neighboring codons accessible by single-base mutations that code for amino acids, only tyrosine and tryptophan mutants were observed. The numbers of rescue mutants found with each codon are indicated at the end of the arrows. c, The evolved phage isolate with the amber codon (TAG) at position 39 of the holin protein was competed against rescue mutants with a tyrosine (TAC) or tryptophan (TGG) at that position. In each competition, lysates of the two phage preconditioned separately were mixed and then passaged on RF0 IodoY cells for three transfers. Sanger sequencing traces at this position show that the amber codon increases in frequency relative to the alternative, indicating that 3-iodotyrosine confers a fitness benefit compared to both the ancestral amino acid and a chemically similar amino acid tolerated at this position.