Infection assay for four λ isolates tested on six E. coli strains. Each panel shows a bacterial lawn with aliquots of the phage applied toit; darker regions indicate successful infections that clear the lawn. The phage isolates include the ancestor and three clones isolated from the same population on day 8 of the initial evolution experiment including one, EvoC, that can use the OmpF receptor. The bacterial strains include mutants that differ in the expression of LamB and OmpF porins on two genomic backgrounds, REL606 (the ancestral strain in the evolution experiments) and BW25113 (a derivative of K12). The malT – strains do not express LamB at appreciable levels.

Mutationsin the λ gene encoding theJ protein in three isolates from the same population on day 8 of the initial evolution experiment. The isolates are shown in rows and the mutations in columns, with the first letterbeing the ancestral nucleotide, the number the nucleotide position, and the last letter the evolved nucleotide. The gray fill indicates that the phage isolate has the corresponding mutation.

Mutations affecting the J protein in phage isolates from 48 independent populations of the large-scale experiment. Isolates are shown in rows (with alternate labels offset for readability) and mutations in columns; gray fill indicates an isolate has the mutation. The top 24 rows show phage isolates that can target the new OmpF receptor; the bottom 24 rows show phage that remain dependent on LamB.

Replay experiments using different combinations of phage and bacteria. For each panel, the y-axis shows the proportion of replicate replays that produced phage able to target the new OmpF receptor. Top panel: Replays were initiated with the ancestral bacteria and six phage isolates. Each combination was replicated 12-fold. Three of the phage (H2, F6, and C4) had mutations shared by multiple lineages that evolved the capacity to target OmpF in the large-scale experiment. The other three (D8, H3, and D10) had mutations that were never observed in phage that targeted OmpF. The latter mutations were candidates for impeding the evolution of the new function, but that hypothesis was not supported. Middle panel: Replays were initiated with phage EvoA (which needs only one more mutation to use OmpF) and six bacterial clones. Each combination was replicated 4-fold. Three clones (EcA8, EcC3, and EcD4) were isolated from flasks in which phage evolved the capacity to target OmpF in the large-scale experiment. The other three (EcH2, EcF6, and EcC4) came from flasks in which phage did not evolve that function. The replay outcomes did not support these categories, but sequencing the bacterial genomes identified mutations that uniquely determined whether the phage would evolve the OmpF function. See text for details. Bottom panel: Replays were initiated using the same phage and bacteria used in the middle panel, except with full bacterial communities rather than individual clones. Each combination was replicated 12-fold. The different outcomes for one bacterial clone (EcA8, middle) and its source community (ComA8, bottom) show the effect of bacterial diversityon phage evolution.

Steps in the coevolution of phage λ and its E. coli host leading to the phage’s ability to target a new receptor, OmpF. (1) The ancestral phage targets the LamB porin using the J protein and injects its DNA into the periplasm, then the DNA is transported into the cytoplasm via the ManXYZ permease. (2) The bacteria evolve resistance by mutations in malT, a positive regulator of LamB expression. (3) However, spontaneous inductions of LamB generate a subpopulation of phenotypically sensitive cells that can sustain the phage population. (4) The phage evolves mutations in the J protein that improve performance on the LamB receptor. Some of these mutations are also required for the phage to target OmpF. (5) The phage eventually evolves the four mutations that enable it to use OmpF. (6) However, the bacteria may evolve additional resistance by mutations in manY or manZ that prevent transport of the phage DNA into the cytoplasm. When these mutants become sufficiently common, there is little or no benefit to mutant phage that can use OmpF.