Gain-of-function experiments with bacteriophage lambda uncover residues under diversifying selection in nature

Maddamsetti, Rohan; Johnson, Daniel T.; Spielman, Stephanie J.; Petrie, Katherine L.; Marks, Debora S.; Meyer, Justin R.

To test whether the three critical mutations were sufficient to confer the OmpF + phenotype, we constructed a synthetic phage with just these mutations. Even though the synthetic phage was viable, it proved unable to exploit cells without the ancestral LamB receptor, demonstrating that at least four J mutations were necessary for OmpF + . To find what further mutations might be needed to confer the OmpF + phenotype, we constructed a second combinatorial library using the phage with the three critical J mutations as the baseline, and random combinations of 19 other J mutations found in the OmpF + λ evolved by Meyer et al. (2012) . These 19 included six of the original 10 mutations. Four mutations were excluded because they occur within the two codons we already modified. Eighty-eight OmpF + isolates were sequenced from this much larger library. One OmpF + isolate had just a single extra mutation at residue 1083 in addition to the three critical mutations (Fig. 1) . However, the majority of the engineered OmpF + phage did not possess this specific mutation, signifying that its function could be substituted by other J mutations. In total, this experiment revealed that four mutations are sufficient to evolve the innovation, but only two specific amino acid changes (at residues 1012 and 1107) are universally required to access OmpF in the context of these laboratory experiments.