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.

Here, we use a harmless virus, bacteriophage λ, to demonstrate how gain-of-function experiments can identify mutations and evolutionary processes that mirror those that occur in nature. Typical laboratory strains of λ infect Escherichia coli by binding to the outer membrane protein LamB (Chatterjee and Rothenberg A960V R989R A990V K992T D996E I997V I997M A1000V S1011R S1012G T1040M V1048D S1049R T1053K A1076V V1077A I1083T H1104Y I1122F L1127P nucleotide C2879T A2966G C2969T A2975C C2988A A2989G T2991G C2999T C3033A A3034G C3119T T3143A C3147G A3158A C3227T T3230C T3248C C3310T G3319A A3320G T3321A A3364T T3380C 1 0 1 0 0 1 1 1 0 1 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 1 0 1 0 1 0 1 0 0 0 0 0 1 1 0 1 0 1 0 0 1 0 0 1 1 1 0 1 0 0 0 0 0 0 0 1 1 0 1 0 1 1 0 0 0 0 1 0 1 0 1 0 1 1 0 0 0 0 1 1 0 1 0 0 1 0 1 0 0 1 1 1 0 1 0 0 0 0 0 0 0 1 1 0 1 0 0 1 0 1 0 0 1 1 1 0 1 0 1 0 0 0 0 0 0 1 0 1 0 0 1 1 0 0 0 1 0 0 0 1 1 1 1 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 1 1 1 1 0 1 0 0 0 0 0 0 1 0 1 1 1 1 0 0 0 0 1 1 1 0 1 0 1 1 0 0 0 0 1 1 0 1 0 2012), but the phage rapidly evolves in the laboratory to exploit a different membrane protein, OmpF (Meyer et al. 2012; Meyer et al. 2016; Petrie et al. 2018) . These experiments are a proxy for the ability of the phage to switch hosts. The evolved gainof-function phenotype in λ, OmpF + , involves multiple nonsynonymous mutations in the host-recognition gene J. Each OmpF + isolate in Meyer et al. (2012) had between 4 and 10 single nucleotide substitutions in J, and none had insertions or deletions (indels). These nucleotide substitutions evolve in parallel across replicate experiments, indicating the action of strong positive selection rather than hypermutability. The 97% of the substitutions in 24 independently evolved OmpF + λ phage occurred in the J protein between residues 960-1132, which we call the "specificity region" of J (Fig. 1A) . Not much is known about the J protein structure and how exactly these mutations modify J. Homology based analyses of the protein reveal that they fall outside two structural domains of the J protein (a Phage-tail 3 domain spanning residues 330-500 and a DUF1983 domain spanning residues 845-925) and overlap with a third: a DUF3672 domain spanning residues 949-1090.