Convergent mutations in phage virion assembly proteins enable evasion of Type I CBASS immunity.

CBASS is an anti-phage defense system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by viral DNA but the stage of phage replication which activates bacterial CBASS remains unclear. Here we define the specificity of Type I CBASS immunity using a comprehensive analysis of 975 operon-phage pairings and show that Type I CBASS operons composed of distinct CD-NTases, and Cap effectors exhibit striking patterns of defense against dsDNA phages across five diverse viral families. We demonstrate that escaper phages evade CBASS immunity by acquiring mutations in structural genes encoding the prohead protease, capsid, and tail fiber proteins. Acquired CBASS resistance is highly operon-specific and typically does not affect overall fitness. However, we observe that some resistance mutations drastically alter phage infection kinetics. Our results define late-stage virus assembly as a critical determinant of CBASS immune activation and evasion by phages.