Resolution of a T1-Like Bacteriophage Outbreak by Receptor Engineering.

Bacteriophage contaminations pose substantial risks to biomolecular production pipelines, and their resolution is especially difficult when the identity of the offending agent is unknown. We recently experienced an outbreak of Escherichia coli culture lysis in our Melbourne-based structural biology labs that halted protein production despite our use of T1-resistant (TonA/FhuA-disrupted) strains. Genetic analysis of the isolated phage yielded a 45,053 bp genome showing 80-90% identity with multiple Rtp-like siphophages, and transmission electron microscopy images were consistent with this classification. Further analysis revealed that our isolate was nearly identical to a highly virulent lytic coliphage MSK, recently isolated in Hangzhou, China, whose host receptor has not been determined. Sequence and structural modelling analysis of its putative receptor-binding protein suggested that its terminal receptor was likely to be LptD, an essential outer membrane protein involved in lipopolysaccharide transport. Based on a recent report of spontaneously arising mutations that blocked infection by other LptD-dependent bacteriophages, we designed a targeted genomic LptD loop deletion that successfully generated resistance to vB_EcoS_OzMSK in E. coli BL21(DE3) without apparent detriment to fitness. Here, we report a CRISPR-based, single-plasmid solution that will benefit other labs or facilities experiencing challenges due to LptD-dependent lytic phage outbreaks.