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.

Recall that 97% of substitutions in OmpF + gain-of-function experiments occur in the specificity region. Likewise, the natural J homologs had disproportionate variation here: 29% of the total amino acid variation occurred in the specificity region, despite it only being 16% of the total length of J ( Fig. 2A) . As we will discuss later, peaks in amino acid variation correspond to peaks in indel variation (Fig. 2B ). This nonrandom clustering of variation in the specificity region strongly suggests that J has experienced substantial diversifying selection on host attachment. Furthermore, the 17 residues with substitutions in the screened OmpF + isolates (excluding a synonymous R989R substitution) were significantly more variable than randomly chosen groups of 17 residues from J (nonparametric bootstrap: P < 10 −5 ) and from the specificity region 960-1132 (nonparametric bootstrap: P = 0.00075) showing the experiments had identified evolutionary hotspots. However, the specific 19 substitutions that evolved at these 17 residues in the laboratory were not common in natural homologs, suggesting that our experiments had the resolution to predict where changes would evolve, but not the exact change (Fig. S1 ). Focusing in on the two residues critical for the OmpF + gain of function, 1012 and 1107, we find that they are more variable than random pairs of sites in J (nonparametric bootstrap: P = 0.00055) and the specificity region (nonparametric bootstrap: P = 0.020). Finally, we calculated evolutionary rates for each site in the specificity region, controlling for recombination (Fig. S3) . We found that the 17 residues studied in the gain-of-function experiments evolve faster than equally sized random samples taken from the specificity region (nonparametric bootstrap, P = 0.012) and residues 1012 and 1107 evolve faster than random pairs of sites sampled from the specificity region (nonparametric bootstrap, P = 0.0086).