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.

The most variable and rapidly evolving regions in the J homologs, including the critical residues 1012 and 1107, are hotspots for in-frame indels. We measured protein indel variation using gap entropy and found that most amino acid sites have none (mode and median gap entropy = 0), with a few notable exceptions. In the specificity region, we see four peaks in gap entropy (Fig. 2B ) that correspond to peaks in sequence entropy (Fig. 2A) . The purple and green peaks overlap residues 1012 and 1107, which are critical for the OmpF + gain of function. The yellow and orange peaks occur at residues 1048 and 1077 that were not essential for OmpF use; however, λ evolved many mutations during the experiment near these positions (residues 1048, 1049, 1053, 1076, 1077, and 1083) . Indels can have large beneficial effects on proteins, including altering specificity by changing surface loops (Chatterjee and Rothenberg 2012; Porcek and Parent 2015) , or causing structural rearrangements that improve function (Arpino et al. 2014) . We hypothesize that the four peaks represent distinct surface loops that affect host specificity, much as influenza's hemagglutinin contains variable-length surface loops that affect binding to avian and human host receptors (Peacock et al. 2017; Tzarum et al. 2017) . Four additional peaks in gap entropy were observed in a region of J that did not evolve in the laboratory. Two particular peaks are notable because they are separated by a plateau caused by one indel nested within another. We are unsure of what consequence this variation has for λ function and evolution.