RT Journal Article SR Electronic T1 Deep metagenomic mining reveals bacteriophage sequence motifs driving host specificity JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.02.07.527309 DO 10.1101/2023.02.07.527309 A1 Phil Huss A1 Kristopher Kieft A1 Anthony Meger A1 Kyle Nishikawa A1 Karthik Anantharaman A1 Srivatsan Raman YR 2023 UL http://biorxiv.org/content/early/2023/02/09/2023.02.07.527309.abstract AB Bacteriophages can adapt to new hosts by altering sequence motifs through recombination or convergent evolution. Where such motifs exist and what fitness advantage they confer remains largely unknown. We report a new method, Bacteriophage Library Informed Sequence Scoring (BLISS), to discover sequence motifs in metagenomic datasets governing phage activity. BLISS uses experimental deep mutational scanning data to create sequence profiles to enable deep mining of metagenomes for functional motifs which are otherwise invisible to searches. We experimentally tested 10,073 BLISS-derived sequence motifs for the receptor-binding protein of the T7 phage. The screen revealed hundreds of T7 variants with novel host specificity with functional motifs sourced from distant families besides other major phyla. Position, substitution and location preferences on T7 dictated different specificities. To demonstrate therapeutic utility, we engineered highly active T7 variants against urinary tract pathogens. BLISS is a powerful tool to unlock the functional potential encoded in phage metagenomes.Competing Interest StatementThe authors have declared no competing interest.