Finally, thank you to all of the authors, the University of Colorado core facilities, and members of the Whiteley lab for making this work possible!

While we investigated two phage triggers in detail, we anticipate that other candidate phage trigger-ECOR pairs will be valuable dataset for determining the molecular mechanisms of activation for many additional antiphage systems.

Together these data show an alternative approach to findings new defense systems and characterizing known defense systems.

Alphafold 3 modeling supported these findings! The major capsid proteins share little sequence identity but have shared structural features, supporting the hypothesis that Avs8 recognizes conserved motifs.

Transposon mutagenesis screening also led to the identification of λ major capsid protein as the activator of Avs8, an antiphage system previously named PD-λ-4. We show direct binding between HK97-fold proteins and Avs8, and activation of nuclease activity.

But how do we now identify the bacterial gene(s) responsible for phage ORF plasmid loss? Transposon mutagenesis! When examining the genetic interaction between ECOR03 and T7 tail fiber (gp17), we identified a novel 2 gene operon that inhibits growth and provides robust phage defense, named PD-T2-1.

Activation of many antiphage systems results in death/slowed growth of the bacterium, which allowed us to use NGS to monitor plasmid abundance. We looked for proteins that selectively inhibited growth of specific ECOR strains. This revealed over 100 candidate phage triggers of antiphage systems.

We cloned over 400 plasmids that expressed every open reading frame (ORF) from 6 representative DNA and RNA phages. This “ORFeome” was transformed into the diverse 72 strains of the E. coli reference (ECOR) collection, each of which harbors a unique arsenal of known antiphage systems.

This work identified a new defense system, explained a known defense system, and provides a wealth of data for future investigations.